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The proteome of spore surface layers in food spoiling bacteria

Abhyankar, W.R.

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Citation for published version (APA):Abhyankar, W. R. (2014). The proteome of spore surface layers in food spoiling bacteria.

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Proteome of Spore Surface Layers

W

ishwas A

bhyankar 2014

The Proteome of Spore Surface Layers in Food Spoiling Bacteria

Wishwas Abhyankar

Uitnodiging

voor het bijwonen van de openbare

verdediging van mijn proefschrift op

woensdag 2 april 2014

om 12;00 uur

in deAgnietenkapel

Oudezijds Voorburgwal 231

Amsterdam

Na afloop bent u van harte welkom op de

receptie.

Wishwas Abhyankar0643125239

[email protected]

Paranimfen

Sacha Stelder0645992393

[email protected]

Linli Zheng0659712898

[email protected]

200905-os-Abhyankar.indd 1 25-02-14 11:53

The Proteome of Spore Surface Layers in

Food Spoiling Bacteria

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

Prof. Dr. D.C. van den Boom

ten overstaan van een door het college voor promoties ingestelde

commissie, in het openbaar te verdedigen in de Agnietenkapel

op woensdag 2 april 2014, te 12:00 uur

door

Wishwas Ravindra Abhyankar

geboren te Pune, India

Promotiecommissie

Promotor: Prof. Dr. C. G. de Koster

Prof. Dr. S. Brul

Co-promotor: Dr. L. J. de Koning

Overige Leden: Prof. Dr. A. Driks

Prof. Dr. E. Ricca

Prof. Dr. M. Peck

Prof. Dr. R. Kort

Prof. Dr. P. J. Schoenmakers

Faculteit der Natuurwetenschappen, Wiskunde en Informatica

The research described in this thesis was carried out in the Mass spectrometry of Biomacromolecules lab as well as in the Molecular Biology and Microbial Food Safety lab of the Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands. The research was funded by the Erasmus Mundus Scholarship programme Window 15 (EMECW15) and the University of Amsterdam.

To raise new questions, new possibilities, to regard old problems from a new angle,

requires creative imagination and marks real advance in science.

Albert Einstein

Contents

Abbreviations 7 Chapter 1 9 Introduction & Outline of the thesis Chapter 2 35 Gel-free proteomic identification of the Bacillus subtilis insoluble spore coat protein fraction Chapter 3 51 In Pursuit of Protein Targets: Proteomic characterization of bacterial spore outer layers Chapter 4 79 Proteomic characterization of spore coat protein mutants of Bacillus subtilis Chapter 5 95 Probing progress in protein cross-linking during spore maturation in Bacillus subtilis Chapter 6 113 Molecular properties of Spore Surface proteins Chapter 7 135 General discussion and outlook Appendix 145 Summary 161 List of Publications 167 Acknowledgements 169

7

Abbreviations

2-DE Two Dimensional Electrophoresis ACN Acetonitrile AMPA Anti-Microbial Peptide assessment Algorithm APEX Absolute Protein Expression ATCC American Type Culture Collection AUC Area Under the Curve BATH test Bacterial Adherence to Hydrocarbons test BHIS medium Brain Heart Infusion Supplemented medium CDAD Clostridium difficile-Associated Diarrhoea CDGS medium Chemically Defined Growth and Sporulation medium COFRADIC Combined Fractional Diagonal Chromatography DNA Deoxyribonucleic Acid DPA Dipicolinic Acid DTT Dithiothreitol DUF Domain of Unknown Function EFSA European Food Safety Authority EM Electron Microscopy emPAI Exponentially Modified Protein Abundance Index ESI Electrospray Ionization FT-ICR Fourier Transform Ion Cyclotron Resonance GFP Green Fluorescent Protein GRAVY Grand Average of Hydropathy HIC Hydrophobic Interaction Chromatography HPLC High Performance Liquid Chromatography IAA Iodoacetamide ICAT Isotope Coded Affinity Tags IEF Isoelectric Focusing IPD Intrinsic Protein Disorder iTRAQ Isobaric Tags for Relative and Absolute Quantification kDa kiloDalton KEGG Kyoto Encyclopedia of Genes and Genomes LC-MS Liquid Chromatography Mass Spectrometry m/z Mass (m) to Charge (z) ratio MAL Muramic Acid Lactam MALDI Matrix-Assisted Laser Desorption/Ionization MHCPEP database Major Histocompatibility Complex-binding Peptide MOPS 3-(N-Morpholino) Propanesulfonic acid MRM Multiple Reaction Monitoring MS Mass Spectrometry MS/MS Tandem Mass Spectrometry MUDPIT Multidimensional Protein Identification Technology NAG N-Acetyl Glucosamine NAM N-Acetyl Muramic acid NCBI National Center for Biotechnology Information NTD N (amino)-Terminal Domain PAI Protein Abundance Index PDB Protein Data Bank pI Isoelectric point

8

PMF Peptide Mass Fingerprinting PMSF Phenyl Methyl Sulfonyl Fluoride POPI Prediction of Peptide Immunogenicity PSI-BLAST Position-Specific Iterated Basic Local Alignment Search Tool Q-ToF Quadrupole Time of Flight SASPs Small Acid Soluble Proteins SC Spectrum Count SCLEs Spore Cortex Lytic Enzymes SDS Sodium Dodecyl Sulfate SDS-PAGE Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis SILAC Stable Isotope Labeling with Amino acids in Cell culture SMC broth Sorbitol MacConkey broth TEP buffer Tris-Ethyl di-amine tetra-acetic acid Phosphate buffer TFA Tri-Fluoroacetic Acid TGY medium Tryptone Glucose Yeast extract medium TIC Total Ion Chromatogram TLC Thin Layer Chromatography TLPs Thermolysin-like peptidases TMHMM Trans-membrane Hidden Markov Model TMHs Trans-membrane Helices TMT Tandem Mass Tags TNF Tumor Necrosis Factor TSB medium Tryptic Soy Broth medium

1 General Introduction

Chapter 1

10

Stress response and Sporulation

The stress response in bacteria enables bacteria to survive extreme and fluctuating conditions in their immediate surroundings. Various bacterial mechanisms recognize different environmental changes and build an appropriate response. A bacterial cell can react simultaneously to a wide variety of stresses and the various stress response systems interact with each other by a complex of global regulatory networks[1]. These regulatory systems govern the expression of more effectors that maintain stability of the cellular equilibrium under the various conditions.

Most culturable bacteria grow and survive in rich media used to cultivate them in the laboratory. Nevertheless, in natural environments, the nutrient availability is a major hurdle for their growth. Nutrient supply is affected by diverse conditions like availability of water, competition with the other bacterial species in the environment etc. Also in certain environments, like oceans, the nutrients are extremely diluted. However, bacteria have evolved some characteristic mechanisms to adapt to such “starvation” conditions. The soil bacterium Bacillus subtilis, other Bacillus spp., anaerobic Clostridium spp. and related organisms can form endospores - small, metabolically dormant cellular structures that are remarkably resistant to heat, desiccation, radiation and chemical insult - in response to nutrient starvation. A variety of alternative responses can occur, including the activation of flagellar motility to search for new food sources by chemotaxis, the production of antibiotics to destroy competing soil microbes, the secretion of hydrolytic enzymes to scavenge extracellular proteins and polysaccharides, or the induction of ‘competence’ for uptake of exogenous DNA for consumption, with the occasional side-effect that new genetic information is stably integrated [2]. It is only after alternative responses have proven to be inadequate to relieve the stress, that sporulation is the fate chosen by a majority of the cells. Sporulation is an irreversible process where as other stress responses can be rapidly reversed by elimination of stress. Figure 1 (A) summarizes the possible fates for Bacillus subtilis cells upon nutrient starvation. The sporulation process holds to the fact that the state of dormancy achieves continuation of life and synchronization of developing stages of life forms with the environments or periods to which they are adapted. Sporulation is set-in as a response to a single stress (e.g. nutrient starvation) but the spores are resistant to many multiple stress conditions. Factors inducing sporulation and the germinants that break the dormancy both can be seen as the indicators of unfavourable and favourable periods, respectively. Thus, spore formation, albeit a stress response, may represent a timing device that perpetuates viability during unfavorable periods.

Sporulation cycle

The master regulator responsible for the decision to differentiate from a starving vegetative cell into a dormant spore is Spo0A[3]. An advanced sensing system orchestrates and directs the stress response into a phosphate flow through a network of kinases, which ends with the accumulation of Spo0A-P (phosphorylated Spo0A [4]). The level of Spo0A-P defines three stages in the decision-making process (see Figure 1 (B)

General Introduction

11

[5]). When the levels of Spo0A-P are low, the AbrB–Rok interaction cascade closes the path towards competence development. The Rap system together with two two-component sensing systems performs the “early assessment stage” [6, 7]. The assessment involves governing the probability to escape towards competence and governing the progression toward sporulation. After Spo0A-P reaches a threshold level S1, the cell enters into the “decision stage”[8], during which the AbrB–Rok circuit opens a time window of opportunity (called a “competence window”) to escape into competence or make a final commitment to sporulation. The third and final “commitment stage,” is reached when Spo0A-P is accumulated above the threshold level S2 [9]. The SinI-SinR signaling circuit is turned on by Spo0A-P leading to commitment to sporulation.

Figure 1. (A) Vegetative dormancy versus Sporulation. The σB-dependent general stress response and competence as components of a survival strategy alternative to sporulation. (Adapted from Hecker & Völker [10]) (B) Role of Spo0A in decision of sporulation. Depending on the levels of Spo0A, a cell goes through 3 stages of decision-making before it commits itself to sporulation. Spo0A* represents phosphorylated Spo0A or Spo0A-P. (Adapted from Schultz et al. [11])

The stages in the sporulation cycle of Bacillus subtilis are shown in Figure 2. The sequence of morphological changes resulting in the formation of a dormant endospore has been demonstrated by electron microscopy in the past and is similar for all species of Bacillus and Clostridium that have been examined. Although the process is continuous, it is convenient to divide it into different stages. Considering the vegetative cells at Stage 0, the sporulation division produces two distinct cells with very different fates, the smaller prespore (also known as the forespore), which develops into the spore, and the mother cell, which is necessary for spore formation but ultimately lyses (programmed cell death). The condensation stage, where the two nuclei of the vegetative cell (post-asymmetric cell division) fuse to form a single axial filament of chromatin, was originally defined as Stage I. Completion of a septum formed by membrane invagination and growth at one pole of the cell takes places in Stage II. Soon after the division, distinct programs of gene expression are initiated in the two cell types. These are directed by sporulation-specific RNA polymerase σ factors, σF in the prespore and σE in the mother cell. Freese [12] has suggested that at least some peptidoglycan synthesis is necessary at this time to give direction to the membrane synthesis and to ensure that a septum is formed. The

(A) (B)

Chapter 1

12

peptidoglycan that is synthesized during septation is then apparently digested away [13] so that the bacterium can proceed to Stage III. In this stage, a protoplast is formed within the mother cell. After division, the prespore is engulfed by the mother cell. On completion of the engulfment, there is another transition in transcription, with σG becoming active in the prespore and σK in the mother cell (Figure 3). Stage IV is the deposition of primordial germ cell wall and cortex between the membranes of the spore protoplast. Deposition of the spore coat around the cortex defines Stage V and Stage VI involves the "maturation" of the spore, at which time it develops its characteristic resistant properties. During Stage VII, the mother cell lyses and releases the completed spore. After the release, the spore undergoes further maturation [14]. These changes in gene regulation along with morphogenesis and the inter-compartmental signaling, finally lead to the development of the resistance characteristics of the mature spore.

Figure 2. Stages in the sporulation cycle. See text for the details.

Endospore structure

The resistance properties of spores are the result of its well assembled multi-layered structure. The outer layers called exosporium and coat are mainly responsible for resistance and transmission of spores. Particularly, the proteinaceous coat surrounding the spore provides much of the chemical and enzymatic resistance. Beneath the coat resides a very thick layer of peptidoglycan called the cortex. Proper cortex formation is needed for dehydration of the spore core, which aids in resistance to high temperature. A germ cell wall resides under the cortex. This layer of peptidoglycan will become the cell wall of the bacterium after the endospore germinates. The inner membrane, under the germ cell wall, is a major permeability barrier against several potentially damaging chemicals. The center of the endospore, the core, exists in a very dehydrated state and houses the cell’s DNA, ribosomes and large amounts of dipicolinic acid (DPA). DPA can comprise up to 10% of the spore’s dry weight and appears to play a role in maintaining spore dormancy. Small acid-soluble proteins (SASPs) are also only found in spores. They tightly bind and condense the DNA, and are in part responsible for resistance to UV light and DNA-damaging chemicals. Distinct spore layers are discussed below (see also Figure 4 (A) & (B)).


13

The core The core is the innermost part of the spore and it contains the cytoplasm with

cytoplasmic, ribosomal proteins and DNA. The physical state of the core cytoplasm is distinct with a water content of only 30-50%, as opposed to 70-88% in a vegetative cell cytoplasm [15]. This dehydrated state plays an important role in spore endurance, dormancy and resistance [16]. The pH in the spore core is ~6.5, which is lower than the pH of the vegetative cell cytoplasm [17]. The core contains large quantities of small acid soluble proteins i.e. SASP [18] which form a complex with the spore DNA and protect it

Figure 3. Morphogenesis and gene regulation during spore formation. (Adapted from Piggot & Hilbert, 2004 [19])

Figure 4. Spore structure (A) B. subtilis, (B) B. anthracis. [Abbreviations: spore core (Cr), cortex peptidoglycan layer (Cx), undercoat region (Uc), inner (Ic) & the outer (Oc) coat layers, exosporium (Ex), basal layer (Bl) and a hair-like glycoprotein nap (Hn)] (Adapted from Henriques & Moran, 2007 [20])

against many types of damages [21] by keeping it in a compressed state [22]. Proteins in the core are largely immobile and the divalent cations in the core, mainly Ca2+ form a complex with the spore-specific compound pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA [23]). DPA also plays a role in wet heat resistance [24] and UV resistance [25]. The amount of DPA in the core accounts for about 5-15% of the total spore weight [26]. DPA and calcium are excreted from the core during the first step of germination, and play important roles in further steps of the germination process [27].

(B)

(A) (A)

(B)

(C)

(D)

Chapter 1

14

The inner membrane The spore core is surrounded by the inner membrane. Upon germination it becomes the plasma membrane of the new vegetative cell and can expand to about 2-fold without any new lipid synthesis. The lipids in the inner membrane of dormant spores are also immobile, while in germinated spores and vegetative cells, the membrane is fluid and membrane lipids are highly mobile [28]. The inner membrane is proposed to be the main permeability barrier of spores. It accommodates the spore germination receptors [29]. Furthermore, the inner membrane is a target for several sporicidal chemicals [30]. The cortex and germ cell wall The cortex is composed of a specifically modified peptidoglycan, layered around the inner membrane. The cortex is of central importance for maintaining the spore core dehydrated and thereby resistance and dormancy characteristics of spores [31]. Recent results indicated that the cortex merely serves as a static structure that maintains spore core dehydration [31]. The cortex peptidoglycan is loosely cross-linked [32] and variation in the degree of cross-linking within the cortex region may play a role in the pressure that the cortex exerts on the core [31]. This mechanistic pressure has been suggested to be important for core dehydration. The cortex peptidoglycan has two unique structural modifications [33] (Figure 5). Approximately, half of the muramic acid in the cortex is present in the form of muramic acid lactam (MAL). These lactam residues are found alternating with NAM (N-acetyl muramic acid) residues within the polyglycan strands. Also, about 25% of the NAM monomers have only an L-alanine side chain [34]. The peptides on the remaining NAM residues are cross-linked with each other [33, 34]. The function of the MAL residues remains unclear. The inner part of the cortex called the germ cell wall lacks the specific modifications that are characteristic of the cortex peptidoglycan. The specific structure of the cortex is conserved among species, and may play a role in spore heat resistance, but it has been shown that the volume ratio of the cortex peptidoglycan to the germ cell wall contributes to the formation of a more heat-resistant spore in C. perfringens [35]. During germination, the cortex peptidoglycan is rapidly degraded by the spore cortex lytic enzymes (SCLEs) already present in the dormant spore. The germ cell wall is not degraded upon germination and forms the initial cell wall of the freshly germinated spore [31]. The outer membrane Around the cortex lies the relatively poorly studied outer membrane. The outer membrane may have a function during spore formation but some studies in the past concluded that the outer membrane is quite permeable even to large molecules [36]. Nonetheless, the outer membrane is also identified as the main permeability barrier in bacterial spores [37]. It is reported to be intact in dormant B. megaterium spores [38] and a functional barrier to the diffusion of large ions in B. subtilis and B. cereus spores [27].


15

Figure 5. Structure of B. subtilis spore peptidoglycan. NAM carries side chains of L-alanine, the tetrapeptide L-ala-γ-D-glu-diaminopimelic acid-D-ala, or the tripeptide L-ala-γ-D-glu-diaminopimelic acid. Approximately 50% of the muramic acid residues have been converted to MAL, which is found with great regularity at every second muramic acid position. (Adapted from Popham et al. [34]) The coat

The coat is the most extensively studied layer of spores [20, 39]. It is built around the outer membrane, and is a dynamic, intricate protein structure generally consisting of three distinct layers - an amorphous undercoat layer, a lamellar inner coat, and an electron-dense striated outer coat [40]. The coat proteins constitute up to 10% of the spore dry weight and up to 25% of the entire spore proteome [41]. The coat protects the cortex peptidoglycan from enzymatic attack [42]. Furthermore, it is involved in resistance to environmental UV radiation [43, 44], to a variety of chemicals including oxidative agents [45, 46] but not significantly in resistance to wet heat [39, 47]. The coat contains enzymes, such as laccases, which may be active even when the spore core is devoid of metabolic activity [48, 49]. These enzymes may have a significant function in spore ecology[50]. The inner layer of the coat carries lytic enzymes, which help degrading the cortex during germination [51, 52]. Several other coat proteins are also involved in spore germination, by facilitating the passage of specific germinant molecules through the coat [53]. Recently, with the application of techniques new to spore-research, such as atomic force microscopy [54] and automated scanning microscopy [55], it has become clear that the coat is a very dynamic structure. However, 30% of the coat proteins constitute an insoluble protein fraction, characterized by extensive inter-protein cross-liking [20], that is resistant to proteolytic enzymes and thus remains to be studied in detail. Researchers have suggested the possible presence of di-tyrosine crosslinks [56, 57], ε-γ-glutamyl-lysine crosslinks [58] and the di-sulfide linkages among the proteins in this fraction. Nevertheless, the role of many coat proteins in cellular physiology remains to be elucidated. The exosporium and the crust

In many species the spore coat is surrounded by a loose, membrane-like, glycoproteinaceous structure called the exosporium (Figure 3). The exosporium is important for spore hydrophobicity and adherence properties [59-61]. Since long, spores of B. cereus, B. anthracis and the Clostridium spp. are known to possess an exosporium [62]. B. subtilis spores are, in contrast, devoid of an exosporium. As it is difficult to

Chapter 1

16

obtain sufficient quantities of the exosporium for study, the structure has received little attention over the years. Nevertheless, with renewed efforts in recent years the exosporium from B. cereus and B. anthracis has been analyzed in considerable detail. To this extent the BclA protein from the exosporium has been studied extensively for its structure as well as it role in spore-macrophage interactions [63, 64]. Similar to the important antigens that could serve for detection and identification [65, 66], the exosporium has been found to contain a number of enzymes, such as alanine racemase, nucleoside hydrolases, immune inhibitors etc. some of which are possibly involved in germination [67-69]. Another interesting finding is the presence of a manganese oxidizing enzyme in the exosporium from a marine Bacillus spp. which, in the natural environment, encases the spore in a metal shell, thereby increasing its resistance [70]. Recently, it was found that the ExsA protein is indispensable for anchoring the exosporium to the coat, while the ExsA equivalent i.e. SafA in B. subtilis 168 is essential for proper coat assembly [71]. Several other proteins of the B. cereus exosporium have homology to B. subtilis coat proteins, and therefore it is foreseen that the exosporium is a specialized and further decorated coat layer [71]. Lastly, a layer similar to the exosporium was assigned recently to the Bacillus subtilis spores named ‘crust’ [72]. This layer is claimed to be a glycoproteinaceous layer surrounding the coat. Interestingly, a layer similar to the B. subtilis crust spore surface was recently identified also from the B. anthracis spore in a freeze-etching study [73] indicating commonalities among the spore structure. Spores in Food Industry

In the late 1700s, Nicolas Appert invented the process of appertization, now known as canning, to extend the food quality, shelf life and to prevent the spoilage of food over a longer time. He believed that the elimination of air was responsible for the stability of canned food. But later in 1875, Cohn & Koch for the first time discovered spores and at the same time Pasteur discovered that spore-forming bacteria caused food spoilage during investigations of butyric acid fermentation in wines. These contributions linked microbial activity with food quality and safety. In the late 1800s and early 1900s Prescott and Underwood (Massachusetts Institute of Technology, USA) along with Russell (University of Wisconsin, USA) found that spore-forming Bacilli caused the spoilage of thermally processed clams, lobsters, and corn [74]. Russell also showed that gaseous swelling with bad odors in canned peas was due to growth of heat-resistant bacterial spores [75]. Since then diseases and spoilage caused by spore formers are associated with thermally processed foods, as heat kills the vegetative cells but allows survival and growth of spore-forming organisms. In comparison to the other food-borne vegetative pathogenic bacteria, spores survive better under conditions prevailing in the food and during food processing and thus causing big problems for the food industries. Three species of spore-formers - Clostridium botulinum, Clostridium perfringens, and Bacillus cereus, are especially notorious toxin producers. Other spore forming species cause spoilage. Sporulating bacteria causing food-borne illness and spoilage are particularly important in low-acid foods (pH ≥ 4.6) packaged in cans id est “canned” foods, which are processed by heat.


17

Other sporeformers cause spoilage of high-acid foods (pH ≤ 4.6). Psychrotrophic sporeformers such as Bacillus weihenstephanensis cause spoilage of refrigerated foods (See Table 1). Meat products are also often contaminated with spores of anaerobic Clostridia [76]. Modeling inactivation of food-borne pathogens has been one of the first achievements in predictive microbiology. Inactivation models were initially focused on the destruction of C. botulinum spores in low acid canned foods [77, 78]. According to the recent report of European Food Safety Authority (EFSA) [79], mild heat treatment (e.g. very lightly cooked food or pasteurization) of a few seconds at 70°C permits several log10 units of inactivation of vegetative bacterial pathogens and parasites, but may not be sufficient for food-borne viruses and will not inactivate bacterial spores nor bacterial toxins formed in foods. Also cooking of foods for several minutes at 90°-100°C practically eliminates vegetative bacterial pathogens, food-borne viruses, and parasites allowing several log10 units of inactivation of spores of non-proteolytic C. botulinum (psychrotolerant C. botulinum), but not of pathogenic Bacillus and other pathogenic Clostridium spp. Additionally the toxins from C. botulinum would be inactivated but not the emetic toxin from B. cereus. In general, heating to 100°C may allow survival of thermoduric spores, while lower temperatures may lead to spore activation. Spores cannot grow in dry fat, but they may grow in formulated products or in formulations with moist materials in processed foods [80]. However, according to the EFSA report a reliable inactivation of spores of pathogenic bacteria in high aw (water activity) foods can be achieved by sterilization treatments (e.g. 3 min at 120°C). Killing of spores by inducing their germination and subsequently inactivating the vegetative cells by pasteurization level thermal treatments are thought to be good preservation options for the food industries. In addition, preservatives like weak organic acids (e.g. sorbic acid), are being studied for their effects on spore germination and outgrowth [81, 82]. In order to minimize or eliminate the problems caused by the spores, it is very important to detect and estimate the amount of spores in the food and from the patient’s sample. Efficient removal of spores is possible when simple and quick spore detection and removal systems are available. To build such systems it becomes necessary to understand the properties of spores as well as to gain the knowledge about the entire sporulation and germination pathways. Efforts in the past have led to the understanding of the mechanism of sporulation and the signaling cascade that initiates the whole cycle yet the complete sporulation models have not been mapped for most of the spore formers. Also the signaling mechanisms that operate when a dormant spore germinates yet remain to be uncovered although the gene-expression processes operative during germination have been described [83, 84]. Medical significance of spores

Bacterial spores came into focus in 2001 after the Anthrax bio-terror attacks in the USA. The attacks used spore powder from B. anthracis and the spores after germination spread the Anthrax disease to people killing 22 of them. According to the Federal Bureau of Investigations (FBI), the ensuing investigation became "one of the largest and most complex in the history of law enforcement". In hospitals, spores from C. difficile are often

Chapter 1

18

Table 1. Spoilage of canned foods by sporeformers.

Adapted from Food Microbiology: Fundamentals and Frontiers, 3rd Ed.[85] responsible for the spread of infections. For instance, C. difficile is responsible for a number of diseases of the intestines, including Clostridium difficile-associated disease (CDAD). Also, C. botulinum is the causative organism of the potentially fatal disease - botulism, caused by an extracellular toxin produced during spore germination. The toxins produced by B. cereus can cause two types of illness: one type characterized by diarrhea and the other, by nausea and vomiting and thus B. cereus is of major concern in the food industry. The primary reservoirs of these pathogens are infected (and colonized) patients in hospitals and healthcare facilities. The hands of healthcare workers, which may become transiently colonized with these spore-formers, are the primary sources, although to a less and more controversial extent environmental surfaces, on which endospores can survive for weeks or months, also appear to play a role in the nosocomial transmissions [86].

Effective decontamination of infectious agents on critical and other hospital surfaces will drastically reduce nosocomial infections and impacts of any biological

Type of spoilage pH Major sporeformers responsible

Spoilage defects

Flat sour ≥ 5.3 B. coagulans B. stearothermophilus

No gas, pH lowered. May have abnormal odor and cloudy liquor.

Thermophilic anaerobe

≥ 4.8 C. thermosaccharolyticum The can swells, may burst. Anaerobic end products give sour, fermented, or butyric odor. Typical foods are spinach, corn.

Sulfide spoilage ≥ 5.3 D. nigrificans C. bifermentans

Hydrogen sulfide produced, giving rotten egg odor. Iron sulfide precipitate gives blackened appearance. Typical foods are corn, peas.

Putrefactive anaerobe ≥ 4.8 C. sporogens Plentiful gas. Disgusting putrid odor. pH often increased. Typical foods are corn, asparagus.

Psychrotrophic Clostridia

> 4.6 C. estertheticum C. algidicarnis

Spoilage of vacuum-packaged chilled meats. Production of gas, off flavors and odors, discoloration.

Aerobic sporeformers ≥ 4.8 Bacillus spp. Gas usually absent except for cured meats; milk is coagulated. Typical foods are milk, meat, beets.

Butyric spoilage ≥ 4.0 C. butyricum C. tertium

Gas, acetic and butyric odor. Typical foods are tomatoes, peas, olives, cucumbers.

Acid spoilage ≥ 4.2 B. thermoacidurans Flat (Bacillus) or gas (butyric anaerobes). Off odors depend on organism. Common foods are tomatoes, tomato products etc.

< 4 Alicyclobacillus acidoterrestris

Flat spoilage with off flavors. Most common in fruit juices, acid vegetables, and also reported to spoil iced tea.


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attack. In a recent study, using simulations of endospore-laden surfaces under laboratory conditions, the effects of exposure time, disinfectant concentration and possible synergies with endospore germination stimulant were evaluated. None of the disinfectants tested was found to be sporicidal [87]. Also, killing the non-sporulated vegetative cells causing the infection does not ensure the death of the spores at the same time. The spores can germinate & further re-initiate the infection later. Hence, for spore forming bacteria, the medical procedure has to be designed to get rid of the bacteria as well as the spores. Mass spectrometry (MS)-driven Proteomics

The word “proteome” is derived from the proteins expressed by a genome, and it refers to all the proteins produced by an organism. Proteins are important players in a living organism, involved in a plethora of activities which eventually lead to a particular cellular phenotype. As a consequence, protein analysis is of major interest in cellular and molecular biology and involves protein identification, quantification, localization as well the study of post-translational modifications. To achieve this, many methods starting from Edman sequencing methodology [88] to mass spectrometry (MS) - based proteomics, have been invented and developed over the past few decades. Prior to mass spectrometry-based proteomics, proteome analysis required separation of proteins followed by protein sequencing through Edman sequencing. Thus several different gel-based techniques have been developed that make use of physicochemical properties of proteins to separate them. These methodologies involved separation based on - the isoelectric potentials of proteins i.e. isoelectric focusing (IEF) [89], the molecular weight of proteins i.e. SDS-poly acrylamide gel electrophoresis (SDS-PAGE) [90] and both isoelectric point as well as molecular weight in a two-dimensional gel electrophoresis (2-DE) approach [91]. After separation, proteins could be visualized on the gel by different means such as Coomassie Brilliant blue staining, silver staining or immunoblotting methods followed by Edman sequencing of the proteins from the spots evident on the gels [92]. This approach required a large amount of protein material allowing in most cases only the abundant proteins to fall in the scope of the method. Through the advent of new ionization methods such as electrospray ionization (ESI) [93], matrix-assisted laser desorption/ionization (MALDI) [94, 95], much more sensitive techniques of mass spectrometry became available for protein detection and analysis, requiring much less protein material. Inside the mass spectrometer, the peptides are fragmented by cleavage of, most commonly, peptide bonds thereby generating fragments ions of different charge states. Cleavage of peptide bond leads to formation of two fragments. With regards to these fragments if one peptide fragment retains the positive charge at the carboxyl (C) - terminus of the peptide ion, the ion is called a y-ion. If the fragment retains the positive charge at the amino (N) - terminus, the ion is known as a b-ion. The amount of the y and b-ions generated from the peptides is generally an indicative of the quality of the spectrum generated for that peptide. Thus protein identification is achieved by matching all the spectra generated from the peptides identified from the protein to the theoretical spectra generated for the peptides with the help of sequence databases. Accurate mass measurements of peptides by mass

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spectrometry essentially developed further as more and more genome and/or protein sequences were made available in databases wherein recorded masses could be matched. In a routine proteomic analysis, after gel-based separation, a protein spot is subjected to in-gel digestion using proteases such as trypsin, after which the resulting peptides are extracted and their masses are determined by mass spectrometry. The list of peptide masses, resulting from a single gel-spot, is then used as a fingerprint to identify the protein using a protein sequence database and a specialized search engine in a method denoted as peptide mass fingerprinting (PMF). The major drawbacks of PMF are that the amino acid sequence of protein cannot be directly determined in addition to the fact that the proteins must be purified prior to digestion. As an alternative, peptide-centric proteomics developed as a new standard [96] for proteome analyses. This approach focuses on the separation of peptides by liquid chromatography rather than the separation of proteins. Chromatography can be performed in-line coupled to tandem mass spectrometry (MS/MS), where resulting spectra allow determination of peptide sequences, which can then be used to infer the parent proteins [97]. In this approach multiple peptides can be utilized to identify proteins and since peptides are less extreme in their physicochemical parameters such as the molecular mass, the hydrophobic or hydrophilic nature, the isoelectric points etc., peptide-centric proteomics becomes highly sensitive. In addition, peptide-centric proteomics also allows qualitative proteomics (comprehensively mapping the presence of all the proteins in the sample) and quantitative proteomics (quantification of changes in protein abundance between samples). Both areas of research are covered in some detail below. Qualitative proteomics

Proteome coverage is mainly influenced by three factors - (a) the sensitivity of the mass spectrometer id est, in this context, the lowest amount of the sample that can be detected; (b) the dynamic range of the instrument or the signal intensity range of the instrument, (c) the duty cycle of the mass spectrometer id est the number of fragmentation spectra (with a fair amount of complementary b and y-ions) that the mass spectrometer can produce within a given time frame. In tandem mass spectrometry (MS/MS), data acquisition with the aid of computers is a routine practice. The data acquisition depends on the ion abundance of the peptides in the sample. Also in a complex peptide mixture, co-elution of certain peptides is routinely observed. Therefore the number of ions co-eluting can significantly affect the number of ions for which tandem mass spectra can be acquired. As a result, the data acquisition can be biased against the low abundant ion signals representing the peptides present at low levels. This also leads to random sampling of ions for fragmentation in the mass spectrometer. Requirement of sample fractionation to reduce complexity of samples prior to the mass spectrometry is also essential for complex proteome sample. Moreover, since a proteomics method does not inherently involve amplification of sample peptides, when a low abundant or any protein is not identified, no inference can be drawn on whether the protein is absent in the sample or it falls outside the detection limits of the instrument used. This has not constrained the qualitative proteomics to produce continuously increasing lists of identified peptides


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thereby increasing the proteome coverage. Over the years, researchers have invented strategies to avoid problems arising due to random sampling and to reduce complexity of the peptide mixture resulting from proteome digestion. The digestion of proteins followed by separation of obtained peptides by liquid chromatography (LC) - MS/MS analysis id est a shotgun proteomics strategy or selection and analysis of only a targeted set of peptides related to proteomic experiment id est a targeted proteomics strategy are the main approaches in proteomics studies. An overview of the proteome composition is readily generated by shotgun proteomics strategies (e.g. Multidimensional Protein Identification Technology (MUDPIT) [98] and Gel LC-MS/MS [99]) in which many proteins are identified by multiple peptide sequences per protein, which increases the reliability of such identifications. Whereas in targeted proteomics strategies (e.g. Combined Fractional Diagonal Chromatography (COFRADIC) and Multiple Reaction Monitoring (MRM) [100-103]) the selection of set of peptides is such that it represents the analyzed proteome or target proteins, respectively. Since a selection of a subset of peptides yields a less dense peptide mixture, random sampling tends to be reduced. In addition, targeted proteomics can also be applied in identification of post-translationally modified peptides and yet many possible protein modifications remain challenge in current proteomics framework. With the development of software tools such as MASCOT [104] and OMSAA [105], the identification of proteins by database searching has becomes more accurate as in such cases probabilistic scoring matrices are used to score for the peptide identity. In routine practice protein or peptide MASCOT scores above 20 are accepted as reliable identifications. To consider the role of post-translational modifications in protein chemistry and to study them in the perspective of “omics” strategies and systems biology, measurement of their abundance in a variety of conditions is mandatory. Quantitative proteomics

Qualitative analysis provides a compositional map of proteins which can be extended with relative or absolute abundance information by quantitative proteomics. Quantitative proteomics deals with samples that may vary in cellular phenotypes, provided stimuli, time durations of the stimulus or many other cellular states in which difference in protein composition can be expected [106]. Quantitative studies are mainly done by two approaches. In one approach, mass tags, such as isotopic labels, are added which allow differentiation between peptides from distinct samples. Such mass tags can be administered into proteins or peptides metabolically, by chemical means and enzymatically [107, 108]. Figure 6 illustrates different quantitative proteomics strategies based on the use of mass tags. As seen, the in vivo techniques involve incorporation of a stable isotope into the proteins which can be achieved via the addition of an isotope of an element (e.g. 13C, 15N, or 18O as salts or amino acids) to the growth media in a form that makes it suitable for incorporation into the cell, tissue or even the entire organism [109]. In these cases, protein identification is carried out by analyzing the fragmentation spectra of at least one of the co-eluting ‘heavy’ and ‘light’ peptides and subsequently the relative quantitation is achieved by comparing the intensities of isotope clusters of the intact

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peptides in the combined LC-MS spectrum. The heavy isotope of nitrogen (15N) is more commonly used as the stable isotope due to the lower number of nitrogen atoms in a peptide compared to the number of carbon atoms. For the same reason, with 13C as a label, the mass shift in the heavy peak compared to its light equivalent can be very large, making quantitative analysis challenging. Higher number of carbon atoms also makes it difficult to get a completely labeled proteome in short time, as a considerable exchange of 12C and 13C takes place within the cell during various cellular activities. Deuterium (2H) labeling, though efficient, is rarely used due to its potential toxic effects on the organism under study [110]. The technique developed by Ong and Mann [111], called stable isotope labeling with amino acids in cell culture (SILAC; Figure 6 (A)), has been widely applied and has proved suitable for a variety of organisms [112-115]. In a routine SILAC experiment, 13C/15N-labeled lysine and/or arginine is used, that gives a fixed mass difference for the peptides when trypsin or Lys-C is used as a protease. However, one of the limitations faced by this technique is that the cells or the organism to be used in these experiments needs to be an auxotroph for the labeled amino acid(s) to confirm that the labeled amino acid is the only source for protein synthesis. Also especially in eukaryotes, arginine can be metabolically converted to proline [116] thereby interfering with the protein quantification if arginine is used for SILAC-labeling [109]. For in vitro labeling (Figure 6 (B)), typically, isotope coded affinity tags (ICAT) that label the cysteines in the proteins can be used but the success of the method depends on the amount of cysteines available in the proteins as well as on good purification methods. The relative abundances of the peptides can then be determined comparing the ratio of the light and heavy forms of each peptide. Likewise, using isobaric tags for relative and absolute quantification (iTRAQ) or tandem mass tags (TMT) that label the N-termini and the Lysine-side chains in the digested peptides, quantification can be achieved. These tags contain four regions - a mass reporter (M), a cleavable linker (F), a mass normalizer (N) and a protein reactive group (R). Structurally all the tags are identical but they differ in the positions of isotope substitutions, such that the mass reporter and mass normalization regions differ in their molecular masses in each tag. These combined M-F-N-R regions of the tags have the same total molecular weights and thus during chromatographic or electrophoretic separation and in single MS mode, molecules labeled with different tags are not distinguished. Further, during peptide fragmentation, the isobaric amine groups, introduced by the iTRAQ and TMT labels, are also fragmented giving reporter ions with distinct m/z ratios. Relative peptide abundances are then estimated by comparing the intensities of these reporter ions [117, 118]. Therefore, for such studies sufficient fragmentation of the labelled peptides is of prime importance. Since, the in vitro labeling is performed after protein extraction it remains independent of the source and preparation of the sample allowing practically any type of biological sample to be labelled. Also, the time needed for the chemical or in vitro tagging is generally much shorter than when a label is incorporated metabolically where it may take weeks to in vivo label the organisms or cells depending on the growth rate. The in vivo labeling techniques are still preferred over the in vitro labeling methods due to their ingrained advantages that (i) the in vivo labeling does not suffer from side reactions or incomplete labeling that might occur in


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Figure 6. (A) In vivo and (B) In vitro approaches for labeling of proteins and/ or peptides for mass spectrometry based quantitative proteomics. See the text for details. chemical derivatization and (ii) the metabolic labeling occurs at the earliest possible moment in the sample preparation process, thus minimizing the errors in quantification. Isotope labeling may involve a number of technical difficulties such as the specific requirements for metabolic labeling or the problems of reproducible chemical labeling. Thus, in the second quantitative proteomics approach, id est label-free quantification, aligned peak intensity profiles from LC-MS or LC-MS/MS analyses are integrated to find the differences in protein abundances. Quantitative protein comparisons are based on the relative intensities of extracted ion chromatograms from their tryptic peptides. Resulting datasets, usually in triplicate, are aligned using peptide mass and LC retention times. The spectral count (SC) based label-free method is easy, fast and correlates well with isotope-labeling quantification [119]. It involves counting the number of peptide MS/MS spectra assigned to a protein in an LC-MS/MS experiment and allows both relative and absolute quantification of protein abundance. The second method takes into account the chromatographic peak area under the curve (AUC) or the signal intensity measurement of the peptide precursor ion in the MS spectra [120]. In an alternate approach proposed by Rappsilber et al. [121], the protein abundance index (PAI) can be calculated by calculating the ratio of the observed unique peptides and theoretical number of tryptic peptides within a given m/z range for a certain protein in the sequence database. This method was further improved by Ishihama and co-workers [122], where the

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exponentially modified PAI (emPAI) was proposed as a direct estimation of the protein amount in the sample. However, the emPAI method fails in case that the sample contains highly abundant proteins or if mass spectrometers with low resolution are used for the analysis [107]. A recently proposed method, called absolute protein expression (APEX) [123], based on the probability of detection of the peptides by MS makes use of a machine learning classification algorithm for peptide length and residue composition. However for this purpose the initial training set of peptide queries for the algorithm is crucial especially if the sample contains unknown proteins. To circumvent these problems the use of the average of the total ion count for the identified tryptic peptides of a the protein id est the total ion chromatogram (TIC) has also been proposed [124]. Albeit all these efforts, the label-free experiments need to be more carefully controlled, due to possible error caused by run-to-run variations in performance of LC and MS. However, the development of highly reproducible nano-HPLC separation, high resolution mass spectrometers and delicate computational tools has been aimed at to improve the reliability and accuracy of label-free, quantification. Yet, the label-free approaches are less accurate among the mass spectrometry based quantification techniques when considering the overall experimental process because all the variations between experiments are reflected in the obtained data. Consequently, the number of experimental steps should be minimum and reproducibility at each step needs to be controlled. Nevertheless, label-free quantification is worth considering for a number of reasons including costs, simpler experimental steps, the proteome coverage of the sample and comparison of multiple samples or experimental conditions. Spore Proteomics

Due to their structure and composition, spores are particularly difficult to lyse. Various enzymatic, chemical and physical methods have been mentioned in the literature to solubilize the spore coat proteins from pure spore preparations. The thick layers of extensively cross-linked coat proteins and the complexity of the protein sample that can be solubilized from wild-type coats pose an analytical challenge. Approximately one-third of total coat protein in wild-type spores is resistant to extraction procedures normally used to solubilize the majority of proteins [125]. Differences have also been found in the solubility of different coat fractions [126] which could complicate the spore protein extraction further. In 1978, Goldman and Tipper thoroughly analyzed the spore coat protein fraction from B. subtilis strain 168. They could solubilize 65% of the protein from the spore coat whereby they identified a wide mass range (9000 – 16000 Da) of low-molecular weight peptides [127]. In their study, Goldman & Tipper used a two-step protein extraction procedure. In the first step, the coat fraction was subjected to 1% SDS & 50 mM dithiothreitol in sodium carbonate buffer (pH 10) followed by incubation at 37°C for 20 min. After centrifugation, the coat fractions were then subjected to 3% SDS & 2% β-mercaptoethanol in tris (hydroxymethyl)-aminomethane (Tris) - hydrochloride buffer (pH 6.8) followed by boiling at 100°C for 3 min. The protein purification, fractionation by classical SDS-gel electrophoresis and Sephadex chromatography was followed by protein characterization by either molecular approaches or more recently by


25

bioinformatics analyses. This effort paved the way for future studies as many groups used strong alkalis, detergents and reducing agents alone or in combination to solubilize, purify, identify and characterize the spore coat proteins [126, 128, 129].

With the advent of GFP-fusion studies, where a copy of GFP is tagged to the protein of interest, as well as with the progress in the transcriptomic i.e. microarray field, studying the expression and localization of spore coat proteins as well as the exosporium proteins in species forming spores with this additional outer layer, was possible. But it was by virtue of mass spectrometry that the study of proteome-wide data became easier. Thus combining all the methods discussed above, proteome characterization of B. megaterium [130, 131], B. cereus [132], B. anthracis [133], B. thuringiensis [134] as well as the members of the Clostridium family like C. perfringens [135], C. novyi [136], C. difficile [137-139] spore coats has been approached successfully. With all these efforts coalesced, till now, at least 70 proteins have been assigned to the coat. However, the spore coat structures from Clostridia (compared to the Bacilli) as well as the insoluble protein fraction from the coat have not yet attracted the focus. This fraction is the focus of our study presented in this thesis. These extensive efforts to study the spore coats have up till now allowed researchers to employ endospores in various applications. Cutting and co-workers have studied the potential of spores as probiotics in food [140, 141] as well as drug vehicles [142]. Use of spores as surface display systems has been suggested [143] and has been shown to be effective [144] in recent years. Further on, applications of spores as platforms for bio-analytical and biomedical applications are very well reviewed by Knecht and co-workers [145]. Food industries are especially in search of efficient, simple and quick methods to detect and control spores from processed food samples. Spore coat and exosporium proteins can prove apt for such purposes as seen from the studies mentioned above and thus it is necessary to pay focus on proteome analysis of the spore surface layers from many different spore formers. Outline of the thesis

Bacterial spores are a major problem in the food industry as well as the hospitals. Their high resistance allows them to escape the food processing treatments designed to inactivate bacteria. In the final food products, the spores can germinate, outgrow and multiply, leading to food-spoilage and intoxication. To reduce spore-related problems, the industries invest a substantial amount in food quality control and special cares are assigned in hospitals. Consumers nowadays prefer less processed food thereby allowing easy survival of spores. Also on the contrary, to kill the spores if food is treated with extreme measures then the nutritious properties as well as the organoleptic characters of food items are lost. Thus more thorough understanding of the resistant mechanisms of spores as well as the possible structural differences in spores of different species might enable the industries to design processes that will more efficiently eliminate spores. This would lead to an extended shelf-life of food products, a reduction of spoilage and poisoning events, cheaper processes and better product quality as well as restricted spore-mediated outbreaks.

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In this chapter the basic knowledge about sporulation, spore structures and the importance of spores are described. Mass spectrometry based proteomics strategies for qualitative and quantitative proteomics are also briefly discussed in this introductory chapter. With regards to the problem of gel-based identification of spore coat proteins, Chapter 2 describes the establishment of a comprehensive proteomics method that allowed us to focus on the insoluble protein fraction of spore coats in a lab-isolate as well as a food isolate. In Chapter 3, the extension of the newly developed method to genetically distinct spore forms that even belong to different bacterial domains (aerobe and anaerobe) is discussed and along with identification of few potential candidate marker proteins the relevance of the identified proteins to the spores is also discussed. Chapter 4 focusses on mass spectrometry based identification of possible interdependences amongst spore coat proteins in B. subtilis. Chapter 5 shows how mass spectrometry can be tailored to monitor the progress in spore coat maturation and thereby stresses the role of spore coat protein cross-linking. Finally, in Chapter 6, molecular properties of the proteins identified in our studies are summarized and placed in the perspective with future directions and a discussion on their practical applications. References

1. Requena JM, editor. Stress Response in Microbiology: Caister Academic Press. ; 2012. 2. Stephens C. Bacterial sporulation: a question of commitment? Current biology : CB. 1998;8(2):R45-8. Epub 1998/03/21. 3. Hoch JA. Regulation of the phosphorelay and the initiation of sporulation in Bacillus subtilis. Annual review of microbiology. 1993;47:441-65. Epub 1993/01/01. 4. Burbulys D, Trach KA, Hoch JA. Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell. 1991;64(3):545-52. Epub 1991/02/08. 5. Fujita M, Gonzalez-Pastor JE, Losick R. High- and low-threshold genes in the Spo0A regulon of Bacillus subtilis. Journal of bacteriology. 2005;187(4):1357-68. Epub 2005/02/03. 6. Dwyer DJ, Kohanski MA, Collins JJ. Networking opportunities for bacteria. Cell. 2008;135(7):1153-6. Epub 2008/12/27. 7. Henke JM, Bassler BL. Bacterial social engagements. Trends in cell biology. 2004;14(11):648-56. Epub 2004/11/03. 8. Hahn J, Roggiani M, Dubnau D. The major role of Spo0A in genetic competence is to downregulate abrB, an essential competence gene. Journal of bacteriology. 1995;177(12):3601-5. Epub 1995/06/01. 9. Bai U, Mandic-Mulec I, Smith I. SinI modulates the activity of SinR, a developmental switch protein of Bacillus subtilis, by protein-protein interaction. Genes & development. 1993;7(1):139-48. Epub 1993/01/01. 10. Hecker M, Volker U. General stress response of Bacillus subtilis and other bacteria. Advances in microbial physiology. 2001;44:35-91. Epub 2001/06/16. 11. Schultz D, Wolynes PG, Ben Jacob E, Onuchic JN. Deciding fate in adverse times: sporulation and competence in Bacillus subtilis. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(50):21027-34. Epub 2009/12/10. 12. Freese E. Sporulation of bacilli, a model of cellular differentiation. Current topics in developmental biology. 1972;7:85-124. Epub 1972/01/01. 13. Guinand M, Michel G, Tipper DJ. Appearance of gamma-D-glutamyl-(L) meso-diaminopimealate peptidoglycan hydrolase during sporulation in Bacillus sphaericus. Journal of bacteriology. 1974;120(1):173-84. Epub 1974/10/01.


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14. Sanchez-Salas JL, Setlow B, Zhang P, Li YQ, Setlow P. Maturation of released spores is necessary for acquisition of full spore heat resistance during Bacillus subtilis sporulation. Applied and environmental microbiology. 2011;77(19):6746-54. Epub 2011/08/09. 15. Setlow P. Mechanisms which contribute to the long-term survival of spores of Bacillus species. Society for Applied Bacteriology symposium series. 1994;23:49S-60S. Epub 1994/01/01. 16. Gould GW, Dring GJ. Heat resistance of bacterial endospores and concept of an expanded osmoregulatory cortex. Nature. 1975;258(5534):402-5. Epub 1975/12/04. 17. Setlow B, Setlow P. Measurements of the pH within dormant and germinated bacterial spores. Proceedings of the National Academy of Sciences of the United States of America. 1980;77(5):2474-6. Epub 1980/05/01. 18. Setlow P. Small, acid-soluble spore proteins of Bacillus species: structure, synthesis, genetics, function, and degradation. Annual review of microbiology. 1988;42:319-38. Epub 1988/01/01. 19. Piggot PJ, Hilbert DW. Sporulation of Bacillus subtilis. Current opinion in microbiology. 2004;7(6):579-86. Epub 2004/11/24. 20. Henriques AO, Moran CP, Jr. Structure, assembly, and function of the spore surface layers. Annual review of microbiology. 2007;61:555-88. Epub 2007/11/24. 21. Setlow P. Mechanisms for the prevention of damage to DNA in spores of Bacillus species. Annual review of microbiology. 1995;49:29-54. Epub 1995/01/01. 22. Douki T, Setlow B, Setlow P. Effects of the binding of alpha/beta-type small, acid-soluble spore proteins on the photochemistry of DNA in spores of Bacillus subtilis and in vitro. Photochemistry and photobiology. 2005;81(1):163-9. Epub 2004/10/02. 23. Powell JF. Isolation of dipicolinic acid (pyridine-2:6-dicarboxylic acid) from spores of Bacillus megatherium. The Biochemical journal. 1953;54(2):210-1. Epub 1953/05/01. 24. Paidhungat M, Setlow B, Driks A, Setlow P. Characterization of spores of Bacillus subtilis which lack dipicolinic acid. Journal of bacteriology. 2000;182(19):5505-12. Epub 2000/09/15. 25. Slieman TA, Nicholson WL. Role of dipicolinic acid in survival of Bacillus subtilis spores exposed to artificial and solar UV radiation. Applied and environmental microbiology. 2001;67(3):1274-9. Epub 2001/03/07. 26. Murrell WG, Warth, A.D. In: Campbell LL, Halvorson, H.O., editor. Spores III. Ann Arbor: ASM Press; 1965. p. 1. 27. Vries YPd, Voort Mvd, Schaik Wv, Hornstra LM, Vos WMd, Abee T. Progress in food-related research focussing on Bacillus cereus. Microbes and Environments. 2004;19(4):265-9. 28. Cowan AE, Olivastro EM, Koppel DE, Loshon CA, Setlow B, Setlow P. Lipids in the inner membrane of dormant spores of Bacillus species are largely immobile. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(20):7733-8. Epub 2004/05/06. 29. Paidhungat M, Ragkousi K, Setlow P. Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca(2+)-dipicolinate. Journal of bacteriology. 2001;183(16):4886-93. Epub 2001/07/24. 30. Cortezzo DE, Koziol-Dube K, Setlow B, Setlow P. Treatment with oxidizing agents damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stress. Journal of applied microbiology. 2004;97(4):838-52. Epub 2004/09/11. 31. Popham DL. Specialized peptidoglycan of the bacterial endospore: the inner wall of the lockbox. Cellular and molecular life sciences : CMLS. 2002;59(3):426-33. Epub 2002/04/20. 32. Popham DL, Setlow P. Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpE operon, which codes for penicillin-binding protein 4* and an apparent amino acid racemase. Journal of bacteriology. 1993;175(10):2917-25. Epub 1993/05/01. 33. Warth AD, Strominger JL. Structure of the peptidoglycan from spores of Bacillus subtilis. Biochemistry. 1972;11(8):1389-96. Epub 1972/04/11. 34. Popham DL, Helin J, Costello CE, Setlow P. Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance. Proceedings of the National Academy of Sciences of the United States of America. 1996;93(26):15405-10. Epub 1996/12/24.

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35. Orsburn B, Sucre K, Popham DL, Melville SB. The SpmA/B and DacF proteins of Clostridium perfringens play important roles in spore heat resistance. FEMS microbiology letters. 2009;291(2):188-94. Epub 2009/02/04. 36. Gerhardt P, Scherrer, R. & Black, S. H. Molecular sieving by dormant spore structures In: Halvorson HO, Hanson R. & Campbell, L. L. , editor. Spores V Washington, DC: American Society for Microbiology.; 1972. p. 68-74. 37. Nakashio S, Gerhardt P. Protoplast dehydration correlated with heat resistance of bacterial spores. Journal of bacteriology. 1985;162(2):571-8. Epub 1985/05/01. 38. Crafts-Lighty A, Ellar DJ. The structure and function of the spore outer membrane in dormant and germinating spores of Bacillus megaterium. The Journal of applied bacteriology. 1980;48(1):135-45. Epub 1980/02/01. 39. Driks A. Bacillus subtilis spore coat. Microbiology and molecular biology reviews : MMBR. 1999;63(1):1-20. Epub 1999/03/06. 40. Costa T, Steil L, Martins LO, Volker U, Henriques AO. Assembly of an oxalate decarboxylase produced under sigmaK control into the Bacillus subtilis spore coat. Journal of bacteriology. 2004;186(5):1462-74. Epub 2004/02/20. 41. Munoz L, Sadaie Y, Doi RH. Spore coat protein of Bacillus subtilis. Structure and precursor synthesis. The Journal of biological chemistry. 1978;253(19):6694-701. Epub 1978/10/10. 42. Driks A. Maximum shields: the assembly and function of the bacterial spore coat. Trends in microbiology. 2002;10(6):251-4. Epub 2002/06/29. 43. Moeller R, Schuerger AC, Reitz G, Nicholson WL. Protective role of spore structural components in determining Bacillus subtilis spore resistance to simulated mars surface conditions. Applied and environmental microbiology. 2012;78(24):8849-53. Epub 2012/10/16. 44. Riesenman PJ, Nicholson WL. Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation. Applied and environmental microbiology. 2000;66(2):620-6. Epub 2000/02/02. 45. Genest PC, Setlow B, Melly E, Setlow P. Killing of spores of Bacillus subtilis by peroxynitrite appears to be caused by membrane damage. Microbiology. 2002;148(Pt 1):307-14. Epub 2002/01/10. 46. Kim HS, Sherman D, Johnson F, Aronson AI. Characterization of a major Bacillus anthracis spore coat protein and its role in spore inactivation. Journal of bacteriology. 2004;186(8):2413-7. Epub 2004/04/03. 47. Koshikawa T, Beaman TC, Pankratz HS, Nakashio S, Corner TR, Gerhardt P. Resistance, germination, and permeability correlates of Bacillus megaterium spores successively divested of integument layers. Journal of bacteriology. 1984;159(2):624-32. Epub 1984/08/01. 48. Hullo MF, Moszer I, Danchin A, Martin-Verstraete I. CotA of Bacillus subtilis is a copper-dependent laccase. Journal of bacteriology. 2001;183(18):5426-30. Epub 2001/08/22. 49. Martins LO, Soares CM, Pereira MM, Teixeira M, Costa T, Jones GH, et al. Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat. The Journal of biological chemistry. 2002;277(21):18849-59. Epub 2002/03/09. 50. Nicholson WL. Ubiquity, longevity, and ecological roles of Bacillus spores. In: Ricca E, Henriques, A. O. and Cutting, S. M. , editor. Bacterial spore formers:probiotics and emerging applications. Wymondham, Norfolk UK: Horizon Bioscience; 2004. p. 1-15. 51. Bagyan I, Setlow P. Localization of the cortex lytic enzyme CwlJ in spores of Bacillus subtilis. Journal of bacteriology. 2002;184(4):1219-24. Epub 2002/01/25. 52. Ragkousi K, Eichenberger P, van Ooij C, Setlow P. Identification of a new gene essential for germination of Bacillus subtilis spores with Ca2+-dipicolinate. Journal of bacteriology. 2003;185(7):2315-29. Epub 2003/03/20. 53. Behravan J, Chirakkal H, Masson A, Moir A. Mutations in the gerP locus of Bacillus subtilis and Bacillus cereus affect access of germinants to their targets in spores. Journal of bacteriology. 2000;182(7):1987-94. Epub 2000/03/14.


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54. Zolock RA, Li G, Bleckmann C, Burggraf L, Fuller DC. Atomic force microscopy of Bacillus spore surface morphology. Micron. 2006;37(4):363-9. Epub 2005/12/27. 55. Westphal AJ, Price PB, Leighton TJ, Wheeler KE. Kinetics of size changes of individual Bacillus thuringiensis spores in response to changes in relative humidity. Proceedings of the National Academy of Sciences of the United States of America. 2003;100(6):3461-6. Epub 2003/02/14. 56. Henriques AO, Melsen LR, Moran CP, Jr. Involvement of superoxide dismutase in spore coat assembly in Bacillus subtilis. Journal of bacteriology. 1998;180(9):2285-91. Epub 1998/05/09. 57. Pandey NK, Aronson AI. Properties of the Bacillus subtilis spore coat. Journal of bacteriology. 1979;137(3):1208-18. Epub 1979/03/01. 58. Kobayashi K, Suzuki SI, Izawa Y, Miwa K, Yamanaka S. Transglutaminase in sporulating cells of Bacillus subtilis. The Journal of general and applied microbiology. 1998;44(1):85-91. Epub 2002/12/27. 59. Faille C, Jullien C, Fontaine F, Bellon-Fontaine MN, Slomianny C, Benezech T. Adhesion of Bacillus spores and Escherichia coli cells to inert surfaces: role of surface hydrophobicity. Canadian journal of microbiology. 2002;48(8):728-38. Epub 2002/10/17. 60. Koshikawa T, Yamazaki M, Yoshimi M, Ogawa S, Yamada A, Watabe K, et al. Surface hydrophobicity of spores of Bacillus spp. Journal of general microbiology. 1989;135(10):2717-22. Epub 1989/10/01. 61. Lequette Y, Garenaux E, Tauveron G, Dumez S, Perchat S, Slomianny C, et al. Role played by exosporium glycoproteins in the surface properties of Bacillus cereus spores and in their adhesion to stainless steel. Applied and environmental microbiology. 2011;77(14):4905-11. Epub 2011/05/31. 62. Matz LL, Beaman TC, Gerhardt P. Chemical composition of exosporium from spores of Bacillus cereus. Journal of bacteriology. 1970;101(1):196-201. Epub 1970/01/01. 63. Bozue J, Cote CK, Moody KL, Welkos SL. Fully Virulent Bacillus anthracis Does Not Require the Immunodominant Protein BclA for Pathogenesis. Infection and Immunity. 2007;75(1):508-11. 64. Réty S, Salamitou S, Garcia-Verdugo I, Hulmes DJ, Le Hegarat F, Chaby R, et al. The crystal structure of the Bacillus anthracis spore surface protein BclA shows remarkable similarity to mammalian proteins. The Journal of biological chemistry. 2005;280(52):43073-8. Epub 2005/10/27. 65. Fox A, Stewart GC, Waller LN, Fox KF, Harley WM, Price RL. Carbohydrates and glycoproteins of Bacillus anthracis and related bacilli: targets for biodetection. Journal of microbiological methods. 2003;54(2):143-52. Epub 2003/06/05. 66. Steichen C, Chen P, Kearney JF, Turnbough CL, Jr. Identification of the immunodominant protein and other proteins of the Bacillus anthracis exosporium. Journal of bacteriology. 2003;185(6):1903-10. Epub 2003/03/06. 67. Charlton S, Moir AJ, Baillie L, Moir A. Characterization of the exosporium of Bacillus cereus. Journal of applied microbiology. 1999;87(2):241-5. Epub 1999/09/04. 68. Redmond C, Baillie LW, Hibbs S, Moir AJ, Moir A. Identification of proteins in the exosporium of Bacillus anthracis. Microbiology. 2004;150(Pt 2):355-63. Epub 2004/02/10. 69. Todd SJ, Moir AJ, Johnson MJ, Moir A. Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium. Journal of bacteriology. 2003;185(11):3373-8. Epub 2003/05/20. 70. Francis CA, Casciotti KL, Tebo BM. Localization of Mn(II)-oxidizing activity and the putative multicopper oxidase, MnxG, to the exosporium of the marine Bacillus sp. strain SG-1. Archives of microbiology. 2002;178(6):450-6. Epub 2002/11/07. 71. Bailey-Smith K, Todd SJ, Southworth TW, Proctor J, Moir A. The ExsA protein of Bacillus cereus is required for assembly of coat and exosporium onto the spore surface. Journal of bacteriology. 2005;187(11):3800-6. Epub 2005/05/20. 72. McKenney PT, Driks A, Eskandarian HA, Grabowski P, Guberman J, Wang KH, et al. A distance-weighted interaction map reveals a previously uncharacterized layer of the Bacillus subtilis spore coat. Current biology : CB. 2010;20(10):934-8. Epub 2010/05/11. 73. Ball DA, Taylor R, Todd SJ, Redmond C, Couture-Tosi E, Sylvestre P, et al. Structure of the exosporium and sublayers of spores of the Bacillus cereus family revealed by electron crystallography. Molecular microbiology. 2008;68(4):947-58. Epub 2008/04/11.

Chapter 1

30

74. Montville TJ, Matthews, K. R. In: Matthews KR, editor. Food Microbiology: An Introduction. 2 ed. Washington,DC: ASM Press; 2008. p. 39-56. 75. Ray B, Bhunia, A. Fundamental Food Microbiology. 4 ed: CRC Press; 2007. p. 3-9. 76. Granum PE. Clostridium perfringens toxins involved in food poisoning. International journal of food microbiology. 1990;10(2):101-11. Epub 1990/03/01. 77. Hersom ACaH, E.D. Canned Foods Thermal processing and microbiology. 7 ed. Edinburgh, London and NewYork: Churchill Livingstone; 1980. 78. Jay JM. Modern Food Microbiology. 4 ed. New York: Chapman & Hall; 1992. 79. (BIOHAZ) EPoBH. Scientific Opinion on Public health risks represented by certain composite products containing food of animal origin. 2012. 80. Gill CO. Microbiology of edible meat by-products Edible Meat By-Products, Advances in Meat Research. London, United Kingdom: Elsevier Applied Science; 1988. 81. Ter Beek A, Keijser BJ, Boorsma A, Zakrzewska A, Orij R, Smits GJ, et al. Transcriptome analysis of sorbic acid-stressed Bacillus subtilis reveals a nutrient limitation response and indicates plasma membrane remodeling. Journal of bacteriology. 2008;190(5):1751-61. Epub 2007/12/25. 82. Pandey R, Ter Beek A, Vischer NO, Smelt JP, Brul S, Manders EM. Live cell imaging of germination and outgrowth of individual Bacillus subtilis spores; the effect of heat stress quantitatively analyzed with SporeTracker. PloS one. 2013;8(3):e58972. Epub 2013/03/29. 83. Keijser BJ, Ter Beek A, Rauwerda H, Schuren F, Montijn R, van der Spek H, et al. Analysis of temporal gene expression during Bacillus subtilis spore germination and outgrowth. Journal of bacteriology. 2007;189(9):3624-34. Epub 2007/02/27. 84. Ter Beek A, Hornstra LM, Pandey R, Kallemeijn WW, Smelt JP, Manders EM, et al. Models of the behaviour of (thermally stressed) microbial spores in foods: tools to study mechanisms of damage and repair. Food Microbiol. 2011;28(4):678-84. Epub 2011/04/23. 85. Setlow P, Johnson, E. A. . In: Doyle MPaB, L. R. , editor. Food Microbiology: Fundamentals and Frontiers. 3 ed. Washington, DC: ASM Press. p. 33-68. 86. Weinstein RA, Hota B. Contamination, Disinfection, and Cross-Colonization: Are Hospital Surfaces Reservoirs for Nosocomial Infection? Clinical Infectious Diseases. 2004;39(8):1182-9. 87. Moench I. Survival and Decontamination of Potential Bio-warfare Agents on Hospital Surfaces: Florida Atlantic University; 2005. 88. Edman P. A method for the determination of amino acid sequence in peptides. Archives of biochemistry. 1949;22(3):475. Epub 1949/07/01. 89. Svensson H, Woldbye F, Lindahl T, Palmstierna H, Sjöberg B, Toft J. Isoelectric Fractionation, Analysis, and Characterization of Ampholytes in Natural pH Gradients. I. The Differential Equation of Solute Concentrations at a Steady State and its Solution for Simple Cases. Acta chemica Scandinavica. 1961;15:325-41. 90. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-5. Epub 1970/08/15. 91. O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. The Journal of biological chemistry. 1975;250(10):4007-21. Epub 1975/05/25. 92. Lauber WM, Carroll JA, Dufield DR, Kiesel JR, Radabaugh MR, Malone JP. Mass spectrometry compatibility of two-dimensional gel protein stains. Electrophoresis. 2001;22(5):906-18. Epub 2001/05/03. 93. Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989;246(4926):64-71. Epub 1989/10/06. 94. Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Analytical Chemistry. 1988;60(20):2299-301. 95. Tanaka K, Waki H, Ido Y, Akita S, Yoshida Y, Yoshida T, et al. Protein and polymer analyses up to m/z 100 000 by laser ionization time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry. 1988;2(8):151-3. 96. Mann M, Wilm M. Error-Tolerant Identification of Peptides in Sequence Databases by Peptide Sequence Tags. Analytical Chemistry. 1994;66(24):4390-9.


31

97. Reisinger F, Martens L. Database on Demand - an online tool for the custom generation of FASTA-formatted sequence databases. Proteomics. 2009;9(18):4421-4. Epub 2009/09/03. 98. Washburn MP, Wolters D, Yates JR, 3rd. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nature biotechnology. 2001;19(3):242-7. Epub 2001/03/07. 99. de Godoy LM, Olsen JV, de Souza GA, Li G, Mortensen P, Mann M. Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system. Genome biology. 2006;7(6):R50. Epub 2006/06/21. 100. Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas GR, et al. Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides. Nature biotechnology. 2003;21(5):566-9. Epub 2003/04/01. 101. Gevaert K, Van Damme J, Goethals M, Thomas GR, Hoorelbeke B, Demol H, et al. Chromatographic isolation of methionine-containing peptides for gel-free proteome analysis: identification of more than 800 Escherichia coli proteins. Molecular & cellular proteomics : MCP. 2002;1(11):896-903. Epub 2002/12/19. 102. Grote E, Fu Q, Ji W, Liu X, Van Eyk JE. Using pure protein to build a multiple reaction monitoring mass spectrometry assay for targeted detection and quantitation. Methods Mol Biol. 2013;1005:199-213. Epub 2013/04/23. 103. Guo B, Chen B, Liu A, Zhu W, Yao S. Liquid chromatography-mass spectrometric multiple reaction monitoring-based strategies for expanding targeted profiling towards quantitative metabolomics. Current drug metabolism. 2012;13(9):1226-43. Epub 2012/04/24. 104. Perkins DN, Pappin DJC, Creasy DM, Cottrell JS. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis. 1999;20(18):3551-67. 105. Geer LY, Markey SP, Kowalak JA, Wagner L, Xu M, Maynard DM, et al. Open mass spectrometry search algorithm. Journal of proteome research. 2004;3(5):958-64. Epub 2004/10/12. 106. Jensen ON. Interpreting the protein language using proteomics. Nature reviews Molecular cell biology. 2006;7(6):391-403. Epub 2006/05/26. 107. Van Oudenhove L, Devreese B. A review on recent developments in mass spectrometry instrumentation and quantitative tools advancing bacterial proteomics. Applied microbiology and biotechnology. 2013;97(11):4749-62. Epub 2013/04/30. 108. Gingras AC, Gstaiger M, Raught B, Aebersold R. Analysis of protein complexes using mass spectrometry. Nature reviews Molecular cell biology. 2007;8(8):645-54. Epub 2007/06/28. 109. Gouw JW, Krijgsveld J, Heck AJ. Quantitative proteomics by metabolic labeling of model organisms. Molecular & cellular proteomics : MCP. 2010;9(1):11-24. Epub 2009/12/04. 110. Kushner DJ, Baker A, Dunstall TG. Pharmacological uses and perspectives of heavy water and deuterated compounds. Canadian journal of physiology and pharmacology. 1999;77(2):79-88. Epub 1999/10/27. 111. Ong SE, Mann M. Mass spectrometry-based proteomics turns quantitative. Nature chemical biology. 2005;1(5):252-62. Epub 2006/01/13. 112. Phillips NJ, Steichen CT, Schilling B, Post DM, Niles RK, Bair TB, et al. Proteomic analysis of Neisseria gonorrhoeae biofilms shows shift to anaerobic respiration and changes in nutrient transport and outermembrane proteins. PloS one. 2012;7(6):e38303. Epub 2012/06/16. 113. Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI, et al. Nucleolar proteome dynamics. Nature. 2005;433(7021):77-83. Epub 2005/01/07. 114. Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell. 2006;127(3):635-48. Epub 2006/11/04. 115. Soufi B, Kumar C, Gnad F, Mann M, Mijakovic I, Macek B. Stable isotope labeling by amino acids in cell culture (SILAC) applied to quantitative proteomics of Bacillus subtilis. Journal of proteome research. 2010;9(7):3638-46. Epub 2010/06/01. 116. Van Hoof D, Pinkse MWH, Oostwaard DW-V, Mummery CL, Heck AJR, Krijgsveld J. An experimental correction for arginine-to-proline conversion artifacts in SILAC-based quantitative proteomics. Nat Meth. 2007;4(9):677-8.

Chapter 1

32

117. Jain S, Graham C, Graham RL, McMullan G, Ternan NG. Quantitative proteomic analysis of the heat stress response in Clostridium difficile strain 630. Journal of proteome research. 2011;10(9):3880-90. Epub 2011/07/27. 118. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Molecular & cellular proteomics : MCP. 2004;3(12):1154-69. Epub 2004/09/24. 119. Li Z, Adams RM, Chourey K, Hurst GB, Hettich RL, Pan C. Systematic comparison of label-free, metabolic labeling, and isobaric chemical labeling for quantitative proteomics on LTQ Orbitrap Velos. Journal of proteome research. 2012;11(3):1582-90. Epub 2011/12/23. 120. Neilson KA, Ali NA, Muralidharan S, Mirzaei M, Mariani M, Assadourian G, et al. Less label, more free: approaches in label-free quantitative mass spectrometry. Proteomics. 2011;11(4):535-53. Epub 2011/01/19. 121. Rappsilber J, Ryder U, Lamond AI, Mann M. Large-scale proteomic analysis of the human spliceosome. Genome research. 2002;12(8):1231-45. Epub 2002/08/15. 122. Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J, et al. Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Molecular & cellular proteomics : MCP. 2005;4(9):1265-72. Epub 2005/06/17. 123. Lu P, Vogel C, Wang R, Yao X, Marcotte EM. Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation. Nature biotechnology. 2007;25(1):117-24. Epub 2006/12/26. 124. Asara JM, Christofk HR, Freimark LM, Cantley LC. A label-free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen. Proteomics. 2008;8(5):994-9. Epub 2008/03/08. 125. Serrano M, Zilhao R, Ricca E, Ozin AJ, Moran CP, Jr., Henriques AO. A Bacillus subtilis secreted protein with a role in endospore coat assembly and function. Journal of bacteriology. 1999;181(12):3632-43. Epub 1999/06/15. 126. Aronson AI, Fitz-James P. Structure and morphogenesis of the bacterial spore coat. Bacteriological reviews. 1976;40(2):360-402. Epub 1976/06/01. 127. Goldman RC, Tipper DJ. Bacillus subtilis spore coats: complexity and purification of a unique polypeptide component. Journal of bacteriology. 1978;135(3):1091-106. Epub 1978/09/01. 128. Donovan W, Zheng LB, Sandman K, Losick R. Genes encoding spore coat polypeptides from Bacillus subtilis. Journal of molecular biology. 1987;196(1):1-10. Epub 1987/07/05. 129. Jenkinson HF, Sawyer WD, Mandelstam J. Synthesis and Order of Assembly of Spore Coat Proteins in Bacillus subtilis. Journal of general microbiology. 1981;123(1):1-16. 130. Setlow P. Identification and localization of the major proteins degraded during germination of Bacillus megaterium spores. The Journal of biological chemistry. 1975;250(20):8159-67. Epub 1975/10/25. 131. Setlow P. Purification and properties of some unique low molecular weight basic proteins degraded during germination of Bacillus megaterium spores. The Journal of biological chemistry. 1975;250(20):8168-73. Epub 1975/10/25. 132. Aronson AI, Horn D. . Characterization of spore coat protein of Bacillus cereus T. In: Halvorson HO, Hanson R., Campbell, L.L. , editor. Spore V. Washington, DC: ASM Press; 1972. p. 19-27. 133. Lai EM, Phadke ND, Kachman MT, Giorno R, Vazquez S, Vazquez JA, et al. Proteomic analysis of the spore coats of Bacillus subtilis and Bacillus anthracis. Journal of bacteriology. 2003;185(4):1443-54. Epub 2003/02/04. 134. Aronson AI, Tyrell DJ, Fitz-James PC, Bulla LA, Jr. Relationship of the syntheses of spore coat protein and parasporal crystal protein in Bacillus thuringiensis. Journal of bacteriology. 1982;151(1):399-410. Epub 1982/07/01. 135. Ryu S, Labbe RG. Coat and enterotoxin-related proteins in Clostridium perfringens spores. Journal of general microbiology. 1989;135(11):3109-18. Epub 1989/11/01.


33

136. Plomp M, McCaffery JM, Cheong I, Huang X, Bettegowda C, Kinzler KW, et al. Spore coat architecture of Clostridium novyi NT spores. Journal of bacteriology. 2007;189(17):6457-68. Epub 2007/06/26. 137. Lawley TD, Croucher NJ, Yu L, Clare S, Sebaihia M, Goulding D, et al. Proteomic and genomic characterization of highly infectious Clostridium difficile 630 spores. Journal of bacteriology. 2009;191(17):5377-86. Epub 2009/06/23. 138. Permpoonpattana P, Phetcharaburanin J, Mikelsone A, Dembek M, Tan S, Brisson MC, et al. Functional characterization of Clostridium difficile spore coat proteins. Journal of bacteriology. 2013;195(7):1492-503. Epub 2013/01/22. 139. Permpoonpattana P, Tolls EH, Nadem R, Tan S, Brisson A, Cutting SM. Surface layers of Clostridium difficile endospores. Journal of bacteriology. 2011;193(23):6461-70. Epub 2011/09/29. 140. Cutting SM. Bacillus probiotics. Food Microbiology. 2011;28(2):214-20. 141. Hong HA, Duc LH, Cutting SM. The use of bacterial spore formers as probiotics. FEMS Microbiology Reviews. 2005;29(4):813-35. 142. Cutting SM, Hong, H.A., Baccigalupi, L., Ricca, E. Oral Vaccine Delivery by Recombinant Spore Probiotics. International Reviews of Immunology. 2009;28(6):487-505. 143. Mauriello EMF, Duc LH, Isticato R, Cangiano G, Hong HA, Felice MD, et al. Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner. Vaccine. 2004;22(9–10):1177-87. 144. Potot S, Serra CR, Henriques AO, Schyns G. Display of Recombinant Proteins on Bacillus subtilis Spores, Using a Coat-Associated Enzyme as the Carrier. Applied and environmental microbiology. 2010;76(17):5926-33. 145. Knecht L, Pasini P, Daunert S. Bacterial spores as platforms for bioanalytical and biomedical applications. Anal Bioanal Chem. 2011;400(4):977-89.

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2 Gel-free proteomic identification of the

Bacillus subtilis insoluble spore coat protein fraction

Wishwas Abhyankar, Alex Ter Beek, Henk Dekker, Remco Kort, Stanley Brul and Chris G. de Koster

Published in Proteomics, 2011, 11(23):4541-50

Supplementary material can be found at

http://onlinelibrary.wiley.com/doi/10.1002/pmic.201100003/suppinfo

Abstract Species from the genus Bacillus have the ability to form endospores, dormant cellular forms that are able to survive heat and acid preservation techniques commonly used in the food industry. Resistance characteristics of spores towards various environmental stresses are in part attributed to their coat proteins. Previously, seventy proteins have been assigned to the spore coat of Bacillus subtilis using SDS-PAGE, 2-DE gel approaches, protein localization studies and genome-wide transcriptome studies. Here we present a “gel-free” protocol that is capable of comprehensive B. subtilis spore coat protein extraction and addresses the insoluble coat fraction. Using LC-MS/MS we identified 55 proteins from the insoluble B. subtilis spore coat protein fraction, of which 21 are putative novel spore coat proteins not assigned to the spore coat until now. Identification of spore coat proteins from a B. subtilis food spoilage isolate corroborated a generic and ‘applied’ use of our protocol. To develop specific and sensitive spore detection and/or purification systems from food stuff or patient material, suitable protein targets can be derived from our proteomic approach. Finally, the protocol can be extended to study cross-linking amongst the spore coat proteins as well as for their quantification.

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Introduction In natural environments, the nutrient availability is a major hurdle for the growth of bacteria. However, bacteria have evolved a number of characteristic mechanisms to adapt to starvation conditions. Species from the genera Bacillus, Clostridium and related organisms, can form endospores when subjected to nutrient starvation. Spore development has been most extensively studied in the Gram positive model organism B. subtilis, the first spore-forming bacterium for which the genome sequence was reported [1]. The basic endospore structure is probably conserved across species. Endospore development consists of a series of complex morphological changes which begin with an asymmetric division producing two cellular compartments of very different size. In B. subtilis, each of the two different cell types formed contains one complete copy of the chromosome, exhibits different programs of gene expression and differs in its developmental fate [2-4]. The smaller prespore is engulfed by the larger mother cell, and ultimately becomes the spore. The engulfed prespore, or forespore, exists as a free protoplast delimited by a double membrane system inside the mother cell compartment. The mature spores consist of a dehydrated core with a copy of the bacterial DNA bound tightly by small acid-soluble proteins, embedded in a chelating complex of dipicolinic acid and divalent cations like Ca2+ and Mg2+ [5-8]. The core is surrounded by the cortex, a peptidoglycan layer synthesized between the forespore inner and outer membranes, that helps to keep the water content in the core low. The outer layer of the spore, the coat, consists of an inner coat and an outer coat both of which are assembled mostly from components present or synthesized in the mother cell [5, 8]. Approximately 30% of the total coat protein content is resistant to extraction and defines an insoluble fraction [9] that contains highly cross-linked material (reviewed by Henriques and Moran [10]). Spores are remarkably resistant to many stresses [11] and proteins from the inner and outer coat layers are presumed to play a major role in spore structure and resistance. The types of association, the cross-linkages amongst the different proteins, the differences found in coat proteins due to strain variation have not been comprehensively described [12, 13]. Spore proteins have been isolated by methods based on alkaline treatments either alone or in combination with reducing agents such as dithiothreitol or β-mercaptoethanol in the presence of ionic detergents [14-16]. These studies have made use of SDS-PAGE gels and/or 2-D electrophoretic (2-DE) approaches generally followed by peptide mass fingerprinting for spore coat protein separation and identification [17, 18]. High-throughput analysis of spore proteomes using 2-DE gels is challenging because the individual protein extraction, digestion, and analysis of each spot from 2-DE gels is a laborious and time-consuming process. Moreover, a 2-DE gel approach is poor at resolving very low abundant proteins as well as low molecular mass, acid and basic proteins. Furthermore, hydrophobic proteins hardly enter the first isofocussing dimension of a 2-DE gel [19]. Here, we present a “gel-free” protocol (using similar principles for coat isolation as Goldman and Tipper [9] and based on the LC-MS/MS method for proteomic analysis of cell walls of Candida albicans developed by de Groot et al.[20]),

Gel-free proteomics of B. subtilis spore coat

37

that is capable of comprehensive B. subtilis spore coat protein analysis. We focused, for the first time, exclusively on the insoluble spore coat fraction in spores of a B. subtilis laboratory strain and a food spoilage isolate.

Spore preparation.

To obtain an exponentially growing and spore-free pre-culture, B. subtilis lab-strain PB2 and food isolate strain A163 were pre-cultured in TSB medium and subsequently transferred to a defined (MOPS buffered) medium. In this defined medium, sporulation was induced by glucose exhaustion. The cells were allowed to sporulate for 96 hours after which the spore crop was harvested (Figure 1). The crop contained >99.9% of the phase-bright spores. After harvesting, the spores were disintegrated by beat beating, the efficiency of which was determined by microscopic observation. Samples of disintegrated spores were characterized microscopically by loose fragments of spore material. No intact spores were noticed.

Figure 1. “Gel-free” approach for spore coat proteomics. See Materials & Methods for details.

Spore peptidoglycan analysis.

Muramic acid is present in the cortex peptidoglycan of spores. To estimate the contamination of the freeze dried insoluble spore material with spore cortex we performed a muramic acid assay. Intact spores from strains PB2 and A163, disintegrated spores before and after NaCl wash (to wash away cytosolic remnant proteins), and the final reduced and alkylated spore extracts were used for the assay. The amount of muramic acid in the final extract (used for MS analysis) compared to that in intact spores was reduced to below detection limit in preparations of B. subtilis strain PB2. This amount, though reduced by ~ 97%, was still detectable in case of strain A163 (data not shown). Thus, the insoluble spore fractions that we analyzed do not, or hardly, contain spore cortex material. Hereon we refer to this material as the insoluble spore coat fraction.

Gel-free proteomic identification of the insoluble protein fraction of the B. subtilis spore coat.

Four biological replicates were carried out for B. subtilis PB2 while for B. subtilis A163 five biological replicates were done. For each biological replicate, three technical replicates were run. The analysis of tryptic digests of the purified B. subtilis PB2 spore coat layer resulted in the identification of 55 spore coat proteins from the insoluble

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fraction, of which 34 are spore coat assigned proteins identified and reviewed in previous studies [10] (Table 1, Figure 2). Moreover, 21 novel putative spore coat proteins were identified, of which 19 proteins were identified from B. subtilis PB2 spore coats (Table 2) and 2 proteins uniquely in food isolate strain A163 (further discussed below). Among the total 55 identified proteins there were three morphogenetic proteins (Figure 2), some enzymes and other spore coat associated proteins with known and unknown functions. Our studies revealed no abundant cytosolic proteins such as ribosomal proteins, elongation factors or glycolytic enzymes in our preparations. This corroborated the notion that our procedure is especially suited for the isolation of insoluble spore coat proteins some of which likely will be covalently cross-linked. Most proteins from the ‘Cot’ family were identified (Table 1). We found all possible tryptic peptides of CotC. Proteins CwlJ and SleB, the two main cell-wall hydrolases important for spore germination, were also identified. Protein SpoIVA, important for the initial sporulation stages and for protein build up in the inner coat, was

Figure 2. Schematic structure of the spore showing possible localization of identified known as well as the putative novel spore coat proteins from B. subtilis lab-strain PB2. Proteins identified in our study and reported for their localization in previous studies [21-24] are shown. In pink, blue, purple, and orange are the proteins regulated by sigma factors σK, σE, σG and σF, respectively. In brown, are the proteins regulated by both σE & σK. CoxA (in red) is regulated by both σE & σG. SpoIVA, SafA & CotE (boxed) are the morphogenetic proteins as reported by McKenney et al. [23]. Out of the putative novel spore coat proteins, proteins YhcN & YjqC (black asterisks) are reported for their localization in previous studies. Proteins with other or unknown regulatory components are shown in grey (also see Table 1). Sigma factor dependency is as reported in the previous studies [25-29].


39

identified from spores of lab-strain PB2. Out of 19 novel putative spore coat proteins from B. subtilis PB2 spore coats, 11 were also identified by Kuwana et al.[28] from whole spore extracts, while 8 were identified for the first time in this study. We detected protein AtcL (YloB) that has been identified as a putative spore coat protein previously through transcriptome analysis [29], but to date had not been physically isolated from spore coat extracts. Proteins CotN (YqfT), YisY and YybI (identified by MASCOT in B. subtilis PB2) were found in A163 but only after detailed manual inspection of the spectra. They were not identified by MASCOT itself. On the other hand, two proteins YckD and YpeB were identified by MASCOT only in B. subtilis A163. YckD is a putative exported protein with an N-terminal signal sequence, regulated by σF and σG [29]. Localization and function of the protein is not known. YpeB was identified by MASCOT only in B. subtilis A163. It is the product of the ypeB gene lying downstream in the genome of the sleB gene. Both genes are part of one operon. The authors believe that YpeB is required for localization, and/or activation of SleB [30, 31]. It was also detected by Kuwana et al.[28]. Fifty of the proteins identified from spores of lab-strain PB2 were also identified from spores of food isolate strain A163 (Table 1 and 2).

Discussion

A comprehensive analysis of the protein composition of the spore coat will provide fundamental information on the molecular interactions in this protective spore layer while also validating transcriptome analyses that suggest a spore coat localization for a given protein. Previous studies provide evidence that, in summation, 70 proteins are associated with the spore coat of B. subtilis [10]. These studies mainly focused on the SDS-extractable fractions of the spore coat protein. However, both the extraction of total proteins and purification of each protein are quite difficult because of the occurrence of substantial cross-linking between proteins [13, 16]. Also, gel-based methods have identified maximally 20-30 spore coat proteins in a single study. Our direct and “gel-free” proteomic strategy (Figure 1) allows for a reproducible analysis of proteins from the insoluble fraction of the spore coat, some of which likely contains Tgl-mediated cross-linked proteins. We identified 55 proteins from B. subtilis PB2, of which 8 are newly detected, putative spore coat proteins. Also, due to the extensive washes of the spore crop and lysed spore preparations with NaCl as well as SDS-extraction thereafter, our spore protein preparations neither contained ribosomal proteins, elongation factors nor glycolytic proteins. These abundant cytosolic proteins from the vegetative or mother cell are generally seen as markers for cytosolic contamination. We performed a muramic acid assay to estimate contamination of our spore coat preparations by spore cortex material. The results showed that our coat preparations of B. subtilis PB2 spores do not contain spore cortex material and confirm that the newly identified proteins are indeed associated with the spore coat. Although unlikely, we cannot fully rule out the possibility that released proteins from degraded cortex peptidoglycan might be trapped in the coat fraction.

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Table 1. Gel-free proteomic identification of previously assigned spore coat proteins (reviewed by Henriques & Moran [10]) from B. subtilis strain PB2.

Protein Descriptiona Total no. of identified peptides

Molecular Mass (Da) a

Possible Regulator b

Identified in B.

subtilis A163

CotA During sporogenesis, multicopper oxidase; pigment production;

18 58499.0 SigK, GerE +

CotB Posttranslational modification dependent on CotG and CotH;

13 42971.5 SigE, SigK, GerE

+

CotC Spore coat protein; N-terminal signal sequence present

6 14644.9 SigK, GerE +

CotE Protein at the inner/outer interface; mutant generates no outer coat; involved in coat assembly


+

CotF Spore coat protein, forms 5- and 8-kDa polypeptides that are component of spore coat; null mutants produce normal looking spores[32]

6 18725.3 SigK +

CotG Outer coat protein required for the incorporation of CotB into the coat; 9 tandem copies of 13 a.a. long sequence; mutant generates outer coat defect


+

CotI (YtaA)

Spore coat protein 7 41245.4 SigK +

CotJA Part of polypeptide composition of the spore coat

3 9739.1 SigE +

CotJC Part of polypeptide composition of the spore coat

4 21695.6 SigE +

CotN (YqfT)

Conserved hypothetical spore coat protein 1 9742.2 SigE +

CotQ (YvdP)

Putative oxidoreductase; spore coat protein

10 50084.7 SigK, GerE +

CotR (YvdO)

Expressed during sporulation, in the late phase of coat protein synthesis[33]; putative sporulation hydrolase

7 35356.5 SigK +

CotS Required for localization of CotSA 10 41084.1 SigK, GerE + CotSA Glycosyltransferase 9 42912.1 SigK, GerE + CotU (YnzH)

Spore coat protein; sequence similarity with CotC protein

6 11424.6 SigK[25] +

CotX Insoluble fraction; mutant generates outer coat defect

3 18601.0 SigK, GerE +

CotY Insoluble fraction; mutant generates outer coat defect

6 17884.4 SigK, GerE +

CotZ Insoluble fraction 4 16533.7 SigK, GerE + CwlJ Cell wall hydrolase involved in the

depolymerization of cortex peptidoglycan during germination; synthesized during sporulation in mother cell compartment, then located in the spore coat [34]

3 16462.7 SigE +

GerQ Protein, Ca-DPA dependent cortex hydrolysis, Tgl-dependent

2 20275.8 SigE +

LipC (YcsK)

Lysophopholipase; involved in spore germination

3 23605.8 SigK, GerE +

OxdD Oxalate decarboxylase; resistance to oxalic acid stress; highly related to plant

2 43554.2 SigK +


41

Protein Descriptiona Total no. of identified peptides

Molecular Mass (Da) a

Possible Regulator b

Identified in B.

subtilis A163

allergens SafA Inner coat morphogenetic protein,

interacts with SpoVID; involved in coat assembly

3 43229.4 SigE +

SodA Superoxide dismutase; transcribed throughout the growth and sporulation; required for the assembly of CotG into the insoluble matrix of the spore; mutants exhibit growth defect and reduced survival at elevated temperature, but has no effect on heat or hydrogen peroxide resistance of spores

3 22489.9 SigB (late exponential

phase) SigF

(Sporulation) [27]

SpoIVA Required for proper spore cortex formation and coat assembly

9 55174.9 SigE +

TcyA (YckK)

Cystine ABC transporter (glutamine-binding protein)

1 29514.3 SigA and/or others[28]

Tgl Transglutaminase; forms an epsilon-(gamma-glutamyl) lysine isopeptide bond between a lysine donor from one GerQ protein and a glutamine acceptor from another GerQ protein

1 28295.7 SigK +

SleL (YaaH)

Spore peptidoglycan hydrolase; required for the L-alanine-stimulated germination pathway; together with CotR assembles spore coat proteins

9 48636.7 SigE +

YdhD Spore cortex lytic enzyme; involved in forespore assembly

2 46864.3 SigE +

YisY Putative hydrolase 1 30559.1 SigE + YjdH Hypothetical protein 2 15201.1 SigE +

YodI Unknown function; N-terminal signal sequence /trans-membrane helix present

2 9193.6 SigK, GerE +

YxeE Spore coat protein; a substrate of YabG protease

2 14714.2 SigK, GerE +

YybI Inner spore coat protein 1 30149.0 SigE + aDetails taken from SubtiList database (http://genodb.pasteur.fr/cgi-bin/WebObjects/GenoList.woa/) bThe possible regulators are as indicated in Steil et al.[29] unless otherwise mentioned.

Spore coat proteins of B. subtilis

We used two strains; B. subtilis lab-strain PB2 and a food isolate B. subtilis A163, for our proteomic analysis. Figure 2 represents the identified spore coat proteins from B. subtilis PB2. From our spore coat preparations we found many proteins whose transcription is regulated by mother cell specific sigma factors σE and σK. Since it is well known that the synthesis of the spore coat proteins is mainly controlled by the mother cell, our findings support the fact that we identified proteins exclusively from spore coats. However, we do not rule out the possibility that these proteins can be distributed across the different spore layers. Unexpectedly, we also found σG and σF regulated proteins. We

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42

Table 2. Putative novel spore coat proteins identified in this study in B. subtilis strain PB2.

Protein Molecular Mass (Da)a

Localization signal b

Total no. of

identified peptides

Possible Regulator c

Description a Identified in

B. subtilis A163

AtcL (YloB)

97292.8 Transmembrane (10)

4 SigE P-type calcium transport ATPase; mutants form spores with less heat resistance

+

CoxA* (YrbB)

22117.2 Signal sequence 1 SigG, SigE [26]

Spore cortex protein; located within the spore integument, mainly in cortex with a part in inner coat region [35]

+

DacF* 43297.4 Signal sequence

3 SigF D-alanyl-D-alanine carboxypeptidase (penicilin binding protein); required for spore cortex synthesis (peptidoglycan biosynthesis); regulation of the low degree of cross-linking of spore peptidoglycan

+

OppA* 61491.5 Signal sequence 3 TnrA[36] Oligopeptide ABC transporter (binding lipoprotein); required for initiation of sporulation

SleB* 34001.5 Signal sequence 1 SigG Spore cortex-lytic enzyme; N-terminal signal sequence present; required for complete spore-cortex hydrolysis during germination; mutant unable to complete L-alanine-mediated germination; probable lytic transglycosylase

+

SpsC 43189.9 1 SigE, SigK Putative glutamine-dependent sugar transaminase; putative enzyme for spore coat polysaccharide synthesis

+

SspG 5270.4 1 SigK, GerE Small acid-soluble spore protein

+

YbfO 51788.5 Signal sequence 1 ND d Putative exported hydrolase +

YdcC* 38136.5 Signal sequence 7 SigE Putative lipoprotein + YhcM* 17030.7 Signal sequence 1 SigG Hypothetical protein;

glutamine-rich protein

YhcN* 21016.3 Signal sequence 3 SigG Putative lipoprotein; detected in the inner spore membrane

+

YhcQ 24791.2 8 SigG Conserved hypothetical protein; similar to spore coat protein; locally rich in methionine; conserved in bacilli

+


43

Protein Molecular Mass (Da)a

Localization signal b

Total no. of

identified peptides

Possible Regulator c

Description a Identified in

B. subtilis A163

YhfD 8405.9 3 ND Hypothetical protein (Record is discontinued from databases)

+

YhxC* 30845.2 1 SigE Putative oxidoreductase YjqC* 31309.5 2 SigK Putative PBSX phage

manganese-containing Catalase, SafA dependent [23]

YkvN 13569.7 1 ND Putative transcriptional regulator

+

YqfX 13901.6 Signal sequence/ Transmembrane

(1)

1 SigG Conserved hypothetical protein

+

YrkC* 21255.0 2 SigK Putative dioxygenase; cupin family

+

YtfJ* 16346.5 2 SigF Conserved hypothetical protein

+

aDetails taken from SubtiList database (http://genodb.pasteur.fr/cgibin/WebObjects/GenoList.woa/) bLocalization signals predicted by SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) & TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) tools with number of transmembrane helices in brackets. c The possible regulators are as indicated in Steil et al.[29] unless otherwise mentioned. d ND: No definite regulator can be suggested. * Proteins detected also by Kuwana et al.[28].

suggest that these proteins might be covalently linked, the nature of covalent bond being unknown or might be cross-linked by o,o-di-tyrosine and (γ)-glutamyl-lysine isopeptide linkages like some other spore coat proteins reported in previous studies [13, 16]. We identified in our fractions many spore coat proteins from the ‘Cot’ family such as CotA and CotB. These proteins are usually found in the SDS-extractable coat fraction. This observation suggests that these proteins are covalently as well non-covalently bound to the spore coat layers. We did not identify CotH, which is in agreement with the fact that CotH has been previously extracted and identified mainly from the SDS fraction on 2-DE gels [17, 18, 37, 38]. CotM is a small protein (~14 kDa) and presumed to be embedded in the cross- linked part of the spore coat [39]. It is possible that we did not detect it because of its small size and low levels of lysine and arginine. On the other hand CotM has not been identified and/or isolated as a spore coat protein in previous studies either. CotV and W were not detected in our analyses. Takamatsu & Watabe [18] did not observe CotV and W in their studies on mature spores either. However, Kuwana et al. [28] did identify CotW in extracts of whole spores. Contrary to us, these authors also identified CotJB. CotO, P, T were not isolated from mature spores [28]. Moreover, CotD has only 2 lysines and 1 arginine residue. Hence it is difficult to identify it by the tryptic digestion approach. Similar to CotD, assigned coat protein YsnD [22] has only 1 lysine and 1 arginine residue. For such proteins, treatments with other proteolytic digestion enzymes like Chymotrypsin [28] or Glu-C(V8) endoproteinase, producing smaller fragments, could be explored. Factors like strain variation, the sporulation medium, and age of harvested spores might contribute to differences in identification of these proteins in previous

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studies compared to ours. In addition, most of the remaining known spore proteins (reviewed by Henriques and Moran [10]) that we did not detect have only transcriptional evidence suggesting spore coat localization, impeding a careful comparative discussion.

Putative novel spore coat proteins

Our approach allowed for the identification of 19 proteins putatively assigned in the earlier studies to the insoluble fraction of spore coats of B. subtilis PB2 (Table 2). Eight of these proteins have never been detected before, while 11 were also identified by Kuwana et al. [28] as spore localized proteins (see Table 1 and 2). DacF, a D, D-carboxypeptidase, is believed to regulate the degree of cross-linking of spore peptidoglycan. It belongs to the penicillin-binding protein family (PBPs), which polymerizes the peptidoglycan on the outer surface of the vegetative cell membrane [40]. The D,D-carboxypeptidase activity, prevents cross-linking of peptides by removing the terminal D-alanine. DacF may only act on a limited amount of peptidoglycan that is synthesized from the forespore side. It is rather unexpected to find DacF in our preparations but not impossible as DacF has a signal sequence. SpsC is a σK & σE regulated protein [25, 29] and is involved in the synthesis of polysaccharide on the spore coat [41]. SspG, a σK regulated protein, though not detected from spore coat in previous studies, is predicted to be a spore coat protein [42]. Mutants lacking SspG sporulated normally and have showed similar resistance to heat and UV radiation as the wild-type. No defect in germination and outgrowth was observed in these mutants [42]. OppA (also known as Spo0K) is an oligopeptide binding factor which is involved in initiation of sporulation [43] and required for competence development [44]. OppA is held in the extracellular face of the cell membrane via a lipid anchor. We suggest that during the process of layering the coats, OppA might be trapped in the coat layers and hence is retrieved from our preparations. Three proteins from the yhc family were identified in our study. YhcN is a putative lipoprotein. The MFGK--K------------QVLASVLLILPLMTGC sequence motif provides the lipid anchorage to the protein and thus it might be located in the coat layers. Interestingly, it also has an asparagine-rich motif similar to that seen in γ-type small, acid-soluble spore proteins (SASP) ubiquitous in the dormant spores of B. subtilis and other related species [45]. The sequence KLEVADE found in YhcN is very similar to the sequence KLEIASE found in α/β-type SASP from B. subtilis [45]. YhcM has a signal sequence as well as a glutamine-rich domain. This is a predicted spore protein in B. pumilus SAFR-032. YhcQ, a conserved protein in bacilli, has sequence similarity to spore coat protein CotF [46] and is rich in methionine. YjqC, in B. pumilus SAFR-032 known to be involved in sporulation, shows an oxidoreductase property [47]. Since its transcription is regulated by σK [29, 44] the protein is likely formed by the mother cell and from there incorporated as part of the spore coat. Protein AtcL (YloB) is a calcium transporting ATPase. Raeymaekers et al.[48] have shown that an insertional mutation of the yloB gene did not affect the growth of vegetative cells, did not prevent the formation of viable spores, and did not significantly affect Ca2+- accumulation during sporulation.


45

However, spores from knockouts were less resistant to heat and had slower rate of germination. It was concluded that this gene is not essential for survival, but assists in the formation of resistant spores.

Immunogenicity predictions and outlook

Our analysis provides concrete protein targets for the development of specific and sensitive spore detection and/or purification systems from food stuff or patient material. A preliminary analysis of the immunogenicity of all identified peptides from B. subtilis PB2 and B. subtilis food isolate A163 was done using the POPI 2.0 immunogenicity prediction server[49] (Table S2 of Supporting Information). While most of the peptides displayed no immunogenicity, a single peptide from protein YhcQ (--DQELLNILDR--) did show high immunogenicity. In summary, our novel gel free proteomics approach allows direct analysis of the proteins from the insoluble cross-linked protein fraction of the spore coat. Covalently attached coat proteins that escaped detection before in methods based on alkali or reducing agent in the presence of detergents extraction could be identified. Our analytical strategy will also be a starting point in future research on quantitative analysis of coat proteins to compare differences between strains or stress conditions. Furthermore, our analysis provides concrete protein targets for the development of specific and sensitive spore detection and/or purification systems from food stuff or patient material. Finally, our protocol will also enable identification of cross-links between coat proteins [50], and contribute to a better understanding of the macromolecular organization of the spore coat.

Materials and methods

Bacterial strains used

The strains used in this study were B. subtilis 168 laboratory wild-type strain PB2 (trpC2) and food spoilage isolate strain A163 [6]. Strain A163 was isolated in Unilever R&D, Vlaardingen, The Netherlands and classified as Bacillus subtilis subsp. subtilis [6]. Spores of spoilage strain B. subtilis A163 can survive temperature treatments of 111°C for 20 min [51].

Growth and sporulation conditions

Cells from a single colony were inoculated in Tryptic Soy Broth (TSB; pH 7.5), cultivated until early exponential phase, and transferred into a defined minimal medium, buffered with 3-(N-Morpholino) propanesulfonic acid (MOPS) to pH 7.4 as described previously[6]. As carbon- and nitrogen- sources, 10 mM glucose and 10 mM NH4Cl were used. Cells were grown until early exponential phase and diluted into 20 ml of fresh MOPS buffered medium. When early exponential phase was reached again, 1% of this final pre-culture was used to inoculate 500 ml MOPS buffered medium. Sporulation was initiated by growing the culture into stationary phase, induced by glucose exhaustion. Sporulation was allowed for 96 hours during which its efficiency was followed using phase contrast microscopy.

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Spore coat isolation

After 96 hours, spores were harvested by washing the crop seven times with sterile Milli-Q water and treating it with 0.01% Tween to lyse remaining vegetative cells. The harvested spore crop contained >99.9% of phase bright spores. Spores were resuspended in cold 10 mM Tris-HCl, pH 7.5 (1011 spores/ml), and disintegrated with 0.1-mm glass beads (BioSpec Products, Inc., USA) in a Bio-Savant Fast Prep 120 machine (Qbiogene, Carlsbad, CA). The machine was set at runs of 40 s at maximum speed of 6. Ten rounds of Fast-prepping were performed. Between the runs the samples were placed on ice for 1 min to prevent protein degradation by over-heating. To remove non-covalently linked proteins and intracellular contaminants, isolated layers of insoluble spore material were washed extensively with 1 M NaCl and thermally treated for 10 min, starting from an ambient temperature water bath and reaching a final temperature of 80°C , with 50 mM Tris-HCl, pH 7.8, containing 2% SDS, 100 mM Na-EDTA, 150mM NaCl and 100 mM β-mercaptoethanol. SDS-treated coats were washed four times with Milli-Q water and freeze dried. Spore cortex peptidoglycan analysis

The purity and thereby the absence of cortex peptidoglycan in the thus obtained spore coat material was checked by estimating its muramic acid content [52]. Sample preparation for MS analysis

The freeze dried samples were reduced with 10 mM dithiothreitol in 100 mM NH4HCO3 (1 hour at 55°C) followed by a reaction with 55 mM iodoacetamide in 100 mM NH4HCO3 for 45 min at room temperature in the dark. The samples were directly digested for 18 hours at 37°C with trypsin (Trypsin gold Promega, Madison, WI) using a 1:50 (w/w) protease: protein ratio. The tryptic digests were desalted using Omix μC18 pipette tips (80 μg capacity, Varian, Palo Alto, CA) according to the manufacturer’s instructions.

Mass spectrometric analysis

Each sample was diluted with 0.1% trifluoroacetic acid to a final concentration of around 7.5 ng/nl and introduced on an Ultimate LC-Packings nano-HPLC system (Dionex, Sunnyvale, CA) equipped with a PepMap C18 reversed phase column (75 μm inner diameter, 25 cm length; Dionex, Sunnyvale, CA). The starting mobile phase A was water + 0.1% formic acid and the mobile phase B was 50% acetronitrile + 0.1% formic acid. The peptides were eluted with a linear gradient from 0%-100% phase B over 30 min with a flow of 300 nl/min and directly ionized by electrospray in a Q-TOF mass spectrometer (Waters, United Kingdom). Each sample was analyzed three times including a survey TOF-MS run followed by multiple experiments with a data dependent acquisition of the abundant peptide ions which were fragmented by collision induced dissociation (MS/MS).

Analysis of mass spectrometric data

The generated spectra were processed using the Masslynx Proteinlynx software (Micromass Ltd., UK). The resulting peak list (.pkl) files were submitted to the MASCOT MS/MS ion search engine both on http://www.matrixscience.com and on an in-house MASCOT B. subtilis database (SwissProt version 2003 downloaded from http://expasy.org/sprot/). In MASCOT, search parameters were as follows: allowance of one missed cleavage, fixed modification of carbamidomethyl cysteine and variable modification of oxidation on methionine, an error tolerance of 0.3 Da for calculated peptides and their corresponding MS/MS spectra. If needed the search was repeated with the same parameters but with semitrypsin as the enzyme. Probabilistic MASCOT scoring was used to evaluate the identified peptides and an individual peptide score of 30 (p<0.05) or higher was considered significant for peptide identification. All identified peptides (Table S1 of Supporting Information) were manually checked on their raw MS/MS data, if


47

necessary (Figures S1-S15 of Supporting Information). The complete gel-free protocol is summarized in Figure 1.

References

1. Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, et al. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature. 1997;390(6657):249-56. Epub 1997/12/31 23:39. 2. Errington J. Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis. Microbiol Rev. 1993;57(1):1-33. Epub 1993/03/01. 3. Johnson WC, Moran CP, Jr., Losick R. Two RNA polymerase sigma factors from Bacillus subtilis discriminate between overlapping promoters for a developmentally regulated gene. Nature. 1983;302(5911):800-4. Epub 1983/04/28. 4. Piggot PJ, Coote JG. Genetic aspects of bacterial endospore formation. Bacteriol Rev. 1976;40(4):908-62. Epub 1976/12/01. 5. Driks A. Bacillus subtilis spore coat. Microbiol Mol Biol Rev. 1999;63(1):1-20. Epub 1999/03/06. 6. Kort R, O'Brien AC, van Stokkum IH, Oomes SJ, Crielaard W, Hellingwerf KJ, et al. Assessment of heat resistance of bacterial spores from food product isolates by fluorescence monitoring of dipicolinic acid release. Appl Environ Microbiol. 2005;71(7):3556-64. Epub 2005/07/08. 7. Setlow P. I will survive: DNA protection in bacterial spores. Trends Microbiol. 2007;15(4):172-80. Epub 2007/03/06. 8. Zheng LB, Losick R. Cascade regulation of spore coat gene expression in Bacillus subtilis. J Mol Biol. 1990;212(4):645-60. Epub 1990/04/20. 9. Goldman RC, Tipper DJ. Bacillus subtilis spore coats: complexity and purification of a unique polypeptide component. J Bacteriol. 1978;135(3):1091-106. Epub 1978/09/01. 10. Henriques AO, Moran CP, Jr. Structure, assembly, and function of the spore surface layers. Annu Rev Microbiol. 2007;61:555-88. Epub 2007/11/24. 11. Setlow P. Mechanisms which contribute to the long-term survival of spores of Bacillus species. Soc Appl Bacteriol Symp Ser. 1994;23:49S-60S. Epub 1994/01/01. 12. Isticato R, Pelosi A, Zilhão R, Baccigalupi L, Henriques AO, De Felice M, et al. CotC-CotU heterodimerization during assembly of the Bacillus subtilis spore coat. J Bacteriol. 2008;190(4):1267-75. Epub 2007/12/11. 13. Ragkousi K, Setlow P. Transglutaminase-mediated cross-linking of GerQ in the coats of Bacillus subtilis spores. J Bacteriol. 2004;186(17):5567-75. Epub 2004/08/20. 14. Aronson AI, Fitz-James PC. Biosynthesis of bacterial spore coats. J Mol Biol. 1968;33(1):199-212. Epub 1968/04/14. 15. Linn T, Losick R. The program of protein synthesis during sporulation in Bacillus subtilis. Cell. 1976;8(1):103-14. Epub 1976/05/01. 16. Pandey NK, Aronson AI. Properties of the Bacillus subtilis spore coat. J Bacteriol. 1979;137(3):1208-18. Epub 1979/03/01. 17. Jenkinson HF, W.D.Sawyer, J. Mandelstam. Synthesis and order of assembly of spore coat proteins in Bacillus subtilis. J Gen Microbiol. 1981(123):1-16. 18. Takamatsu H, Watabe K. Assembly and genetics of spore protective structures. Cell Mol Life Sci. 2002;59(3):434-44. Epub 2002/04/20. 19. Gorg A, Weiss W, Dunn MJ. Current two-dimensional electrophoresis technology for proteomics. Proteomics. 2004;4(12):3665-85. Epub 2004/11/16. 20. de Groot PW, de Boer AD, Cunningham J, Dekker HL, de Jong L, Hellingwerf KJ, et al. Proteomic analysis of Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins. Eukaryot Cell. 2004;3(4):955-65. Epub 2004/08/11.

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21. Imamura D, Kuwana R, Takamatsu H, Watabe K. Localization of proteins to different layers and regions of Bacillus subtilis spore coats. J Bacteriol. 2010;192(2):518-24. Epub 2009/11/26. 22. Kim H, Hahn M, Grabowski P, McPherson DC, Otte MM, Wang R, et al. The Bacillus subtilis spore coat protein interaction network. Mol Microbiol. 2006;59(2):487-502. Epub 2006/01/05. 23. McKenney PT, Driks A, Eskandarian HA, Grabowski P, Guberman J, Wang KH, et al. A distance-weighted interaction map reveals a previously uncharacterized layer of the Bacillus subtilis spore coat. Curr Biol. 2010;20(10):934-8. Epub 2010/05/11. 24. Wang KH, Isidro AL, Domingues L, Eskandarian HA, McKenney PT, Drew K, et al. The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis. Mol Microbiol. 2009;74(3):634-49. Epub 2009/09/25. 25. Eichenberger P, Fujita M, Jensen ST, Conlon EM, Rudner DZ, Wang ST, et al. The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis. PLoS Biol. 2004;2(10):e328. Epub 2004/09/24. 26. Eichenberger P, Jensen ST, Conlon EM, van Ooij C, Silvaggi J, Gonzalez-Pastor JE, et al. The σE regulon and the identification of additional sporulation genes in Bacillus subtilis. J Mol Biol. 2003;327(5):945-72. Epub 2003/03/29. 27. Inaoka T, Matsumura Y, Tsuchido T. Molecular cloning and nucleotide sequence of the superoxide dismutase gene and characterization of its product from Bacillus subtilis. J Bacteriol. 1998;180(14):3697-703. Epub 1998/07/11. 28. Kuwana R, Kasahara Y, Fujibayashi M, Takamatsu H, Ogasawara N, Watabe K. Proteomics characterization of novel spore proteins of Bacillus subtilis. Microbiology. 2002;148(Pt 12):3971-82. Epub 2002/12/14. 29. Steil L, Serrano M, Henriques AO, Volker U. Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis. Microbiology. 2005;151(Pt 2):399-420. Epub 2005/02/09. 30. Boland FM, Atrih A, Chirakkal H, Foster SJ, Moir A. Complete spore-cortex hydrolysis during germination of Bacillus subtilis 168 requires SleB and YpeB. Microbiology. 2000;146 ( Pt 1):57-64. Epub 2000/02/05. 31. Chirakkal H, O'Rourke M, Atrih A, Foster SJ, Moir A. Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination. Microbiology. 2002;148(Pt 8):2383-92. Epub 2002/08/15. 32. Cutting S, Zheng LB, Losick R. Gene encoding two alkali-soluble components of the spore coat from Bacillus subtilis. J Bacteriol. 1991;173(9):2915-9. Epub 1991/05/01. 33. Little S, Driks A. Functional analysis of the Bacillus subtilis morphogenetic spore coat protein CotE. Mol Microbiol. 2001;42(4):1107-20. Epub 2001/12/12. 34. Bagyan I, Setlow P. Localization of the cortex lytic enzyme CwlJ in spores of Bacillus subtilis. J Bacteriol. 2002;184(4):1219-24. Epub 2002/01/25. 35. Takamatsu H, Hiraoka T, Kodama T, Koide H, Kozuka S, Tochikubo K, et al. Cloning of a novel gene yrbB, encoding a protein located in the spore integument of Bacillus subtilis. FEMS Microbiol Lett. 1998;166(2):361-7. Epub 1998/10/14. 36. Yoshida K, Yamaguchi H, Kinehara M, Ohki YH, Nakaura Y, Fujita Y. Identification of additional TnrA-regulated genes of Bacillus subtilis associated with a TnrA box. Mol Microbiol. 2003;49(1):157-65. Epub 2003/06/26. 37. Naclerio G, Baccigalupi L, Zilhão R, De Felice M, Ricca E. Bacillus subtilis spore coat assembly requires cotH gene expression. J Bacteriol. 1996;178(15):4375-80. Epub 1996/08/01. 38. Zilhão R, Naclerio G, Henriques AO, Baccigalupi L, Moran CP, Jr., Ricca E. Assembly requirements and role of CotH during spore coat formation in Bacillus subtilis. J Bacteriol. 1999;181(8):2631-3. Epub 1999/04/10. 39. Henriques AO, Beall BW, Moran CP, Jr. CotM of Bacillus subtilis, a member of the alpha-crystallin family of stress proteins, is induced during development and participates in spore outer coat formation. J Bacteriol. 1997;179(6):1887-97.


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40. Popham DL, Gilmore ME, Setlow P. Roles of low-molecular-weight penicillin-binding proteins in Bacillus subtilis spore peptidoglycan synthesis and spore properties. J Bacteriol. 1999;181(1):126-32. Epub 1998/12/29. 41. Knurr J, Benedek O, Heslop J, Vinson RB, Boydston JA, McAndrew J, et al. Peptide ligands that bind selectively to spores of Bacillus subtilis and closely related species. Appl Environ Microbiol. 2003;69(11):6841-7. Epub 2003/11/07. 42. Bagyan I, Setlow B, Setlow P. New small, acid-soluble proteins unique to spores of Bacillus subtilis: identification of the coding genes and regulation and function of two of these genes. J Bacteriol. 1998;180(24):6704-12. Epub 1998/12/16. 43. Perego M, Higgins CF, Pearce SR, Gallagher MP, Hoch JA. The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation. Mol Microbiol. 1991;5(1):173-85. Epub 1991/01/01. 44. Rudner DZ, LeDeaux JR, Ireton K, Grossman AD. The spo0K locus of Bacillus subtilis is homologous to the oligopeptide permease locus and is required for sporulation and competence. J Bacteriol. 1991;173(4):1388-98. Epub 1991/02/01. 45. Setlow P. Small, acid-soluble spore proteins of Bacillus species: structure, synthesis, genetics, function, and degradation. Annu Rev Microbiol. 1988;42:319-38. Epub 1988/01/01. 46. Noback MA, Terpstra P, Holsappel S, Venema G, Bron S. A 22 kb DNA sequence in the cspB-glpPFKD region at 75 degrees on the Bacillus subtilis chromosome. Microbiology. 1996;142 ( Pt 11):3021-6. Epub 1996/11/01. 47. Gioia J, Yerrapragada S, Qin X, Jiang H, Igboeli OC, Muzny D, et al. Paradoxical DNA repair and peroxide resistance gene conservation in Bacillus pumilus SAFR-032. PLoS One. 2007;2(9):e928. Epub 2007/09/27. 48. Raeymaekers L, Wuytack E, Willems I, Michiels CW, Wuytack F. Expression of a P-type Ca2+-transport ATPase in Bacillus subtilis during sporulation. Cell Calcium. 2002;32(2):93. Epub 2002/08/06. 49. Tung CW, Ho SY. POPI: predicting immunogenicity of MHC class I binding peptides by mining informative physicochemical properties. Bioinformatics. 2007;23(8):942-9. Epub 2007/03/27. 50. Back JW, Notenboom V, de Koning LJ, Muijsers AO, Sixma TK, de Koster CG, et al. Identification of Cross-Linked Peptides for Protein Interaction Studies Using Mass Spectrometry and 18O Labeling. Analytical Chemistry. 2002;74(17):4417-22. 51. Oomes SJCM, S. Brul. The effect of metal ions commonly present in food on gene expression of sporulating Bacillus subtilis cells in relation to spore wet heat resistance. Innovat Food Sci Emerg Technol. 2004;5(3):307-16. 52. Hadžija O. A simple method for the quantitative determination of muramic acid. Anal Biochem. 1974;60(2):512-7. Epub 1974/08/01.

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3 In Pursuit of Protein Targets: Proteomic

characterization of Bacterial Spore Outer Layers.

Wishwas Abhyankar, Abeer H. Hossain, André Djajasaputra, Patima Permpoonpattana, Alexander Ter Beek, Henk L. Dekker, Simon M. Cutting, Stanley Brul, Leo J. de Koning,

Chris G. de Koster

Published in Journal of Proteome Research, 2013, 12, 4507−4521

Supplementary material can be found at

http://pubs.acs.org/doi/suppl/10.1021/pr4005629

Abstract Bacillus cereus, responsible for food poisoning and Clostridium difficile, causative agent of Clostridium difficile-associated diarrhoea (CDAD) are both spore forming pathogens involved in food spoilage, food intoxication and other infections in humans and animals. The proteinaceous coat and the exosporium layers from spores are important for their resistance and pathogenicity characteristics. The exosporium additionally provides an ability to adhere to surfaces eventually leading to spore survival in food. Thus studying these layers and identifying suitable protein targets for rapid detection and removal of spores is of utmost importance. In this study, we identified 100 proteins from B. cereus spore coat, exosporium and 54 proteins from the C. difficile coat insoluble protein fraction. In an attempt to define a universal set of spore outer layer proteins we identified 11 superfamily domains common to the identified proteins from two Bacilli and a Clostridium species. The evaluated orthologue relationships of identified proteins across different spore formers resulted in a set of 13 coat proteins conserved across the spore formers and 12 exosporium proteins conserved in the B. cereus group which could be tested for quick and easy detection or targeted in strategies aimed at removal of spores from surfaces.

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Introduction

Studies with Bacillus subtilis have provided good insights in the sporulation and germination processes of spores. However, studies of more pathogenic species like aerobic or facultatively anaerobic Bacillus cereus and strictly anaerobic Clostridium difficile are needed to gain more insights in the spore structure and germination characteristics of these spores. The B. cereus group (also known as B. cereus sensu lato), is comprised of several closely related species: Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus [1]. B. cereus is mainly known as a food-borne pathogen causing two types of gastrointestinal diseases: emesis and diarrhea [2]. B. cereus related emesis is caused by the cereulide toxin, which once produced remains stable upon enzyme, heat or acid treatment. Diarrhea, on the other hand, is caused by enterotoxins haemolysin BL (Hbl), non-haemolytic enterotoxin (Nhe) and cytotoxin K (CytK) [2, 3] that are produced by the vegetative cells. B. cereus strains have also been reported in few cases to be associated with non-gastrointestinal diseases including: respiratory tract infections, endophthalmitis, central nervous system infections, gas gangrene-like infections, cutaneous infections, endocarditis, osteomyelitis and urinary tract infections [4]. Spore forming Clostridium spp. (e.g. Clostridium botulinum, Clostridium tetani), like Bacilli, are also known for the infections and diseases caused by them. Pathogenic C. difficile can colonize the intestinal tracts of humans and other mammals [5, 6]. Routine treatments containing use of antibiotics such as Metronidazole, Vancomycine have led to emergence of resistant strains [7, 8] and thus prolonged treatment with such antibiotics can result in C. difficile overgrowth and can lead to diseases ranging from diarrhea to life-threatening pseudomembranous colitis, especially in immunocompromised people [9, 10]. C. difficile is reported to be a continuously evolving species [11, 12] and in recent times the organism has emerged as one of the major causes of nosocomial diarrhea (Clostridium difficile-associated diarrhea; CDAD). CDAD is mainly caused by the secretion of two cytotoxic, enterotoxic and proinflammatory toxins known as toxin A (TcdA) and toxin B (TcdB) [13]. CDAD is particularly problematic to treat and to avert because of the robust endospores that can persist and be easily transferred, person-to-person, in a hospital environment and thus the morbidity and mortality rates have been increasing in recent years. Endospores, are metabolically dormant multi-layered cellular forms which upon germination, lose their protective external layers and resume vegetative growth [14]. In B. subtilis the outermost thick concentric proteinaceous layers - the inner and the outer spore coat [15] - aid in resistance against variety of environmental assaults. Additionally in spores of many Bacillus and Clostridium species surrounding the coats an external loosely-fitting, hydrophobic, glycosylated, balloon-like layer - the exosporium - is present, which assists in adherence to the surfaces [16]. Though the cortex layer from spores might have evolved from the cell walls homologous to those of vegetative cells, the proteinaceous coat is quite unique. The immense resistance of the coat to attack by microorganisms and by free enzymes is unmatched in the bacterial domain. The wide variety of germination signals amongst the closely related species suggests that

Spore surface layer proteins from B. cereus & C. difficile

53

germination receptor assembly is a relatively later event in the evolution of the spore structure as the ancestral Bacilli and Clostridia moved to different niches after their divergence about 2.3 billion years ago [17]. Albeit the evolutionary distance, the ability to form spores and the overall sporulation process are conserved in these organisms with significant differences still existing both in the regulation of spore formation and in the spore structure [18]. Thus it is imperative to study the spore coat and exosporium layers from the spores of these two organisms in order to obtain detailed knowledge about the spore structure as well as to design quick and simple techniques for detection and thereby facilitating eradication of spores form the environment. Previous extensive research has led to the identification of up to 70 different proteins from the spore coat layers in B. subtilis. The exosporium is a relatively less studied layer, possibly due to the difficulties in obtaining its large quantities, and is reported to comprise of at least 25 proteins as studied from B. cereus and B. anthracis spores [15, 19-21]. Mostly these studies have focused on the soluble fraction of proteins. Our “gel-free” method [22] allowed us for the first time to focus on the insoluble protein fraction from the spore coat, which makes up to 30% of the proteins [15], and is characterized by extensive inter-protein cross-linking and thus is difficult to analyze using conventional PAGE gels. We have showed that our gel-free method is comprehensive in isolating and identifying proteins from the insoluble protein fraction of B. subtilis spore coats with identification of 19 new proteins. Here, we have extended our method to B. cereus and C. difficile to identify protein targets for early and rapid detection of spores and have characterized for the first time the insoluble proteome of the coat and exosporium of B. cereus ATCC 14579 spores and of the coat layers of C. difficile 630 spores. Recently, McKenney et al. [23] mentioned that the coat morphogenetic proteins may be the targets for evolutionary adaptation for spore formers. Therefore to obtain a universal set of spore coat and exosporium proteins we also assessed different spore formers for the conservation of the spore surface proteins that were identified from B. subtilis [22], B. cereus and C. difficile in our studies. Results and Discussion

Spore preparation

The harvested B. cereus ATCC 14579 spore crop contained >95% purified spores and for the C. difficile strain 630 the sporulation efficiency was noted to be ~75%. In contrast to the B. cereus spores, the presence of exosporium in harvested C. difficile spores was inconsistent and if present only remnants of the exosporium were seen under the phase contrast microscope, as observed previously [24]. Spore peptidoglycan analysis

Protein isolation and extensive washing of the pellets with salt reduced the amount of cortex muramic acid below1% compared to the intact spores. However the final reduced, alkylated pellet unexpectedly showed a slight increase in the amount of muramic acid. This could be due to the release of sugar moieties post-digestion. Lysozyme

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treatment did not further lower the muramic acid content of the pellet material. Deglycosylation of the reduced and alkylated pellet did significantly reduce the muramic acid content to values below 0.05% (results not shown). In conclusion, the final pellets used for proteomic analysis hardly contained any cortex muramic acid contamination. Conserved Domain Search

Amongst all the identified proteins from both species, 148 different superfamily domains including 22 distinct Domains of Unknown Function (DUFs) were identified by a domain searching tool from NCBI (see Supplementary Table 1). The hydrolases and peptidases identified from B. cereus ATCC 14579 and C. difficile 630 in this study might be important for spore germination and spore structure. Amongst the proteins identified from C. difficile, a preference towards proteins with catalase-domains, ferritin-like domains and metal-binding domains that might have a role in resistance towards oxidative stress (see below), was observed. There were 11 superfamily domains that emerged as common to the identified proteins from B. subtilis 168[22], B. cereus ATCC 14579 and C. difficile 630 (Figure 1).

Figure 1. Venn diagramme of conserved superfamily domains in the identified spore coat & exosporium proteins from B. subtilis 168, B. cereus ATCC 14579 and C. difficile 630. A total of 148 superfamily domains were assigned to the identified proteins from the three species indicated. The numbers correspond to the superfamily domains from the identified proteins. The eleven superfamily domains common to the three organisms are listed in the box.

Protein identification and Orthologue evaluation i.e. PSI-BLAST analysis

Orthologues are genes (in different species) evolved from a common ancestral gene by speciation. This suggests that orthologues retain the same function in the course of evolution. Evolutionary pressure however is not equal on all residues of a protein. For instance, buried residues at an active site or at a binding site are generally more conserved than residues in loops. Also evolutionary pressure may force addition or deletion of gene segments from the genomes leading to different gene and protein sizes. Orthologue identifications has been done by various different approaches amongst which a Best-Reciprocal BLAST Hit (BBH) or Bi-directional Best BLAST hit (BDBH) approach has


55

been considered the best[25]. However, this approach has its own flaws in the sense that only one-to-one orthologous pairs are found, for duplicated genes or paralogues only a single hit is found. The PSI-BLAST search tool helps in identification of regions of importance (not variable) and gives them more weight in subsequent comparisons. The PSI-BLAST tool may also detect subtle relationships between proteins that are distant structural or functional homologues. Such relationships are often not detected by a simple BLAST search. Our current study focused for the first time on the insoluble fraction and led to the identification of total 100 proteins from the spore coat and exosporium layers of B. cereus ATCC 14579 and 54 proteins from the spore coat layers of C. difficile 630 (Table 1 & 2). These proteins were then used for orthologue evaluation using PSI-BLAST search (see Supplementary Tables 2, 3, 4 and 5). As mentioned above, due to the possibility of various sizes of proteins, we assigned the sequence identity threshold at 30% and an E-value of 0.005 was the default value chosen. The experimentally identified proteins with conserved orthologues in two Bacilli and a Clostridium species analyzed by us are listed in Table 3. As seen proteins CotJC, DacF, SpoIVA and YisY were identified experimentally from all the three organisms. For further comparative analysis, we selected five Bacillus species (11 different strains) and four Clostridium species (total 13 different strains) available on the Genolist database (http://genolist.pasteur.fr/GenoList). Proteins SafA (BC_4420), CotE (BC_3770), YhcN (BC_4419), YusW (BC_0212), CotD (BC_1560), YxeE (BC_3534) and others (Table 1) are among the proteins identified only from coat + exosporium (fraction 1) and coat (fraction 2) confirming their localization in the spore coat while proteins InA (BC_1284), CalY (BC_1279) and BC_1591 were among those identified only from fraction 1 & exosporium (fraction 3). As seen there was a considerable overlap between fractions 2 & 3 id est between spore coat and exosporium fractions implying two possibilities - either these proteins may be spread across both the layers or the removal of the exosporium layer was inefficient. Five proteins - BC_0944, SodF (BC_1468), YppG (BC_1559), BC_2237 and BclB (BC_2382) - were identified only by a semi-trypsin enzyme search. In case of C. difficile 630, CdeC [26] (CD1067), CD1581, CotA (CD1613), CotB (CD1511), CotJB1/CotCA (CD0597), CotJC1/CotCB (CD0598), CotJC2/CotD (CD2401), CotE (CD1433), CotF (CD0596), CotJB2 (CD2400), Rbr (CD2845), SipL (CD3567), were among the high scoring proteins with > 10 peptides identified per protein. In total, from the identified C. difficile proteins 24 proteins have been identified previously [27] from the whole spore protein extracts. We did not study a separate exosporium fraction for C. difficile 630 as the stability of the exosporial layer may depend on the spore preparation method as well as their storage and in our sample the presence of exosporium was inconsistent possibly due to its loss during spore harvest. (see Materials & Methods).

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Table 1. Identified spore coat and exosporium proteins from Bacillus cereus ATCC 14579.

a Gene name a Uniprot ID

a Protein description a Mass (Da)

Spore coat & Exosporium

Spore coat Exosporium

bFDR= 1.77% FDR= 1.62% FDR= 1.99% cScore cno.

pept Score no.

pept Score no.

pept (I) Proteins involved in spore coat morphogenesis and other known spore coat proteins

BC_4420 (safA) d Q812R2 SpoVID-dependent spore coat assembly factor SafA 65354 1286 46 2357 70

BC_2872 (cotX) Q81CA3 Spore coat protein X 16345 1145 28 1288 32 216 5

BC_2030g Q81EF0 Spore coat protein G 24013 1141 36 1309 41 471 30

BC_0389 (cotB) Q81IJ7 Spore coat protein B 19768 1109 67 2763 100 524 23

BC_3770 (cotE) d Q81A24 Spore coat protein E 20615 971 37 122 5 BC_0390 (cotB) Q81IJ6 Spore coat protein B 17339 744 29 1563 39 328 11

BC_4419 (yhcN) d Q817V9 Putative uncharacterized protein 24526 672 21 628 21 BC_1222 Q81GH8 Spore coat protein Y 17596 486 19 729 21 5668 188

BC_5056 d Q815S6 Collagen adhesion protein 34657 383 19 1427 37 BC_2874 (cotX) Q81CA1 Spore coat protein X 21850 306 12 293 12 36 1 BC_1509 (spoIVA) Q81FR0 Stage IV sporulation protein A 55636 242 11 BC_4640 (ytfJ) d Q817B7 Putative uncharacterized protein 13968 237 10 81 3 BC_1560 (cotD) d Q81FM0 Spore coat protein D 15393 214 10 565 16 BC_0212 (yusW) d Q81IY1 Putative uncharacterized protein 17725 122 3 204 6

BC_4075 (dacF) d Q819B1 D-alanyl-D-alanine carboxypeptidase 44032 85 3 150 5

BC_1279 (cotN) Q81GC8 Spore coat-associated protein N 21848 79 3 BC_1245 Q81GF8 Putative uncharacterized protein 15287 39 2 BC_0822 (cotJB) Q81HI6 CotJB protein 10523 45 1 BC_0821 (cotJC) Q81HI7 CotJC protein 21694 37 1 BC_0823 (cotJA) Q81HI5 CotJA protein 8475 30 1 BC_3534 (yxeE) d Q81AM8 IG hypothetical 17193 16298 21 1 33 1 BC_2095 (ytfJ) d Q81E93 Putative uncharacterized protein 15087 120 5 32 1 BC_0063 (yabP) Q81J89 Putative uncharacterized protein 11627 45 1 BC_2677 Q81CR9 L-alanyl-D-glutamate peptidase 31439 41 1 BC_0047 (yabG) Q81JA4 Sporulation-specific protease

YabG 33219 23 1 BC_1559(yppG)f Q81FM1 Spore coat protein 22228 23 1

(II) Spore coat proteins likely to play a role in spore resistance

BC_4047 (cotα) Q819D8 Putative uncharacterized protein 14344 519 24 680 28 310 18

BC_4639 d Q817B8 Thiol peroxidase 18055 158 8 48 2 BC_1391 (yqfX) d Q81G20 Putative uncharacterized protein 13457 187 5 69 1 BC_2099 (yqfX) d Q81E89 Putative uncharacterized protein 12239 26 1 67 1 BC_4774 (yisY) d Q816P9 Non-heme chloroperoxidase 30166 54 1 62 1

(III) Exosporium proteins likely to be involved in attachment to surfaces

BC_1218 (exsY) Q81GI1 Spore coat protein Y 16761 640 26 1447 46 2274 106

BC_2493 (exsK) Q81D85 Putative uncharacterized protein 13556 1128 26 1372 34 10874 236

BC_3712 (bclC) Q812Y5 Hypothetical Membrane Spanning Protein 75838 673 37 483 21 820 30

BC_1221 (bxpB) Q813V0 Exosporium basal layer protein 17390 425 19 448 21 1775 65


57






pept Score no.

pept Score no.

pept BC_3547 Q81AL6 Cell surface protein 97913 199 5 713 16 848 32

BC_2374 (exsFB) Q813L4 Hypothetical Membrane Spanning Protein 17496 141 6 88 3 164 5

BC_2382 (bclB)f Q81DI4 Putative uncharacterized protein 39119 50 1

BC_2639 d Q81CV2 Cell surface protein 521640 80 1 29 1 BC_3345 Q81B46 Collagen-like triple helix repeat

protein 77508 116 2

BC_2569 Q81D14 Collagen triple helix repeat protein 53405 73 3 24 1 85 2

BC_2149 (bxpA) d Q81E43 Putative uncharacterized protein 32484 47 1 412 8 (IV) Exosporium proteins possibly involved in pathogenicity

BC_1284 (inA) e Q81GC3 Immune inhibitor A 85713 920 41 137 5

BC_2267 Q81DT6 Putative uncharacterized protein 21872 274 11 433 14 261 10

BC_2266 Q81DT7 Putative uncharacterized protein 20239 41 2 144 4 42 2

BC_1281 (calY) e Q81GC6 Cell envelope-bound metalloprotease (Camelysin) 21801 409 18 21 1

(V) Spore coat proteins involved in spore germination

BC_3607 (yaaH) d Q81AG3 Spore peptidoglycan hydrolase (N-acetylglucosaminidase) 48167 1185 54 1235 41

BC_5391 (gerQ) d Q814N4 Putative uncharacterized protein 16090 587 21 227 8 BC_0264 (alr1) Q81IT5 Alanine racemase 1 43789 382 22 678 18 1393 47

BC_2889 (iunH) Q81C90 Inosine-uridine preferring nucleoside hydrolase 36552 306 11 580 20 1797 61

BC_2207 Q81DY9 Sporulation-specific N-acetylmuramoyl-L-alanine amidase 35924 129 8

BC_5390 (cwlJ) d Q814N5 Cell wall hydrolase cwlJ 16467 93 5 45 3 BC_4319 (gpr) Q818E2 Germination protease 40450 90 3 BC_1591 e Q81FJ1 Putative uncharacterized protein 58965 87 2 5360 170

BC_3552 (iunH) Q81AL1 Inosine-uridine preferring nucleoside hydrolase 34507 27 1

BC_2752 (ypeB) Q813I5 Spore germination protein 50109 81 2 BC_2753 (sleB) d P0A3V0 Spore cortex-lytic enzyme 28297 66 2 32 1

(VI) Other putative spore coat and/or exosporium proteins

BC_0987 Q81H38 Putative uncharacterized protein 14633 736 33 1800 39 878 30

BC_0996 Q81H29 Putative uncharacterized protein 15501 536 23 725 31 366 11

BC_5135 (eno) d Q815K8 Enolase 46400 516 16 95 4 BC_p0002 d Q814F0 Putative uncharacterized protein 17989 333 11 97 4 BC_2858 Q81CB5 Putative uncharacterized protein 9626 266 12 232 7 52 1

BC_2426 Q81DE1 Putative uncharacterized protein 26265 263 16 365 21 139 5

BC_2026 Q81EF3 Oligopeptide-binding protein oppA 63075 237 11 BC_1613 d Q81FH4 Zn-dependent hydrolase 62004 193 8 187 5 BC_4410 (yajC) d Q817W7 Protein translocase subunit YajC 9486 132 6 72 2 BC_0337 d Q814A8 Hypothetical Membrane Spanning

Protein 14189 126 8 151 9 BC_3195 Q813E6 Hypothetical Cytosolic Protein 15025 113 7 74 3 109 5

BC_3787 Q81A08 Zinc protease 48972 112 3 BC_3786 Q81A09 Zinc protease 49288 112 2 BC_3586 Q81AI2 Oligopeptide-binding protein oppA 63759 104 3

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pept Score no.

pept Score no.

pept BC_3133 Q81BM0 Putative hydrolase 42323 98 3 BC_1424 Q81FY8 Ferredoxin--nitrite reductase 60770 97 3 BC_3986 d Q812V3 Hypothetical Cytosolic Protein 9170 95 3 61 2 BC_4480 (tig) d Q812Q9 Trigger factor 47316 88 4 35 1 BC_0344 (rocA) d Q81IP0 1-pyrroline-5-carboxylate

dehydrogenase 56418 84 4 23 1 BC_2375 Q81DJ0 Putative uncharacterized protein 9489 70 2 BC_0825 Q81HI3 Putative uncharacterized protein 30284 65 1 BC_2745 Q81CL1 Putative uncharacterized protein 43460 62 3 BC_1029 d Q81GZ8 IG hypothetical 18063 33466 57 4 25 1 BC_3090 d Q81BR0 Putative uncharacterized protein 18565 44 4 48 2 BC_5181 d Q815H3 UPF0145 protein BC_5181 11143 42 1 51 1 BC_3992 Q819I6 Putative uncharacterized protein 12700 30 1 44 2 22 1

BC_2969 Q81C15 Putative uncharacterized protein 12769 30 1 BC_2481 Q81D93 Putative uncharacterized protein 12069 28 1 BC_1456 Q81FV9 Putative uncharacterized protein 16272 27 2 BC_1708 Q81F89 Putative uncharacterized protein 25183 27 1 BC_3977 Q819J9 Zn-dependent hydrolase 62055 27 1 BC_3515 d Q813A3 Hypothetical Glycosyltransferase 82417 24 1 38 1 BC_0395 Q81IJ1 Metal-dependent hydrolase 23560 23 1 BC_3784 Q81A11 IG hypothetical 16623 9209 195 5 BC_4387 Q817Y7 Putative uncharacterized protein 4832 58 1 BC_3582 (yodI) Q81AI6 Putative uncharacterized protein 14601 213 4 25 1

BC_1334 Q813U4 Hypothetical Exported Protein 28367 106 1 BC_2878 Q81C97 Putative uncharacterized protein 29564 42 2 66 4

BC_0263 Q81IT6 Putative uncharacterized protein 38513 42 1 BC_1468 (sodF)f Q81FV0 Superoxide dismutase 26798 29 1 BC_0944 f Q81H77 Putative uncharacterized protein 61227 28 1

BC_2237f Q81DW2 Putative uncharacterized protein 74111 26 1

BC_2427 Q81DE0 Putative uncharacterized protein 34256 26 1 aDetails obtained from Uniprot database (www.uniprot.org/). bPeptide False Discovery Rate. cMASCOT MudPIT score & total number of identified peptides over three biological replicates. d Proteins identified only from coat + exosporium & coat fractions. eProteins identified only from coat + exosporium & exosporium fractions. fProteins identified only be semitrypsin search. g Phosphorylation sites identified by the error-tolerant MASCOT search. None of the other proteins were identified with modifications.

We divided the identified proteins from both species into six different categories based on their possible functions as indicated by their descriptions in the database - (1) proteins involved in spore coat morphogenesis and other known spore coat proteins; (2) spore coat proteins with a possible role in spore resistance; (3) exosporium proteins likely to be involved in attachment to surfaces; (4) exosporium proteins possibly involved in pathogenicity; (5) spore coat proteins possibly involved in spore germination and (6) other putative spore coat and/or exosporium proteins.


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Table 2. Identified spore proteins from Clostridium difficile 630.

a Gene name a

Uniprot ID


Spore coat bFDR= 1.72%

cScore cno. pept

(I) Proteins involved in spore coat morphogenesis and other known spore coat proteins

CD1067(cdeC)d, e Q18AS2 Putative uncharacterized protein 46845 6392 173

CD1581 e Q186D6 Putative uncharacterized protein 20043 2572 103

CD1613 (cotA)d, e Q186G8 Putative uncharacterized protein 34834 1680 50

CD2401 (cotJC2/cotD) d, e Q181Y5 Spore coat peptide assembly protein CotJC 2 21497 1231 39

CD1433 (cotE) d, e Q18BV5 Putative bifunctional protein: peroxiredoxin/chitinase 82019 957 46

CD3567 (sipL) e Q181G7 Putative phage cell wall hydrolase 59373 869 44

CD0598 (cotJC1/cotCB) d, e Q189E4 Spore-coat protein 21577 823 38

CD2400 (cotJB2) e Q181Y6 Spore coat peptide assembly protein CotJB 2 10663 722 16

CD0597 (cotJB1/cotCA) d, e Q189E5 Spore coat peptide assembly protein 10756 389 15

CD1511 (cotB) d ,e Q18C29 Putative uncharacterized protein 35056 370 16

CD0596 (cotF) d, e Q189D6 Putative uncharacterized protein 8844 336 13

CD2399 e Q181Y3 Putative uncharacterized protein 8247 169 5

CD2598 Q182T7 Putative oligosaccharide deacetylase 29780 70 2

CD2629 (spoIVA) e Q182W3 Stage IV sporulation protein A 55539 55 6

CD3569 (yabG) Q181G9 Sporulation-specific protease 32137 33 1

CD0213 (cotF) Q18CV2 Putative spore coat protein 10927 31 3

(II) Spore coat proteins likely to play a role in spore resistance

CD2845 (rbr) e Q183T4 Rubrerythrin 22603 373 12

CD2864 (yisY) e Q183V0 Putative hydrolase 30520 243 13

CD1524 Q18C45 Putative rubrerythrin 20319 126 9

CD1623 Q186H7 Putative oxidoreductase 94987 110 5

CD0825 (rbr) e Q18A24 Rubrerythrin 20829 64 3

CD0116 Q18CK8 Putative ferredoxin/flavodoxin oxidoreductase,alpha subunit 39290 106 4

CD0117 Q18CK7 Putative ferredoxin/flavodoxin oxidoreductase,beta subunit 27031 61 2

CD1567 (cotG) d, e Q186C1 Putative manganese catalase 25329 45 3

CD0176 Q18CR6 Putative oxidoreductase, NAD/FAD binding subunit 45889 38 2

(III) Exosporium proteins likely to be involved in attachment to surfaces

CD0332 (bclA1)e Q18D69 Putative exosporium glycoprotein 68114 85 5

(IV) Exosporium proteins possibly involved in pathogenicity

-- -- -- -- -- -- --

(V) Spore coat proteins involved in spore germination

CD0551 (sleC) e Q188Z5 Spore cortex-lytic enzyme pre-pro-form 47426 227 10

CD3102 Q184U1 Putative peptidase, M20 family, peptidase V related 53995 29 2

(VI) Other putative spore coat and/or exosporium proteins

CD3032 Q184M0 Putative pyridoxal phosphate-dependent transferase 47977 332 18

CD3620 Q181M3 Putative uncharacterized protein 14256 232 11

CD1133 Q18AZ2 Putative uncharacterized protein 23857 217 10

CD3580 Q181I4 Putative uncharacterized protein 29071 157 5

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a Gene name a

Uniprot ID


Spore coat bFDR= 1.72%

cScore cno. pept

CD0855 (oppA) Q18A51 ABC-type transport system, oligopeptide-family extracellular solute-binding protein 58649 126 9

CD3664 Q181R8 Putative aminotransferase 44997 119 7

CD1536 Q18C55 Ferredoxin--NADP(+) reductase subunit alpha 33353 94 3

CD3522 Q181C2 Putative uncharacterized protein 50689 91 4

CD1291 (dacF) e Q18BF4 D-alanyl-D-alanine carboxypeptidase 42194 90 3

CD2808 Q183P2 Putative uncharacterized protein 23705 87 1

CD1463 Q18BY4 Putative uncharacterized protein 17988 68 5

CD3652 Q181Q6 Putative peptidase, M1 family 54828 62 5

CD3170 (eno) e Q181T5 Enolase 46289 67 3


CD2865 Q183U9 Putative bacterioferritin 20747 54 3

CD3613 Q181L4 Putative uncharacterized protein 17425 42 3

CD2431 Q182B6 Putative nitrite/sulphite reductase 58684 40 1

CD0115 Q18CK1 Putative 4Fe-4S ferredoxin, iron-sulfur binding domain protein, delta subunit 8140 40 2

CD1622 Q186H8 Putative uncharacterized protein 23461 39 1

CD3232 Q17ZX7 UPF0597 protein CD630_32320 45787 39 1

CD3457 Q180V4 Putative uncharacterized protein 15859 37 2

CD1063.1 e Q18AR2 Putative uncharacterized protein 8221 35 2

CD0894 Q18A86 Putative iron-dependent hydrogenase 56384 32 1


CD0279 Q18D22 Putative uncharacterized protein 14922 24 1

CD2477 e D5Q2G9 Putative uncharacterized protein 36070 22 1 aDetails obtained from Uniprot database (www.uniprot.org/). bPeptide False Discovery Rate. cMASCOT MudPIT score & total number of identified peptides over three biological replicates. dRenamed in the previous studies[24, 26, 28]. eProteins identified previously from the whole spore protein extract[27]. 1. Proteins involved in spore coat morphogenesis and other known spore coat proteins.

In the previous studies in B. subtilis different morphogenetic proteins important for the morphogenesis of the spore coat were assigned. These proteins include, starting from the cortex and moving towards the outer coat, SpoVM, SpoIVA, SpoVID, SafA and CotE. For the biogenesis of the inner coat SpoIVA and SafA are important while CotE plays a pivotal role in outer coat development [15, 23]. In our current study we identified CotE (BC_3770), SafA (BC_4420) and SpoIVA (BC_1509) from B. cereus ATCC 14579 and SpoIVA (CD2629) as well as SipL (CD3567, recently identified [29] morphogenetic protein) from C. difficile 630. The CotE orthologue of B. subtilis was not found by protein sequence comparison in the C. difficile 630 proteome. However CD1433 renamed as CotE [24], was identified. Orthologues of other morphogenetic proteins i.e. SpoVID, SpoVM were not identified but for these two proteins it is important to keep in mind that


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Table 3. Spore coat and/or exosporium proteins identified in our studies from B. subtilis 168, B. cereus ATCC 14579 and C. difficile 630.

Spore coat and/or exosporium proteins identified in our studies and having conserved orthologues (> 30% protein identity) in the three spore formers. A letter Y indicates experimental identification of the proteins. Proteins CotJC, DacF, SpoIVA and YisY were identified experimentally from all the three organisms. they are mostly identified in the soluble fraction of spores from B. subtilis [30, 31]. In B.subtilis, coat proteins CotC, CotG and CotU are known to be important for the coat structure wherein cross-linking among these proteins plays a major role [32-35]. Genes for CotC, CotG, CotS, CotT and CotW are not present in the B. cereus genome [15]. Likewise, orthologues of SpoVID, SpoVM, CotC, CotG, CotT, CotU and CotW are not observed in the C. difficile genome indicating a possible difference in the spore structure. Spore coat G (BC_2030) identified in our study should not be confused with CotG from B. subtilis as it is 89% identical to the highly phosphorylated exosporium protein ExsB from B. anthracis. The phosphorylated region of BC_2030 is 96% identical to the region identified from ExsB from B. anthracis [36]. Identical to B. anthracis ExsB [36], we identified Threonine residues at positions 76, 79, 82, 105, 114, 117, 129 from BC_2030 as phosphorylated in our error-tolerant MASCOT search. Identified protein CD2399 is a paralogue of CD0596 (renamed as CotF [28]) also identified in the current study. Both these proteins contain a CotJA superfamily domain of spore proteins and thus could function as the CotJA orthologues. Two copies of each, spore coat B (BC_0389 and BC_0390), spore coat Y (BC_1218 and BC_1222) and spore coat X (BC_2872 and BC_2874), are present in the genome of B. cereus ATCC 14579, and these were all identified in this study. Considering the Bacillus species in which the orthologues of these proteins are found and based on the fact that B. subtilis has an orthologue of one of each duo (as predicted from the GenoList database), it is possible that these proteins are

Protein Bacillus cereus ATCC 14579 Clostridium difficile 630 Bacillus subtilis 168

YabG Y Y YabP Y YdbD Y Y CotJC Y Y Y CotJB Y Y SpoIVA Y Y Y SleB Y Y RnjA Y YmfH Y YmfF Y DacF Y Y Y Gpr Y Tig Y YtfJ Y Y YtjP Y YisY Y Y Y Eno Y Y CwlJ Y Y

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localized either in the spore coat, the exosporium or in the basal layer of the exosporium. For instance, protein spore coat Y (BC_1218) is in fact a close orthologue of exosporium morphogenetic protein BAS1141 or ExsY from B. anthracis [37]. Another structural protein identified in our study was spore coat protein D (BC_1560), which is an orthologue of CotD - an inner coat protein in B. subtilis identified prominently from the soluble fraction. This indicates a possible structural difference amongst B subtilis and B. cereus spore coats. From B. cereus, we also identified protein BC_4419, an orthologue of YhcN from B. subtilis (< 30% sequence identity and thus left out of the orthologue data (Supplementary Table 2)) that contains a Spore_YhcN_YlaJ motif. This motif is reported to be found in lipoproteins present in spores and not in vegetative cells. In B. subtilis 168 spores YhcN is suggested to be located in the inner membrane or integument of the spore [38]. Although the expression of YhcN in B. subtilis is controlled by the transcription factor σG (expressed late in the forespore during sporulation [39]) due to the signal sequence carried by the protein it may be identified in the insoluble coat fraction. The collagen adhesion protein (BC_5056) contains a TQxA domain and is conserved amongst the B. cereus group. TQxA domain containing proteins have been reported to be associated with LPXTG-containing sortase target domains. Additionally, LPXTG proteins are located at the cell wall peptidoglycan [40]. It is plausible that BC_5056 is in the spore coat or in the integument region connecting cortex and the coat. Another interesting protein is the L-alanyl-D-glutamate peptidase BC_2677, an orthologue of CwlK which is thought to be involved in cell wall peptidoglycan hydrolysis in B. subtilis [41]. This protein has a VanY domain that is present in proteins involved in cell wall biosynthesis. Additionally, BC_2677 also has SH3 superfamily domain. Proteins belonging to the SH3 family are involved in the formation of multi-protein complex assemblies [42]. This is an essential feature in spore coat build-up and this protein could be an interesting candidate for studying structural built-up in B. cereus spores. Superoxide dismutase (SodA) also plays an important role in spore coat assembly in B. subtilis by mediating protein cross-linking. We did not identify the enzyme in B. cereus ATCC 14579. By a semitrypsin enzyme search with MASCOT we identified SodF (BC_1468) from the spore coat fraction. The superoxide dismutase (CD1631) from the genome of C. difficile was not identified in our study but, to our surprise, PSI-BLAST with one iteration did not predict orthologue for SodA either. In fact CD1631 is more identical to SodF identified from exosporium fractions previously (Supplementary Table 5). 2. Spore coat proteins with a role in spore resistance.

CotA from B. subtilis is known to play a role in UV-resistance of spores. Interestingly CotA from B. subtilis has orthologues present in C. botulinum A ATCC 19397, C. botulinum A ATCC 3502 and C. botulinum strain A Hall but not in any other species and/or strains considered for orthologue evaluation (Supplementary Table 4). On the other hand CD1613 (renamed as CotA) suggested to be involved in assembly of the outer coat in C. difficile 630 spores[28] was identified. The newly identified protein BC_4047 is an orthologue of the outer coat protein Cotα (BAS3957) in B. anthracis


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Sterne and contains a DUF3915 domain of unknown function. There are three Cysteine residues at the amino terminus (first 20 residues) and similar to the case in B. anthracis spores [43], these are likely to be cross-linked. The absence of Cotα is reported to affect visual appearance of the outer spore coat as well as the chemical resistance and sensitivity properties of the spores towards phenol, chloroform, and hypochlorite [43]. In anaerobic organisms defense against oxidative stress is of prime importance. Since spores are important in the dissemination of the clostridial species it is likely that proteins with ability to scavenge the oxidative radicals are present in the outermost layers of the spores. This is also evident from the identified catalases (CotJC1/CotCB (CD0598), CotG (CD1567), ferritin like proteins, ruberythrins (CD0825, CD1524, CD2845) and oxidoreductases (CD0117, CD0176, CD1623) from C. difficile spores (Supplementary Table 1). These proteins may play a dual role - one in structural built-up of spores coat layers by mediating dityrosine cross-linking among proteins and/or second in resistance against oxidative stress [44, 45] or they may have some other hitherto unknown functions. For instance, in addition to the radical scavenging activity, the oxidoreductase CD1623 also has a predicted β-lactamase domain and thus might be important in antibiotic resistance. 3. Exosporium proteins likely to be involved in the attachment to the surfaces.

The exosporium layer has been less extensively studied than the spore coat. BclA from B. anthracis is one of the few exosporium proteins that is well characterized and is an important structural component of the hair-like nap in exosporium [46, 47]. We could not identify this protein (accession no. HM071986.1[16]) in our B. cereus exosporium preparations but proteins BC_3345 & BC_2569 (56% and 58% identical to HM071986.1 respectively) and BclC (BC_3712) were identified in this study. These Bcl-family proteins, in general, form an extended rod-like structure in the regions that possess the (Gly-X-Y)n motifs, which is characteristic for a triple helix, rod-like structure formation [48]. A recent study [49] showed that the BC_2569 protein, similar to BclA, contains a potential NTD attachment motif (-INPDLLGPTLPAI-) necessary to dock this protein in the basal layer of the exosporium. From C. difficile, CD0332 (BclA1) was identified which indicates that the presence of the exosporium in spores was inconsistent in our study. As opposed to the predictions of algorithms used by databases such as KEGG (www.genome.jp/kegg/), orthologues for Bcl family of proteins were not identified to be conserved by the PSI-BLAST analysis by a single PSI-BLAST iteration from any other clostridial species considered in this study. Recently other exosporium proteins BxpA, BxpB, BclB [19, 50, 51] have also been identified in B. anthracis. Protein BxpA (BC_2149) has an amino acid sequence unusually rich in glutamine and proline [52]. Interestingly, VrrA protein in B. anthracis is also rich in glutamine and proline residues which are suggested to be involved in cross-linking of protein subunits [53]. BxpB is an exosporium basal layer protein in B. anthracis Sterne [50] and its orthologue BC_1221 in B. cereus ATCC 14579 was identified in the current study. In B. anthracis BxpB has a paralogue ~80% identical, named ExsFB (BAS2303) and the identified protein BC_2374 is an orthologue of ExsFB.

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Both BC_1221 & BC_2374 are 75% identical to BxpB (BAS1144) and ExsFB (BAS2303) as shown by a Clustal Omega alignment tool. This suggests that BC_1221 and BC_2374 may be localized in the basal layer of the B. cereus ATCC 14579 exosporium in providing attachment sites for BclA-like proteins. In bxpB mutants of B. anthracis, the function of BxpB could not be complemented with BAS2303, implying a different function for BAS2303 in B. anthracis spores[50]. Orthologues for BxpA (BC_2149), BxpB (BC_1221) and ExsFB (BC_2374) are well conserved in the B. cereus group. Protein BC_2493 is an orthologue of exosporium protein ExsK identified from the exosporium fraction of B. anthracis spores (BA_2554) in a study of Redmond et al.[54] Identified cell surface proteins BC_2639 and BC_3547 (523.5 kDa and 97.9 kDa, respectively) contain multiple unknown domains named DUF11 and non-specific domains called B_ant_repeat domains. These B_ant_repeat domain containing proteins are shown to be encoded by one, two or three very conserved large genes in the B. cereus group [55]. These genes are expressed in the last developmental time waves during sporulation [56]. Although a function is unknown it has been reported that these proteins may have a similar function to that of proteins that form ribbon-like appendage structure on the exosporium of Clostridium taeniosporum spores [55]. These proteins denoted P29a and P29b in C. taeniosporum are smaller in size than the proteins we identified, but the size may be extremely variable between species. These appendage-like structures may be common in spores of Bacilli and of Clostridia [57] and may facilitate spore dissemination, assist in spore nutrition during formation, or have no function and result from a deranged metabolism [58]. 4. Exosporium proteins possibly involved in pathogenicity.

Since the exosporium is the outermost layer of the spores in many species, it is the initial point of contact with the host cells and tissues. Therefore, in addition to providing unique adherence and hydrophobic properties to spores, it may express molecules that play a significant role in spore attachment and pathogenicity. The BclA protein is suggested to be the immunodominant epitope on the surface of Bacillus anthracis spores and contains a C-terminal TNF-like domain [47, 59]. Though not required for pathogenesis [60] BclA may mediate internalization of spores by host cells via the C1q/TNF-like domain. Putative uncharacterized proteins BC_2266 and BC_2267, identified in our study both possess a domain belonging to the TNF superfamily and has a weak homology with the C1q domain. The C1q domain has been reported to play a role in spore attachment and in host entry mechanisms where spores are suggested to bind to the gC1qR receptors on the host cell via some unidentified molecules, in a Ca2+ dependent manner [61]. Thus, BC_2266 and BC_2267 could very well be mediators involved in a spore attachment and host entry process. Tertiary structure modeling with Phyre2 tool for both molecules showed that they have 99-100% homology with TNF in the region from amino acids 70 to 150. About 79% to 84% of the sequence could be modeled with over 90% confidence. Further studies are required to understand the precise location and the function (e.g. using animal models) of these two proteins.


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5. Spore coat proteins involved in spore germination.

While the spores of Bacillus and Clostridium species can survive for years in the dormant state, they can be rapidly converted to a metabolically active state via the process of spore germination and outgrowth. A variety of small molecules can trigger spore germination preliminary by binding to the germination receptors in the inner membrane. L-alanine is known to be a potent germinant of B. cereus spores, whereas D-alanine inhibits germination. Alanine racemase (Alr1; BC_0264) by converting L-alanine to its stereoisomer thereby preventing germination [62] and the inosine-uridine preferring nucleoside hydrolase (IunH; BC_2889) by converting the germinant inosine to D-ribose could play a role in preventing germination[63]. Once germination is triggered, the coat needs to be broken down and peptidases identified in this study (Supplementary Table 3) may be involved in germination. Subsequent spore outgrowth requires that the spore cortex is degraded immediately once germination is triggered. Cortex lytic enzymes (CLEs) like CwlJ and SleB play an important role in cortex degradation and are located in the B. subtilis spore coat. In B. cereus ATCC 14579 we identified SleB (BC_2753) and CwlJ (BC_5390). Identified protein BC_5391 is an orthologue of GerQ/YwdL that is involved in proper localization of the cortex lytic enzyme CwlJ in the B. subtilis spore coat [64]. However, in other studies GerQ/YwdL and CwlJ were identified in the exosporium fraction of B. cereus ATCC 10876 and B. anthracis spores, respectively [52, 65]. Another enzyme identified from the insoluble fraction in this study and reported to be involved in spore cortex degradation [66] during spore germination was YaaH orthologue BC_3607. Newly identified BC_1591 contains a predicted pectate lysase domain, which is related to the family of glycosyl hydrolase proteins and may therefore be involved in peptidoglycan modification, degradation or synthesis. In addition there were also other hydrolases identified in our study that can be involved in spore germination (Supplementary Table 3). From C. difficile the predominant cortex lytic enzyme SleC (CD0551) was identified in this study. Of further interest, the transcriptomic data of Dembek and colleagues [67] shows that genes cd0115, cd0116, cd0117, cd0176, cd0279, cd0587, cd0825, cd0855, cd1133, cd1524, cd1536, cd1622, cd1623, cd3232, cd3664 are down-regulated while genes cd0213, cd1511, cd1613, cd2845, cd3567 are up-regulated upon initiation of germination. It is noteworthy that genes cd0115, cd0116, cd0117, cd0176, cd1536, cd1623 (all encoding oxidoreductases) and cd0825, cd1524, cd2845 (all encoding ruberythrins) may play a role in energy-dependent germination [68] of Clostridium spores. We identified all these gene products and thus it could be worthwhile to study their role in spore germination. 6. Other putative spore coat and/or exosporium proteins.

Another newly identified protein in our study could be an important target for the B. cereus group of spore formers. It is an orthologue of BAS5303 in B. anthracis Sterne [69] and BA5699 in B. anthracis Ames, identified previously in the exosporium fraction in the Ames strain [52] and also shown to be immunogenic in animal models [69, 70]. BC_0996 could therefore also be located in the exosporium of B. cereus ATCC 14579,

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although its function remains unclear. Protein BC_0987 is much less conserved in the B. cereus group and could very well be a candidate marker for the B. cereus spores. There were many peptidases and hydrolases identified (Supplementary Table 1 & 2) which we could not assign to any particular category and were classified as putative coat and or exosporium proteins. The plasmid encoded BC_p0002 was found to be unique to the B. cereus ATCC 14579 strain. Notably, this protein might have a distant relation (20% sequence identity, E-value 1.8) with the envelope glycoprotein GP120 (residues 193 - 372) from Human Immunodeficiency Virus (HIV). The highly abundant [27] and cysteine-rich (~9%) protein CD1067, recently shown to be important for exosporium assembly [26], was identified and could very well be a potential marker for this species. Another abundant protein CD1581 was identified as well and is uniquely present in C. difficile 630. Identified protein CD3613 belongs to the Stay-green (SGR) protein family. The SGR family is conserved in plants and is involved in chlorophyll degradation. Distant SGR homologs are also present in algae and, unexpectedly, in species of Bacillus and Clostridium, but not in other bacterial genomes [71]. However, the exact role of these proteins in bacterial species remains to be studied. Protein CD3652 from C. difficile 630 has a characteristic GluZincin protease domain. The GluZincin family (thermolysin-like peptidases or TLPs) includes several zinc-dependent metallopeptidases that contain His-Glu-X-X-His and Glu-X-X-X-Asp motifs as part of their active site. Interestingly, the light-chain (LC, 448 a.a from the N-terminus of the protein) of the potent neurotoxin BoNT/A from C. botulinum also has this domain [72]. Previous studies have confirmed that zinc-binding plays an essential role in the catalytic activity of the light chain of BoNT/A. Thus protease CD3652 is an interesting target to study for its role in spore-mediated infections. As mentioned above, for orthologue evaluation using PSI-BLAST and to study the conservation of coat and exosporium proteins we used the identified proteins from B. cereus ATCC 14579 (this study), C. difficile 630 (this study) and B. subtilis 168[15, 22] as the initial dataset. Figure 2 shows the distribution of orthologues respective to the three species mentioned. As seen, identified B. subtilis coat proteins are very unique to the organism with 25 being only present in B. subtilis. Although B. cereus and B. subtilis are evolutionary distant, 53 B. subtilis coat proteins had orthologues present in B. cereus ATCC 14579 with > 30% sequence identity. From the identified B. subtilis 168 spore coat protein dataset, 9 proteins - CotJB, CotJC, DacF, SpoIVA, SleB, YabG, YhxC, and YtfJ were found to be conserved in selected Bacilli and Clostridia (Figure 2, Supplementary Table 4). Focusing on the identified proteins from B. cereus ATCC 14579, it was seen that 77 proteins are conserved in the B. cereus group while 44 had orthologues in B. subtilis 168 and 11 in C. difficile 630. Eleven of the identified proteins emerged to be conserved in all of the spore forming species considered for comparative analysis. Amongst the known exosporium proteins, 27 proteins have conserved orthologues (Supplementary Table 5) in the B. cereus group including the Bcl-family of proteins. Alanine racemase is conserved throughout the spore formers while in addition to BclA, only SodF has an orthologue present in C. difficile and few strains of C. botulinum. From the C. difficile 630 identified proteins, 13 are unique to this strain, 12 have orthologues in B. subtilis 168, 13 have orthologues in B. cereus and 8 are conserved


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across the spore formers considered for comparison. Our orthologue predictions for certain B. subtilis and C. difficile proteins were also supported by previous transcriptomic [73] and the phylogenetic studies [74] making our orthologue identification method confident. Figure 3 summarizes the orthologue conservation results highlighting potential protein targets for easy detection and removal of spores. There are 13 coat proteins conserved in all spore formers considered in this study. From the exosporium proteins, 11 are conserved in the B. cereus group and only Alr has orthologues in all Clostridia. The exact localization of three proteins BC_0987, BC_0996 both conserved in the B. cereus group and CD3613 conserved in Clostridia is not known. Lastly, despite severe and harsh treatments to the sample pellets, we identified a number of cytosolic proteins including highly abundant ribosomal proteins and elongation factors (Supplementary Table 8). We think that since in both species the exosporium was not completely removed, these proteins might have been trapped in the interspace region between the coat and exosporium layers during sporulation. The fact that Liu et al.[52] also identified orthologues of many of these proteins from the exosporium fraction of B. anthracis indicates that the process of exosporium attachment to the coat occurs in a highly regulated and precise manner inside the mother cell cytoplasm. It is suggested that efficient removal of the exosporium is a must to diminish the contribution of cytosolic proteins. Recently an effort has been made to remove exosporium from C. difficile 630 spores [75] but in Bacillus spp. efficient removal of exosporium has not been achieved [76]. For the same reason, it is also difficult to classify the identified proteins as solely coat or exosporium proteins. Concluding Remarks

In conclusion, our gel-free method performs comprehensively by identifying coat and exosporium proteins from different aerobic and anaerobic spore formers. We provide potential protein targets emerging from this study, for use in food and health sectors for selective removal of spores from the samples, and these targets need to be studied in detail. Also our study outlines the general classes of proteins that are necessary for the spore integrity namely - the structural proteins (eg. SpoIVA, SafA, CotE), proteins involved in resistance mechanisms (eg. oxidoreductases, catalases, ferritin like proteins) and proteins that facilitate germination (eg. hydrolases, peptidases). Additionally, we provide a wide view of protein conservation in the structure of spores from different aerobic and anaerobic pathogenic spore formers in an attempt to derive a universal set of spore surface proteins. We propose that proteins CotJB, CotJC, DacF, Gpr, Eno, SleB, SpoIVA, RnjA, YabG, YabP, YhxC, YloB, YtfJ make up the universally conserved set of proteins in bacterial spore formers. Nevertheless, functional annotation of the genomes, localization studies for the identified putative spore coat and exosporium proteins as well as quantitative studies are necessary for gaining more detailed insights of the spore structure.

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Figure 2. Orthologue distribution for identified spore coat & exosporium proteins from B. subtilis 168, B. cereus ATCC 14579 and C. difficile 630. Query protein sequences used for orthologue identification comprised of proteins identified from B. subtilis 168 (92 proteins) [15],[22], B. cereus ATCC 14579 (100 proteins; this study) and C. difficile 630 (54 proteins; this study). The bars indicate the number of proteins from the respective query datasets that have orthologues present in the species or groups indicated below. *Number of proteins exclusive for indicated organisms studied in our work (See Supplementary Tables 2, 3 & 4 for details).


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Figure 3. Potential target proteins from spore coat and/or exosporium layers emerging from this study. Spore coat proteins presented are conserved in the spore formers considered for the comparison. a Exosporium proteins conserved in the B. cereus group (Alr is conserved in Bacilli as well as in Clostridia). b Localization of the proteins is unknown. BC_0987 & BC_0996 are conserved in the B. cereus group and CD3613 is conserved in Clostridia. Materials and Methods

Media and bacterial strains used

The strains used in this study were B. cereus ATCC 14579 and C. difficile 630 (tcdA+ tcdB+). For B. cereus ATCC 14579, growth and sporulation were carried out in Tryptic Soy Broth (TSB) medium as well as a chemically defined growth and sporulation (CDGS) medium, as described previously[77]. The CDGS medium consisted of the following components (final concentrations): 10 mM D-glucose, 20 mM L-glutamic acid, 6 mM leucine, 2.6 mM L-valine, 1.4 mM L-threonine, 0.47 mM L-methionine, 0.32 mM L-histidine, 5 mM D/L-lactic acid, 1 mM acetic acid, 50μM FeCl3, 2.5 μM CuCl2, 12.5 mM ZnCl2, 66 μM MnSO4, 1 mM MgCl2, 5 mM (NH4)2SO4, 2.5 μM Na2MoO4, 2.5 μM CoCl2, 1 mM Ca(NO3)2, buffered to pH 7.2 using 100 mM potassium phosphate buffer. C. difficile 630 was routinely grown in vegetative state on Brain-Heart Infusion Supplemented (BHIS) agar plates and in Tryptone Glucose Yeast extract (TGY)-vegetative medium (3% TSB, 2% glucose, 1% yeast extract, 0.1% L-cysteine). Pre-culturing was done in SMC broth (90 g peptone, 5 g proteose peptone, 1 g (NH4)2SO4, 1.5 g Tris) containing 0.1% L-cysteine, while sporulation was initiated on SMC agar plates [24]. Conditions for growth and sporulation

For B. cereus ATCC 14579 and C. difficile 630 respectively four and three independent biological spore crops were made. A B. cereus culture was grown overnight in 50 mL TSB medium (pH 7.5) at 30⁰C in a shaker-incubator at 200 rpm. Cells were harvested by centrifugation at 10000 rpm for 30 min at 4⁰C, resuspended in 500 mL CDGS medium, and incubated at 30⁰C for 96 h at 200 rpm. After 4 days, the spores were harvested by centrifugation. To lyse the remaining vegetative cells, the spore pellet was treated twice with 0.1% (final concentration) Tween-20 and subsequently washed five times with sterile Milli-Q water. From each B. cereus spore crop, three sample fractions were prepared. The first fraction contained spores with intact exosporium and coat (Fraction 1), the second fraction contained spores with exosporium removed (spore coat sample; Fraction 2) and the third fraction contained the concentrated exosporium fragments (Fraction 3). For C. difficile, a colony from a BHIS agar plate was inoculated into 10 ml TGY medium and incubated at 37°C overnight. This TGY culture was then subcultured into SMC broth and incubated until the cells achieved the logarithmic phase, and then plated onto SMC agar plates. The plates were further

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incubated for 7 days at 37°C. After 7 days, the spores were harvested by washing twice in water and followed by suspension in phosphate-buffered saline (PBS) containing 125 mM Tris, 200 mM EDTA, 0.3 mg/ml proteinase K (Fermentas), and 1% sarcosyl with further incubation and gentle shaking at 37°C for 2 h. Finally, spores were centrifuged; the pellets were resuspended in water, and washed a further 10 times [24]. Only the coat fraction was analyzed from C. difficile in this study. Removal of exosporium

To separate the exosporium layer from B. cereus spores, spores (1010 spores / mL) were resuspended in 50 mM Tris-HCl, 0.5 mM Na-EDTA (pH 7.5) and passaged 4 times through a French pressure cell (Thermo Fisher Scientific, MA, USA) at 20000 psi. Spores were separated from exosporium fragments by centrifugation at 10000 rpm for 15 min at 4⁰C. The supernatant (containing loose exosporium fragments) was centrifuged one more time, at 10000 rpm for 15 min at 4oC and pressed through a 0.22 μm PES filter (VWR International, PA, USA) to remove any remaining spores and stored at 4oC. The pelleted spore fraction was resuspended in sterile Milli-Q water and stored at 4oC until further use. The protocol was adapted from the work of Todd and co-workers [78]. Isolation of spore coat

The spore fraction of B. cereus ATCC 14579 (obtained after French press treatment) and C. difficile 630 was centrifuged and the spore pellets were resuspended in 10 mM Tris-HCl (pH 7.5). The spores were disintegrated by bead-beating with 0.1 mm Zirconia-Silica beads (BioSpec Products, USA) using a Precellys 24 homogenizer (Bertin Technologies, Aix en Provence, France). Nine rounds of bead-beating were performed, with each round consisting of a 40 s cycle at 6000 rpm and a 1 min interval between each round for cooling. After every three rounds of beating, 10 min cooling periods (on ice) were allowed to prevent protein degradation by overheating. The disintegrated spore pellets were then washed 5-6 times with 1 M NaCl to remove non-covalently bound proteins and intracellular contaminants. Spore coat protein extraction was done by thermal heating for 10 min using a water bath, starting at ambient temperature and reaching a final temperature of 80⁰C in SDS extraction buffer consisting of (in final concentrations): 50 mM Tris-HCl (pH 7.8), 100 mM Na-EDTA, 150 mM NaCl, 0.2% SDS and 100 mM β-mercaptoethanol. After protein extraction, the remaining SDS insoluble spore coat protein pellets were washed four times with sterile Milli-Q water to remove SDS. The pellets were then frozen in liquid nitrogen and freeze-dried overnight and stored at -80⁰C. Concentration and analysis of exosporium

For B. cereus spores, the suspension (obtained after French press) containing the exosporium fragments, was concentrated by ultracentrifugation at 40000 rpm for 1 h at 4⁰C. The pellet containing exosporium fragments was then washed in 1 M NaCl and subsequently twice in TEP buffer, consisting of 50 mM Tris-HCl (pH 7.5), 10 mM Na-EDTA, 0.2% SDS and 2 mM β-mercaptoethanol. After washing in TEP buffer, the pellet was washed once in TEP buffer without SDS. Exosporium protein extraction was done as mentioned above for spore coat protein extraction. After protein extraction, the remaining SDS insoluble exosporium pellet was washed four times with sterile Milli-Q water to remove residual SDS. The obtained pellet was then frozen in liquid nitrogen, freeze-dried overnight and stored at -80 ⁰C. Though C. difficile spores have exosporium, its presence in our sample was variable. This is likely because of its loss during spore harvest. Therefore we did not proceed to isolate exosporium from C. difficile spores. The method was adapted from the work of Redmond et al [54]. Analysis of spore cortex peptidoglycan

The muramic acid from the spore cortex peptidoglycan is an efficient marker to estimate the purity (i.e. the absence of cortex peptidoglycan) of the spore coat sample. Therefore muramic acid assays were performed where the muramic acid is estimated based on the absorbance measurements as described


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previously[79, 80]. To eliminate any possible interference in the assay from the exosporial sugars (indicating inefficient exosporium removal as reported previously by Thompson & co-workers[76]) deglycosylation of the sample pellets was done by β-elimination according to the manufacturer’s instructions (GlycoProfile™ IV Chemical Deglycosylation Kit, Sigma-Aldrich Co. LLC). Lysozyme is known to act on the 1,4-β-linkage between N-acetylmuramic acid and N-acetyl-D-glucosamine from the vegetative bacterial cell wall thus we also subjected the pellets to lysozyme (Sigma-Aldrich Chemie B.V.; final concentration 250 μg/ml) treatment[81] to remove the remaining muramic acid residues, if any. Intact spores, disintegrated spores before and after NaCl wash, and the final reduced and alkylated spore extracts were used for the assay. Preparation for MS analysis

Preparation of samples from spore coat & exosporium fraction, spore coat fraction and exosporium fraction was done separately in the following manner. For reduction of disulfide bridges, the freeze-dried samples were incubated with 10 mM dithiothreitol (DTT) in 100 mM NH4HCO3 for 1 h at 55⁰C. The reducing reaction was followed by an alkylation treatment with 55 mM iodacetamide (IAA) in 100 mM NH4HCO3 for 45 min. at room temperature in the dark. The samples were immediately digested for 18 h at 37⁰C with trypsin (Trypsin Sequencing Grade Promega, Madison, WI, USA) using 1:60 w/w protease: protein ratio. The tryptic digests were desalted using Omix μC18 pipette tips (80 μg capacity, Varian, Palo Alto, CA, USA) according to the manufacturer’s instructions and the peptides were collected in 25μL 50% acetonitrile (ACN), 0.1 % trifluoroacetic acid (TFA) and stored at -80⁰C. Before analysis a fraction of eluted peptide material was freeze-dried and concentrated in 10 μL of 0.1% TFA and peptide concentration was measured at 205 nm [82] with a Nanodrop ND1000 spectrophotometer (Isogen Life Sciences, De Meern, The Netherlands).

LC-FT-ICR MS/MS analysis

LC-MS/MS data were acquired with an Bruker ApexUltra Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a 7 T magnet and a nano-electrospray Apollo II DualSource™ coupled to an Ultimate 3000 (Dionex, Sunnyvale, CA, USA) HPLC system. Samples containing up to 60 ng of the tryptic peptide mixtures were injected as a 10 μl 0.1% TFA, 3% ACN aqueous solution and loaded onto a PepMap100 C18 (5-μm particle size, 100-Å pore size, 300-μm inner diameter x 5 mm length) precolumn. Following injection, the peptides were eluted via an Acclaim PepMap 100 C18 (3-μm particle size, 100-Å pore size, 75-μm inner diameter x 250 mm length) analytical column (Thermo Scientific, Etten-Leur, The Netherlands) to the nano-electrospray source. Gradient profiles of up to 120 minutes were used from 0.1% formic acid / 3% CH3CN / 97% H2O to 0.1% formic acid / 50% CH3CN / 50% H2O at a flow rate of 300 nL/min. Data dependent Q-selected peptide ions were fragmented in the hexapole collision cell at an Argon pressure of 6x10-6 mbar (measured at the ion gauge) and the fragment ions were detected in the ICR cell at a resolution of up to 60000. In the MS/MS duty cycle, 3 different precursor peptide ions were selected from each survey MS. The MS/MS duty cycle time for 1 survey MS and 3 MS/MS acquisitions was about 2 s. Instrument mass calibration was better than 1 ppm over a m/z range of 250 to 1500. Raw FT-MS/MS data were processed with the MASCOT DISTILLER program, version 2.4.3.1 (64bits), MDRO 2.4.3.0 (MATRIX science, London, UK), including the Search toolbox and the Quantification toolbox. Peak-picking for both MS and MS/MS spectra were optimized for the mass resolution of up to 60000. Peaks were fitted to a simulated isotope distribution with a correlation threshold of 0.7, with minimum signal to noise of 2. The processed data, combined from the three independent biological replicates, were searched in a MudPIT approach with the MASCOT server program 2.3.02 (MATRIX science, London, UK) against a complete B. cereus ATCC 14579 and C. difficile 630 ORF translation database (Uniprot 2011 update, downloaded from http://www.uniprot.org/uniprot), respectively. The databases were complemented with their corresponding decoy data bases for statistical analyses of peptide false discovery rate (FDR). Trypsin was used as enzyme and 2 missed cleavages were allowed. Carbamidomethylation of cysteine was used as a fixed modification and oxidation of methionine as a

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variable modification. The peptide mass tolerance was set to 30 ppm and the peptide fragment mass tolerance was set to 0.03 Dalton. If needed the search was repeated with the same parameters but with semitrypsin as the enzyme. Error-tolerant MASCOT search was also done to identify possible modification in the peptides. In addition, MS/MS data were matched, allowing semi-tryptic peptides. MASCOT MudPIT peptide identification score was set to a cut-off of 20. At this cut-off and based on the number of assigned decoy peptide sequences, a peptide false discovery rate (FDR) of ~2% for all analyses was obtained. The identified coat and/or exosporium proteins of Bacillus cereus ATCC 14579 and Clostridium difficile 630 are listed in Table 1 and 2 respectively. The identified cytosolic proteins are listed in Supplementary Table 6.

Tertiary structure prediction and Sequence alignment

For tertiary structure prediction Phyre2 tool (www.sbg.bio.ic.ac.uk/phyre2/) was used in the default mode. Sequence alignment was done using the Clustal Omega tool (http://www.ebi.ac.uk/Tools/msa/clustalo/) with the default parameters. Conserved Domain Search

The annotation of protein sequences for the identification of domains is important in the protein sequence analysis. The identification of a conserved domain may aid in understanding the cellular and molecular function of a protein, as well as the evolutionary history of proteins. Identification of conserved domains from the identified proteins was performed using the automatic mode of the Conserved Domain (CD)-search tool from NCBI database (http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). The default parameters were used. Orthologue identification and PSI-BLAST analysis

For orthologue identification, PSI-BLAST (Position-Specific Iterated Basic Local Alignment Search Tool) option from BLASTP tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) version 2.2.27+ was used. The thresholds for orthologue identification: maximum identity ≥30%, E-value ≤0.005. Only single iteration for PSI-BLAST was performed. Proteins were said to be conserved if orthologues were found in at least 80% of the strains selected for comparison.

References

1. Tourasse NJ, Helgason E, Okstad OA, Hegna IK, Kolsto AB. The Bacillus cereus group: novel aspects of population structure and genome dynamics. Journal of applied microbiology. 2006;101(3):579-93. Epub 2006/08/16. 2. Stenfors Arnesen LP, Fagerlund A, Granum PE. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS microbiology reviews. 2008;32(4):579-606. Epub 2008/04/22. 3. Lund T, De Buyser ML, Granum PE. A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Molecular microbiology. 2000;38(2):254-61. Epub 2000/11/09. 4. Bottone EJ. Bacillus cereus, a volatile human pathogen. Clinical microbiology reviews. 2010;23(2):382-98. Epub 2010/04/09. 5. Bartlett JG, Willey SH, Chang TW, Lowe B. Cephalosporin-associated pseudomembranous colitis due to Clostridium difficile. JAMA : the journal of the American Medical Association. 1979;242(24):2683-5. Epub 1979/12/14. 6. Songer JG, Anderson MA. Clostridium difficile: an important pathogen of food animals. Anaerobe. 2006;12(1):1-4. Epub 2006/05/17. 7. Huang H, Weintraub A, Fang H, Nord CE. Antimicrobial resistance in Clostridium difficile. International Journal of Antimicrobial Agents. 2009;34(6):516-22. 8. Razavi B, Apisarnthanarak A, Mundy LM. Clostridium difficile: emergence of hypervirulence and fluoroquinolone resistance. Infection. 2007;35(5):300-7. Epub 2007/09/22.


73

9. Bartlett JG. New drugs for Clostridium difficile infection. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2006;43(4):428-31. Epub 2006/07/14. 10. Brazier JS, Raybould R, Patel B, Duckworth G, Pearson A, Charlett A, et al. Distribution and antimicrobial susceptibility patterns of Clostridium difficile PCR ribotypes in English hospitals, 2007-08. Euro surveillance : bulletin europeen sur les maladies transmissibles = European communicable disease bulletin. 2008;13(41). Epub 2008/10/18. 11. Cairns MD, Stabler RA, Shetty N, Wren BW. The continually evolving Clostridium difficile species. Future microbiology. 2012;7(8):945-57. Epub 2012/08/24. 12. Dawson LF, Valiente E, Wren BW. Clostridium difficile--a continually evolving and problematic pathogen. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases. 2009;9(6):1410-7. Epub 2009/06/23. 13. Carter GP, Rood JI, Lyras D. The role of toxin A and toxin B in Clostridium difficile-associated disease: Past and present perspectives. Gut microbes. 2010;1(1):58-64. Epub 2010/07/29. 14. Setlow P. Spore germination. Current opinion in microbiology. 2003;6(6):550-6. Epub 2003/12/10. 15. Henriques AO, Moran CP, Jr. Structure, assembly, and function of the spore surface layers. Annual review of microbiology. 2007;61:555-88. Epub 2007/11/24. 16. Lequette Y, Garénaux E, Tauveron G, Dumez S, Perchat S, Slomianny C, et al. Role played by exosporium glycoproteins in the surface properties of Bacillus cereus spores and in their adhesion properties to stainless steel. Applied and environmental microbiology. 2011. 17. Paredes CJ, Alsaker KV, Papoutsakis ET. A comparative genomic view of clostridial sporulation and physiology. Nature reviews Microbiology. 2005;3(12):969-78. Epub 2005/11/02. 18. de Hoon MJ, Eichenberger P, Vitkup D. Hierarchical evolution of the bacterial sporulation network. Current biology : CB. 2010;20(17):R735-45. Epub 2010/09/14. 19. Leski TA, Caswell CC, Pawlowski M, Klinke DJ, Bujnicki JM, Hart SJ, et al. Identification and Classification of bcl Genes and Proteins of Bacillus cereus Group Organisms and Their Application in Bacillus anthracis Detection and Fingerprinting. Applied and environmental microbiology. 2009;75(22):7163-72. 20. Thompson BM, Hoelscher BC, Driks A, Stewart GC. Localization and assembly of the novel exosporium protein BetA of Bacillus anthracis. Journal of bacteriology. 2011;193(19):5098-104. Epub 2011/08/09. 21. Waller LN, Stump MJ, Fox KF, Harley WM, Fox A, Stewart GC, et al. Identification of a second collagen-like glycoprotein produced by Bacillus anthracis and demonstration of associated spore-specific sugars. Journal of bacteriology. 2005;187(13):4592-7. Epub 2005/06/22. 22. Abhyankar W, Beek AT, Dekker H, Kort R, Brul S, de Koster CG. Gel-free proteomic identification of the Bacillus subtilis insoluble spore coat protein fraction. Proteomics. 2011;11(23):4541-50. Epub 2011/09/10. 23. McKenney PT, Driks A, Eichenberger P. The Bacillus subtilis endospore: assembly and functions of the multilayered coat. Nature reviews Microbiology. 2013;11(1):33-44. Epub 2012/12/04. 24. Permpoonpattana P, Tolls EH, Nadem R, Tan S, Brisson A, Cutting SM. Surface layers of Clostridium difficile endospores. Journal of bacteriology. 2011;193(23):6461-70. Epub 2011/09/29. 25. Abecasis AB, Serrano M, Alves R, Quintais L, Pereira-Leal JB, Henriques AO. A genomic signature and the identification of new sporulation genes. Journal of bacteriology. 2013. Epub 2013/02/12. 26. Barra-Carrasco J, Olguin-Araneda V, Plaza-Garrido A, Miranda-Cardenas C, Cofre-Araneda G, Pizarro-Guajardo M, et al. The Clostridium difficile Exosporium Cysteine (CdeC) Rich Protein is Required for Exosporium Morphogenesis and Coat Assembly. Journal of bacteriology. 2013. Epub 2013/06/25. 27. Lawley TD, Croucher NJ, Yu L, Clare S, Sebaihia M, Goulding D, et al. Proteomic and genomic characterization of highly infectious Clostridium difficile 630 spores. Journal of bacteriology. 2009;191(17):5377-86. Epub 2009/06/23. 28. Permpoonpattana P, Phetcharaburanin J, Mikelsone A, Dembek M, Tan S, Brisson MC, et al. Functional Characterization of Clostridium difficile Spore Coat Proteins. Journal of bacteriology. 2013. Epub 2013/01/22.

Chapter 3

74

29. Putnam EE, Nock AM, Lawley TD, Shen A. SpoIVA and SipL Are Clostridium difficile Spore Morphogenetic Proteins. Journal of bacteriology. 2013;195(6):1214-25. Epub 2013/01/08. 30. Cutting S, Anderson M, Lysenko E, Page A, Tomoyasu T, Tatematsu K, et al. SpoVM, a small protein essential to development in Bacillus subtilis, interacts with the ATP-dependent protease FtsH. Journal of bacteriology. 1997;179(17):5534-42. Epub 1997/09/01. 31. Ozin AJ, Henriques AO, Yi H, Moran CP, Jr. Morphogenetic proteins SpoVID and SafA form a complex during assembly of the Bacillus subtilis spore coat. Journal of bacteriology. 2000;182(7):1828-33. Epub 2000/03/14. 32. Henriques AO, Melsen LR, Moran CP, Jr. Involvement of superoxide dismutase in spore coat assembly in Bacillus subtilis. Journal of bacteriology. 1998;180(9):2285-91. Epub 1998/05/09. 33. Isticato R, Esposito G, Zilhao R, Nolasco S, Cangiano G, De Felice M, et al. Assembly of multiple CotC forms into the Bacillus subtilis spore coat. Journal of bacteriology. 2004;186(4):1129-35. Epub 2004/02/06. 34. Isticato R, Pelosi A, Zilhao R, Baccigalupi L, Henriques AO, De Felice M, et al. CotC-CotU heterodimerization during assembly of the Bacillus subtilis spore coat. Journal of bacteriology. 2008;190(4):1267-75. Epub 2007/12/11. 35. Zilhão R, Serrano M, Isticato R, Ricca E, Moran CP, Jr., Henriques AO. Interactions among CotB, CotG, and CotH during assembly of the Bacillus subtilis spore coat. Journal of bacteriology. 2004;186(4):1110-9. Epub 2004/02/06. 36. McPherson SA, Li M, Kearney JF, Turnbough CL, Jr. ExsB, an unusually highly phosphorylated protein required for the stable attachment of the exosporium of Bacillus anthracis. Molecular microbiology. 2010;76(6):1527-38. Epub 2010/05/07. 37. Boydston JA, Yue L, Kearney JF, Turnbough CL, Jr. The ExsY protein is required for complete formation of the exosporium of Bacillus anthracis. Journal of bacteriology. 2006;188(21):7440-8. Epub 2006/08/29. 38. Bagyan I, Noback M, Bron S, Paidhungat M, Setlow P. Characterization of yhcN, a new forespore-specific gene of Bacillus subtilis. Gene. 1998;212(2):179-88. Epub 1998/06/05. 39. Errington J. Regulation of endospore formation in Bacillus subtilis. Nature reviews Microbiology. 2003;1(2):117-26. Epub 2004/03/24. 40. Aucher W, Davison S, Fouet A. Characterization of the sortase repertoire in Bacillus anthracis. PloS one. 2011;6(11):e27411. Epub 2011/11/15. 41. Fukushima T, Yao Y, Kitajima T, Yamamoto H, Sekiguchi J. Characterization of new L,D-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in Bacillus subtilis. Molecular genetics and genomics : MGG. 2007;278(4):371-83. Epub 2007/06/26. 42. Mirey G, Soulard A, Orange C, Friant S, Winsor B. SH3 domain-containing proteins and the actin cytoskeleton in yeast. Biochemical Society transactions. 2005;33(Pt 6):1247-9. Epub 2005/10/26. 43. Kim HS, Sherman D, Johnson F, Aronson AI. Characterization of a major Bacillus anthracis spore coat protein and its role in spore inactivation. Journal of bacteriology. 2004;186(8):2413-7. Epub 2004/04/03. 44. Lehmann Y, Meile L, Teuber M. Rubrerythrin from Clostridium perfringens: cloning of the gene, purification of the protein, and characterization of its superoxide dismutase function. Journal of bacteriology. 1996;178(24):7152-8. Epub 1996/12/01. 45. Riebe O, Fischer RJ, Wampler DA, Kurtz DM, Jr., Bahl H. Pathway for H2O2 and O2 detoxification in Clostridium acetobutylicum. Microbiology. 2009;155(Pt 1):16-24. Epub 2009/01/02. 46. Brahmbhatt TN, Janes BK, Stibitz ES, Darnell SC, Sanz P, Rasmussen SB, et al. Bacillus anthracis exosporium protein BclA affects spore germination, interaction with extracellular matrix proteins, and hydrophobicity. Infection and immunity. 2007;75(11):5233-9. Epub 2007/08/22. 47. Kailas L, Terry C, Abbott N, Taylor R, Mullin N, Tzokov SB, et al. Surface architecture of endospores of the Bacillus cereus/anthracis/thuringiensis family at the subnanometer scale. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(38):16014-9. Epub 2011/09/08.


75

48. Beck K, Brodsky B. Supercoiled protein motifs: the collagen triple-helix and the alpha-helical coiled coil. Journal of structural biology. 1998;122(1-2):17-29. Epub 1998/09/02. 49. Tan L, Turnbough CL, Jr. Sequence motifs and proteolytic cleavage of the collagen-like glycoprotein BclA required for its attachment to the exosporium of Bacillus anthracis. Journal of bacteriology. 2010;192(5):1259-68. Epub 2009/12/30. 50. Steichen CT, Kearney JF, Turnbough CL, Jr. Characterization of the exosporium basal layer protein BxpB of Bacillus anthracis. Journal of bacteriology. 2005;187(17):5868-76. Epub 2005/08/20. 51. Thompson BM, Hoelscher BC, Driks A, Stewart GC. Assembly of the BclB glycoprotein into the exosporium and evidence for its role in the formation of the exosporium 'cap' structure in Bacillus anthracis. Molecular microbiology. 2012;86(5):1073-84. Epub 2012/09/20. 52. Liu H, Bergman NH, Thomason B, Shallom S, Hazen A, Crossno J, et al. Formation and composition of the Bacillus anthracis endospore. Journal of bacteriology. 2004;186(1):164-78. Epub 2003/12/18. 53. Andersen GL, Simchock JM, Wilson KH. Identification of a region of genetic variability among Bacillus anthracis strains and related species. Journal of bacteriology. 1996;178(2):377-84. Epub 1996/01/01. 54. Redmond C, Baillie LW, Hibbs S, Moir AJ, Moir A. Identification of proteins in the exosporium of Bacillus anthracis. Microbiology. 2004;150(Pt 2):355-63. Epub 2004/02/10. 55. Walker JR, Gnanam AJ, Blinkova AL, Hermandson MJ, Karymov MA, Lyubchenko YL, et al. Clostridium taeniosporum spore ribbon-like appendage structure, composition and genes. Molecular microbiology. 2007;63(3):629-43. Epub 2007/02/17. 56. Reiter L, Tourasse NJ, Fouet A, Loll R, Davison S, Okstad OA, et al. Evolutionary history and functional characterization of three large genes involved in sporulation in Bacillus cereus group bacteria. Journal of bacteriology. 2011;193(19):5420-30. Epub 2011/08/09. 57. Driks A. Surface appendages of bacterial spores. Molecular microbiology. 2007;63(3):623-5. Epub 2007/02/17. 58. Rode LJ, Crawford MA, Williams MG. Clostridium spores with ribbon-like appendages. Journal of bacteriology. 1967;93(3):1160-73. Epub 1967/03/01. 59. Réty S, Salamitou S, Garcia-Verdugo I, Hulmes DJ, Le Hegarat F, Chaby R, et al. The crystal structure of the Bacillus anthracis spore surface protein BclA shows remarkable similarity to mammalian proteins. The Journal of biological chemistry. 2005;280(52):43073-8. Epub 2005/10/27. 60. Bozue J, Cote CK, Moody KL, Welkos SL. Fully virulent Bacillus anthracis does not require the immunodominant protein BclA for pathogenesis. Infection and immunity. 2007;75(1):508-11. Epub 2006/11/01. 61. Ghebrehiwet B, Tantral L, Titmus MA, Panessa-Warren BJ, Tortora GT, Wong SS, et al. The exosporium of B. cereus contains a binding site for gC1qR/p33: implication in spore attachment and/or entry. Advances in experimental medicine and biology. 2007;598:181-97. Epub 2007/09/26. 62. Chesnokova ON, McPherson SA, Steichen CT, Turnbough CL, Jr. The spore-specific alanine racemase of Bacillus anthracis and its role in suppressing germination during spore development. Journal of bacteriology. 2009;191(4):1303-10. Epub 2008/12/17. 63. Liang L, He X, Liu G, Tan H. The role of a purine-specific nucleoside hydrolase in spore germination of Bacillus thuringiensis. Microbiology. 2008;154(Pt 5):1333-40. Epub 2008/05/03. 64. Ragkousi K, Eichenberger P, van Ooij C, Setlow P. Identification of a new gene essential for germination of Bacillus subtilis spores with Ca2+-dipicolinate. Journal of bacteriology. 2003;185(7):2315-29. Epub 2003/03/20. 65. Terry C, Shepherd A, Radford DS, Moir A, Bullough PA. YwdL in Bacillus cereus: its role in germination and exosporium structure. PloS one. 2011;6(8):e23801. Epub 2011/09/03. 66. Lambert EA, Popham DL. The Bacillus anthracis SleL (YaaH) protein is an N-acetylglucosaminidase involved in spore cortex depolymerization. Journal of bacteriology. 2008;190(23):7601-7. Epub 2008/10/07.

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67. Dembek M, Stabler RA, Witney AA, Wren BW, Fairweather NF. Transcriptional Analysis of Temporal Gene Expression in Germinating Clostridium difficile 630 Endospores. PloS one. 2013;8(5):e64011. Epub 2013/05/22. 68. Ando Y, Tsuzuki T. Energy-dependent activation of spore-lytic enzyme precursor by germinated spores of Clostridium perfringens. Biochemical and biophysical research communications. 1984;123(2):463-7. Epub 1984/09/17. 69. Cybulski RJ, Jr., Sanz P, McDaniel D, Darnell S, Bull RL, O'Brien AD. Recombinant Bacillus anthracis spore proteins enhance protection of mice primed with suboptimal amounts of protective antigen. Vaccine. 2008;26(38):4927-39. Epub 2008/07/29. 70. Walper S, Lee P, Anderson G, Goldman E. Selection and Characterization of Single Domain Antibodies Specific for Bacillus anthracis Spore Proteins. Antibodies. 2013;2(1):152-67. 71. Barry CS. The stay-green revolution: Recent progress in deciphering the mechanisms of chlorophyll degradation in higher plants. Plant Science. 2009;176(3):325-33. 72. Fu Z, Chen S, Baldwin MR, Boldt GE, Crawford A, Janda KD, et al. Light chain of botulinum neurotoxin serotype A: structural resolution of a catalytic intermediate. Biochemistry. 2006;45(29):8903-11. Epub 2006/07/19. 73. Galperin MY, Mekhedov SL, Puigbo P, Smirnov S, Wolf YI, Rigden DJ. Genomic determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation-specific genes. Environmental microbiology. 2012;14(11):2870-90. Epub 2012/08/14. 74. Traag BA, Pugliese A, Eisen JA, Losick R. Gene conservation among endospore-forming bacteria reveals additional sporulation genes in Bacillus subtilis. Journal of bacteriology. 2013;195(2):253-60. Epub 2012/11/06. 75. Escobar-Cortes K, Barra-Carrasco J, Paredes-Sabja D. Proteases and sonication specifically remove the exosporium layer of spores of Clostridium difficile strain 630. Journal of microbiological methods. 2013. Epub 2013/02/07. 76. Thompson BM, Binkley JM, Stewart GC. Current physical and SDS extraction methods do not efficiently remove exosporium proteins from Bacillus anthracis spores. Journal of microbiological methods. 2011;85(2):143-8. Epub 2011/02/23. 77. de Vries YP, Hornstra LM, de Vos WM, Abee T. Growth and sporulation of Bacillus cereus ATCC 14579 under defined conditions: temporal expression of genes for key sigma factors. Applied and environmental microbiology. 2004;70(4):2514-9. Epub 2004/04/07. 78. Todd SJ, Moir AJ, Johnson MJ, Moir A. Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium. Journal of bacteriology. 2003;185(11):3373-8. Epub 2003/05/20. 79. Hadžija O. A simple method for the quantitative determination of muramic acid. Analytical biochemistry. 1974;60(2):512-7. Epub 1974/08/01. 80. Taylor KCC. A simple colorimetric assay for muramic acid and lactic acid. Appl Biochem Biotechnol. 1996;56(1):49-58. 81. Klobutcher LA, Ragkousi K, Setlow P. The Bacillus subtilis spore coat provides "eat resistance" during phagocytic predation by the protozoan Tetrahymena thermophila. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(1):165-70. Epub 2005/12/24. 82. Scopes RK. Measurement of protein by spectrophotometry at 205 nm. Analytical biochemistry. 1974;59(1):277-82. Epub 1974/05/01.


77

Chapter 3

78

4 Proteomic characterization of spore coat

protein mutants of Bacillus subtilis

Wishwas Abhyankar, Linli Zheng, Stanley Brul, Leo J. de Koning, Chris G. de Koster

Abstract Spore coat proteins play an important role in maintaining spore structure as well as the resistive capacity of the spores. Spore morphogenetic proteins are responsible for layering the proteinaceous layers during spore morphogenesis. Previous studies have analyzed the dependence of certain coat proteins on the well-known morphogenetic proteins such as SpoIVA, CotE, CotH etc. Yet many coat proteins remain to be studied for regulation of their assembly and their dependence on the morphogenetic or other coat proteins. With the aim to map the dependence of coat protein deposition on CotA, CotE, Tgl, we studied protein profiles from the ΔcotA, ΔcotE and Δtgl spores. We identified a subset of coat proteins dependent and/or affected by the absence of the candidate protein genes cotE and tgl in these spores. ΔcotA spores did not show an effect on the overall protein composition of spore coat. The thermals stress resistance tests of ΔcotE and Δtgl mutant spores compared to the wild-type spores showed that Δtgl spores are more resistant to thermal stress than ΔcotE spores. Δtgl spores are compared to the equally thermal resistant spores from control wild-type cells, under our test conditions.

Chapter 4

80

Introduction

Spore coat, crust and exosporium are the outermost layers of bacterial endospores. Spores are formed by bacteria belonging to the genera Bacillus and Clostridium in response to environmental stress conditions. Spore formation is a highly controlled processed where each layer of spore is deposited progressively leading to further maturation of the spores. The process starts with an initial asymmetric division of the bacterial cell, followed by separation of the larger mother cell and the smaller pre-spore and finally ending up into the lysis of the mother cell. Chapter 1 discusses the details of the sporulation process as well as the details of the different spore layers. The coat acts as a shield protecting the spores from degradative enzymes, chemicals and reactive oxygen species [1]. The coat is also important for germination as it houses the spore cortex lytic enzymes (SCLEs) required for degrading the cortex during initial stages of germination [2, 3]. The coat is also capable of accommodating changes in spore volume that occur during spore formation and germination [4, 5]. The coat layer is unique to bacterial spores, as is the majority of the coat proteins from B. subtilis. This uniqueness is confirmed by the fact that these proteins do not have homologues except among the Bacilli and Clostridia. The molecular mechanisms behind coat and exosporium assembly are only partially understood with at least 70 proteins known to form the coat. With the advents of new methods such as protein-GFP tagging with fluorescence microscopy, Atomic Force Microscopy (AFM), mass spectrometry, access to the as yet unknown coat and exosporium proteins has become possible. In such an effort, as discussed in Chapter 2 and Chapter 3 we identified 21 putative novel coat proteins from B. subtilis 168 using our gel-free technique. Given that only a small subset of coat proteins play essential roles in the determination of coat morphology [6, 7] it is essential to know the function of these proteins. Moreover, the identification of novel proteins could be important to uncover putative mediators of spore resistance and novel compounds important to the spore structure. A group of spore coat proteins listed in Table 1 controls the assembly of the spore coat proteins in a supra-molecular structure. These proteins are called morphogenetic proteins [6]. Although their functions in coat assembly have been characterized to some extent, the complete understanding of their role in establishing the protein interaction network in the coat layers has not been achieved. Essentially, the coat morphogenetic proteins only control the assembly of proteins but do not affect the gene expression. The coat-associated transglutaminase (tgl) [8] and superoxide dismutase (sod) [9] also have been shown to play a role in modulating coat layer by introduction of glutamyl-lysine and dityrosine cross-links respectively. As an extension to these findings, we attempted to map the inter-protein dependences in the coat proteins from B. subtilis. Also, several reports about the role of laccases in protein cross-linking are available. Though the spore coat laccase CotA in Bacillus subtilis is known to be involved in a melanin-like pigment production[10] (during sporulation) and spore-resistance against UV radiation[11], we hypothesize its participation in protein cross-linking in our study. We discuss the findings of the proteomic characterization of ΔcotE, ΔcotA and Δtgl mutants of B. subtilis in this chapter.

Spore coat protein mutants of B. subtilis

81

Table 1. The known morphogenetic proteins in the spore coat of Bacillus subtilis.

Results and Discussion

Protein CotE affects the assembly of the outer coat proteins.

It is well known from the prior research that protein CotE plays a pivotal role in assembly of the outer coat [14]. A possible protein interaction network was predicted by Kim and co-workers [17] based on their study performed using cell biological (GFP-tagging) and protein biochemical (SDS-PAGE) methods. Some of their findings were confirmed in the current study. As seen in Table 2, peptide identifications of highly abundant coat proteins CotA, CotB, CotZ and YqfA were significantly affected by the absence of CotE. Interestingly only 1 to 2 peptides were identified from the inner coat morphogenetic protein SpoIVA in the ΔcotE spores as opposed to the wild-type. Little and Driks[14] showed that the C-terminal region of CotE directs deposition of CotA, CotB, CotG, CotSA, CotS, CotR and YaaH (Figure 1). The C-terminus of CotE does not contain any tryptic cleavage sites and therefore no tryptic peptides would be identified from this region. On the contrary, this region is rich in glutamic acid and aspartic acid residues (Figure 1). These residues might be good candidates for isopeptide linkages or the salt bridge interactions discussed in Chapter 7. In agreement with Kim and colleagues[17] as well as Little & Driks[14], outer coat proteins CotSA, CotS, CotI and inner coat protein CotH were not identified from the ΔcotE spores indicating dependence of these proteins on CotE for their assembly in the outer coat. It is noteworthy, that CotS and CotSA have been identified previously to depend either on CotH or on CotE for their assembly. Other findings of the current study are shown in Table 2 where a selected group of proteins is represented. The complete list of identified proteins can be found in Appendix Table I. On the contrary to the above mentioned observations, certain coat proteins were indicative of easy extraction from the coat as predicted from the higher number of peptide MS/MS spectra identified and the MASCOT scores (Table 1). Of these proteins SafA has been predicted to depend on the presence of CotE for its extractability[14]. Although less likely, an up regulation of these proteins in an effort to compensate for the loss of most of the other coat proteins might take place. Though proteins CotG, CotR, YaaH all are dependent on CotE for their proper coat deposition,

Protein Description

SpoIVA Attaches the coat to the forespore membranes and is suggested to be required for formation of the cortex[12]

SpoVID Required for attachment of the coat to the forespore[12]

SafA The safA mutants are defective in germination and possess a defective coat that fails to protect the cortex from lysozyme[13]

CotE Layered between the inner and outer coat, directs assembly of a subset of coat proteins including most if not all of the outer coat proteins[14]

CotH Directs assembly of CotG[14, 15] CotO Directs assembly of a subset of coat proteins that overlaps with the CotH-controlled

proteins[16]

Chapter 4

82

they were identified as their dependence is specific to the C-terminal end of CotE. In fact, in our ΔcotE spores, the N-terminal of CotE has been replaced by the chloramphenicol resistance marker while the C-terminus is intact [18]. Despite the aforementioned mutation the oligomerization sequence is still present in the cotE gene of our mutant and yet it appears that CotE fails to form multimers indicating importance of the N-terminus of CotE in overall protein biochemistry. Thus, it is also intriguing that though not completely structured and produced at low levels, CotE can still control the deposition of these proteins. Previously researchers have tested the resistance behaviour of ΔcotE spores towards thermal stress and lysozyme. These researchers found that ΔcotE spores are, in spite of a loose coat structure, resistant to thermal stress but are sensitive towards lysozyme [18]. Overall decrease in the protein identification due to absence of functional CotE is shown in Figure 2. As seen in ΔcotE spores, from the insoluble protein fraction, proteins with molecular mass of 20kDa and proteins with molecular masses in the range of 40-50 kDa are missed in identification. Little & Driks[14], in their study on soluble coat protein fraction, also observed smear bands in the region of 30 kDa and 43 kDa indicative of multiple protein species which they could not identify using a MALDI-TOF-MS peptide mass fingerprinting approach. In our study, there were a few putative novel proteins identified yet further studies are needed to confirm their dependence on CotE. Intriguingly, in the absence of an electron dense outer coat as observed under electron microscope (EM), ΔcotE spores are also seen to be affected in their germination rates with spores germinating slower in presence of L-alanine and faster in presence of dodecylamine compared to the wild-type [19]. The proteins and the signal mechanisms that are affected by the absence of outer coat still remain to be a mystery. Future studies, including gene knock-out studies as well as protein-protein cross-linking studies, will aid in solving the inter-protein dependence to a deeper extent. Transglutaminase (Tgl) mediated cross-links may only affect assembly of a fraction of spore coat proteins.

It is known from previous studies that deletion of one protein from the spore coat does not alter the structure of the coat extensively unless the deleted gene encodes a morphogenetic protein as seen in the cotE mutants above. One such important protein that renders certain coat proteins insoluble is the enzyme transglutaminase encoded by the gene tgl in B. subtilis. Transglutaminases are known to catalyze covalent bond formation between a free amine group (such as from lysine) and an acyl group (such as from glutamine) at the end of peptide side chain. Such glutamyl-lysine bonds are resistant to proteolytic digestion. A previous study by Ragkousi and Setlow [20] showed that Tgl-mediated cross-linking is involved in the assembly of the spore coat protein GerQ. Though the presence of glutamine-lysine cross-links has been predicted in the spore coat, no other proteins except GerQ have been identified yet to be cross-linked by Tgl-mediated cross-linking. In the current study, we also analyzed the protein composition of spore coats from Δtgl mutants of B. subtilis. It appeared that only a subset of proteins was


83

Figure 1. Structural regions of CotE involved in assembly and deposition of spore coat proteins. The N-terminal region (a.a. 1 to 101) underlined in black is required for deposition of certain coat proteins. The region shown in bold and orange lettering (a.a. 58 to 75) is responsible for oligomerization of CotE. The region shown in bold and blue lettering is required for targeting CotE to the spore. Deposition of protein CotA, CotR and YaaH is dependent on the C-terminal region highlighted in grey (a.a. 156 to 158), of proteins CotG, CotB is dependent on region highlighted in green (a.a. 159 to 169) and of proteins CotS and CotSA on the region highlighted in yellow (a.a. 176 to 178). The C-terminal region rich in aspartic acid and glutamic acid is underlined in red colour. The regions are demarcated based on the study by Little and Driks[14].

Figure 2. Comparison of protein identification in wild type (WT) and ΔcotE spores of B. subtilis PY79. Results of three independent biological replicates (R1, R2 and R3) are shown in terms of frequency distributions of number of proteins identified with respective molecular masses. These results are consistent with previous findings[14].

MSEYREIITK AVVAKGRKFT QCTNTISPEK KPSSILGGWI INHKYDAEKI

GKTVEIEGYY DINVWYSYAD NTKTEVVTER VKYVDVIKLR YRDNNYLDDE

HEVIAKVLQQ PNCLEVTISP NGNKIVVQAE REFLAEVVGE TKVVVEVNPD

WEEDDEEDWE DELDEELEDI NPEFLVGDPE E

Cha

pter

4

84

Tab

le 2

. Ide

ntifi

catio

n of

spor

e co

at p

rote

ins i

n Δc

otE

spor

es o

f B. s

ubtil

is. T

he w

ild ty

pe st

rain

use

d w

as B

. sub

tilis

PY

79.

Prot

ein

Des

crip

tion

Mas

s (D

a)

Wild

Typ

e Δc

otE

Rep

licat

e1

Δcot

E R

eplic

ate2

Δc

otE

Rep

licat

e3

MA

SCO

T Sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T Sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T Sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T Sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a Pr

otei

ns su

gges

ted

to b

e di

rect

ly d

epen

dent

on

Cot

E fo

r th

eir

asse

mbl

y C

otA

Sp

ore

coat

pro

tein

A

5869

0 19

47

42

27

1 64

1

Cot

B

Spor

e co

at p

rote

in B

42

946

1771

37

26

6 8

87

3 C

otE

Spor

e co

at p

rote

in E

21

078

1400

31

38

1

Cot

SA

Spor

e co

at p

rote

in S

A

4305

6 10

45

27

Cot

Z Sp

ore

coat

pro

tein

Z

1709

3 10

05

15

219

4 53

9 8

374

5 C

otS

Spor

e co

at p

rote

in S

41

286

940

24

Ydc

C

Spor

ulat

ion

prot

ein

3817

0 65

4 11

C

otI

Spor

e co

at p

rote

in I

4144

7 48

8 12

A

tcL

Cal

cium

-tran

spor

ting

ATP

ase

9751

6 44

9 12

Y

qfA

U

PF03

65 p

rote

in

3567

6 43

5 10

80

2

Dac

F D

-ala

nyl-D

-ala

nine

car

boxy

pept

idas

e 43

327

402

8 Y

qfX

U

ncha

ract

eriz

ed p

rote

in

1389

3 33

1 4

SpoI

VA

St

age

IV sp

orul

atio

n pr

otei

n A

55

197

330

9 47

1

27

1 84

2

SodA

Su

pero

xide

dis

mut

ase

[Mn]

22

476

307

5 C

otH

In

ner s

pore

coa

t pro

tein

H

4284

3 26

4 6

Yfk

D

Unc

hara

cter

ized

pro

tein

29

569

235

5 C

otN

Sp

ore

coat

-ass

ocia

ted

prot

ein

N

2828

7 19

9 4

Prot

eins

sugg

este

d to

be

easil

y ex

trac

ted/

dige

sted

or

up

regu

late

d ba

sed

on #

MS/

MS

Cot

Q

Unc

hara

cter

ized

FA

D-li

nked

oxi

dore

duct

ase

5016

7 56

2 15

54

6 19

28

5 7

880

27

Cot

R

Puta

tive

spor

ulat

ion

hydr

olas

e 35

335

497

12

619

15

165

6 72

8 23

C

otC

Sp

ore

coat

pro

tein

C

8868

36

6 13

17

0 9

338

13

591

25

SleB

Sp

ore

corte

x-ly

tic e

nzym

e 34

151

315

6 29

5 7

31

1 47

0 12

Y

jqC

Unc

hara

cter

ized

pro

tein

31

403

281

6 45

3 11

18

7 4

500

13

Yga

K

Unc

hara

cter

ized

FA

D-li

nked

oxi

dore

duct

ase

5103

9 25

1 7

570

16

167

5 60

9 17

C

otJC

Pr

otei

n C

otJC

21

739

221

5 43

5 12

20

6 3

424

10

Yjd

H

Unc

hara

cter

ized

pro

tein

15

249

215

4 36

5 9

258

7 49

6 11

C

otU

U

ncha

ract

eriz

ed p

rote

in

1161

2 31

9 6

84

3 51

1

268

8 Y

odI

Unc

hara

cter

ized

pro

tein

92

45

154

6 39

2 19

16

4 8

328

20

Yxe

E U

ncha

ract

eriz

ed p

rote

in

1470

5 76

1

486

13

504

12

805

17

SafA

Sp

oIV

D-a

ssoc

iate

d fa

ctor

A

4342

9 46

2

688

21

380

10

784

19


85

affected by the deletion of this protein. In fact a set of glutamine and/or lysine rich proteins was only identified from the Δtgl mutant. These results point to fact that these selected group of proteins (Table 3) could be made accessible for digestion by the absence of Tgl. It was observed previously that the protein GerQ could be completely extracted from the spore coat in the absence of Tgl. We confirmed this observation by identifying all possible C-terminal peptides from this protein but the number of MS/MS spectra for peptides was significantly reduced when compared to the wild type. The N-terminal region of GerQ is rich in glutamine (Q) and does not have a suitable tryptic cleavage site and hence with the use of trypsin as the protease no peptide was identified from this region. Proteins CotM and CotX have been suggested as other targets for Tgl-mediated cross-linking [8] however we did not identify the CotM protein in our analyses also from the wild type. We identified CotX in the wild type as well as in the tgl-mutant however the number of peptide MS/MS was slightly lower in mutants and hence no conclusion can be drawn (Appendix Table II). Interestingly, proteins CotN, GerE, YxeD, YuzC - all rich in glutamine and/or lysine, were identified in the wild type but not in the Δtgl mutant in our study. It will be interesting to study the precise location of these proteins in spores as well the effect of deletion of these proteins on the thermal and chemical resistance properties of spores.

Deletion of cotA does not affect the spore coat protein composition

The cotA gene is expressed under the control of σK, with GerE being a transcriptional repressor. Protein CotA is a copper-dependent laccase. Laccases are multicopper oxidases known to take part in oxidative reactions [21]. CotA plays an important role in the biosynthesis of the brown pigment, which is thought to be a melanin-like product[10]. The ΔcotA spores also lack the brown pigment. The absence of brown pigment is generally diagnostic of a block in spore formation, however in our experiments we obtained intact phase-bright spores. It has been thought that, the production of pigment is somehow tightly coupled to sporulation [22]. The ΔcotA spores have been shown to be sensitive to UV-B as well as UV-A radiations. In the functional study of CotA, Hullo et al. [11] recognized the role of CotA in UV-resistance where the brown melanin-like pigment produced by CotA during sporulation is said to play a role. Though, in contrast to tyrosinases, most laccases are not known to oxidize tyrosines; it is still not known if CotA has any oxidase activity in B. subtilis. Therefore to assess a possible role of CotA in oxidative linking of proteins (mostly via di-tyrosine formation [21]) we tested the protein profile of ΔcotA spores. Our results did not show a significant effect on the spore coat protein composition and the protein identification profile of mutants was similar to that of the wild type. Hullo and co-researchers [11] have reported that CotA is a classical laccase without tyrosinase activity. Nevertheless proteins CotS, CotSA and CotQ were identified with reduced peptide MS/MS spectra pointing to lower protein abundance or extraction efficiency (Table 4). Incidentally, these three proteins are rich in tyrosine. It is not clear if CotA-mediated cross-linking plays any role in assembly of spore coat proteins and thus detailed studies are required. Contrary to these

Cha

pter

4

86

Tab

le 3

. Ide

ntifi

catio

n of

spor

e co

at p

rote

ins i

n Δt

gl sp

ores

of B

. sub

tilis

. The

wild

type

stra

in u

sed

was

tryp

toph

an p

roto

troph

B. s

ubtil

is 1

68.

Prot

ein

Des

crip

tion

Mas

s (D

a)

Wild

type

Δt

gl R

eplic

ate

1 Δt

gl R

eplic

ate

2 Δt

gl R

eplic

ate

3

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a Id

entif

ied

prot

eins

aff

ecte

d by

del

etio

n of

tgl

Ger

E Sp

ore

germ

inat

ion

prot

ein

8583

70

3

Ger

Q

Spor

e co

at p

rote

in

2026

3 24

7 16

26

4 5

442

7 21

8 6

Tgl

Prot

ein-

glut

amin

e ga

mm

a-gl

utam

yl tr

ansf

eras

e 28

278

172

10

Yab

P Sp

ore

prot

ein

1139

6 42

2

Iden

tifie

d pr

otei

ns p

ossib

ly d

epen

dent

on

Tgl-m

edia

ted

links

Y

fkD

U

ncha

ract

eriz

ed p

rote

in

2956

9 11

2 4

145

3 72

2

Yku

J U

ncha

ract

eriz

ed p

rote

in

9296

29

1

126

3 70

1

Yci

C

Puta

tive

met

al c

hape

rone

45

734

54

2 76

1

Yhf

M

Unc

hara

cter

ized

pro

tein

14

998

52

1 87

1

Yhc

X

UPF

0012

hyd

rola

se

6063

6 28

1

Yku

S U

PF01

80 p

rote

in

8716

14

0 1

Ytx

J U

ncha

ract

eriz

ed p

rote

in

1245

1 92

1

Yne

T U

ncha

ract

eriz

ed p

rote

in

1503

7 75

3

Yus

N

Unc

hara

cter

ized

pro

tein

13

187

57

1 Y

tzB

U

ncha

ract

eriz

ed p

rote

in

1171

6 53

1

Yxe

D

Unc

hara

cter

ized

pro

tein

13

793

44

1 63

2

Yur

Z U

ncha

ract

eriz

ed p

rote

in

1389

8 43

2

57

2 Y

urT

Unc

hara

cter

ized

pro

tein

14

485

39

1 Y

uzM

U

ncha

ract

eriz

ed p

rote

in

9676

30

1

41

1 Y

lbN

U

ncha

ract

eriz

ed p

rote

in

2009

5 29

1

Ykt

B U

PF06

37 p

rote

in

2465

8 23

1

Spor

e co

at p

rote

in m

utan

ts of

B. s

ubtil

is 87

Tab

le 4

. Ide

ntifi

catio

n of

spor

e co

at p

rote

ins i

n Δc

otA

spor

es o

f B. s

ubtil

is. T

he w

ild ty

pe st

rain

use

d w

as B

. sub

tilis

PY

79.

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Chapter 4

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observations, a group of proteins with very less or no tyrosine was identified in wild type but not in mutants. Since these proteins were identified with very few MS/MS spectra per protein it is still premature to derive any firm conclusion. The complete list of proteins identified from ΔcotA spores can be seen in Appendix Table III.

Thermal resistance of ΔcotE, Δtgl spores

Wet-heat resistance of spores is the most studied survival challenge of wild-type and mutant spores. Zheng and colleagues [18] studied the thermal resistance of ΔcotE spores and found that these spores did not show any deviation in terms of survival level when the bahaviour was compared to the wild type spores. In our study, we tested the ΔcotE spores for their thermal resistance and observed that ΔcotE spores possessed lower resistivity to the thermal stress conditions used in our experiment (Figure 3). It is noteworthy that, these spores lack or contain lower levels of structural coat proteins such as CotA, CotB, SpoIVA etc. indicating the role of individual spore coat layers in spore heat resistance. Additionally, proteins CotB, CotI, CotS, CotSA appear to be involved in spore maturation (Chapter 5). Absence of these proteins in ΔcotE spores can thus also be linked to the lower thermal resistance of these spores. In a similar experiment, the Δtgl spores did not show any effect on their resistant nature towards thermal stress as compared to the wild type spores (Figure 3). Sanchez-Salas et al. [23] also showed that heat stress did not affect the survival of Δtgl spores. Impressively, in the absence of the transglutaminase (inducer of cross-links) the resistance levels of spores are not affected. We did not study the thermal resistance of ΔcotA spores but these spores have been reported to possess normal heat resistance that is comparable to the wild-type [21]. Hence we conclude that while outer coat spore proteins are important for proper spore assembly and wet-heat resistance, the formation of glutamine-lysine cross-links plays no role of major importance in spore thermal stress resistance. This observation also implies the presence of other types of cross-links, as discussed in Chapter 7, which might be involved in spore maturation which in turn is linked with spore wet-heat resistance. The role of small set of possibly CotA-dependent proteins in UV-resistance as well as effect of UV on the ΔcotE and Δtgl mutant spores remains a topic of future research.

Figure 3. Effect of thermal stress, at 85°C for 10 min., on wild-type, ΔcotE sand Δtgl spores of B. subtilis. All the spore samples were normalized to their control samples. All samples were heat activated at 70°C for 30 min. prior to the heat stress (n = 1).


89

Concluding remarks

Analysis of spore coat protein mutants in our study revealed a small group of coat proteins that were affected by the mutation in question. CotH, CotI, CotS, CotSA previously suggested to be directly or indirectly dependent on CotE for their assembly were not identified in our study confirming the original hypothesis. These proteins along with a few others are seen to participate in spore maturation. A loss of these proteins also appears to be linked to a lower heat resistance as seen in our study. Similar to SafA, we also suggest the extractability of certain coat proteins to depend on CotE as observed from our results. Absence of transglutaminase affects the oligomerization of GerQ but in addition we suggest that proteins GerE, YabP as well as other glutamine/lysine rich putative novel proteins are also affected by the absence of transglutaminase activity. CotA is seen to affect the pigmentation phenotype in mutant spores and only few putative coat proteins likely depend on CotA for their localization and interaction in spore coat. Heat resistance tests of Δtgl spores argue a presence of other types of cross-links in the spore coat.

Materials and Methods

Bacterial strain and sporulation conditions

Bacillus subtilis 168 (Trp+) lab-strain and Bacillus subtilis PY79 wild-type were the background strains used in this study. The ΔcotE, ΔcotA (both with PY79 background) and Δtgl (B. subtilis PS832 (Trp+) background) mutant strains were obtained from the Eichenberger lab (New York University, USA). Bacteria were pre-cultured and sporulated as described previously [24]. The pre-culturing of mutant strains was done in Luria Bertani (LB) broth medium (pH 7.5). For ΔcotE and ΔcotA mutant strains the pre-culture medium was supplemented with chloramphenicol (5 mg/L) and the Δtgl mutant strain was pre-cultured in medium supplemented with erythromycin (1 mg/L). For sporulation a defined minimal medium, buffered with 3-(N-morpholino) propanesulfonic acid (MOPS) to pH 7.4, was used[25]. Three independent biological replicates for both wild-type and mutants were analyzed. After 96 hours, the spores were harvested as described elsewhere[24].

Spore coat isolation and protein extraction

The harvested spores were subjected to spore coat isolation & protein extraction as described previously [24, 26]. The isolated coat material was freeze-dried overnight and immediately used for mass spectrometric analysis.

Measurements of thermal resistance

Thermal resistance of spores to wet heat was assessed using the previously used screw-cap tube method[25]. In short, a 1 ml (heat-activated (70°C, 30 min); OD600 ~2) spore suspension in sterile milli-Q water, for each time point, was injected with a syringe into a preheated metal screw-cap tube containing 9.0 ml of sterile milli-Q water. The heat activation helped to kill all the remaining vegetative cells in the sample. The tube was heated by immersing it completely in a glycerol bath (85°C for 10 min). After 10 min the tube was transferred to ice-water. Dilution series of spore suspension were prepared in sterile milli-Q water and 100 μl of sample was spread on Tryptic Soy agar plates. The number of colonies was counted after 24 hours of incubation at 37°C. The thermal resistance of spores was thus determined by the loss of their ability to germinate and form colonies (i.e., viability counts). As a control a same dilution series for

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non-heat stressed spores ware plated and the final colony counts from the stress samples were normalized with those from the control sample. The significance of the thermal resistance tests was tested by one-way ANOVA test.

Sample preparation for MS analysis



LC-MS/MS data were acquired with an Bruker ApexUltra Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a 7 T magnet and a nano-electrospray Apollo II DualSource™ coupled to an Ultimate 3000 (Dionex, Sunnyvale, CA, USA) HPLC system. Samples containing up to 160 ng of the tryptic peptide mixtures were injected as a 10 μl 0.1% TFA, 3% ACN aqueous solution and loaded onto a PepMap100 C18 (5-μm particle size, 100-Å pore size, 300-μm inner diameter x 5 mm length) precolumn. Following injection, the peptides were eluted via an Acclaim PepMap 100 C18 (3-μm particle size, 100-Å pore size, 75-μm inner diameter x 250 mm length) analytical column (Thermo Scientific, Etten-Leur, The Netherlands) to the nano-electrospray source. Gradient profiles of up to 120 min were used from 0.1% formic acid / 3% CH3CN / 97% H2O to 0.1% formic acid / 50% CH3CN / 50% H2O at a flow rate of 300 nL /min. Data dependent Q-selected peptide ions were fragmented in the hexapole collision cell at an Argon pressure of 6x10-6 mbar (measured at the ion gauge) and the fragment ions were detected in the ICR cell at a resolution of up to 60000. In the MS/MS duty cycle, 3 different precursor peptide ions were selected from each survey MS. The MS/MS duty cycle time for 1 survey MS and 3 MS/MS acquisitions was about 2 s. Instrument mass calibration was better than 1 ppm over a m/z range of 250 to 1500. Raw FT-MS/MS data were processed with the MASCOT DISTILLER program, version 2.4.3.1 (64bits), MDRO 2.4.3.0 (MATRIX science, London, UK), including the Search toolbox and the Quantification toolbox. Peak-picking for both MS and MS/MS spectra were optimized for the mass resolution of up to 60000. Peaks were fitted to a simulated isotope distribution with a correlation threshold of 0.7, with minimum signal to noise of 2. The processed data, from the three independent biological replicates, were searched with the MASCOT server program 2.3.02 (MATRIX science, London, UK) against a complete B. subtilis 168 ORF translation database (Uniprot 2011 update, downloaded from http://www.uniprot.org/uniprot). The database was complemented with its corresponding decoy data base for statistical analyses of peptide false discovery rate (FDR). Trypsin was used as enzyme and 1 missed cleavage was allowed. Carbamidomethylation of cysteine was used as a fixed modification. The peptide mass tolerance was set to 25 ppm and the peptide fragment mass tolerance was set to 0.025 Dalton. The threshold for MASCOT peptide identification score was set to 20. At this cut-off and based on the number of assigned decoy peptide sequences, a peptide false discovery rate (FDR) of ~2% for all analyses was obtained. For each sample > 200 proteins were identified of which 90 were known as well as candidate coat proteins (see Appendix Tables I,II and III) while the rest were remnants of cytosolic proteins from the mother cell. References

1. Young SB, Setlow P. Mechanisms of killing of Bacillus subtilis spores by Decon and Oxone, two general decontaminants for biological agents. Journal of applied microbiology. 2004;96(2):289-301. Epub 2004/01/16. 2. Paidhungat M, Ragkousi K, Setlow P. Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca2+-dipicolinate. Journal of bacteriology. 2001;183(16):4886-93. Epub 2001/07/24.


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3. Setlow P. Spore germination. Current opinion in microbiology. 2003;6(6):550-6. Epub 2003/12/10. 4. Chada VG, Sanstad EA, Wang R, Driks A. Morphogenesis of Bacillus spore surfaces. Journal of bacteriology. 2003;185(21):6255-61. Epub 2003/10/18. 5. Westphal AJ, Price PB, Leighton TJ, Wheeler KE. Kinetics of size changes of individual Bacillus thuringiensis spores in response to changes in relative humidity. Proceedings of the National Academy of Sciences of the United States of America. 2003;100(6):3461-6. Epub 2003/02/14. 6. Henriques AO, Moran CP, Jr. Structure and assembly of the bacterial endospore coat. Methods. 2000;20(1):95-110. Epub 1999/12/28. 7. Driks A. Proteins of the spore core and coat. In: Sonenshein AL, Hoch, J.A., Losick, R., editor. Bacillus subtilis and Its Closest Relatives. Washington, DC: American Society of Microbiology (ASM) Press; 2002. p. 527–36. 8. Zilhão R, Isticato R, Martins LO, Steil L, Volker U, Ricca E, et al. Assembly and function of a spore coat-associated transglutaminase of Bacillus subtilis. Journal of bacteriology. 2005;187(22):7753-64. Epub 2005/11/04. 9. Henriques AO, Melsen LR, Moran CP, Jr. Involvement of superoxide dismutase in spore coat assembly in Bacillus subtilis. Journal of bacteriology. 1998;180(9):2285-91. Epub 1998/05/09. 10. Schaeffer P. Sporulation and the production of antibiotics, exoenzymes, and exotonins. Bacteriological reviews. 1969;33(1):48-71. Epub 1969/03/01. 11. Hullo MF, Moszer I, Danchin A, Martin-Verstraete I. CotA of Bacillus subtilis is a copper-dependent laccase. Journal of bacteriology. 2001;183(18):5426-30. Epub 2001/08/22. 12. Driks A, Roels S, Beall B, Moran CP, Jr., Losick R. Subcellular localization of proteins involved in the assembly of the spore coat of Bacillus subtilis. Genes & development. 1994;8(2):234-44. Epub 1994/01/01. 13. Ozin AJ, Henriques AO, Yi H, Moran CP, Jr. Morphogenetic proteins SpoVID and SafA form a complex during assembly of the Bacillus subtilis spore coat. Journal of bacteriology. 2000;182(7):1828-33. Epub 2000/03/14. 14. Little S, Driks A. Functional analysis of the Bacillus subtilis morphogenetic spore coat protein CotE. Molecular microbiology. 2001;42(4):1107-20. Epub 2001/12/12. 15. Naclerio G, Baccigalupi L, Zilhão R, De Felice M, Ricca E. Bacillus subtilis spore coat assembly requires cotH gene expression. Journal of bacteriology. 1996;178(15):4375-80. Epub 1996/08/01. 16. McPherson DC, Kim H, Hahn M, Wang R, Grabowski P, Eichenberger P, et al. Characterization of the Bacillus subtilis spore morphogenetic coat protein CotO. Journal of bacteriology. 2005;187(24):8278-90. Epub 2005/12/03. 17. Kim H, Hahn M, Grabowski P, McPherson DC, Otte MM, Wang R, et al. The Bacillus subtilis spore coat protein interaction network. Molecular microbiology. 2006;59(2):487-502. Epub 2006/01/05. 18. Zheng LB, Donovan WP, Fitz-James PC, Losick R. Gene encoding a morphogenic protein required in the assembly of the outer coat of the Bacillus subtilis endospore. Genes & development. 1988;2(8):1047-54. Epub 1988/08/01. 19. Ghosh S, Setlow B, Wahome PG, Cowan AE, Plomp M, Malkin AJ, et al. Characterization of Spores of Bacillus subtilis That Lack Most Coat Layers. Journal of bacteriology. 2008;190(20):6741-8. 20. Ragkousi K, Setlow P. Transglutaminase-mediated cross-linking of GerQ in the coats of Bacillus subtilis spores. Journal of bacteriology. 2004;186(17):5567-75. Epub 2004/08/20. 21. Enguita FJ, Martins LO, Henriques AO, Carrondo MA. Crystal structure of a bacterial endospore coat component. A laccase with enhanced thermostability properties. The Journal of biological chemistry. 2003;278(21):19416-25. Epub 2003/03/15. 22. Donovan W, Zheng LB, Sandman K, Losick R. Genes encoding spore coat polypeptides from Bacillus subtilis. Journal of molecular biology. 1987;196(1):1-10. Epub 1987/07/05. 23. Sanchez-Salas JL, Setlow B, Zhang P, Li YQ, Setlow P. Maturation of released spores is necessary for acquisition of full spore heat resistance during Bacillus subtilis sporulation. Applied and environmental microbiology. 2011;77(19):6746-54. Epub 2011/08/09.

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24. Abhyankar W, Ter Beek A, Dekker H, Kort R, Brul S, de Koster CG. Gel-free proteomic identification of the Bacillus subtilis insoluble spore coat protein fraction. Proteomics. 2011;11(23):4541-50. Epub 2011/09/10. 25. Kort R, O'Brien AC, van Stokkum IHM, Oomes SJCM, Crielaard W, Hellingwerf KJ, et al. Assessment of Heat Resistance of Bacterial Spores from Food Product Isolates by Fluorescence Monitoring of Dipicolinic Acid Release. Applied and environmental microbiology. 2005;71(7):3556-64. 26. Abhyankar W, Hossain AH, Djajasaputra A, Permpoonpattana P, Ter Beek A, Dekker HL, et al. In Pursuit of Protein Targets: Proteomic Characterization of Bacterial Spore Outer Layers. Journal of proteome research. 2013;12(10):4507-21.


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5 Monitoring the progress in cross-linking of

spore coat proteins during maturation of Bacillus subtilis spores.

Wishwas Abhyankar, Rachna Pandey, Alexander Ter Beek, Stanley Brul, Leo J. de Koning, Chris G. de Koster

Submitted to Food microbiology

Abstract Resistance characteristics of the bacterial endospores towards various environmental stresses such as chemicals and heat are in part attributed to their coat proteins. Heat resistance is developed in a late stage of sporulation and during maturation of released spores. Using our gel-free proteomic approach and LC-FT-ICR-MS/MS analysis we have monitored the efficiency of the tryptic digestion of proteins in the coat during spore maturation over a period of 10 days, using metabolically 15N labeled mature spores as a reference. The results showed that during spore maturation the loss of digestion efficiency of outer coat and crust proteins synchronized with the increase in heat resistance. This implicates that spore maturation involves chemical cross-linking of outer coat and crust layer proteins leaving the inner coat layer proteins unmodified. It appears that digestion efficiencies of spore surface proteins can be linked to their location within the coat and crust layers. We also attempted to study a possible link between spore maturation and the observed heterogeneity in spore germination.

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Introduction

Food spoilage is commonly characterized by various sensory features such as stale colour, foul odour etc. In most cases, such offensive changes are brought about by microorganisms that are common inhabitants of soil and water and are dispersed through air or water. Since past, techniques such as thermal processing, high pressure treatments, irradiations etc. to inactivate spoilage microorganisms in food have been employed. These procedures are sufficient to kill vegetative cells, however bacterial endospores, if present, may escape these processes thereby leading to spoilage or in some instances food intoxication. Bacterial endospores are dormant, multilayered and highly resistant cellular structures formed in response to stress by certain Gram-positive bacteria belonging to the genera Bacillus and Clostridium and other related organisms. On return of more favorable conditions and in presence of nutrients they germinate and grow out as normal vegetative cells via the process of germination and outgrowth [1, 2]. Two properties that make the spores unique and incomparable are their resistance characteristics and the heterogeneity amongst them especially with regards to their germination.

Spore inactivation can be effectively achieved by wet-heat treatment but spores are generally resistant to high temperatures when compared to vegetative cells. In a previous study by Sanchez-Salas and colleagues [3], it was concluded that a further maturation of spores, after being released from the mother cells, is necessary for acquiring thermal resistance. In the same study it was also suggested that changes in coat structure especially with regards to the inter-protein cross-linking could also be one of the possibilities for maturation. Additionally, the extreme heat resistance of spores has also been attributed to - the small, acid soluble proteins (SASPs) protecting the spore DNA, the structure of the coat and the cortex layers, and the Ca2+-DPA as well as the water contents of the spore core [2, 4, 5]. Moreover, in a separate study it was shown that there could be a significant heterogeneity in the heat resistance within a same spore population [6]. Since spore forming organisms like Bacillus anthracis, Clostridium botulinum etc. are pathogenic and toxigenic, the detailed study of spore resistance mechanisms is of high relevance.

It is reported that ~30% of the protein fraction from the spore coat is characterized by extensive inter-protein cross-linking [1]. Three types of cross-links, namely, the dityrosine links, the ε-(γ)-glutamyl-lysine isopeptide linkages and the disulfide bonds - are predicted to render this fraction insoluble [1]. Both the tyrosine and cysteine rich proteins and the transglutaminase enzyme, capable of forming the glutamyl-lysine cross-links, have been identified from the coat and thus provide strong evidence for such chemical reactions occurring in the spore coat. Although the spore coat protein composition in released spores is presumably constant, there can be differences in the level of cross-linking amongst the proteins. It is hypothesized that the higher the proteins are cross-linked the higher would be the resistance behavior of spores. Thus, from the proteomics point of view, the higher the coat proteins are cross-linked the lower would be the efficiency with which they are digested by a protease. To investigate this hypothesis, we used a batch fermenter setup that allowed growth and sporulation of B. subtilis. Using 15N-labeled, 8-day mature spores as a reference, we monitored the loss of digestion

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97

efficiency of spore coat proteins as a function of their tryptic peptide 14N/15N isotopic ratios. In other words, the progress in the spore coat protein cross-linking in the query id est 14N-labeled spores was monitored with reference to the 8-day mature 15N reference spores. For the first time, the effect of spore maturation on the protein digestion efficiency of spore coat proteins from B. subtilis was monitored over a period of 10 days, relative to the 15N-labeled mature spores. The spore maturation was seen to be coupled to protein cross-linking indicated by the loss of digestion efficiency of certain proteins and increased heat-resistance. Compared to the matured spores, the younger spores showed enhanced protein digestion efficiency from the inner to the outer coat to the crust proteins reflecting the extent of cross-linking in these layers. This study, for the first time, identifies the spore coat proteins that could prove critical in the spore maturation process. We also suggest that the level of enhancement of digestion of individual proteins in young spores may indicate their location in the spore layers. Finally, with an established single spore live-imaging method [7], we monitored germination behavior of young and mature spores to map a possible linkage between spore maturation and germination time in spores. Results

Protein digestion efficiency during spore maturation reflects the extent of protein cross-linking.

Protein digestion efficiency during spore maturation was monitored for 89 proteins over a period of 10 days, relative to metabolically 15N labeled coat proteins of reference mature spores and the numbers of the identified proteins were plotted as a function of their (14N/15N) isotopic ratios for each time point (Figure 1). Out of the three independent biological replicates, in the first replicate the spores were allowed to mature for 10 days post-inoculation while in the other two replicates the maturation was allowed for 8 days. All the protein ratios in our datasets appeared to be normally distributed (Figure 1). The distribution for the young (2-day) spores showed a distinct group of proteins with isotopic ratios near to as well as > 1.0. The smaller groups (highlighted green and blue, Figure 1(A)) were centered more towards the ratio values of ~1.5 and 2.0 whereas the larger group (highlighted red, Figure 1(A)) was more centered around ratios of 1.0 - 1.2. The farthest cluster (cluster highlighted green, Figure 1(A)) comprised of outer coat proteins CotG and YurS as well as outer coat and crust proteins such as CotB, CotG, CotC, CotU, CotY, CotZ (cluster highlighted blue, Figure 1(A)). The inner coat proteins such as CotF, CotJA, CotJC, CwlJ, SleB, YdhD, Tgl were identified in the larger cluster (clusters highlighted red and in grey band, Figure 1(A)). As the maturation period increased, the distributions for relatively matured (4-day, 6-day) and matured (8-day, 10-day) spores demonstrated a considerable shift towards the ratios of 1.0 and the two clusters (highlighted blue and red, Figure 1(A)) dissolved to some extent giving a unimodal distribution for 4-day mature spores. For 6-, 8- and 10-day matured spores, the above mentioned clusters reappeared however they were now centered on ratios lower than 1.0 or < 0.3 (Figure 1(A)). This maturation behavior has been reproduced for all the

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three replicates. However it appears from the results that the maturation timings relative to the analytical reference (8-day 15N spores) may vary from one replicate to the other. The cytosolic proteins were also identified but they were not calculated for their 14N/15N ratios.

Figure 1. Protein digestion efficiency as a function of 14N:15N ratios during spore maturation. (A) Three groups of identified proteins are indicated by red, blue and green highlighted regions. The proteins that showed uniform digestion efficiencies over 8 to 10-day period are shaded in grey bar. (B) The proteins belonging to the three groups (red, blue, green) are indicated for their location in spores of B. subtilis. (C) The proteins, from the three groups (red, blue, green), observed for change in their digestion efficiencies when compared to day 2 and day 10 spores are shown. The colour in each column represents the group of the protein in Figure 1(A).


99

Young spores show enhanced protein digestion efficiency from inner to outer coat to crust proteins.

The digestion efficiency appeared to depend on likely the cross-linking of the spore outer coat and crust proteins. Proteins CotG, YurS, CotU, CotI, CotZ, CotY, CotB and CotC were identified to become more protease resistant over time thereby playing a role in spore maturation. Proteins CotG, CotC, CotU have been identified to be cross-linked in the spore coat [8-10]. Also CotX, CotY and CotZ are said to be a part of the insoluble cluster of spore coat proteins [11]. In our analyses it appeared that the outer coat proteins such as CotG, CotU and crust proteins such as CotY and CotZ are more efficiently digested in young (2-day) spores. Proteins CotG and CotU appeared to be more difficult to digest in 8-day and 10-day mature spores whereas the inner coat and spore morphogenetic proteins SpoIVA and SafA appeared to be digested with similar efficiencies in query (14N) and reference (15N) spores over the period of 10 days as the 14N/15N ratios stabilized around 1.0 in the mature spores (Figure 2). Protein CotC, a CotU (YnzH) homologue, also seemed to be involved in the maturation behaviour albeit to a lower extent as compared to CotU. The spore maturation protein CgeA, which is localized in the crust layer [12], was identified and did not show any appreciable change in its digestion efficiency (Table 1). No other member of the CgeABCDE family was identified. The crust proteins CotY and CotZ seemed to be affected by spore maturation in their digestion patterns. CotX however did not show an appreciable change in its digestion efficiency over the period of 8-10 days. Manual inspection of identified peptides from individual proteins showed that the outer coat and the crust proteins carried a large variation in the digestion efficiencies of the peptides. As seen in Figure 3, the coat protein YaaH and inner coat protein CotJC showed uniform digestion efficiencies for all the peptides used for quantification. Contradictorily for most of the peptides identified from the outer coat and crust proteins (represented in Figure 3) the digestion efficiencies for each peptide varied to a great extent. In most cases, the peptides that showed the highest digestion efficiencies in the young spores were seen to be least efficiently digested in the older spores.

Spore maturation is coupled to protein cross-linking and spore thermal resistance.

From the assessment of the viable counts of thermally stressed spores at 85°C for 10 min it appeared that 8-day or mature spores had acquired more heat resistance when compared to 2-day or younger spores confirming the previous notion [3] (Figure 3). In fact, the decrease in the protein digestion efficiency synchronized well with the increase in the thermal stress resistance of spores (P<0.05).

Spore maturation and heterogeneity in spore germination times.

Analysis by Spore Tracker live imaging tool showed that there was a slight , but significant (P <0.05) delay in the germination times of 8-day mature spores when compared to 2-day old spores (Figure 4). The young spore population appeared to comprise of spores germinating early as well as late while 8-day mature spore population

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Table 1. 14N/15N isotopic ratios of identified spore surface proteins from B. subtilis PY79.

Protein Description Mass (Da)

14N/15N ratiosa Day

2 Day

4 Day

6 Day

8 Day 10

CotZ Spore coat protein Z 17093 1.42 0.86 0.79 0.69 0.66

Crust CotY Spore coat protein Y 18728 1.35 0.85 0.83 0.65 0.68

CgeA Protein CgeA 14149 1.19 1.07 1.28 1.12 1.18

CotG Spore coat protein G 24399 1.98 0.75 0.64 0.34 0.44

YurS Uncharacterized protein 10483 1.92 1.05 0.86 0.51 0.38

CotI Spore coat protein I 41447 1.53 1.10 1.06 0.98 0.78

CotU Uncharacterized protein 11612 1.46 0.96 0.67 0.31 0.33

CotW Spore coat protein W 12486 1.44 1.13 1.02 0.89 0.86

CotA Spore coat protein A 58690 1.30 1.07 1.00 0.90 0.83

CotSA Spore coat protein SA 43056 1.30 0.99 0.98 0.86 0.80

CwlJ Cell wall hydrolase 16680 1.26 1.01 1.13 1.08 1.04

Outer CotB Spore coat protein B 42946 1.23 0.81 0.82 0.56 0.56

Coat CotS Spore coat protein S 41286 1.22 0.99 0.94 0.74 0.73

CotE Spore coat protein E 21078 1.20 0.83 0.78 0.67 0.68

CatX Catalase X 62383 1.15 1.26 0.79 1.05 0.89

CotR Putative sporulation hydrolase 35335 1.15 0.99 1.03 0.90 0.75

CotX Spore coat protein X 18989 1.15 0.93 0.92 0.89 0.84

SleB Spore cortex-lytic enzyme 34569 1.08 1.02 1.00 1.11 1.02

CotC Spore coat protein C 8868 0.99 0.81 0.68 0.37 0.38

CotQ Uncharacterized FAD-linked oxidoreductase 50167 0.91 0.81 0.78 0.59 0.56

Gpr Germination protease 40315 1.45 1.00 1.14 0.92 0.87

SafA SpoIVD-associated factor A 43429 1.37 1.21 1.09 1.11 1.00

YxeE Uncharacterized protein 14705 1.34 1.37 1.08 1.08 1.08

Tpx Probable thiol peroxidase 18318 1.32 1.12 1.14 1.05 0.99

YtfJ Uncharacterized spore protein 16336 1.20 0.99 0.99 1.04 1.05

LipC Spore germination lipase 24038 1.19 1.08 0.97 0.85 0.78

Tgl Protein-glutamine gamma-glutamyl transferase

28392 1.15 0.99 0.96 0.97 0.95

DacF D-alanyl-D-alanine carboxypeptidase 43327 1.03 1.05 0.99 1.00 0.99

SodM Superoxide dismutase [Mn] 22476 1.03 1.01 0.99 0.99 0.95

Inner OxdD Oxalate decarboxylase 44113 1.01 1.01 1.07 0.92 0.96

Coat YaaH Spore germination protein 48607 1.00 1.00 1.00 1.00 1.00

CotF Spore coat protein F 18714 0.97 0.98 1.00 0.94 0.91

CotJB Protein CotJB 10214 0.97 0.99 1.17 0.97 1.02

CotJC Protein CotJC 21993 0.95 1.05 1.07 1.01 1.00

SodF Probable superoxide dismutase [Fe] 33513 0.92 0.93 1.03 1.01 1.00

YdhD Putative sporulation-specific glycylase 47411 0.92 0.92 0.87 0.95 0.93

CotH Inner spore coat protein H 42843 0.90 0.94 0.99 0.69 0.63

CotJA Protein CotJA 9790 0.89 1.06 1.03 0.98 0.93

GerQ Spore coat protein 20263 0.84 0.99 1.06 1.02 0.95

SpoIVA Stage IV sporulation protein A 55197 0.83 1.09 1.08 1.06 1.06

Other YqfX Uncharacterized protein 13893 1.29 1.16 1.05 1.07 1.04

Coat YabP Spore protein 11396 1.23 1.19 1.23 1.13 1.11

Proteins YpeB Sporulation protein 51210 1.22 1.12 1.13 1.09 1.07


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Protein Description Mass (Da)

14N/15N ratiosa Day

2 Day

4 Day

6 Day

8 Day 10

YhcM Uncharacterized protein 17020 1.19 1.18 0.92 1.01 1.02

YkuD Putative L,D-transpeptidase 17910 1.16 1.02 1.28 1.23 1.13

YrkC Uncharacterized protein 21299 1.14 1.02 1.38 0.86 0.93

YisY AB hydrolase superfamily protein 30540 1.09 1.04 1.08 1.00 1.09

Other YhcQ Spore coat protein F-like protein 25246 1.08 1.07 0.98 0.96 0.91

Coat YckD Uncharacterized protein 12782 1.05 1.01 1.08 1.15 0.99

Proteins YodI Uncharacterized protein YodI 9245 1.02 0.88 0.82 0.78 0.77

YhbB Uncharacterized protein 36095 0.94 1.03 1.05 1.10 1.00

YhxC Uncharacterized oxidoreductase 31307 0.90 0.99 0.85 0.79 0.85

YjqC Uncharacterized protein 31403 0.87 0.87 0.80 0.79 0.74

YdcC Sporulation protein 38170 0.76 1.01 1.01 1.02 1.01

YrbF UPF0092 membrane protein 9897 1.63 1.21 1.13 1.20 1.13

YyxA Uncharacterized serine protease 42762 1.44 1.20 1.55 2.35 2.70

OppA Oligopeptide-binding protein 61543 1.36 1.38 1.89 1.42 1.36

YkzQ Uncharacterized protein 8889 1.35 1.19 1.77 0.81 0.37

YaaQ Uncharacterized protein 11959 1.31 1.36 1.42 1.28 1.16

YmfF Probable inactive metalloprotease 48794 1.28 0.88 0.81 0.86 0.81

YkuS UPF0180 protein 8716 1.27 1.19 1.04 0.89 0.95

YuzA Uncharacterized membrane protein 8518 1.25 1.05 1.01 1.00 1.10

YkuU Thioredoxin-like protein 20787 1.19 1.06 1.12 1.18 1.07

YrzQ Uncharacterized protein 5039 1.19 1.06 1.06 1.06 1.05

YkuJ Uncharacterized protein 9296 1.17 0.97 0.91 1.58 1.01

YhcN Lipoprotein 21061 1.16 1.12 0.98 0.98 1.03

Putative YfkD Uncharacterized protein 29929 1.16 0.95 1.13 1.07 1.10

Spore YfkO Putative NAD(P)H nitroreductase 25669 1.16 1.03 1.08 0.92 0.91

Coat YhfW Putative Rieske 2Fe-2S iron-sulfur protein 58264 1.15 1.05 1.16 0.97 1.03

Proteins YsdC Putative amino peptidase 39249 1.13 1.00 1.01 0.98 0.87

YmfH Uncharacterized zinc protease 49089 1.09 1.06 1.08 1.04 0.97

YwfI UPF0447 protein 29886 1.08 1.04 1.36 1.23 0.97

YqfA UPF0365 protein 35676 1.07 0.95 0.89 0.93 0.88

YmxG Uncharacterized zinc protease 46137 1.05 1.11 1.13 0.78 0.57

YqgO Uncharacterized protein 6907 1.05 0.94 0.98 0.90 1.03

YjdH Uncharacterized protein 15249 1.04 0.93 1.28 1.00 1.02

YurZ Uncharacterized protein 13898 1.00 1.15 0.79 1.00 0.94

AtcL Calcium-transporting ATPase 98677 0.99 1.05 1.05 0.97 1.00

YgaK Uncharacterized FAD-linked oxidoreductase 51661 0.98 0.97 1.03 0.72 1.07

YqiG Probable NADH-dependent flavin oxidoreductase

40894 0.97 1.13 1.60 1.01 0.81

DacB D-alanyl-D-alanine carboxypeptidase 43561 0.93 1.07 1.07 1.21 1.02

YhcB Uncharacterized protein 19400 0.93 1.02 1.53 1.11 1.09

YxeD Uncharacterized protein 13793 0.81 0.80 0.77 0.88 0.74

Fer Ferredoxin 9092 0.58 0.51 0.62 0.33 0.38

Coreb SspA Small, acid-soluble spore protein A 7066 1.21 1.20 0.98 1.15 1.15

Proteins SspB Small, acid-soluble spore protein B 6975 0.78 0.75 0.61 0.71 0.86 a Normalized ratios of all the identified coat proteins in one biological replicate are tabulated. The coat and crust proteins speculated to take part in spore maturation are highlighted in bold. The results for replicate 2 and 3 can be found in the supplementary data. b Core proteins SspA and SspB are not involved in maturation and serve as additional internal control.

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Figure 2. Isotope ratios, averaged over the identified peptides, from the crust, outer coat and inner coat proteins against the % survival of thermally stressed young to old spores. The primary Y-axis shows the change in the digestion extent of proteins as a function of 14N/15N ratios (line plots) while the secondary Y-axis shows the increase in the thermal resistance of spores as estimated by % survival rate (grey bars) of thermally stressed spores on tryptic soy agar plate after incubation for 24 hours at 37°C. Compared to younger spores reduced digestion extent for crust and outer coat proteins and higher thermal resistance (P<0.05) is seen in more mature spores. Proteins CotG, YurS, CotI, CotU, CotB, CotC are localized in the outer coat while SafA, SpoIVA are inner coat proteins. CotY and CotZ are located in the crust. was more homogeneous in their germination behaviour. Thermally stressed spores showed a delay in germination times as for the young spores the shift in germination times was larger but this shift in germination times was small for the un-stressed and stressed mature spores (Figure 4). Discussion

Bacterial spores are well-known foes of the food-industry. The immense resistance characteristics of spores, towards the routine food-processing practices such as pasteurization, high pressure treatment etc., pose problems in acquiring food safety. Efforts are being made to prohibit spores from entering the food stuff or else to initiate quick germination of spores present in the food to kill the germinating spoiler cells. In these attempts importance of coat layer in the resistance properties [13], importance of structure of spores in maintenance of spore integrity [14], and use of coat or exosporium proteins as targets to design quick spore-detection systems [15, 16] etc. have been researched. A recent study concluded that spore maturation, after the spore’s release from the mother cell, is an important factor in acquiring wet-heat resistance in spores [3]. On the same lines, our research has allowed us to identify a group of spore outer coat and


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Figure 3. Digestion efficiency of the peptides from the spore coat proteins. The x-axis shows the position of the identified peptide within the protein and the y-axis shows the digestion efficiency of the peptides over time compared to the 15N-labeled reference spores. Symbol (■) represents peptides identified from 2-day young spores while symbol (▲) represents the same identified peptides from 8-day mature spores. Proteins YaaH and CotJC represent the inner coat proteins, CotB, CotE, CotG represent the outer coat and CotZ represents the crust proteins.

Figure 4. Heterogeneity in the germination times of young and mature spores. A frequency distribution of number of spores germinating at different germination times is plotted. A slight but significant delay in the germination times (speed) of young and mature un-stressed spores can be seen (black lines). No significant difference in the germination times of thermally-stressed spores is seen (blue lines).

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crust layer proteins affected, most likely due to the extent of their cross-linking, during spore maturation. In our research, the 15N-labeled 8-day mature spores were used as the reference to which the spores prepared in 14N-labeled medium were compared. The query spores were harvested at different time points and mixed with the reference spores in 1:1 ratio based on the OD600 of samples. Since no new proteins are synthesized per se once the spore is released from the mother cell, the relative composition of spore coat protein in the spores is constant. However, we show that the 14N/15N ratios of proteins and peptides thereof display a characteristic distribution when 2-day, 4-day, 6-day and 8-day query (14N) spores are compared to the 8-day mature reference (15N) spores. Also, the proteins from 8-day query spores should exhibit the same digestion efficiencies as in the 8-day reference spores. However, the lack of this similarity in ratios suggests that the maturation in the query and reference spores could have progressed to different extents. Nevertheless, a detailed analysis of the peptides identified from proteins facilitates the use of 14N/15N ratios as a measure of the extent to which these proteins were digested.

Our research clarifies that the inner spore coat proteins are not affected during spore maturation. As seen in Table 1, the 14N/15N ratios for most of the inner coat proteins, averaged over all the identified peptides used for quantification are close to 1.0. Manual inspection of the peptides identified from the inner coat proteins, exemplified by CotJC in Figure 3, clearly shows that the inner coat proteins are digested to the same extent in both the query and the reference spores. The inner coat morphogenetic proteins SpoIVA and SafA, as shown in Figure 2, are indeed involved in the morphogenesis of the inner coat and are affected in their digestion efficiencies in young spores. However, as spores mature these proteins show digestion efficiencies that coincide in the 14N and 15N spores. Based on these results it is possible that the identified putative spore coat proteins, such as YsdC, YqgO, YxeD, with 14N/15N ratios near to the window in which the inner coat proteins fall, may also be localized in the inner coat.

Compared to the inner coat, the outer coat and crust proteins are critical for spore maturation. Most of the outer coat proteins show decreasing 14N/15N ratios, averaged over the identified peptides, when compared between 2- and 8-day old spores (Table 1). Proteins CotG, YurS, CotU, CotI, CotZ, CotY, CotB and CotC are the most affected proteins with regards to their protease resistance over time. Incidentally these proteins are rich in tyrosine, glutamine, lysine and/or cysteine. This fact coincides with the predicted dityrosine, ε-(γ)-glutamyl-lysine and disulfide linkages in the spore surface layer proteins [1]. The strongest maturation effect is seen in case of protein CotG, an outer coat protein which carries 9 repeats of 13 amino acid residues -H/Y-KKS-Y-R/C-S/T-H/Y-KKSRS- at the central core of the protein [17]. These repeats are rich in lysine and tyrosine. In our previous study [18] as well as in the current study we could not identify any peptides from this region. This may be due to two reasons - abundance of lysine giving rise to small peptides that escape the mass spectrometric detection and/or the tyrosine residues that could be cross-linked via an oxidation dependent mechanism mediated by SodA also present in the spore coat [8]. Nevertheless, we identify the peptides from the C- & N-termini of CotG which show a significant decrease in their digestion efficiencies from 2- to 8-day spores (Figure 3). YurS is an uncharacterized


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protein that appears to be involved in spore coat maturation in this study. This protein is a product of gene yurS which is reported to be co-transcribed with another coat protein gene sspG [19]. SspG was a putative coat protein identified from our previous study. We did not identify it in the current study suggesting a strain-dependent variation in coat protein composition. Protein CotU (YnzH) also appears to participate in spore maturation. Though we identified only the C-terminal peptides the digestion efficiencies of these peptides are significantly lower in 8-day spores when compared to 2-day spores. Similarly, the spore morphogenetic protein CotE, responsible for building of the outer coat layer, became more protease resistant in 8- and/or 10-day old spores. CotC, CotE, CotG, CotU are all known to be involved in inter-protein cross-links previously [8-10, 20]. Thus the decrease in the digestion efficiencies of these proteins over the time is indicative of spore maturation coupled to the progress in protein-protein cross-linking. Protein CotB, which is dependent on CotG for its assembly, is rich in serine residues at its C-terminal. Serine residues are target for glycosylation. Glycosylation is a post-translational modification and could also be involved in spore maturation. We did not identify any peptide from this region. This is possible since this region is also rich in lysines giving rise to small peptides undetectable for mass spectrometer. It is also likely that CotB carries glycosyl moieties in this region and due to lack of suitable modification information we do not identify the peptides. Yet, CotB also showed the same behaviour with less digestion efficiency in 8-day spores suggesting its involvement in spore maturation. Outer coat protein CotA is an abundant protein that encodes for a copper-dependent laccase [21]. Laccases can also mediate the protein-protein cross-linking via an oxidation based reaction [22, 23]. Although no such evidence for CotA has been obtained, our results show that spore maturation also has a minor effect on the digestion efficiency of CotA. Its role in enhancing structural integrity of spores needs to be studied in more detail as suggested in Chapter 4.

The difference in the extent of inter-protein cross-linking amongst different spores from a single population could also be a contributing factor to the inherent heterogeneity in spore germination. Therefore we analysed young (2-day) and mature (8-day) spores for their germination behaviour. In the current context, young spores may show heterogeneity in the germination times as optimum maturation has not yet been achieved. In contrast, 8-day spores appear to be a more mature spore population. Also, the germination times measured in our analysis differed significantly (P-value <0.05) when young (2-day) and 4-day, 6-day and 8-day mature spores were compared. Thermal treatment did not show a significant difference in the germination times of young and mature spores as suggested by the observed results. Since, thermal stress can damage the spores, the heat-sensitive young spores may require more time to heal the damage prior to germination. The heat-resistant mature spores are speculated to suffer less damage and therefore they require less time to initiate germination. Whether this hypothesis is true needs to be confirmed by further experiments with a higher number of spores. Also transcriptional analyses of the germinating young and mature spores could help reveal the differences in the two spore populations at the genetic level.

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Conclusion

The spore outer coat and the crust layers are favored targets for spore maturation with proteins CotG, YurS, CotU, CotI, CotZ, CotY, CotB and CotC being critical for the process and likely subjected to cross-linking. The inner coat proteins are not involved in spore maturation except in the young spores where SpoIVA and SafA play a pivotal role in stabilizing this layer before the spore is released out of the mother cell. The level of enhancement of digestion of individual spore proteins in young spores may be an indication of their location in the spore layers.

Materials and Methods

Bacterial strain and sporulation conditions

Bacillus subtilis wild-type strain PY79 was obtained from the Eichenberger lab (New York University, USA) and was used for preparing 14N (Light) and 15N (Heavy)-labeled spores. Bacteria were pre-cultured and sporulated as described previously [18]. For sporulation a defined minimal medium, buffered with 3-(N-morpholino)propanesulfonic acid (MOPS) to pH 7.4, was used [24]. The in-house bench fermenter setup consisted of 4 autoclavable ½ L glass bioreactors, equipped with Tamson T1000 waterbath (Gemini BV, Apeldoorn, The Netherlands). Each bioreactor contained a double-layered glass jacket through which water was continuously flown to maintain the growth temperature at 37°C. Sterile air was continuously plunged through at a constant rate (0.5 L/hr. at 200 rpm). B. subtilis cells from a single pre-culture and with OD600 of 0.4 were inoculated from the same pre-culture to all the bioreactors and allowed to sporulate and mature to a maximum of 8-10 days post inoculation. The query cultures were grown and sporulated in presence of 14NH4Cl while the reference cultures in 15NH4Cl as the sole nitrogen source. The final stock of reference spores consisted of spores pooled from three independent biological replicates while three independent biological replicates were separately analyzed for the query culture. One biological replicate of 14N spores was allowed to mature for 10 days post-inoculation.

Spore harvesting

The 14N-query spores were harvested as described elsewhere [18] on day 2, day 4, day 6, day 8 and day 10 post-inoculation whereas the 15N-labeled reference spores were only harvested on day 8 post inoculation. For 2-day old spores the harvesting procedure also involved a final step of Histodenz gradient (Sigma Chemical Co., St. Louis, MO) centrifugation to get rid of the non-sporulated cells. For these spores the washed and harvested spore pellet fraction was suspended in a small volume of 20% Histodenz and then layered on 50% Histodenz medium. The tubes were centrifuged at 15°C for 45 min at 15,000 × g. By this procedure the free spores were pelleted down which were used for further work.

Measurements of thermal resistance

Thermal resistance of spores to wet heat was assessed using the previously used screw-cap tube method [24]. In short, a 1 ml (heat-activated (70°C, 30 min); OD600 ~2) spore suspension in sterile milli-Q water, for each time point, was injected with a syringe into a preheated metal screw-cap tube containing 9.0 ml of sterile milli-Q water. The heat activation helped to kill all the remaining vegetative cells in the sample. The tube was heated by immersing it completely in a glycerol bath (85°C for 10 min). After 10 min the tube was transferred to ice-water. Dilution series of spore suspension were prepared in sterile milli-Q water and 100 μl of sample was spread on Tryptic Soy agar plates. The number of colonies was counted after 24 hours of incubation at 37°C. The thermal resistance of spores was thus determined by the loss of their ability to germinate and form colonies (i.e., viability counts). As a control a same dilution series for non-heat stressed spores ware plated and the final colony counts from the stress samples were normalized


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with those from the control sample. The significance of the thermal resistance tests was tested by one-way ANOVA test.

Mixing of 14N and 15N-labeled spores, spore coat isolation and protein extraction

The harvested 14N-spores were immediately mixed in 1:1 ratio with 15N-reference spores based on OD600. After mixing the samples were further subjected to spore coat isolation & protein extraction as described previously [15, 18]. The isolated coat material was freeze-dried overnight and immediately used for mass spectrometric analysis.

Sample preparation for MS analysis



LC-MS/MS data were acquired with an Bruker ApexUltra Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a 7 T magnet and a nano-electrospray Apollo II DualSource™ coupled to an Ultimate 3000 (Dionex, Sunnyvale, CA, USA) HPLC system. Samples containing up to 200 ng of the tryptic peptide mixtures were injected as a 10 μl 0.1% TFA, 3% ACN aqueous solution and loaded onto a PepMap100 C18 (5-μm particle size, 100-Å pore size, 300-μm inner diameter x 5 mm length) precolumn. Following injection, the peptides were eluted via an Acclaim PepMap 100 C18 (3-μm particle size, 100-Å pore size, 75-μm inner diameter x 250 mm length) analytical column (Thermo Scientific, Etten-Leur, The Netherlands) to the nano-electrospray source. Gradient profiles of up to 120 min were used from 0.1% formic acid / 3% CH3CN / 97% H2O to 0.1% formic acid / 50% CH3CN / 50% H2O at a flow rate of 300 nL /min. Data dependent Q-selected peptide ions were fragmented in the hexapole collision cell at an Argon pressure of 6x10-6 mbar (measured at the ion gauge) and the fragment ions were detected in the ICR cell at a resolution of up to 60000. In the MS/MS duty cycle, 3 different precursor peptide ions were selected from each survey MS. The MS/MS duty cycle time for 1 survey MS and 3 MS/MS acquisitions was about 2 s. Instrument mass calibration was better than 1 ppm over a m/z range of 250 to 1500. Raw FT-MS/MS data were processed with the MASCOT DISTILLER program, version 2.4.3.1 (64bits), MDRO 2.4.3.0 (MATRIX science, London, UK), including the Search toolbox and the Quantification toolbox. Peak-picking for both MS and MS/MS spectra were optimized for the mass resolution of up to 60000. Peaks were fitted to a simulated isotope distribution with a correlation threshold of 0.7, with minimum signal to noise of 2. The processed data, from the three independent biological replicates, were searched with the MASCOT server program 2.3.02 (MATRIX science, London, UK) against a complete B. subtilis 168 ORF translation database (Uniprot 2011 update, downloaded from http://www.uniprot.org/uniprot). The parameters for Quantification using 15N-Metabolic labeling were used. Trypsin was used as enzyme and 2 missed cleavages were allowed. Carbamidomethylation of cysteine was used as a fixed modification and oxidation of methionine as a variable modification. The peptide mass tolerance was set to 30 ppm and the peptide fragment mass tolerance was set to 0.03 Dalton. Using the quantification toolbox, the isotopic ratios for all identified proteins were determined as an average of the isotopic ratios of the corresponding light over heavy tryptic peptides. Selected critical settings were: require bold red: on, significance threshold: 0.05: Protocol type: precursor; Correction: Element 15N; Value 99.4; Report ratio L/H; Integration method: Simpson’s integration method; Integration source: survey; Allow elution time shift: on; Elution time delta: 20 seconds; Std. Err. Threshold: 0.15,

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Correlation Threshold (Isotopic distribution fit): 0.98; XIC threshold: 0.1; All charge states: on; Max XIC width: 200 seconds; Threshold type: at least homology; Peptide threshold value: 0.05; unique pepseq: on. Data normalization

In order to correct for the possible errors in the 1:1 mixing of the 14N cultures with the 15N reference cultures the protein isotopic ratios were normalized for the data set of each time point by setting the ratio for a normalization protein to 1. The protein for normalization was chosen to be YaaH. This protein is localized in the inner spore coat and it is one of the hydrolases important during germination. The expression levels of YaaH, during sporulation, have been found to be constant in a previous study [25]. Since, germination requires complete hydrolysis of cortex peptidoglycan [26] the likelihood of YaaH to be involved in inter-protein cross-linking is therefore minimal. Also the relative peptide ratios for the protein were found to be stable over the duration of 8 days in our work. The identified proteins and their respective isotopic ratios over the period of ten days are mentioned in Table 1.

Slide preparation for time-lapse microscope

A special microscope slide with a closed air containing chamber developed by Pandey and co-workers was used for phase contrast image acquisition [7]. The slides were prepared as described by the authors. 1 μl of spore solution was loaded on a thin agarose pad made by using two siliconized (24 x 32 mm) cover slips. The agarose-medium pad was placed in an upright position on the Gene Frame® and pressure was applied for complete sealing. This chamber was used for time-lapse microscopy. Time-lapse series were made, using a temperature-controlled boxed incubation system for live imaging set at 37°C and observing the specimens with a 100X/1.3 plane Apochromatic objective (Axiovert-200 Zeiss, Jena, Germany). Phase-contrast time-lapse series were recorded at a sample frequency of 1 frame per min for 5 h for control and 10 h for heat-treated (85°C for 10 min spores). In each field of view, on average 8 spores were identified and followed in time. Maximally 9 areas (fields of view) were recorded in parallel. This resulted in the analysis of approximately 70 spores from the start of each imaging experiment. One biological replicate for control and stress condition was performed.

Microscopic data analysis

Effect of spore maturation on spore germination time was analysed using a semi-automatic image analysis macro called Spore Tracker[7], a plugin for ObjectJ (http://simon.bio.uva.nl/objectj), which runs under ImageJ (http://rsb.info.nih.gov/ij). The germination time (speed) was marked by Spore Tracker [7]. Frequency distribution plots of the germination times of individual spores from day 2, day 4, day 6 and day 8 were generated. Differences in the variance between different sample day and treatment were tested with one-way ANOVA test and student’s T-test was performed to test differences in the averages.

References

1. Henriques AO, Moran CP, Jr. Structure, assembly, and function of the spore surface layers. Annual review of microbiology. 2007;61:555-88. Epub 2007/11/24. 2. Setlow P. I will survive: DNA protection in bacterial spores. Trends in microbiology. 2007;15(4):172-80. Epub 2007/03/06. 3. Sanchez-Salas JL, Setlow B, Zhang P, Li YQ, Setlow P. Maturation of released spores is necessary for acquisition of full spore heat resistance during Bacillus subtilis sporulation. Applied and environmental microbiology. 2011;77(19):6746-54. Epub 2011/08/09. 4. Nicholson WL, Munakata N, Horneck G, Melosh HJ, Setlow P. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiology and molecular biology reviews : MMBR. 2000;64(3):548-72. Epub 2000/09/07.


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5. Setlow P. Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. Journal of applied microbiology. 2006;101(3):514-25. Epub 2006/08/16. 6. Xu H, He X, Gou J, Lee HY, Ahn J. Kinetic evaluation of physiological heterogeneity in bacterial spores during thermal inactivation. The Journal of general and applied microbiology. 2009;55(4):295-9. Epub 2009/08/25. 7. Pandey R, Ter Beek A, Vischer NOE, Smelt JPPM, Brul S, Manders EMM. Live Cell Imaging of Germination and Outgrowth of Individual Bacillus subtilis Spores; the Effect of Heat Stress Quantitatively Analyzed with SporeTracker. PLoS ONE. 2013;8(3):e58972. 8. Henriques AO, Melsen LR, Moran CP. Involvement of Superoxide Dismutase in Spore Coat Assembly in Bacillus subtilis. Journal of Bacteriology. 1998;180(9):2285-91. 9. Isticato R, Esposito G, Zilhão R, Nolasco S, Cangiano G, De Felice M, et al. Assembly of Multiple CotC Forms into the Bacillus subtilis Spore Coat. Journal of Bacteriology. 2004;186(4):1129-35. 10. Isticato R, Pelosi A, Zilhão R, Baccigalupi L, Henriques AO, De Felice M, et al. CotC-CotU Heterodimerization during Assembly of the Bacillus subtilis Spore Coat. Journal of Bacteriology. 2008;190(4):1267-75. 11. Zhang J, Fitz-James PC, Aronson AI. Cloning and characterization of a cluster of genes encoding polypeptides present in the insoluble fraction of the spore coat of Bacillus subtilis. Journal of Bacteriology. 1993;175(12):3757-66. 12. Imamura D, Kuwana R, Takamatsu H, Watabe K. Proteins Involved in Formation of the Outermost Layer of Bacillus subtilis Spores. Journal of Bacteriology. 2011;193(16):4075-80. 13. Riesenman PJ, Nicholson WL. Role of the Spore Coat Layers in Bacillus subtilis Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation. Applied and environmental microbiology. 2000;66(2):620-6. 14. Ghosh S, Setlow B, Wahome PG, Cowan AE, Plomp M, Malkin AJ, et al. Characterization of Spores of Bacillus subtilis That Lack Most Coat Layers. Journal of Bacteriology. 2008;190(20):6741-8. 15. Abhyankar W, Hossain AH, Djajasaputra A, Permpoonpattana P, Ter Beek A, Dekker HL, et al. In Pursuit of Protein Targets: Proteomic Characterization of Bacterial Spore Outer Layers. Journal of proteome research. 2013;12(10):4507-21. 16. Walper SA, Anderson GP, Brozozog Lee PA, Glaven RH, Liu JL, Bernstein RD, et al. Rugged Single Domain Antibody Detection Elements for Bacillus anthracis Spores and Vegetative Cells. PLoS ONE. 2012;7(3):e32801. 17. Sacco M, Ricca E, Losick R, Cutting S. An additional GerE-controlled gene encoding an abundant spore coat protein from Bacillus subtilis. Journal of Bacteriology. 1995;177(2):372-7. 18. Abhyankar W, Ter Beek A, Dekker H, Kort R, Brul S, de Koster CG. Gel-free proteomic identification of the Bacillus subtilis insoluble spore coat protein fraction. Proteomics. 2011;11(23):4541-50. Epub 2011/09/10. 19. Bagyan I, Setlow B, Setlow P. New small, acid-soluble proteins unique to spores of Bacillus subtilis: identification of the coding genes and regulation and function of two of these genes. J Bacteriol. 1998;180(24):6704-12. Epub 1998/12/16. 20. Kim H, Hahn M, Grabowski P, McPherson DC, Otte MM, Wang R, et al. The Bacillus subtilis spore coat protein interaction network. Molecular Microbiology. 2006;59(2):487-502. 21. Hullo MF, Moszer I, Danchin A, Martin-Verstraete I. CotA of Bacillus subtilis is a copper-dependent laccase. J Bacteriol. 2001;183(18):5426-30. Epub 2001/08/22. 22. Elegir G, Bussini D, Antonsson S, Lindstrom ME, Zoia L. Laccase-initiated cross-linking of lignocellulose fibres using a ultra-filtered lignin isolated from kraft black liquor. Applied microbiology and biotechnology. 2007;77(4):809-17. Epub 2007/10/24. 23. Steffensen CL, Andersen ML, Degn PE, Nielsen JH. Cross-linking proteins by laccase-catalyzed oxidation: importance relative to other modifications. Journal of agricultural and food chemistry. 2008;56(24):12002-10. Epub 2008/12/05. 24. Kort R, O'Brien AC, van Stokkum IHM, Oomes SJCM, Crielaard W, Hellingwerf KJ, et al. Assessment of Heat Resistance of Bacterial Spores from Food Product Isolates by Fluorescence Monitoring of Dipicolinic Acid Release. Applied and environmental microbiology. 2005;71(7):3556-64.

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25. Nicolas P, Mader U, Dervyn E, Rochat T, Leduc A, Pigeonneau N, et al. Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis. Science. 2012;335(6072):1103-6. Epub 2012/03/03. 26. Setlow P. Spore germination. Current Opinion in Microbiology. 2003;6(6):550-6.


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6 Molecular properties of Spore surface proteins

Abstract Analysis of bacterial spore surface proteomes has led to identification of > 90 different proteins however functional characterization has not been achieved for most of these proteins. In addition to their discovery, a few proteins have been utilized for biotechnological applications. Spore longevity and their escape from immune surveillance still remain challenges for the food security and medical fields. This chapter reasons out the peculiarities of spore surface proteins that might be decisive in these respects. Emerging bioinformatic tools have made protein structure and function predictions possible. In this context, using such tools the study of amino acid distributions of proteins highlighted some characteristics about structures of these proteins whereas molecular mass, pI and GRAVY distributions focussed on the potential role of these proteins in spore surface adhesion. Finally, the potential of certain peptide sequences, from the identified proteins, in drug development against resistant pathogens was examined.

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Introduction

Spores are of major concern when it comes to food safety and pathogenic infections where they act as mediators. Thus their persistence and the germination mechanisms are considerably important for human infections. As discussed extensively in Chapter 1, spore structure plays a crucial role in their survival. In spite of the many efforts to unravel the details behind spore integrity, longevity and survival, especially with regards to the proteins from the spore outer layers, the functional and structural studies of these proteins have been scarce. X-ray crystallographic structures (with resolutions < 3Å and R-values < 20%) of only 12 proteins are available in the Protein Data Bank (http://www.rcsb.org/pdb/home/ home.do). From B. subtilis, structures of proteins CotA [1], CotI (YtaA [2]), GerBC [3], GerE [4], PdaA [5], SpoVT [6], SpsA [7] and YkuD [8] are available in the PDB data bank while from B. cereus and B. anthracis structures of proteins SleB (catalytic domain [9, 10]), Alr (BA_0252 [11]), BclA [12] are accessible. Recently the structure of CspB protease involved in Clostridium spore germination was also published [13]. Evidently, the knowledge of the molecular mechanisms indispensable for the assembly of the bacterial spore coat is incomplete and more detailed studies are needed to understand the means through which proteins are targeted to the basic coat layer. Along with the fundamental knowledge of the spore proteins and the spore structure, the resistant as well as the adhesive properties of spores also need to be focused upon in order to culminate spores. In an attempt to uncover the structural details of these proteins, their amino acid compositions and other discernible molecular properties that were marked in our studies are discussed below. Furthermore, the potential therapeutic applications of the spores in view of their component coat or exosporium proteins are also discussed. 1. Fundamentals of spore surface proteins

(a) Amino acid compositions

Since 1950s the relation between the protein amino acid sequence and the protein structure is well known. Subsequently, the propensities of different amino acids in formation of the secondary protein structures namely, α helices, β sheets, turns were studied and it was found that, for example, amino acids such as alanine, glutamate, leucine tend to be present in α helices, amino acids such as valine, isoleucine tend to prefer β sheet structures whereas amino acids glycine, asparagine and proline have preference towards turns. More recently it was also shown that the amino acid sequence also determines the intrinsic protein disorder (IPD) that renders a particular region of the protein unstructured thereby enlightening the functional variety in the proteins. These studies concluded that presence of low sequence complexity and amino acid compositional bias, with low content of hydrophobic, bulky amino acids (Val, Leu, Ile, Met, Phe, Trp and Tyr), and a high proportion of charged amino acids (Gln, Ser, Pro, Glu, Lys, Gly and Ala) were indicative of a probable intrinsic disorder [14]. It has also been found that IPDs generally are: resistant to boiling temperature; insensitive to denaturing chemicals; and susceptible to proteolytic cleavage [15]. Due to the intrinsic disorders

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these proteins are also difficult to crystallize in X-ray crystallographic studies. Thus protein sequence comparison has become a powerful tool to characterize the protein sequences and possibly their functional analysis. The amino acid sequence is therefore the most important feature of a protein for its localization, structure and function. In these respects, the spore coat or exosporium proteins have peculiar amino acid sequences such as the collagen-like repeat sequences, histidine-rich or serine-rich C-termini of proteins, Proline-Glycine-Tyrosine rich protein sequences etc. The Uniprot protein database (http://www.uniprot.org/) illustrates the relative contribution of each of the 20 natural amino acids to the entire protein compliment, across all the prokaryotic and eukaryotic organisms studied till date. In comparison with this relative contribution of amino acids, it was observed that the proteomes of individual species - B. subtilis 168, B. cereus ATCC 14579 and C. difficile 630 follow a similar trend and barring certain amino acids such as glutamine, threonine etc. the general amino acid spectrum of spore outer layer proteins strongly resembles that of the vegetative cell proteins. In general, the spore surface proteins appear to be rich in aliphatic hydrophobic and acidic amino acids suggesting a possibility that spore surfaces are hydrophobic and the charged residues could be involved in cross-linking amongst different proteins. Figure 1 shows the relative amino acid composition of the spore surface proteomes from B. subtilis 168 [16, 17], B. cereus ATCC 14579 [18] and C. difficile 630 [18]. Clearly, the anaerobic C. difficile vegetative cell proteome appears to diverge from the aerobic Bacilli in case of isoleucine, asparagine, glutamine and lysine distributions. Interestingly, though these proteins have higher lysine contents the arginine contents are considerably low. Higher amounts of lysine, glutamine, and tyrosine in B. subtilis are in agreement with the predictions of ε-γ-glutamyl-lysine[19] and di-tyrosine crosslinks [20] in the coat proteins. The highest histidine content in B. subtilis spores might indicate their role in protein stability[21], protein interactions[22], or in protein function (the histidine residues in CotA are involved in stabilization of the copper moieties thereby establishing catalytic function of the B. subtilis laccase [1]) or as a cleavage site for proteases to convert precursor proteins into their mature forms [23]. On the other hand the B. cereus ATCC 14579 coat and exosporium proteome appear to be rich in threonine, glycine, proline and asparagine. The Bcl-family proteins identified from the exosporium of B. cereus are well represented by the higher glycine, proline (GXX repeats) and threonine (potential glycosylation site) contribution. Earlier work performed by Aronson and Fitz-James [24] indicated that except in B. licheniformis the spores from other Bacillus spp. are rich in glycine and lysine and our observations for amino acid distributions for the identified proteins illustrate the same trend. The C. difficile 630 proteomes for vegetative cells and spore surfaces emerged to be highly similar except for isoleucine and leucine contents. This higher leucine content in the vegetative cells could result from the cell surface proteins that contain leucine rich repeat (LRR) motives. These proteins may play a role in adhesion of cells to surfaces as well as in host pathogen interactions [25]. The spore surface proteins identified from C. difficile 630 in our study are extremely acidic but are also rich in cysteine, tyrosine, asparagine and lysine again confirming the previous observations [26]. A combination of charged residues of the surface proteins also suggest a possibility of salt-bridge formation, as in case of viruses [27], amongst proteins thus

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adding to the stability of the coat structure. Intriguingly, all the amino acid residues mentioned above have high propensities [28] to be present either on the exposed protein surface or in the protein-protein binding site. Additionally, some amino acid biases were identified in the spore surface proteins in the form of single amino acid repeats or internal tandem repeat sequences (Table 1) that have escaped the focus since past. The repeat sequences, in general, are abundant in proteins and vital for protein structure, function and evolution. Single amino acid repeats are mainly responsible for unstructured regions of proteins. Such repeats have been shown to be the cause of many diseases such as Huntington’s disease in humans [29]. It is hypothesized that such single amino acid repeats can formulate the structure of that particular region of the protein and may also be involved in protein aggregation [30]. The later property is beneficial for rendering spore coat a proteinaceous structure. The tandem repeat sequences on the other hand indicate strong biological relevance wherein the phylogenetically conserved repeats among the orthologous proteins should have a conserved function. These conserved properties are generally responsible for maintaining the essential functions whereas the varying regions may arise due to the effects on environment or due to ecological pressures. Using computational tools previously, tandem repeats have been identified from the archaebacterial cell surface proteins [31, 32]. The authors identified 6 different tandem repeat sequences from the cell surface proteins of Methanosarcina acetivorans. Progressing further it was also shown by the same group that these repeats could be involved in formation of the β-propeller fold structure. Proteins that can form the β-propeller fold are universally found in nature from viruses to bacteria, prokaryotes to eukaryotes, invertebrates to mammals [32]. Also in protozoan parasites proteins with tandem repeats have been found to act as the target of B-lymphocyte response. Thus in another study, efforts were taken to identify the tandem repeat protein sequences from protozoan Trypanosoma cruzi [33]. These examples indicate the significance of mining the protein sequences in search of unique sequence regions. We analyzed the proteins identified in our studies for the presence of the repeat sequences and we found that both single amino acid as well as tandem repeat sequences exist in spore surface proteins. We used the recently published algorithm T-REKS [34] for this purpose. Some representative proteins from the three species are shown in Table 1. In addition, the B. subtilis, proteins CotT and YmaG are rich in proline, glycine and tyrosine. Their sequences show homology with the PGY-rich protein from rice (Oryza sativa). OsPGYRP contains GYPPX-repeat at N-terminus and a cysteine rich region at the C-terminus of the protein [35]. On the contrary, CotT has PYYYPRPYYPF and GYGG repeats and YmaG has GRPFGF repeats. The OsPGYRP is expressed in response to cold, salt or osmotic stress but overexpression of this gene was reported to enhance cold stress in E. coli [35]. The role of tandem repeats in CotT, YmaG and the other spore surface proteins could provide the knowledge about their role in maintaining the structural integrity of spores. Apart from the tandem repeat sequences some spore surface proteins also have single amino acid repeats. For instance the inner coat protein YeeK from B. subtilis spores contains a region with 11 histidine (H) residues towards its C-terminal end (in the

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Table 1. Internal tandem repeat sequences found in spore surface proteins in B. subtilis 168, B. cereus ATCC 14579 & C. difficile 630. Psim similarity coefficient for the repeats.

Protein Sequence Repeat No. of Residues Psim length repeats From To B.

subt

ilis 1

68

YirY (SbcC) EQAEKVL 7 2 216 229 0.86 EQAEARL

CotT PYYYPRPRPPFYP

11 2 21 44 0.85 PYYYPRPYYPF--

CotU(YnzH) KYYDNDKKHYDCD-

12 3 21 59 0.86 KYYDHDKKHYDYDK KYDDHDKKYYD-D-

CotC DYDKKYD

7 3 33 53 0.86 DYDKKYY DHDKKDY

CotB ---SKRSLKSSDYQS 14 2 317 342 0.80 SKSSKRSPRSSDYQ-

CotG

KS-RSHK-

7 15 58 156 0.84

KSFCSHK- KS-RSHK- KSYCSHK- KS-RSHK- KSYRSHK- KS-RSYK- KSYRSYK- KS-RSYK- KSCRSYK- KS-RSYK- KSYCSHKK KS-RSYK- KSCRTHK- KSYRSHK-

B. c

ereu

s ATC

C 1

4579

BC_1559 (YppG) NQAQQPQQQQQPYV

12 3 54 105 0.81 NQAQQPQQQQ-PYV NQAQQSQAQQ-PYM

BC_1560 (CotD) SPFGPGPNV

9 3 100 125 0.83 SPFGPGPNV SPFLPN-NV

BC_2030 (CotG) -VKHCTFVTKCTH-

13 3 60 97 0.83 -VKKWTFVTKCTRV RVQKWTFVTKVTR-

BC_2874 (CotX)

EPKKRVP

7 4 21 48 0.86 EPKKEDC EPKKEDC ELKKEDC

BC_3582 SCGFGCGG-GFS

10 3 77 107 0.81 SC--GCGGRGCS SC--GCGGWGGG

BC_4420 (SafA)

QKEVQV-KP

9 6 109 157 0.91

QKEMQV-KP QKEVQV-KP QKEMQV-KP QKEVQV-KP QKEVQKEQP PNIQMPI-MDNNQP---

13 9 357 482 0.84

PNI-MPI-MDNSQP--- PNI-MPI-MDNNQP--- PNI-MPI-MDNNQP--- PNI-MPI-MDNNQM--- PNM-MPI-MDNNQM--- PNM-MPI-MDNNQM--- PNI-MPI-MDNNKP--- PNM-MPYQMPYQQPMMP

C.

diffi

cile

63

0 CD1067 (CdeC) CNPCKPNP

8 2 64 79 1.00 CNPCKPNP CD3664

PEGLVFTH PEGGLFTW 8 2 313 328 0.81


119

sequence region 110-125) and CotB protein has a stretch of serine residues (potential glycosylation sites) towards its C-terminal. The His- rich region of YeeK may be necessary for the overall protein stability [36] but in addition there are also other repeats in the N-terminal as well as the middle region of the protein and YeeK is speculated to play a role in survival in certain environmental conditions [37]. In B. cereus ATCC 14579, proteins BC_3582 and BC_3992, BC_4420 (SafA) contain glycine, proline and methionine repeat regions respectively while proteins BC_2149 and BC_5391 contain glutamine repeats. Also protein CD3522 from C. difficile is rich in Glutamic acid (13 residues) in the sequence region 226-238 of the protein. Repeats with complex patterns known as domain repeats are also observed in spore surface proteins. The best examples representing the domain repeats are YXY isodityrosine motifs [38] in proteins CotC and CotU from B. subtilis and the GXX repeats from the Bcl-family of exosporium proteins identified from B. cereus spores. Isodityrosine, though not yet identified in spores, has been identified from the plant cell wall glycoprotein and is said to play a role in protein cross-linking [39]. All these findings point towards the unconventional proteins sequence characteristics and thereby the structures of spore surface proteins. (b) Localization signals & Trans-membrane Helices (TMHs)

Amino acid sequences of proteins are also important for the protein sorting assemblies in cells. In prokaryotes and eukaryotes, exported proteins are usually synthesized as precursors with an amino-terminal signal peptide, which is recognized by a cellular sorting and translocation machinery and which guides a protein to its destination. Once the protein is delivered to its destination these short stretches of signal peptides are cleaved off by special signal peptidases (SPases) resulting in the release of mature protein from the protein translocation machinery. The signal peptides generally contain three distinct domains. The N-domain (amino terminal domain) of signal peptides, suggested to interact with the translocation machinery and the phospholipids in the lipid bilayer of the cell membrane, contains at least one arginine or lysine residue. The hydrophobic domain (H-domain), following the N-domain, is a stretch of hydrophobic residues that may form an α-helix in the membrane [40]. Glycine or proline residues that may act as helix-breakers are frequently present in the middle of this hydrophobic core. The following residues (C-domain) might allow the signal peptide to take up a hairpin-like structure in order to insert into the membrane. Signal peptides can be cleaved of by SPase type I or II, the latter being active in pre-lipoproteins that contain the lipobox 4 amino acid consensus sequence [(L/V/I)-(A/S/T/V/I)-(G/A/S)-C] [41]. The SPase-I cleaved pre-proteins can be transported by the regular Sec pathway of protein transport. Some of such SPaseI cleaved proteins may contain a so called twin arginine motif (RR-motif), and thus are transported via the Tat pathway. During sporulation the communication between the mother cell and the forespore requires protein transport. Previously two proteins SpoIIR and SpoIIG have been shown to be transported across the forespore to interact with the mother cell membrane [42, 43]. Since the coat and exosporium layers are synthesized inside the mother cell cytoplasm, these layers are assembled with most of the proteins being independent of a signaling peptide for their localization. However certain σG and/or

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σF regulated proteins from B. subtilis 168 require these signals for their incorporation in the spore coat as mentioned in Chapter 2. B. cereus ATCC 14579 and C. difficile 630 spore surface proteins also have some candidates that have signal sequences, as predicted by SignalP v. 4.1 tool and confirmed with Uniprot database and these are collectively shown in Table 2(A). As seen only a small fraction of proteins appear to depend on their transport by translocation pathways. However, it is also evident from the work of van Ooij and colleagues [44] that certain coat proteins firstly form rings that then encircle the entire spore. Also the work of Stelma (Jr.) and co-workers [45] has shown that coat formation, development of spore body and deposition of coat onto the spores could possibly be independent events. Therefore the details of the pathways of the spore coat assembly and the role of signal-peptides for the assembly of certain proteins (for instance those mentioned in Table 2(A)) into the coat still remain elusive. Table 2. (A) Proteins containing signal sequences. *Proteins with signal peptides containing a Twin-arginine motif (R-R-X-#-#). (B) Proteins containing transmembrane helices (TMHs).

(A) (B)

Gene Name Uniprot ID Protein Length (a.a.)

No. of TMHs

B. subtilis 168 YdcC P96619 338 1 AtcL (YloB) O34431 890 10 YodI O34654 83 1 YpeB P38490 450 1 YqfX P54481 129 2 B. cereus ATCC 14579 BC_0337 Q814A8 125 2 BC_1391 Q81G20 124 2 BC_1456 Q81FV9 142 1 BC_2099 Q81E89 112 2 BC_2382 (BclB)

Q81DI4 401 5

BC_2481 Q81D93 107 1 BC_2752 (YpeB)

Q813I5 446 1

BC_3582 Q81AI6 131 1 BC_3992 Q819I6 113 1 BC_4410 (YajC)

Q817W7 86 1

C. difficile 630 CD1063.1 Q18AR2 68 2 CD1463 Q18BY4 148 1 CD2434 Q182C1 200 1 CD2598 Q182T7 259 1 CD2808 Q183P2 209 1 CD3457 Q180V4 138 1

Gene Name Uniprot ID Residues in

signal peptide B. subtilis 168 YbfO O31455 1-28 YckD P42402 1-23 TcyA (YckK) P42199 1-19 YhcN P54598 1-20 OppA P24141 1-20 SleB P50739 1-29 DacF P38422 1-23 CoxA P94446 1-21 B. cereus ATCC 14579 BC_0212 Q81IY1 1-24 BC_0263* Q81IT6 1-22 BC_1279( Q81GC8 1-29 BC_1281 Q81GC6 1-16 BC_1284 (InA) Q81GC3 1-23 BC_1334 Q813U4 1-26 BC_2026 Q81EF3 1-28 BC_2375* Q81DJ0 1-24 BC_2481 Q81D93 1-33 BC_2753 (SleB) P0A3V0 1-34 BC_3586 Q81AI2 1-31 BC_4075 (DacF) Q819B1 1-23 BC_4419 (YhcN)

Q817V9 1-20

BC_5056 Q815S6 1-28 C. difficile 630 CD0855 (OppA) Q18A51 1-31 CD1291 (DacF) Q18BF4 1-23 CD1622 Q186H8 1-20


121

Along with the signalling sequences the transmembrane segments are also important to be considered for identification of possible transmembrane anchored proteins (Table 2 (B)). The transmembrane helical segments in membrane proteins are usually composed of hydrophobic amino acids [46]. In contrast, water soluble proteins are composed of both polar and apolar amino acids. In the trans-membrane helical (TMH) domains, there is a large variety of conserved sequences. Therefore, there are a significant number of transmembrane segments that potentially interact with the lipid bilayer and in many different modes [47]. Though there are very less transmembrane proteins identified from the spore integument region (region of the boundary between outer forespore membrane and the inner spore coat) some proteins like hydrolase, Ca2+ transporters and ATPases do contain trans-membrane domains. Protein SpoVM, mostly identified from the soluble coat protein fraction is also identified from the integument region and has been shown to responsible for anchoring the coat protein SpoIVA to the spore outer membrane [48]. Thus the proteins discussed in this table could either be important for germination or speculatively may serve as anchors for subsequent proteins layers. 2. Adhesive properties of spores

Bacterial adhesion to surfaces has been reasoned as a possible virulence factor for many pathogenic microorganisms that are important in the medical, pharmaceutical, and food industries. Many cell surface proteins from bacteria are known to be involved in adhesion processes [49]. Physical properties such as surface energy, texture, and relative charge distribution are the basis of adhesion of proteins to surfaces. Larger proteins are more likely to adhere and remain attached to a surface due to the higher number of contact points between amino acids and the surface. In addition, the role of hydrophobic interactions in bacterial cell adhesion to surfaces has previously been examined by using several methods, based on the precipitation of cells by salts, hydrophobic interaction chromatography (HIC [50]), and adherence to various liquid hydrocarbons including hexadecane (BATH test[51]). Separation using HIC is carried out due to the reversible interaction between a protein and the hydrophobic cells or ligands bound to the chromatography matrix whereas in the BATH test, the hydrophobic cells associate more to the hydrocarbon or the oil layer when cells are mixed into a hydrocarbon/ oil : water system. In both cases the estimate, of cells that are more hydrophobic than the rest of the cell population, can be interpreted in % values. For BATH assays, the % hydrophobicity is defined as the average % decrease in the A440 of the aqueous phases after partitioning and for HIC, it is defined as the average % decrease in the A440 of the spore suspensions eluded from duplicate Sepharose columns (Figure 2). Each of these methods has some limitations. The two-phase separation technique, id est the BATH test, may alter surface hydrophobicity [52] of B. subtilis and B. thuringiensis spores. For HIC, spore aggregation can be a problem as the clumps may be trapped in the matrix thereby interfering with the hydrophobicity measurements. Compared to the vegetative cells, spores are more adhesive in nature however their surface hydrophobicity has not been studied in good detail. Koshikawa and colleagues established a relationship between spore surface hydrophobicity and the presence of an exosporium layer in their study using the BATH

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assay where more than 70% of the spores from species that showed presence of an exosporium were found to associate with the hexadecane layer [53]. Later Wiencek and his group [54] showed that spores possessed significantly higher hydrophobic surfaces compared to the vegetative cells. The authors suggested that amongst the species considered for their study, spore form B. subtilis strain A possessed the least surface hydrophobicity when measured by the BATH assay (Figure 2).

The chemical and morphological characteristics of spores from different species may relate to the large variations in the hydrophobicity behaviours of spores. Large differences in the chemical composition of spore coats have been found in spores of Bacillus species [55]. We analysed the identified spore surface proteins for their hydropathy characteristics (hydrophilicity or hydrophobicity) and molecular masses. The GRAVY index indicates the solubility of the proteins. Therefore the identified protein set from the spore surface layers from three species was subjected to GRAVY (Grand Average of hydropathY) index analysis using the ProtParam tool from the ExPASy proteomics server (http://web.expasy.org/protparam/). The results are compiled in Figure 3. For the three organisms studied, all the proteins were < 100kDa in size, except BC_2639 (~520kDa). The mean size of identified pore surface layer proteins increases from 29 kDa for B. subtilis 168, 33 kDa for C. difficile 630 to 36 kDa for B. cereus ATCC 14579. Most of the identified proteins were relatively hydrophilic (negative GRAVY indices). The mean GRAVY-index was -0.28 for B. cereus ATCC 14579, -0.37 for C. difficile 630, and -0.61 for B. subtilis 168. However previous studies indicated that spore surfaces are hydrophobic and also the proteins were found to contain high number of hydrophobic amino acid residues (Figure 1) in their structure. It is plausible that the spore surface proteins form a cross-linked matrix in the coat and exosporium and along with the other reported components such as lipids, phosphorous, sugar moieties[56, 57] these layers become hydrophobic. To address the question of spore adhesion further, it is desirable to select a group of coat and exosporium proteins and study their role in spore adhesion e.g. by mutational studies. The small size of proteins suggests importance of quantitative studies in order to get the knowledge about the abundance and distribution of these proteins in the surface layers.

The maximum adhesiveness of spores due to surface hydrophobicity has been proposed to be at pH 3 [58]. Husmark & Rönner found that pH 3 correlated well with the isoelectric point of the spore surface and thus the differences in the number of adhered spores to surfaces was attributed to the differing charges of the spore surface. The differing charge will change the electrostatic attractions between the spores and the external surface as well as the steric stability of the adhesion will be altered. To gain insights into the role of spore surface proteins in the pH dependent adhesive properties we analyzed the proteins for their respective pI values. The hydrophilic proteins were spread over both the acidic and basic pH ranges. The mean pI of the identified B. cereus spore coat & exosporium proteome was 6.46, while the mean pI for the identified spore coat proteomes from C. difficile 630 and B. subtilis 168 were respectively 5.79 & 6.99. In general, the pI distribution of identified proteins was found to be unimodal for C. difficile 630, and bimodal for both the Bacillus spp. Based on a study performed by Schwartz and colleagues [59] in vegetative bacteria as well as eukaryotic cells, we speculate that this


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modality behavior may reflect the localization of proteins in the spore layers. Interestingly in all three organisms, the pI values for most of the outer coat, crust and exosporium proteins were in the range of 3 - 5 while some abundant proteins (CotG, CotB, ExsK, and BclC etc.) peculiarly possessed pI values in the range of 6 - 11. If these

Figure 2. Hydrophobicities of spores and vegetative cells of Bacillus and Clostridium spp. For BATH assays, the % hydrophobicity after partitioning with 0.1, 0.2, 0.6, and 1.0 ml of hexadecane. For HIC, the % hydrophobicity of the spore suspensions eluded from duplicate Sepharose columns. The data is adapted from Wiencek et al. [54] (n = 2 for all experiments). proteins are important for spore adhesion then according to the study performed by Husmark & Rönner, at pH 3 all these proteins will be charged and therefore the adhesiveness of spores at this pH will be decreased due to electrostatic repulsions between the charged spore surface and the charged stainless steel surface. Spore adhesion is a major problem in dairy industries. In a typical dairy industry the storage tanks, centrifuges, pasteurizers, heat exchangers, packaging machines and many other equipments used are made of principally two varieties of stainless steel - 304 and/or 316. According to a study performed by Gispert and others [60], the surface charge of 316 variety of stainless steel is near 0 at pH 6.0. Therefore in their study the protein bovine serum albumin (BSA) did not adhere to the stainless steel surface as much as it adhered to the alumina surface which is more positively charged. The authors speculated that the Ca2+ and Mg2+ ions present in the solvent, used in this study, may have bridged the negatively charged BSA surface and the stainless steel surface. Ca2+ and Mg2+ ions along with many other proteins are also present in the milk (pH ~7). At this pH, in case of B. subtilis many of the spore coat proteins will carry a negative charge leading to

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electrostatic repulsion with the stainless steel surface making the spores less adhesive however hydrophobic surfaces might still induce adhesion. In case of B. cereus spores, the components such as lipids and phospholipids impart hydrophobicity to the exosporium; sugars from glycoproteins tend to make exosporium sticky and bulky and proteins from the hair-like nap (e.g. BclC in B. cereus) will potentially carry a positive charge at pH 7 thus overcoming the electrostatic repulsion forces as predicted by Busscher & Weerkamp [61]. Thus B. cereus spores may adhere more strongly to the stainless steel surfaces compared to the B. subtilis spores. Therefore it is strongly recommended that the stainless steel surfaces should be replaced or treated to obtain more hydrophilic surfaces thereby minimizing the risk of spore adhesion.

3. Therapeutic applications of spores surface proteins

Since past the conventional means for identifying bioactive or antimicrobial peptides have been tedious and time consuming. Also from past few years emergence of antibiotic resistance amongst the bacterial domain has been a major problem and it still continues to threaten the medical field. Thus pharmaceutical industries are now in search of quick methods to identify and synthesize potent antimicrobial agents to control the bacterial infections. The field of bioinformatics has emerged as a supportive tool to solve this problem. Past few years have produced a lot of bioinformatics based prediction data for antibacterial compounds as well as for potential immunogenic behavior of proteins and peptides [62-66]. However all such prediction based methods strongly demand clinical trials and studies in animal models for their further use. As discussed in Chapter 1, spores and spore surface proteins can serve beneficial in many other ways. Spores have been used as drug vehicles, in probiotic treatments whereas spore coat proteins from B. subtilis have been used in surface display systems. For the identified set of proteins from the spore surface of three spore formers we analysed the potential peptides that can be used as antimicrobial agents as well as are immunogenic in behavior.

Anti-microbial peptides (AMPs)

Antimicrobial peptides (AMPs) are relatively short polypeptides (12-100 amino acids) that are positively charged (net charge of +2 to +9, most commonly +4 to +6) and amphiphilic. Such peptides may play a role in innate and adaptive immunity, including e.g. immuno-modulation, chemotaxis, inflammatory response, and wound repair [67] and therefore such peptides could have applications as therapeutics against biologically harmful agents. Usually the curative measures taken against bacterial or viral infections include vaccination, passive antibody therapy and antibiotic treatments. Antimicrobial peptides such as Nisin have been shown to affect the spore outgrowth [68] and thus can be used to control spore-mediated infections. In a similar way we ventured the antimicrobial potential of the set of peptides identified from the three spore forming organisms in our study. Using the AMPA automated web tool [69], A total of 63 peptides from 57 proteins (across three organisms) were identified to have 0-5% probability of being non-antimicrobial peptides id est a probability that the predicted stretch of amino


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Figure 3. Plot of molecular weight and pI versus GRAVY-score. The pI and molecular mass are plotted for the GRAVY-scores of the identified spore coat & exosporium proteins of B. cereus ATCC 14579, C. difficile 630, and B. subtilis 168 using IBM SPSS statistical tool v. 20. GRAVY-scores were calculated according to the values from Kyte and Doolittle [46].

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acid sequence is found by chance in a non-antimicrobial protein. The AMPA algorithm is based on the antimicrobial propensity scale obtained from high-throughput screening results from bactenecin 2A [70]. Of the 63 peptides, 20 peptides had probability of 5%, 14 had probability values of 0% i.e. highest chance of being an antimicrobial peptide stretch (Table 3) and remaining 29 peptides had probabilities in the range 1% - 4%. Notably, the AMPA server predicted 4% probability for the Nisin sequence stretch. Also the predicted average antimicrobial index for Nisin was 0. 227 and this value for all the peptides shown in Table 3 ranged from minimum of 0. 182 (for CotG) to maximum of 0.215 (for BclC).

Immunogenicity predictions for the identified peptides

We also predicted the immunogenic potential of the identified tryptic peptides using the automated server called POPI v. 2.0 [66]. Considering about 428 human MHC class I binding peptides belonging to four classes of immunogenicity (established by MHCPEP database) as the initial dataset and based on the propensities of amino acids occurring in these peptides with regards to 23 physicochemical properties such as secondary structure, molecular volume, codon diversity, electrostatic charge etc., POPI predicts the overall possibility of a peptide being immunogenic [66]. In Chapter 1 the results for B. subtilis are discussed. We extended the same analysis to the peptides identified from more pathogenic species i.e. B. cereus and C. difficile. Most of the peptides were predicted to have no or little immunogenic potential towards cytotoxic (CTLs) and helper (HTLs) T-cells. From B. cereus a single peptide - LDILGIVAEYGNVSR- from inosine-uridine preferring nucleoside hydrolase (IunH/BC_2889) showed a likelihood of being highly immunogenic to both CTLs and HTLs. Both organisms contained similar number of peptides with moderate to high immunogenic potential towards CTLs and HTLs (Table 4). Concluding remarks

The biological role of spores is well known and discussed in details in this thesis. Their impeccable resistance characters, their unique structure have been studied and described elsewhere in the literature. Protein structure prediction has become an important application for bioinformatics and the knowledge of the structure is important in understanding the function of a protein. In an attempt to characterize the identified spore surface proteins, in situ, for their potential roles in spore integrity a detailed bioinformatics analysis was undertaken. The results showed that spore surface proteins have peculiar sequence properties and therefore structures or surface topologies. There were also certain amino acid biases in the sequences of spore surface proteins. These amino acids could be very important in imparting the spores with such resistant outer layers. Dependence of only a small fraction of proteins on the protein translocation and


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Table 3. Prediction of anti-microbial peptides (AMPs) from the identified spore surface proteins.

Only the peptides with probability value of 0% are shown. Note: here probability is the misclassification probability value, id est the probability that the predicted stretch is found by chance in a non-antimicrobial protein. NA not available. Table 4. Prediction for the immunogenic potential of identified peptides.

Only the no. of peptides predicted to have moderate and high immunogenic potentials are shown. High (PD50 < 1 nM) and Moderate (PD50 = 100 nM - 1 nM). PD50 is the protective dose that protects 50% of the animals challenged.

Protein name Uniprot ID Residues Avg. anti- Predicted AMP sequence

From To microbial index

B. subtilis 168

YabQ P37559 186 210 0.197 KGAGFLKKKKKLLITIRTTITRFLK

YdhD O05495 407 419 0.205 GPWLLRKFFTIRK YhfD BG13050 44 66 NA PCEKKKKKHHCFSCKKHRHSCCH CoxA P94446 185 197 0.213 DKGLFRKLHKMNN

CotG P39801 32 166 0.182

KKRSHKKSHRTHKKSRSHKKSYCSHKKSRSHKKSFCSHKKSRSHKKSYCSHKKSRSHKKSYRSHKKSRSYKKSYRSYKKSRSYKKSCRSYKKSRSYKKSYCSHKKKSRSYKKSCRTHKKSYRSHKKYYKKPHHHC

B. cereus ATCC 14579

BC_2030 (cotG)

Q81EF0 38 125 0.189

HHCTTGCKCTTRGKCPRTRCTRVKHCTFVTKCTHVKKWTFVTKCTRVRVQKWTFVTKVTRRKECVLVTKRTRRKHCTFITKCIRFEKK

BC_2639 Q81CV2 4988 5009 0.212 GERFIRITPRNIRSYLWAWIWW BC_2753 (sleB)

P0A3V0 237 258 0.202 TATSKWIWTRPQIKKIGKHIFC

BC_3547 Q81AL6 923 940 0.206 VVNACFTRRGRSRTWIPI BC_3712 (bclC)

Q812Y5 805 817 0.215 TGNTNIRLTVFRI

BC_4387 Q817Y7 25 41 0.191 VFSKRNMKKARKMIKSY C. difficile 630 CD1581 Q186D6 141 159 0.192 GNNNNCCHKCHKCNCNCCR CD2399 Q181Y3 57 68 0.196 LYAKPKKYRGGR CD3170 (eno) Q181T5 416 429 0.212 QARYCGLKSFYNLK

No. of peptides (in %) showing immunogenic potential towards CTLs HTLs

Moderate High Moderate High B. subtilis 168 28.0 5.2 11.0 5.2 B. cereus ATCC 14579 19.7 9.0 13.0 6.5 C. difficile 630 17.0 6.5 10.0 5.6

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localization machinery in cells may make spore formation a relatively easy process. The study of the hydrophobicity patterns and pI distributions of the proteins is important to understand the surface character of spores as exemplified by the example of the dairy industry. Lastly, bioinformatic predictions of the bioactivity of peptides could lead to fast and relatively simple drug designing processes thereby aiding in the control of resistant pathogens.

Bioinformatics tools used in this study

Derivation of amino acid composition

The sequences of identified proteins were subjected to the Amino acid calculator (http://proteome.gs. washington.edu/cgi-bin/aa_calc.pl). The obtained numbers for each amino acid were converted to % manually and averaged over all the proteins from the organism. Identification of internal tandem repeats

The protein sequences were submitted to automated T-REKS server [34] http://bioinfo.montp.cnrs.fr/?r=t-reks/. Default parameters were used for repeat identification.

Localization & signal peptide estimation

The sequences of proteins were submitted to TMHMM server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) for identification of trans-membrane helices [71]. SignalP v. 4.1 (http://www.cbs.dtu.dk/services/SignalP/) tool[72] was used for prediction of signal sequences.

Prediction of hydropathy nature and pI

The hydropathy nature and pI values were predicted by using ProtParam tool http://web.expasy.org/protparam/ from the ExPASy proteomics server [73].

Identification of antimicrobial peptides.

An automated server AMPA (http://tcoffee.crg.cat/apps/ampa/do) [69] was used for prediction of antibacterial character of identified peptides. Default threshold parameters were used.

Immunogenicity predictions.

Automated server POPI v. 2.0 (http://iclab.life.nctu.edu.tw/POPI/) [66] was used for immunogenicity predictions of identified peptides.

References

1. Enguita FJ, Martins LO, Henriques AO, Carrondo MA. Crystal structure of a bacterial endospore coat component. A laccase with enhanced thermostability properties. The Journal of biological chemistry. 2003;278(21):19416-25. Epub 2003/03/15. 2. Scheeff ED, Axelrod HL, Miller MD, Chiu HJ, Deacon AM, Wilson IA, et al. Genomics, evolution, and crystal structure of a new family of bacterial spore kinases. Proteins. 2010;78(6):1470-82. Epub 2010/01/16. 3. Li Y, Setlow B, Setlow P, Hao B. Crystal structure of the GerBC component of a Bacillus subtilis spore germinant receptor. Journal of molecular biology. 2010;402(1):8-16. Epub 2010/07/27.


129

4. Ducros VM, Lewis RJ, Verma CS, Dodson EJ, Leonard G, Turkenburg JP, et al. Crystal structure of GerE, the ultimate transcriptional regulator of spore formation in Bacillus subtilis. Journal of molecular biology. 2001;306(4):759-71. Epub 2001/03/13. 5. Blair DE, van Aalten DM. Structures of Bacillus subtilis PdaA, a family 4 carbohydrate esterase, and a complex with N-acetyl-glucosamine. FEBS letters. 2004;570(1-3):13-9. Epub 2004/07/15. 6. Asen I, Djuranovic S, Lupas AN, Zeth K. Crystal structure of SpoVT, the final modulator of gene expression during spore development in Bacillus subtilis. Journal of molecular biology. 2009;386(4):962-75. Epub 2008/11/11. 7. Tarbouriech N, Charnock SJ, Davies GJ. Three-dimensional structures of the Mn and Mg dTDP complexes of the family GT-2 glycosyltransferase SpsA: a comparison with related NDP-sugar glycosyltransferases. Journal of molecular biology. 2001;314(4):655-61. Epub 2001/12/06. 8. Bielnicki J, Devedjiev Y, Derewenda U, Dauter Z, Joachimiak A, Derewenda ZS. B. subtilis ykuD protein at 2.0 A resolution: insights into the structure and function of a novel, ubiquitous family of bacterial enzymes. Proteins. 2006;62(1):144-51. Epub 2005/11/16. 9. Jing X, Robinson HR, Heffron JD, Popham DL, Schubot FD. The catalytic domain of the germination-specific lytic transglycosylase SleB from Bacillus anthracis displays a unique active site topology. Proteins. 2012;80(10):2469-75. Epub 2012/07/11. 10. Li Y, Jin K, Setlow B, Setlow P, Hao B. Crystal structure of the catalytic domain of the Bacillus cereus SleB protein, important in cortex peptidoglycan degradation during spore germination. Journal of bacteriology. 2012;194(17):4537-45. Epub 2012/06/26. 11. Au K, Ren J, Walter TS, Harlos K, Nettleship JE, Owens RJ, et al. Structures of an alanine racemase from Bacillus anthracis (BA0252) in the presence and absence of (R)-1-aminoethylphosphonic acid (L-Ala-P). Acta crystallographica Section F, Structural biology and crystallization communications. 2008;64(Pt 5):327-33. Epub 2008/05/06. 12. Réty S, Salamitou S, Garcia-Verdugo I, Hulmes DJ, Le Hegarat F, Chaby R, et al. The crystal structure of the Bacillus anthracis spore surface protein BclA shows remarkable similarity to mammalian proteins. The Journal of biological chemistry. 2005;280(52):43073-8. Epub 2005/10/27. 13. Adams CM, Eckenroth BE, Putnam EE, Doublie S, Shen A. Structural and functional analysis of the CspB protease required for Clostridium spore germination. PLoS pathogens. 2013;9(2):e1003165. Epub 2013/02/15. 14. Romero P, Obradovic Z, Li X, Garner EC, Brown CJ, Dunker AK. Sequence complexity of disordered protein. Proteins. 2001;42(1):38-48. Epub 2000/11/28. 15. Tompa PF, A. Structure and Function of Intrinsically Disordered Proteins. Boca Raton, Florida,US: Chapman and Hall/CRC Press; 2010. 359 p. 16. Abhyankar W, Beek AT, Dekker H, Kort R, Brul S, de Koster CG. Gel-free proteomic identification of the Bacillus subtilis insoluble spore coat protein fraction. Proteomics. 2011;11(23):4541-50. Epub 2011/09/10. 17. Henriques AO, Moran CP, Jr. Structure, assembly, and function of the spore surface layers. Annual review of microbiology. 2007;61:555-88. Epub 2007/11/24. 18. Abhyankar W, Hossain AH, Djajasaputra A, Permpoonpattana P, Ter Beek AS, Dekker HL, et al. In Pursuit of Protein Targets: Proteomic characterization of Bacterial Spore Outer Layers. Journal of Proteome Research. 2013. 19. Kobayashi K, Kumazawa Y, Miwa K, Yamanaka S. ε-(γ-Glutamyl)lysine cross-links of spore coat proteins and transglutaminase activity in Bacillus subtilis. FEMS Microbiology Letters. 1996;144(2-3):157-60. 20. Pandey NK, Aronson AI. Properties of the Bacillus subtilis spore coat. Journal of bacteriology. 1979;137(3):1208-18. Epub 1979/03/01. 21. Graciani NR, Tsang KY, McCutchen SL, Kelly JW. Amino acids that specify structure through hydrophobic clustering and histidine-aromatic interactions lead to biologically active peptidomimetics. Bioorganic & Medicinal Chemistry. 1994;2(9):999-1006. 22. Liao S-M, Du Q-S, Meng J-Z, Pang Z-W, Huang R-B. The multiple roles of histidine in protein interactions. Chemistry Central Journal. 2013;7(1):44.

Chapter 6

130

23. Munoz L, Sadaie Y, Doi RH. Spore coat protein of Bacillus subtilis. Structure and precursor synthesis. The Journal of biological chemistry. 1978;253(19):6694-701. Epub 1978/10/10. 24. Aronson AI, Fitz-James PC. Biosynthesis of bacterial spore coats. Journal of molecular biology. 1968;33(1):199-212. Epub 1968/04/14. 25. Noël CJ, Diaz N, Sicheritz-Ponten T, Safarikova L, Tachezy J, Tang P, et al. Trichomonas vaginalis vast BspA-like gene family: evidence for functional diversity from structural organisation and transcriptomics. BMC genomics. 2010;11:99. Epub 2010/02/11. 26. Murrell WG. Chemical composition of spores and spore structures. In: Gould GWH, A., editor. The Bacterial Spore. London: Academic Press; 1969. p. 215-74. 27. Gertsman I, Fu CY, Huang R, Komives EA, Johnson JE. Critical salt bridges guide capsid assembly, stability, and maturation behavior in bacteriophage HK97. Molecular & cellular proteomics : MCP. 2010;9(8):1752-63. Epub 2010/03/25. 28. Ma B, Elkayam T, Wolfson H, Nussinov R. Protein-protein interactions: structurally conserved residues distinguish between binding sites and exposed protein surfaces. Proceedings of the National Academy of Sciences of the United States of America. 2003;100(10):5772-7. Epub 2003/05/06. 29. Spires TL, Hannan AJ. Molecular mechanisms mediating pathological plasticity in Huntington's disease and Alzheimer's disease. Journal of neurochemistry. 2007;100(4):874-82. Epub 2007/01/16. 30. Subirana JA, Palau J. Structural features of single amino acid repeats in proteins. FEBS letters. 1999;448(1):1-3. Epub 1999/04/27. 31. Adindla S, Inampudi KK, Guruprasad K, Guruprasad L. Identification and Analysis of Novel Tandem Repeats in the Cell Surface Proteins of Archaeal and Bacterial Genomes Using Computational Tools. Comparative and Functional Genomics. 2004;5(1):2-16. 32. Adindla S, Inampudi KK, Guruprasad L. Cell surface proteins in archaeal and bacterial genomes comprising “LVIVD”, “RIVW” and “LGxL” tandem sequence repeats are predicted to fold as β-propeller. International Journal of Biological Macromolecules. 2007;41(4):454-68. 33. Goto Y, Carter D, Reed SG. Immunological Dominance of Trypanosoma cruzi Tandem Repeat Proteins. Infection and Immunity. 2008;76(9):3967-74. 34. Jorda J, Kajava AV. T-REKS: identification of Tandem REpeats in sequences with a K-meanS based algorithm. Bioinformatics. 2009;25(20):2632-8. Epub 2009/08/13. 35. Li H, Yang J, Wang Y, Chen Z, Tu S, Feng L, et al. Expression of a novel OSPGYRP (rice proline-, glycine- and tyrosine-rich protein) gene, which is involved in vesicle trafficking, enhanced cold tolerance in E. coli. Biotechnology letters. 2009;31(6):905-10. Epub 2009/02/12. 36. Sancho J, Serrano L, Fersht AR. Histidine residues at the N- and C-termini of .alpha.-helixes: perturbed pKas and protein stability. Biochemistry. 1992;31(8):2253-8. 37. Takamatsu H, Imamura D, Kuwana R, Watabe K. Expression of yeeK during Bacillus subtilis sporulation and localization of YeeK to the inner spore coat using fluorescence microscopy. Journal of bacteriology. 2009;191(4):1220-9. Epub 2008/12/09. 38. Cannon MC, Terneus K, Hall Q, Tan L, Wang Y, Wegenhart BL, et al. Self-assembly of the plant cell wall requires an extensin scaffold. Proceedings of the National Academy of Sciences. 2008;105(6):2226-31. 39. Fry SC. Isodityrosine, a new cross-linking amino acid from plant cell-wall glycoprotein. The Biochemical journal. 1982;204(2):449-55. Epub 1982/05/15. 40. Tjalsma H, Bolhuis A, Jongbloed JD, Bron S, van Dijl JM. Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiology and molecular biology reviews : MMBR. 2000;64(3):515-47. Epub 2000/09/07. 41. Babu MM, Priya ML, Selvan AT, Madera M, Gough J, Aravind L, et al. A Database of Bacterial Lipoproteins (DOLOP) with Functional Assignments to Predicted Lipoproteins. Journal of bacteriology. 2006;188(8):2761-73. 42. Hofmeister A. Activation of the proprotein transcription factor pro-sigmaE is associated with its progression through three patterns of subcellular localization during sporulation in Bacillus subtilis. Journal of bacteriology. 1998;180(9):2426-33. Epub 1998/05/09.


131

43. Hofmeister AE, Londono-Vallejo A, Harry E, Stragier P, Losick R. Extracellular signal protein triggering the proteolytic activation of a developmental transcription factor in B. subtilis. Cell. 1995;83(2):219-26. Epub 1995/10/20. 44. van Ooij C, Eichenberger P, Losick R. Dynamic Patterns of Subcellular Protein Localization during Spore Coat Morphogenesis in Bacillus subtilis. Journal of bacteriology. 2004;186(14):4441-8. 45. Stelma GN, Jr., Aronson AI, Fitz-James PC. A Bacillus cereus mutant defective in spore coat deposition. Journal of general microbiology. 1980;116(1):173-85. Epub 1980/01/01. 46. Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. Journal of molecular biology. 1982;157(1):105-32. Epub 1982/05/05. 47. Lemmon MA, Engelman DM. Specificity and promiscuity in membrane helix interactions. FEBS letters. 1994;346(1):17-20. Epub 1994/06/06. 48. de Francesco M, Jacobs JZ, Nunes F, Serrano M, McKenney PT, Chua M-H, et al. Physical Interaction between Coat Morphogenetic Proteins SpoVID and CotE Is Necessary for Spore Encasement in Bacillus subtilis. Journal of bacteriology. 2012;194(18):4941-50. 49. Takeichi M. Functional correlation between cell adhesive properties and some cell surface proteins. The Journal of cell biology. 1977;75(2 Pt 1):464-74. Epub 1977/11/01. 50. Doyle R, Nedjat-Haiem F, Singh J. Hydrophobic characteristics of Bacillus spores. Current Microbiology. 1984;10(6):329-32. 51. Rosenberg M. Bacterial adherence to hydrocarbons: a useful technique for studying cell surface hydrophobicity. FEMS Microbiology Letters. 1984;22(3):289-95. 52. Mozes N, Rouxhet PG. Methods for measuring hydrophobicity of microorganisms. Journal of Microbiological Methods. 1987;6(2):99-112. 53. Koshikawa T, Yamazaki M, Yoshimi M, Ogawa S, Yamada A, Watabe K, et al. Surface hydrophobicity of spores of Bacillus spp. Journal of general microbiology. 1989;135(10):2717-22. Epub 1989/10/01. 54. Wiencek KM, Klapes NA, Foegeding PM. Hydrophobicity of Bacillus and Clostridium spores. Applied and environmental microbiology. 1990;56(9):2600-5. Epub 1990/09/01. 55. Kondo M, Foster JW. Chemical and Electron Microscope Studies on Fractions Prepared From Coats of Bacillus Spores. Journal of general microbiology. 1967;47(2):257-71. 56. Matz LL, Beaman TC, Gerhardt P. Chemical Composition of Exosporium from Spores of Bacillus cereus. Journal of bacteriology. 1970;101(1):196-201. 57. Warth AD, Ohye DF, Murrell WG. The composition and structure of bacterial spores. The Journal of cell biology. 1963;16:579-92. Epub 1963/03/01. 58. Husmark U, Rönner U. Forces involved in adhesion of Bacillus cereus spores to solid surfaces under different environmental conditions. The Journal of applied bacteriology. 1990;69(4):557-62. Epub 1990/10/01. 59. Schwartz R, Ting CS, King J. Whole proteome pI values correlate with subcellular localizations of proteins for organisms within the three domains of life. Genome research. 2001;11(5):703-9. Epub 2001/05/05. 60. Gispert MP, Serro AP, Colaço R, Saramago B. Bovine serum albumin adsorption onto 316L stainless steel and alumina: a comparative study using depletion, protein radiolabeling, quartz crystal microbalance and atomic force microscopy. Surface and Interface Analysis. 2008;40(12):1529-37. 61. Busscher HJ, Weerkamp AH. Specific and non-specific interactions in bacterial adhesion to solid substrata. FEMS Microbiology Letters. 1987;46(2):165-73. 62. Jacob L, Vert JP. Efficient peptide-MHC-I binding prediction for alleles with few known binders. Bioinformatics. 2008;24(3):358-66. Epub 2007/12/18. 63. Khalidi K. Bioinformatics approaches for identifying new therapeutic bioactive peptides in food. The Journal of Functional Foods in Health and Disease. 2012;2(10):325-38. 64. Mooney C, Haslam NJ, Holton TA, Pollastri G, Shields DC. PeptideLocator: prediction of bioactive peptides in protein sequences. Bioinformatics. 2013;29(9):1120-6. Epub 2013/03/19. 65. Torrent M, Andreu D, Nogués VM, Boix E. Connecting Peptide Physicochemical and Antimicrobial Properties by a Rational Prediction Model. PLoS ONE. 2011;6(2):e16968.

Chapter 6

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66. Tung C-W, Ho S-Y. POPI: predicting immunogenicity of MHC class I binding peptides by mining informative physicochemical properties. Bioinformatics. 2007;23(8):942-9. 67. Seebah S, Suresh A, Zhuo S, Choong YH, Chua H, Chuon D, et al. Defensins knowledgebase: a manually curated database and information source focused on the defensins family of antimicrobial peptides. Nucleic acids research. 2007;35(Database issue):D265-8. Epub 2006/11/09. 68. Gut IM, Blanke SR, van der Donk WA. Mechanism of inhibition of Bacillus anthracis spore outgrowth by the lantibiotic nisin. ACS chemical biology. 2011;6(7):744-52. Epub 2011/04/27. 69. Torrent M, Di Tommaso P, Pulido D, Victòria Nogués M, Notredame C, Boix E, et al. AMPA: An automated web server for prediction of protein antimicrobial regions. Bioinformatics. 2011. 70. Torrent M, Nogués V, Boix E. A theoretical approach to spot active regions in antimicrobial proteins. BMC Bioinformatics. 2009;10(1):1-9. 71. Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. Journal of molecular biology. 2001;305(3):567-80. Epub 2001/01/12. 72. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature methods. 2011;8(10):785-6. Epub 2011/10/01. 73. Gasteiger EH, C.; Gattiker, A.; Duvaud, S.;Wilkins, M.R.; Appel, R.D.; Bairoch, A. Protein Identification and Analysis Tools on the ExPASy Server. In: Walker JM, editor. The Proteomics Protocols Handbook: Humana Press; 2005. p. 571-607.


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134

7 General Discussion & Outlook

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136

Throughout this thesis, the sub-proteomes of the surface layers of the bacterial spores have been analysed and discussed extensively. Beginning with the development of a gel-free method (Chapter 2) that focussed on the insoluble fraction of the spore coat protein from B. subtilis, this focus was broadened from the laboratory strain and a heat resistant food-isolate to the sub-proteomes of the spore coat and exosporium layers of two important pathogenic species - Bacillus cereus, a Gram positive, facultatively anaerobic organism and Clostridium difficile, a Gram positive, anaerobic organism (Chapter 3). These studies revealed that spore coat protein isolation and identification has not yet reached the ultimate and many more proteins can still be identified with the aid of newly developed technology provided precise and meticulous experimentation is done. Also the study on B. cereus and C. difficile strain shed light on the absolute requirement of proper and accurate genome annotations. Much of the identified B. cereus and C. difficile proteins have not been studied for their functional roles thereby opening the door for, yet unknown, possible mechanisms of spore survival and integrity. In order to examine the possible interdependence of spore coat and exosporium proteins, some mutants in spore coat proteins of B. subtilis were also analysed. This study (Chapter 4), in agreement with the previous literature, reveals that the deletion of a single coat protein gene, with the exception of morphogenetic protein coding genes, does not significantly change the spore coat architecture. As seen in our study, in case of the cotE mutant the outer coat proteins are considerably reduced in amounts when compared to the wild-type strain. Such effects are not apparent in any other mutants analysed. Inter-protein cross-linking, spore maturation and spore resistance to thermal stress are proposed to be linked together and therefore we also have probed the progress in spore coat protein cross-linking using 15N-labeled mature spores as a reference (Chapter 5). This study, for the first time, reveals a set of proteins that are really affected during spore maturation with regards to their protease resistance. These proteins are all said to be involved in protein cross-linking thus providing a strong evidence for the role of protein cross-linking in spore’s resistance towards thermal stress. During all the studies that shaped this thesis, there are certain molecular properties of the spore coat and exosporium proteins that become evident from our investigations. In Chapter 6 such molecular properties are discussed by taking spore structure, resistance properties and potential applications of spores into consideration. This bioinformatics-based study made some hypothesis regarding the protein pI, surface charge of spores, the inherent protein sequences and repeats. These hypotheses need to be proved by further studies. Though the comprehensiveness of our research has been demonstrated through this thesis there are certain observations that can be further acted upon in the future. These points are discussed below.

General Discussion and Outlook

137

1. Study of cross-linked proteins in the bacterial spore coat

(a) Protein digestion

As mentioned in Chapter 6, the coat proteins are very peculiarly structured and functionally oriented. Though we developed a method for characterizing the insoluble fraction, the nature and abundance of cross-links has not been studied in detail. Also the stoichiometry of coat proteins involved in cross-linking needs to be focussed upon further. For the complete characterization of the insoluble fraction it is desirable to first achieve complete or maximum digestion of the insoluble fraction. The method of acid hydrolysis has been used previously by researchers to digest Bacillus spore proteins [1-3]. Unfortunately, these studies were limited only up to the estimation of amino acid compositions of these proteins and did not venture the possibility of identifying the cross-links. The chemical cleavage with acid provides an effective alternative to enzymatic digestion but also leads to formation of unspecific cleavage products post-digestion [4, 5]. Heijnis et al. [6] have shown the use of trypsin and glu-C treatments for the identification of di-tyrosine crosslinks from α-lactalbumin. In the work presented in this thesis we have used trypsin to achieve the protein digestion but use of such multiple protein digestion steps will allow the study of cross-linked proteins. (b) Identification of cross-linked peptides

The insoluble fraction of spore coat proteins is said to comprise of three types of cross-links: di-tyrosine cross-links, the glutamyl-lysine cross-links and the disulfide linkages (Figure 1). Our bioinformatic analysis of amino acid compositions of spore surface proteins has shown that these proteins are, in general, rich in amino acids tyrosine, glutamic acid, glutamine, aspartic acid, asparagine, lysine and cysteine. All these amino residues reflect the possibilities of the presence of the above mentioned cross-links amongst the spore coat proteins. In a previous study [2] the authors have suggested the presence of di-tyrosine residues amongst the coat proteins by studying the emission spectra of di-tyrosine (synthesized in vitro) along with the spore coat samples that were subjected to performic acid oxidation prior to the analysis. A shoulder in the region 390 - 400 nm, observed in the emission spectrum, was attributed to di-tyrosine and ~3 di-tyrosine moieties were predicted per 60000 Da of amino residues. However, the authors also have suggested that the increase in the di-tyrosine content of oxidized coat material might be due to the earlier performic acid oxidation. These authors also made use of acid hydrolysis in their study. Therefore, using a similar approach along with the spectroscopic methods the di-tyrosine estimation is possible. Di-tyrosine moiety has an absorption maximum centered around 318 nm [7] therefore the use of fluorescence spectroscopy is recommended for more precise determinations. It is also possible to detect di-tyrosine by performing amino acid analysis with or without further mass spectrometric analysis. In such experiments though, a chromatographic separation of the peptide material is needed. Alternative approaches that can be used involve use of antibodies against di- tyrosine [8], thin-layer chromatography

Chapter 7

138

Figure 1. The types of cross-links predicted to comprise the spore coat insoluble fraction.

(TLC)-based as well as HPLC-based methods [9] and use of mass spectrometric quantification of di-tyrosine based on gas chromatography mass spectrometry (GC-MS), selected ion monitoring (SIM)-GC-MS as well as LC-ESI-MS/MS methods [10]. These approaches are rather costly, and require extensive sample preparation. Moreover, each of these methods has its own pitfalls which are comprehensively reviewed in the literature [10] and all the above methods are only applicable for preparative-scale experiments, to limited quantities of biological samples and can separate di-tyrosine from other hydrolyzed amino acids and are not applicable to a heterogeneous and complex protein sample such as the spore coat. The second type of linkages id est isopeptide linkages have been identified recently in the exosporium proteins [11]. With the use of mass spectrometry identification and quantification of isopeptide linkages has also been achieved [12, 13]. An MS-based method that can be a candidate to identify the cross-linked peptides from spore coat samples is the use of 18O-labelled water. This strategy facilitates cross-link analysis by providing peptides with a tag that enables distinction in a mass spectrum of signals belonging to linear and cross-linked proteins. Cross-linked peptides can be distinguished in mass spectra by a characteristic shift of 8 amu compared to the 16O-labelled ones. Cross-linked peptides contain two carboxy-termini and thus will show a shift of 8 amu when digested in H2

18O due to the incorporation of two 18O atoms in each C-terminus post-digestion. Therefore, normal linear peptides are characterized in a mass spectrum by a 4 amu shift, while cross-linked peptides digested in H2

18O will shift 8 amu [14]. Identification of the so-detected cross-linked peptide is then achieved through


139

interpretation of the MS/MS data of these cross-linked peptides. It needs to be investigated if this isotopic labeling method can be applied to insoluble cross-linked spore coat proteins. (c) Additional oxidative coupling products of tyrosine residues

In several cases, di-tyrosine has been shown to be present in the structural proteins [15, 16]. The presence of YXY isodityrosine motifs [17] in the coat proteins as mentioned in Chapter 6 and richness of certain coat proteins in amino residues proline, glycine and tyrosine raises a question if additional oxidative tyrosine coupling takes place. A range of additional oxidative coupling is shown in Figure 2. Isodityrosine, trityrosine, pulcherosine have all been identified to be present in structural protein components in various organisms. Isodityrosine has been observed in the cell wall glycoproteins of tomato [18], pulcherosine has been identified from plant cell wall proteins [19], trityrosine from the protein resilin in arthropods [20] and in the fertilization envelops of sea urchin [21], isotrityrosine from the second-molt cuticle of Haemonchus contortus infective larvae of sheep [22]. Although the distribution of amino acids in the spore coat proteins suggests that these cross-links are likely to be present also in and between the spore coat proteins they have not been identified yet. Therefore there is a need to focus on identification of additional oxidative coupling. As mentioned previously, fluorescence

Figure 2. Additional oxidative coupling products of tyrosine residues. Figure is adapted from the work of Braddy et al. [19] Tyr = tyrosine; Idt = isodityrosine

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140

spectroscopy is a powerful tool to distinguish tyrosine moieties from di-tyrosine. However this method is not suitable to differentiate between the above mentioned oxidation products as all these products fall in the same range of excitation and emission maxima (Table 1). More efforts to develop new methodologies are required in order to distinguish and further characterize these crosslinks.

Table 1. Fluorescence spectroscopic properties of Tyrosine-derived cross-Links.

Data adapted from Nomura et al. [21]. NA = not available.

II. Salt-bridge linkages between the spore coat proteins

The MS analyses of the tryptic digests of the spore coat described in this thesis have shown very frequent occurrence of peptides with a glutamic acid (E) at the C-terminus. These peptides cannot be formed by proteolytic cleavage using trypsin or chymotrypsin. Cleavage of the glutamine acid peptide bond can be realized using Glu-C protease or in strongly acidic media at elevated temperatures and will cleave the protein both before and after glutamic acid. This does not explain the observed specific protein cleavage after glutamic acid. Glutamic (E) and aspartic (D) acid as well as the basis amino acid lysine (K) appear to be overpopulated in many coat proteins as shown in Chapter 6. This suggests that these residues are involved in the assembly of the spore coat. Maturation of the spore coat is associated with the formation of cross-links in the outer layer and crust as described in Chapter 5. The hypothesis is that during initial maturation the dityrosine and ε-(γ)-glutamyl-lysine link are formed. As the number of cross-links grows, the water is squeezed out between the proteins. In water free environment the acidic residues D and E will transfer a protein to the basic lysine (K) residue resulting in an ionic bond shown in Figure 3. The large number of E, D and K residues in the outer layer proteins may result in many ionic bonds between the proteins which will contribute significantly to the assembly and stability of the spore coat. However, during the processing of the coat for MS analysis the bead beating event induces heat and mechanical stress. This stress may drive a reaction which cleaves the peptide bond after glutamic acid as depicted in Figure 3 (A). The cleavage is activated by the ionic bond which facilitates the negatively charged carboxyl oxygen to undergo a BAC2 reaction with the peptide carbonyl via an energetically favorable 6-member ring transition state. The resulting cleavage of the peptide bond after the glutamic acid residue yield a cyclic anhydride, which under the

Tyrosine derived oxidative coupling products

Fluorescence Excitation- Emission maxima (nm) pH 2.0 pH 12.0

Pulcherosine (natural) 281 - 420 316 – 420 Pulcherosine (synthetic) 281 - 420 318 – 422 Di-tyrosine (synthetic) 283 - 410 3 15 – 422 Trityrosine (synthetic) 288 - 412 323 – 409 Isodityrosine NA NA Isotrityrosine NA NA


141

bead beating aqueous conditions will by hydrolyzed to C-terminal glutamic acid. This explains the occurrence of many glutamic acid C-terminal peptides after tryptic digestion of the coat proteins. Likewise, also aspartic acid (D) can form an ionic bond with the basic lysine residue. Yet, no aspartic acid C-terminal peptides have been identified after tryptic digestion of the coat proteins. This is consistent with the expected higher activation energy for the BAC2 reaction with the peptide carbonyl which for the aspartic acid residue proceeds via an energetically unfavorable 5-membered ring transition state as depicted in Figure 3 (B). Amino acid sequences of coat proteins such as CotC, CotU, CotT suggest a lack of ordered secondary structure. Thus these proteins may function as molecular glue by sealing the gaps in the coat via cross-links and ionic bond formation (Figure 4). These molecular glues may mimic a zipper structure and this zip can be opened once the water enters the spore upon initiation of germination which breaks the ionic bond, leading further to activation of cortex degrading enzymes. The supra-molecular-structure based on above mentioned hypothesis is shown in Figure 4.

Figure 3. Cleavage of the peptide bond after (A) glutamic acid and (B) aspartic acid activated by the ionic bond between carboxylic group and amino group of lysine.

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142

Figure 4. Model of the supra-molecular structure of the spore coat. The neighbouring coat proteins (dark blue, green, red) are stabilized by di-tyrosine, glutamyl-lysine and ionic bonds. Small proteins, such as CotC, can act as molecules glues (faint blue) in the coat.

Outlook

The studies presented in this thesis have led to the identification of many putative proteins from the insoluble coat fraction. For the most interesting protein candidates based on criteria such as amino acid sequence, secondary structure, size as well as dependency on the known morphogenetic proteins, their localization and function can be analysed. To achieve this, the use of GFP-tags and point mutations in the protein sequences, respectively, has been proven successful [11, 23] In Chapter 5 the role has been discussed of a subset of coat proteins that are found to participate in spore maturation by assembly of cross-links. The progress of this cross-linking is shown to be coupled with the increase in wet-heat resistance. It will be interesting to monitor this set of cross-linking coat proteins during spore germination. A recent ICAT-based quantitative proteomics study [24] of B. anthracis has already shown that the protein levels of CotJA, CotJB, CotJC, CoxA etc. are decreased during spore germination. The15N-metabolic labeling method discussed in Chapter 5, can be used to monitor the digestion efficiency and hence the density of cross-links during germination. For this, a short pulse of germinants can be given to the spores followed by quenching the germination by addition germination inhibitor such as phenyl methyl sulfonyl fluoride (PMSF [24]). In conclusion, the bio-information on the proteome of spore coats of food spoiling bacteria obtained with developed proteomic methods described in this thesis has provided novel insights in the molecular structure and assembly of spore coats and offers new opportunities for further studies to unravel the mysteries of bacterial sporulation and germination.


143

References

1. Carroll AM, Plomp M, Malkin AJ, Setlow P. Protozoal digestion of coat-defective Bacillus subtilis spores produces "rinds" composed of insoluble coat protein. Applied and environmental microbiology. 2008;74(19):5875-81. Epub 2008/08/12. 2. Pandey NK, Aronson AI. Properties of the Bacillus subtilis spore coat. Journal of bacteriology. 1979;137(3):1208-18. Epub 1979/03/01. 3. Swatkoski S, Russell SC, Edwards N, Fenselau C. Rapid Chemical Digestion of Small Acid-Soluble Spore Proteins for Analysis of Bacillus Spores. Analytical Chemistry. 2005;78(1):181-8. 4. Li A, Sowder RC, Henderson LE, Moore SP, Garfinkel DJ, Fisher RJ. Chemical Cleavage at Aspartyl Residues for Protein Identification. Analytical Chemistry. 2001;73(22):5395-402. 5. Pickering MV, Newton P. Amino acid hydrolysis: old problems, new solutions. LC-GC. 1980;8(10). 6. Heijnis WH, Dekker HL, de Koning LJ, Wierenga PA, Westphal AH, de Koster CG, et al. Identification of the Peroxidase-Generated Intermolecular Dityrosine Cross-Link in Bovine α-Lactalbumin. Journal of Agricultural and Food Chemistry. 2010;59(1):444-9. 7. Michon T, Wang W, Ferrasson E, Gueguen J. Wheat prolamine crosslinking through dityrosine formation catalyzed by peroxidases: improvement in the modification of a poorly accessible substrate by "indirect" catalysis. Biotechnology and bioengineering. 1999;63(4):449-58. Epub 1999/04/01. 8. Fukuchi Y, Miura Y, Nabeno Y, Kato Y, Osawa T, Naito M. Immunohistochemical detection of oxidative stress biomarkers, dityrosine and N(epsilon)-(hexanoyl)lysine, and C-reactive protein in rabbit atherosclerotic lesions. Journal of atherosclerosis and thrombosis. 2008;15(4):185-92. Epub 2008/09/09. 9. Malencik DA, Anderson SR. Dityrosine as a product of oxidative stress and fluorescent probe. Amino acids. 2003;25(3-4):233-47. Epub 2003/12/09. 10. DiMarco T, Giulivi C. Current analytical methods for the detection of dityrosine, a biomarker of oxidative stress, in biological samples. Mass spectrometry reviews. 2007;26(1):108-20. Epub 2006/10/05. 11. Tan L, Li M, Turnbough CL, Jr. An unusual mechanism of isopeptide bond formation attaches the collagenlike glycoprotein BclA to the exosporium of Bacillus anthracis. mBio. 2011;2(3):e00084-11. Epub 2011/06/02. 12. Kang HJ, Middleditch M, Proft T, Baker EN. Isopeptide bonds in bacterial pili and their characterization by X-ray crystallography and mass spectrometry. Biopolymers. 2009;91(12):1126-34. Epub 2009/02/20. 13. Schäfer C, Schott M, Brandl F, Neidhart S, Carle R. Identification and quantification of epsilon-(gamma-glutamyl)lysine in digests of enzymatically cross-linked leguminous proteins by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). J Agric Food Chem. 2005;53(8):2830-7. Epub 2005/04/14. 14. Back JW, Notenboom V, de Koning LJ, Muijsers AO, Sixma TK, de Koster CG, et al. Identification of Cross-Linked Peptides for Protein Interaction Studies Using Mass Spectrometry and 18O Labeling. Analytical Chemistry. 2002;74(17):4417-22. 15. Garcia-Castineiras S, Dillon J, Spector A. Detection of bityrosine in cataractous human lens protein. Science. 1978;199(4331):897-9. Epub 1978/02/24. 16. Waykole P, Heidemann E. Dityrosine in collagen. Connective tissue research. 1976;4(4):219-22. Epub 1976/01/01. 17. Cannon MC, Terneus K, Hall Q, Tan L, Wang Y, Wegenhart BL, et al. Self-assembly of the plant cell wall requires an extensin scaffold. Proceedings of the National Academy of Sciences. 2008;105(6):2226-31. 18. Brady JD, Fry SC. Formation of Di-Isodityrosine and Loss of Isodityrosine in the Cell Walls of Tomato Cell-Suspension Cultures Treated with Fungal Elicitors or H2O2. Plant physiology. 1997;115(1):87-92. Epub 2002/09/12. 19. Brady JD, Sadler IH, Fry SC. Pulcherosine, an oxidatively coupled trimer of tyrosine in plant cell walls: Its role in cross-link formation. Phytochemistry. 1998;47(3):349-53. 20. Andersen SO. The Cross-Links in Resilin Identified as Dityrosine and Trityrosine. Biochimica et biophysica acta. 1964;93:213-5. Epub 1964/10/09.

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21. Nomura K, Suzuki N, Matsumoto S. Pulcherosine, a novel tyrosine-derived, trivalent cross-linking amino acid from the fertilization envelope of sea urchin embryo. Biochemistry. 1990;29(19):4525-34. Epub 1990/05/15. 22. Fetterer RH, Rhoads ML. Tyrosine-derived cross-linking amino acids in the sheath of Haemonchus contortus infective larvae. The Journal of parasitology. 1990;76(5):619-24. Epub 1990/10/01. 23. Little S, Driks A. Functional analysis of the Bacillus subtilis morphogenetic spore coat protein CotE. Molecular microbiology. 2001;42(4):1107-20. Epub 2001/12/12. 24. Jagtap P, Michailidis G, Zielke R, Walker AK, Patel N, Strahler JR, et al. Early events of Bacillus anthracis germination identified by time-course quantitative proteomics. Proteomics. 2006;6(19):5199-211. Epub 2006/08/24.

Appendix

Appendix

App

endi

x

146

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I: P

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Unc

hara

cter

ized

pro

tein

Yck

D

1278

2 68

3

22

1

59

2

ydcC

Sp

orul

atio

n pr

otei

n Y

dcC

38

170

654

11

App

endi

x

148

G

ene

nam

e Pr

otei

n M

ass

(Da)

Wild

type

R

eplic

ate

1 R

eplic

ate

2 R

eplic

ate

3 M

ASC

OT

Scor

e #

pept

ide

MS/

MS

spec

tra

MA

SCO

T Sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T Sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T Sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a yd

hD

Puta

tive

spor

ulat

ion-

spec

ific

glyc

osyl

ase

Ydh

D

4683

5 86

2

276

8 18

2 4

435

11

yfkD

U

ncha

ract

eriz

ed p

rote

in Y

fkD

29

569

235

5

yf

kO

Puta

tive

NA

D(P

)H n

itror

educ

tase

Y

fkO

25

669

103

2

51

1

ygaK

U

ncha

ract

eriz

ed F

AD

-link

ed

oxid

ored

ucta

se Y

gaK

51

039

251

7 57

0 16

16

7 5

609

17

yhbB

U

ncha

ract

eriz

ed p

rote

in Y

hbB

36

095

73

1

yh

cB

Unc

hara

cter

ized

pro

tein

Yhc

B

1917

4 14

0 3

65

2 yh

cM

Unc

hara

cter

ized

pro

tein

Yhc

M

1702

0 63

1

91

2 yh

cN

Lipo

prot

ein

Yhc

N

2106

1 14

9 3

368

9

24

3 7

yhcQ

Sp

ore

coat

pro

tein

F-li

ke p

rote

in

Yhc

Q

2494

6 47

4 15

37

3 8

111

3 72

4 20

yhfW

Pu

tativ

e Ri

eske

2Fe

-2S

iron-

sulfu

r pr

otei

n Y

hfW

57

587

113

2

yhxC

U

ncha

ract

eriz

ed o

xido

redu

ctas

e Y

hxC

30

940

351

11

124

3 51

1

163

4 yi

sY

AB

hydr

olas

e su

perf

amily

pro

tein

Y

isY

30

540

44

1 11

7 3

107

4

yjdH

U

ncha

ract

eriz

ed p

rote

in Y

jdH

15

249

215

4 36

5 9

258

7 49

6 11

yj

qC

Unc

hara

cter

ized

pro

tein

Yjq

C

3140

3 28

1 6

453

11

187

4 50

0 13

yk

aA

UPF

0111

pro

tein

Yka

A

2400

0 38

1

ykuD

Pu

tativ

e L,

D-tr

ansp

eptid

ase

Yku

D

1768

9 73

1

ykuJ

U

ncha

ract

eriz

ed p

rote

in Y

kuJ

9296

59

1

ykuS

U

PF01

80 p

rote

in Y

kuS

87

16

ykuU

Th

iore

doxi

n-lik

e pr

otei

n Y

kuU

20

548

58

1

yl

oB

Cal

cium

-tran

spor

ting

ATP

ase

97

516

449

12

ymfF

Pr

obab

le in

activ

e m

etal

lopr

otea

se

4823

1 54

2

App

endi

x

149

Gen

e na

me

Prot

ein

Mas

s (D

a)

Wild

type

R

eplic

ate

1 R

eplic

ate

2 R

eplic

ate

3 M

ASC

OT

Scor

e #

pept

ide

MS/

MS

spec

tra

MA

SCO

T Sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T Sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T Sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a Y

mfF

ym

fH

Unc

hara

cter

ized

zin

c pr

otea

se Y

mfH

49

089

38

2

52

1

ymxG

U

ncha

ract

eriz

ed z

inc

prot

ease

Ym

xG

4613

7 65

3

67

2 yn

eT

Unc

hara

cter

ized

pro

tein

Yne

T

1503

7 61

1

ynzH

U

ncha

ract

eriz

ed p

rote

in Y

nzH

11

612

319

6 84

3

51

1 26

8 8

yodI

U

ncha

ract

eriz

ed p

rote

in Y

odI

9245

15

4 6

392

19

164

8 32

8 20

yp

eB

Spor

ulat

ion

prot

ein

Ype

B

5121

0 37

4 8

83

2

23

0 6

yqfA

U

PF03

65 p

rote

in Y

qfA

35

676

435

10

80

2 yq

fX

Unc

hara

cter

ized

pro

tein

Yqf

X

1389

3 33

1 4

yqgO

U

ncha

ract

eriz

ed p

rote

in Y

qgO

69

07

62

1 54

2

103

2 yq

iG

Prob

able

NA

DH

-dep

ende

nt fl

avin

ox

idor

educ

tase

Yqi

G

4089

4 64

1

38

1

yrkC

U

ncha

ract

eriz

ed p

rote

in Y

rkC

21

299

140

2

yr

zQ

Unc

hara

cter

ized

pro

tein

Yrz

Q

5039

84

2

118

2 ys

dC

Puta

tive

amin

opep

tidas

e Y

sdC

39

249

75

2 yt

fJ

Unc

hara

cter

ized

spor

e pr

otei

n Y

tfJ

1633

6 16

6 4

136

4 11

2 3

257

7 yu

rS

Unc

hara

cter

ized

pro

tein

Yur

S

1048

3 64

2

50

1

yu

rZ

Unc

hara

cter

ized

pro

tein

Yur

Z

1389

8

36

1

yuzA

U

ncha

ract

eriz

ed m

embr

ane

prot

ein

Yuz

A

8518

yvdP

U

ncha

ract

eriz

ed F

AD

-link

ed

oxid

ored

ucta

se Y

vdP

50

167

562

15

546

19

285

7 88

0 27

ywfI

U

PF04

47 p

rote

in Y

wfI

29

543

38

1 yx

eD

Unc

hara

cter

ized

pro

tein

Yxe

D

1379

3 62

1

yxeE

U

ncha

ract

eriz

ed p

rote

in Y

xeE

14

705

76

1 48

6 13

50

4 12

80

5 17

yy

xA

Unc

hara

cter

ized

serin

e pr

otea

se Y

yxA

42

762

App

endi

x

150

T

able

II: P

rote

ins i

dent

ified

from

Δtg

l spo

re c

oats

of B

acill

us su

btili

s.

Gen

e na

me

Prot

ein

Mas

s (D

a)

Wild

type

R

eplic

ate

1 R

eplic

ate

2 R

eplic

ate

3

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a cg

eA

Prot

ein

CgeA

14

149

64

2 co

tA

Spor

e co

at p

rote

in A

58

462

770

29

944

28

1341

36

13

52

35

cotB

Sp

ore

coat

pro

tein

B

4294

6 57

3 29

98

3 32

16

62

35

1210

32

co

tC

Spor

e co

at p

rote

in C

88

11

340

18

382

16

300

10

530

18

cotE

Sp

ore

coat

pro

tein

E

2096

4 10

57

56

1687

58

16

53

38

2045

45

co

tF

Spor

e co

at p

rote

in F

18

714

694

20

416

12

715

14

587

14

cotG

Sp

ore

coat

pro

tein

G

2394

2 42

6 24

19

8 11

21

7 10

19

9 10

co

tI

Spor

e co

at p

rote

in I

41

219

118

7 12

7 8

354

10

348

12

cotJ

A

Prot

ein

Cot

JA

9733

64

4

33

1 18

3 4

103

3 co

tJC

Pr

otei

n C

otJC

21

682

160

12

49

2 40

3 7

186

4 co

tR

Puta

tive

spor

ulat

ion

hydr

olas

e C

otR

35

335

122

6 14

2 6

470

10

145

4 co

tS

Spor

e co

at p

rote

in S

41

058

294

12

516

16

398

13

537

19

cotS

A

Spor

e co

at p

rote

in S

A

4288

5 43

3 23

25

7 10

36

0 13

38

0 13

co

tT

Spor

e co

at p

rote

in T

10

125

162

3 co

tW

Spor

e co

at p

rote

in W

12

329

53

1 co

tX

Spor

e co

at p

rote

in X

18

589

966

32

536

17

846

28

821

22

cotY

Sp

ore

coat

pro

tein

Y

1787

2 14

21

44

513

16

733

18

489

14

cotZ

Sp

ore

coat

pro

tein

Z

1652

3 93

9 20

17

7 5

682

11

489

9 co

xA

Spor

ulat

ion

corte

x pr

otei

n C

oxA

22

161

111

2 92

3

70

2 cw

lJ

Cel

l wal

l hyd

rola

se C

wlJ

16

452

201

5 96

2

338

7 25

2 6

dacF

D

-ala

nyl-D

-ala

nine

car

boxy

pept

idas

e da

cF

4327

0 11

0 5

162

8 17

6 5

150

7 ge

rE

Spor

e ge

rmin

atio

n pr

otei

n ge

rE

8583

70

3

gerQ

Sp

ore

coat

pro

tein

ger

Q

2026

3 24

7 16

26

4 5

442

7 21

8 6

App

endi

x

151

Gen

e na

me

Prot

ein

Mas

s (D

a)

Wild

type

R

eplic

ate

1 R

eplic

ate

2 R

eplic

ate

3

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a lip

C

Spor

e ge

rmin

atio

n lip

ase

lipC

23

591

141

12

190

9 16

7 5

197

8 op

pA

Olig

opep

tide-

bind

ing

prot

ein

oppA

61

486

396

15

995

21

65

3 ox

dD

Oxa

late

dec

arbo

xyla

se o

xdD

43

527

56

4 97

4

246

6 91

3

safA

Sp

oIV

D-a

ssoc

iate

d fa

ctor

A

4320

1 21

1 10

12

7 3

40

2 33

1

sleB

Sp

ore

corte

x-ly

tic e

nzym

e

3398

0 23

0 7

148

5 47

7 9

182

6 so

dA

Supe

roxi

de d

ismut

ase

[Mn]

22

476

203

10

78

1 56

6 12

so

dF

Prob

able

supe

roxi

de d

ism

utas

e [F

e]

3345

6 13

0 7

172

7 28

0 7

155

4 sp

oIVA

St

age

IV sp

orul

atio

n pr

otei

n A

55

140

288

13

119

8 54

5 15

17

1 9

tcyA

L-

cyst

ine-

bind

ing

prot

ein

tcyA

29

553

76

1 tg

l Pr

otei

n-gl

utam

ine

gam

ma-

glut

amyl

trans

fera

se

2827

8 17

2 10

tp

x

Prob

able

thio

l per

oxid

ase

18

204

53

1 41

2

382

8 36

1

yaaH

Sp

ore

germ

inat

ion

prot

ein

yaaH

48

607

397

19

377

13

849

22

727

23

yabG

Sp

orul

atio

n-sp

ecifi

c pr

otea

se y

abG

33

468

31

1 ya

bP

Spor

e pr

otei

n ya

bP

1139

6 42

2

yceE

U

ncha

ract

eriz

ed p

rote

in Y

ceE

20

935

143

3 33

1

yciC

Pu

tativ

e m

etal

cha

pero

ne Y

ciC

45

734

54

2 76

1

ycnJ

C

oppe

r tra

nspo

rt pr

otei

n Y

cnJ

5975

7 28

1

ydcC

Sp

orul

atio

n pr

otei

n yd

cC

3811

3 22

1 7

101

3 22

1 4

111

4 yd

hD

Puta

tive

spor

ulat

ion-

spec

ific

glyc

osyl

ase

ydhD

46

835

61

2 64

2

283

6 66

3

yezF

U

ncha

ract

eriz

ed m

embr

ane

prot

ein

yezF

85

54

88

2 44

1

yfkD

U

ncha

ract

eriz

ed p

rote

in y

fkD

29

569

112

4 14

5 3

72

2 yf

kO

Puta

tive

NA

D(P

)H n

itror

educ

tase

yfk

O

2561

2 11

1 5

198

5 17

3 4

62

2

ygaK

U

ncha

ract

eriz

ed F

AD

-link

ed o

xido

redu

ctas

e yg

aK

5086

8 12

7 8

92

4 12

3 4

45

3

yhbB

U

ncha

ract

eriz

ed p

rote

in y

hbB

36

095

55

1 82

1

App

endi

x

152

G

ene

nam

e Pr

otei

n M

ass

(Da)

Wild

type

R

eplic

ate

1 R

eplic

ate

2 R

eplic

ate

3

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a yh

bJ

Puta

tive

efflu

x sy

stem

com

pone

nt y

hbJ

2360

8 73

2

136

3 82

2

yhcB

U

ncha

ract

eriz

ed p

rote

in y

hcB

19

003

213

4 yh

cM

Unc

hara

cter

ized

pro

tein

yhc

M

1702

0 78

3

74

2 13

2 3

105

2 yh

cN

Lipo

prot

ein

yhcN

21

004

261

11

338

11

539

10

224

5 yh

cQ

Spor

e co

at p

rote

in F

-like

pro

tein

Yhc

Q

2477

5 29

4 9

543

19

864

23

686

25

yhcX

U

PF00

12 h

ydro

lase

yhc

X

6063

6 28

1

yhdG

U

ncha

ract

eriz

ed a

min

o ac

id p

erm

ease

Yhd

G

4990

6 42

1

yhfE

Pu

tativ

e am

inop

eptid

ase

yhfE

38

712

68

5 29

1

yhfK

U

ncha

ract

eriz

ed su

gar e

pim

eras

e yh

fK

2274

6 74

2

yhfM

U

ncha

ract

eriz

ed p

rote

in y

hfM

14

998

52

1 87

1

yhfN

U

ncha

ract

eriz

ed m

etal

lopr

otea

se y

hfN

48

901

27

1 49

1

yhfW

Pu

tativ

e Ri

eske

2Fe

-2S

iron-

sulfu

r pro

tein

yhf

W

5707

4 53

1

36

2 48

1

40

2 yh

jR

Unc

hara

cter

ized

pro

tein

yhj

R

1733

3 17

8 3

59

2 yh

xC

Unc

hara

cter

ized

oxi

dore

duct

ase

yhxC

30

826

169

9 13

9 5

221

6 24

0 8

yisY

A

B hy

drol

ase

supe

rfam

ily p

rote

in y

isY

30

540

81

3 28

1

125

2 39

1

yitG

U

ncha

ract

eriz

ed M

FS-ty

pe tr

ansp

orte

r yitG

45

763

89

2 28

1

yjcG

U

PF04

77 p

rote

in y

jcG

19

649

128

6 36

1

185

2 yj

dH

Unc

hara

cter

ized

pro

tein

yjd

H

1519

2 27

3 7

270

5 27

4 5

184

4 yj

fA

Unc

hara

cter

ized

pro

tein

yjfA

17

097

36

1 yj

qC

Unc

hara

cter

ized

pro

tein

Yjq

C

3128

9 62

2

94

5 15

9 4

158

6 yk

aA

UPF

0111

pro

tein

yka

A

2400

0 33

1

49

1 yk

gB

Unc

hara

cter

ized

pro

tein

Ykg

B

3850

1 47

1

yktB

U

PF06

37 p

rote

in y

ktB

24

658

23

1 yk

uD

Puta

tive

L,D

-tran

spep

tidas

e Y

kuD

17

689

22

1 37

1

App

endi

x

153

Gen

e na

me

Prot

ein

Mas

s (D

a)

Wild

type

R

eplic

ate

1 R

eplic

ate

2 R

eplic

ate

3

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a yk

uJ

Unc

hara

cter

ized

pro

tein

yku

J 92

96

29

1 12

6 3

70

1 yk

uS

UPF

0180

pro

tein

yku

S

8716

14

0 1

ykuU

Th

iore

doxi

n-lik

e pr

otei

n yk

uU

2054

8 28

1

120

3 43

1

ykvP

Sp

ore

prot

ein

ykvP

46

266

56

6 11

7 3

39

1 yk

wC

Unc

hara

cter

ized

oxi

dore

duct

ase

ykw

C

3080

6 35

1

ylbB

U

ncha

ract

eriz

ed p

rote

in y

lbB

16

230

67

1 yl

bN

Unc

hara

cter

ized

pro

tein

ylb

N

2009

5 29

1

yloB

C

alci

um-tr

ansp

ortin

g A

TPas

e

9723

1 71

3

81

4 51

7 9

240

6 ym

fJ

Unc

hara

cter

ized

pro

tein

ym

fJ

9642

64

1

35

1 yn

eT

Unc

hara

cter

ized

pro

tein

yne

T

1503

7 75

3

ynzH

U

ncha

ract

eriz

ed p

rote

in y

nzH

11

555

469

12

131

4 41

6 8

435

9 yo

dH

Unc

hara

cter

ized

met

hyltr

ansf

eras

e yo

dH

2628

0 47

1

yodI

U

ncha

ract

eriz

ed p

rote

in y

odI

9188

10

6 6

156

6 28

2 12

26

6 11

yo

xD

Unc

hara

cter

ized

oxi

dore

duct

ase

yoxD

25

283

71

1 yp

eB

Spor

ulat

ion

prot

ein

ypeB

51

153

182

9 49

3 14

52

5 16

52

8 15

yq

fA

UPF

0365

pro

tein

yqf

A

3561

9 15

7 8

142

5 49

2 11

20

8 5

yqfX

U

ncha

ract

eriz

ed p

rote

in y

qfX

13

893

80

5 21

1

408

6 41

8 7

yqhO

U

ncha

ract

eriz

ed p

rote

in y

qhO

32

896

38

1 yq

hY

Unc

hara

cter

ized

pro

tein

Yqh

Y

1462

5 59

2

48

1 23

1

yqiG

Pr

obab

le N

AD

H-d

epen

dent

flav

in o

xido

redu

ctas

e yq

iG

4078

0 54

2

22

1 54

1

39

1

yqjE

U

ncha

ract

eriz

ed p

rote

in y

qjE

39

622

87

4 93

1

ysdC

Pu

tativ

e am

inop

eptid

ase

ysdC

39

192

53

1 47

1

142

3 76

2

ytfJ

U

ncha

ract

eriz

ed sp

ore

prot

ein

ytfJ

16

336

243

6 25

6 6

267

7 10

4 7

ytxJ

U

ncha

ract

eriz

ed p

rote

in y

txJ

1245

1 92

1

App

endi

x

154

G

ene

nam

e Pr

otei

n M

ass

(Da)

Wild

type

R

eplic

ate

1 R

eplic

ate

2 R

eplic

ate

3

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a yt

xO

Unc

hara

cter

ized

pro

tein

ytx

O

1654

3 44

3

39

2 86

4

ytzB

U

ncha

ract

eriz

ed p

rote

in y

tzB

11

716

53

1 yt

zL

Unc

hara

cter

ized

pro

tein

ytz

L

6091

49

1

27

1 yu

rT

Unc

hara

cter

ized

pro

tein

yur

T

1448

5 39

1

yurZ

U

ncha

ract

eriz

ed p

rote

in y

urZ

13

898

43

2 57

2

yusN

U

ncha

ract

eriz

ed p

rote

in y

usN

13

187

57

1 yu

tF

Unc

hara

cter

ized

hyd

rola

se y

utF

28

053

23

1 yu

zA

Unc

hara

cter

ized

mem

bran

e pr

otei

n yu

zA

8461

26

2

69

3 yu

zC

Unc

hara

cter

ized

pro

tein

yuz

C

1419

0 48

1

yuzM

U

ncha

ract

eriz

ed p

rote

in y

uzM

96

76

30

1 41

1

yvdP

U

ncha

ract

eriz

ed F

AD

-link

ed o

xido

redu

ctas

e yv

dP

5005

3 40

0 17

37

2 14

40

9 12

26

1 11

yv

dQ

Unc

hara

cter

ized

pro

tein

yvd

Q

1868

2 18

9 4

yxeD

U

ncha

ract

eriz

ed p

rote

in y

xeD

13

793

44

1 63

2

yxeE

U

ncha

ract

eriz

ed p

rote

in y

xeE

14

705

228

7 11

1 2

110

3 14

2 3

yybI

U

ncha

ract

eriz

ed p

rote

in y

ybI

3013

0 68

2

42

1 75

2

yyxA

U

ncha

ract

eriz

ed se

rine

prot

ease

yyx

A

4276

2 51

1

193

3

App

endi

x

155

Tab

le II

I: P

rote

ins i

dent

ified

from

Δco

tA sp

ore

coat

s of B

acill

us su

btili

s.

Gen

e na

me

Prot

ein

Mas

s (D

a)

Wild

type

R

eplic

ate

1 R

eplic

ate

2 R

eplic

ate

3

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a co

tA

Spor

e co

at p

rote

in A

58

690

1947

42

co

tB

Spor

e co

at p

rote

in B

42

946

1771

37

77

6 18

74

7 19

10

57

29

cotC

Sp

ore

coat

pro

tein

C

8868

36

6 13

21

9 8

421

15

578

25

cotE

Sp

ore

coat

pro

tein

E

2107

8 14

00

31

1453

37

11

82

29

1128

34

co

tF

Spor

e co

at p

rote

in F

18

714

583

11

856

16

598

13

609

13

cotG

Sp

ore

coat

pro

tein

G

2439

9 23

3 9

139

5 42

7 13

44

4 14

co

tH

Inne

r spo

re c

oat p

rote

in H

42

843

264

6 30

1

cotI

Sp

ore

coat

pro

tein

I

4144

7 48

8 12

23

6 8

167

6 22

8 9

cotJ

A

Prot

ein

Cot

JA

9790

36

1

67

2 co

tJB

Pr

otei

n C

otJB

10

214

44

1 29

1

cotJ

C

Prot

ein

Cot

JC

2173

9 22

1 5

326

7 75

1

274

8 co

tN

Spor

e co

at-a

ssoc

iate

d pr

otei

n N

28

287

199

4 co

tR

Puta

tive

spor

ulat

ion

hydr

olas

e C

otR

35

335

497

12

394

13

211

6 13

8 6

cotS

Sp

ore

coat

pro

tein

S

4128

6 94

0 24

37

3 14

36

0 10

42

3 13

co

tSA

Sp

ore

coat

pro

tein

SA

43

056

1045

27

39

4 10

28

6 9

414

13

cotW

Sp

ore

coat

pro

tein

W

1232

9 50

2

cotX

Sp

ore

coat

pro

tein

X

1898

9 62

5 18

65

5 19

39

8 9

936

26

cotY

Sp

ore

coat

pro

tein

Y

1872

8 89

5 25

95

4 27

56

7 17

36

6 11

co

tZ

Spor

e co

at p

rote

in Z

17

093

1005

15

58

0 11

46

5 9

587

14

cwlC

Sp

orul

atio

n-sp

ecifi

c N

-ace

tylm

uram

oyl-L

-ala

nine

am

idas

e

2713

0 60

3

cwlJ

C

ell w

all h

ydro

lase

Cw

lJ

1668

0 27

2 5

94

3 57

1

186

4 da

cB

D-a

lany

l-D-a

lani

ne c

arbo

xype

ptid

ase

Dac

B

4305

3 70

3

42

1 70

3

dacF

D

-ala

nyl-D

-ala

nine

car

boxy

pept

idas

e D

acF

43

327

402

8 24

4 9

76

3 22

4 9

App

endi

x

156

G

ene

nam

e Pr

otei

n M

ass

(Da)

Wild

type

R

eplic

ate

1 R

eplic

ate

2 R

eplic

ate

3

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a fe

r Fe

rred

oxin

90

92

22

1 ge

rE

Spor

e ge

rmin

atio

n pr

otei

n G

erE

85

83

96

2 ge

rQ

Spor

e co

at p

rote

in G

erQ

20

263

145

3 22

9 6

130

3 18

3 7

gpr

Ger

min

atio

n pr

otea

se

4031

5 51

2

40

1 40

1

katX

C

atal

ase

X

6238

2 49

2

39

1 31

1

oppA

O

ligop

eptid

e-bi

ndin

g pr

otei

n O

ppA

61

543

426

9 30

8 9

85

4 82

3

oxdD

O

xala

te d

ecar

boxy

lase

Oxd

D

4358

4 12

8 5

292

6 58

1

32

1 sa

fA

SpoI

VD

-ass

ocia

ted

fact

or A

43

429

46

2 11

7 2

118

4 10

1 2

sleB

Sp

ore

corte

x-ly

tic e

nzym

e

3415

1 31

5 6

307

9 23

7 6

170

4 so

dA

Supe

roxi

de d

ismut

ase

[Mn]

22

476

307

5 18

1 6

162

6 21

3 7

sodF

Pr

obab

le su

pero

xide

dis

mut

ase

[Fe]

33

513

124

3 20

1 7

108

3 14

7 4

spoI

VA

Stag

e IV

spor

ulat

ion

prot

ein

A

5519

7 33

0 9

411

13

315

10

229

9 ss

pB

Smal

l, ac

id-s

olub

le sp

ore

prot

ein

B

6975

85

1

tgl

Prot

ein-

glut

amin

e ga

mm

a-gl

utam

yltra

nsfe

rase

28

392

222

6 17

9 8

120

4 24

1 8

tig

Trig

ger f

acto

r 47

458

33

1 tp

x

Prob

able

thio

l per

oxid

ase

18

318

89

2 10

0 4

29

1 78

3

yaaH

Sp

ore

germ

inat

ion

prot

ein

Yaa

H

4860

7 40

5 12

40

1 14

28

2 10

39

1 12

ya

aQ

Unc

hara

cter

ized

pro

tein

Yaa

Q

1195

9 10

0 2

27

1 44

1

yabP

Sp

ore

prot

ein

Yab

P

1139

6 61

1

102

3 33

1

yckD

U

ncha

ract

eriz

ed p

rote

in Y

ckD

12

782

68

3 97

4

84

2 13

3 5

ydcC

Sp

orul

atio

n pr

otei

n Y

dcC

38

170

654

11

490

13

387

10

405

9 yd

hD

Puta

tive

spor

ulat

ion-

spec

ific

glyc

osyl

ase

Ydh

D

4683

5 86

2

216

4 10

5 3

198

4 yf

kD

Unc

hara

cter

ized

pro

tein

Yfk

D

2956

9 23

5 5

178

4 30

1

yfkO

Pu

tativ

e N

AD

(P)H

nitr

ored

ucta

se Y

fkO

25

669

103

2 49

1

99

2 13

7 3

ygaK

U

ncha

ract

eriz

ed F

AD

-link

ed o

xido

redu

ctas

e Y

gaK

51

039

251

7 10

4 3

97

2 11

2 3

App

endi

x

157

Gen

e na

me

Prot

ein

Mas

s (D

a)

Wild

type

R

eplic

ate

1 R

eplic

ate

2 R

eplic

ate

3

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a yh

bB

Unc

hara

cter

ized

pro

tein

Yhb

B

3609

5 73

1

95

2 79

2

yhcB

U

ncha

ract

eriz

ed p

rote

in Y

hcB

19

174

140

3 13

9 2

52

1 11

0 2

yhcM

U

ncha

ract

eriz

ed p

rote

in Y

hcM

17

020

63

1 17

0 4

116

3 13

0 4

yhcN

Li

popr

otei

n Y

hcN

21

061

149

3 39

6 9

274

5 33

2 8

yhcQ

Sp

ore

coat

pro

tein

F-li

ke p

rote

in Y

hcQ

24

946

474

15

692

17

507

13

536

16

yhfW

Pu

tativ

e Ri

eske

2Fe

-2S

iron-

sulfu

r pro

tein

Yhf

W

5758

7 11

3 2

87

3 yh

xC

Unc

hara

cter

ized

oxi

dore

duct

ase

Yhx

C

3094

0 35

1 11

16

1 5

70

2 18

2 5

yisY

A

B hy

drol

ase

supe

rfam

ily p

rote

in Y

isY

30

540

44

1 33

1

28

1 yj

dH

Unc

hara

cter

ized

pro

tein

Yjd

H

1524

9 21

5 4

160

3 16

0 4

87

3 yj

qC

Unc

hara

cter

ized

pro

tein

Yjq

C

3140

3 28

1 6

139

3 61

2

146

4 yk

aA

UPF

0111

pro

tein

Yka

A

2400

0 38

1

53

1 yk

uD

Puta

tive

L,D

-tran

spep

tidas

e Y

kuD

17

689

73

1 yk

uJ

Unc

hara

cter

ized

pro

tein

Yku

J 92

96

59

1 58

2

50

2 yk

uS

UPF

0180

pro

tein

Yku

S

8716

86

1

73

1 yk

uU

Thio

redo

xin-

like

prot

ein

Yku

U

2054

8 58

1

47

1 54

1

52

1 yl

oB

Cal

cium

-tran

spor

ting

ATP

ase

97

516

449

12

379

14

128

4 24

0 9

ymfF

Pr

obab

le in

activ

e m

etal

lopr

otea

se Y

mfF

48

231

54

2 53

1

39

1 ym

fH

Unc

hara

cter

ized

zin

c pr

otea

se Y

mfH

49

089

38

2 18

4 7

79

3 13

9 5

ymxG

U

ncha

ract

eriz

ed z

inc

prot

ease

Ym

xG

4613

7 65

3

46

1 40

1

35

1 yn

eT

Unc

hara

cter

ized

pro

tein

Yne

T

1503

7 61

1

67

1 38

1

32

1 yn

zH

Unc

hara

cter

ized

pro

tein

Ynz

H

1161

2 31

9 6

30

1 76

2

277

7 yo

dI

Unc

hara

cter

ized

pro

tein

Yod

I 92

45

154

6 14

8 5

94

5 16

1 5

ypeB

Sp

orul

atio

n pr

otei

n Y

peB

51

210

374

8 53

7 14

33

7 9

361

11

yqfA

U

PF03

65 p

rote

in Y

qfA

35

676

435

10

261

7 15

0 4

246

8 yq

fX

Unc

hara

cter

ized

pro

tein

Yqf

X

1389

3 33

1 4

134

2 13

2 2

131

2

App

endi

x

158

G

ene

nam

e Pr

otei

n M

ass

(Da)

Wild

type

R

eplic

ate

1 R

eplic

ate

2 R

eplic

ate

3

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a

MA

SCO

T sc

ore

# pe

ptid

e M

S/M

S sp

ectr

a yq

gO

Unc

hara

cter

ized

pro

tein

Yqg

O

6907

62

1

66

2 77

3

92

2

yqiG

Pr

obab

le N

AD

H-d

epen

dent

flav

in o

xido

redu

ctas

e Y

qiG

40

894

64

1

yr

kC

Unc

hara

cter

ized

pro

tein

Yrk

C

2129

9 14

0 2

50

2 24

1

yrzQ

U

ncha

ract

eriz

ed p

rote

in Y

rzQ

50

39

154

2 77

1

123

2 ys

dC

Puta

tive

amin

opep

tidas

e Y

sdC

39

249

51

1 58

1

61

1 yt

fJ

Unc

hara

cter

ized

spor

e pr

otei

n Y

tfJ

1633

6 16

6 4

385

9 21

0 4

276

7 yu

rS

Unc

hara

cter

ized

pro

tein

Yur

S

1048

3 64

2

yurZ

U

ncha

ract

eriz

ed p

rote

in Y

urZ

13

898

43

2 35

1

40

1 yu

zA

Unc

hara

cter

ized

mem

bran

e pr

otei

n Y

uzA

85

18

79

3 50

2

46

2 yv

dP

Unc

hara

cter

ized

FA

D-li

nked

oxi

dore

duct

ase

Yvd

P

5016

7 56

2 15

21

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Appendix

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Appendix

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Summary

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Summary The bacterial stress response enables bacteria to survive extreme environmental assaults. Different bacterial mechanisms perceive different environmental changes and mount a congruous response. A bacterial cell can react simultaneously to a wide variety of stresses and the various stress response systems act together via a complex of global regulatory networks. These regulatory systems maintain the stability of the cellular equilibrium under these conditions. Most bacteria can be grown and cultivated in a laboratory. Nonetheless, in environmental niches, the nutrient availability is a major hurdle for their growth. Nutrient supply is affected by diverse conditions. However, bacteria have evolved some characteristic mechanisms to adapt to such starvation conditions. One such mechanism is sporulation. Species from the bacterial group of Firmicutes, especially belonging to the genera Bacillus and Clostridium, have the ability to form endospores - dormant cellular forms capable of survival under the heat and acid preservation techniques commonly used in the food industry. Since some of these species are highly pathogenic spore research has gained an immense importance in biological fraternity. Spores are multilayered entities with proteinaceous surface layers, id est the spore coat and the exosporium. Resistance characteristics of spores towards various environmental stresses are in part attributed to the surface layer proteins. With the use of SDS-PAGE, 2-DE gel approaches, protein localization studies and genome-wide transcriptome studies more than seventy proteins have been assigned to the soluble fraction of spore coat proteins in Bacillus subtilis. The spore coat also contains an insoluble protein fraction, characterized by protein-protein cross-links, which is difficult to analyze by gel-based proteomics approaches. In Chapter 2 a “gel-free” protocol is presented capable of comprehensively extracting and identifying the B. subtilis spore coat proteins addressing the insoluble protein fraction. Using LC-MS/MS 55 proteins have been identified from the insoluble B. subtilis spore coat protein fraction, of which 21 are putative novel spore coat proteins not assigned to the spore coat until now. Identification of spore coat proteins from a B. subtilis food spoilage isolate has corroborated a generic and ‘applied’ use of our protocol. Similarly the Gram-positive Bacillus cereus, responsible for food poisoning and Clostridium difficile, causative agent of Clostridium difficile-associated diarrhoea (CDAD) are pathogenic spore formers involved in food spoilage, food intoxication and other infections in humans and animals. The exosporium provides an ability to adhere to surfaces eventually leading to spore survival on surfaces thereby providing an added benefit during pathogenicity. Thus studying the coat and exosporium layers and identifying suitable protein targets for rapid detection and removal of spores is of utmost importance. In the study described in Chapter 3, 100 proteins have been identified from the B. cereus spore coat, exosporium and 54 proteins from the C. difficile coat insoluble protein fraction. To define a universal set of spore outer layer proteins, 11 superfamily domains have been identified common to the identified proteins from two Bacilli and a Clostridium species. The evaluated orthologue relationships of identified proteins across different spore formers define a group of 13 coat proteins conserved across the spore

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formers and 12 exosporium proteins conserved in the B. cereus group. These proteins are candidates that can be tested for readily detection for spore screening purposes or targeted in strategies for the removal of spores from surfaces.

Spore coat formation is dependent on the spore coat morphogenetic proteins. Previous studies have analyzed the dependence of protein incorporation of a subset of spore coat proteins on well-known morphogenetic proteins such as SpoIVA, CotE, CotH etc. Yet many coat proteins remain to be studied for the regulation of their assembly and localization into spore layers. With the aim to map the inter-protein dependence, Chapter 4 describes the study of protein profiles of the ΔcotA, ΔcotE and Δtgl spores. A subset of spore coat proteins have been identified dependent on and/or affected by the absence of the candidate protein genes cotE and tgl. The thermal resistance tests of these spores shows that Δtgl spores are more resistant to thermal stress than ΔcotE spores. CotA is a laccase and since laccases can participate in cross-link formation the effect of cotA deletion on spore coat protein profile was studied. No effect on spore coat protein profiles is detected in the absence of CotA.

Wet-heat resistance in spores is developed in the later stages of sporulation and during maturation of the released spores. The efficiency of the tryptic digestion of proteins in the spore coat is monitored during maturation over a period of 10 days using our gel-free method and LC-FT-ICR-MS/MS quantitative analysis with metabolically 15N labeled mature spores as a reference. The results are shown and discussed in Chapter 5. During spore maturation the loss of digestion efficiency of outer coat and crust proteins synchronized with the increase in heat resistance. This implicates that spore maturation involves chemical cross-linking of outer coat and crust layer proteins with the inner coat proteins remaining unmodified. The digestion efficiencies of spore surface proteins are linked to their location within the coat and crust layers. A possible link between spore maturation and the heterogeneity in the rate of spore germination was also studied using a single-spore live imaging microscopy technique.

Analysis of bacterial spore surface proteomes demands functional characterization of putative newly identified spore surface proteins. Although a few of these proteins have already been utilized for biotechnological applications, the spore longevity and their escape from immune surveillance remain challenges for the food security and the medical fields. Chapter 6 discusses the specific molecular properties of spore surface proteins that can be used to select suitable targets, to design accurate spore detection and removal systems. Using bioinformatic software tools reveal the specific amino acid distributions while molecular mass, pI and GRAVY hydrophobicity distributions emphasise the role of these proteins in spore surface adhesion. Finally, the potential of given peptide sequences, associated with the identified proteins, in drug development against resistant pathogens has been examined using in silico algorithms.

In the presented studies identification and quantification of proteins involved in spore maturation has contributed to new insights with respect to their functions, abundances and nature of inter-protein cross-links. Further research on spores is required to gain more detailed molecular information on sporulation of bacteria, spore layer assembling and spore maturation as well as spore germination.

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Samenvatting Een veelvoud aan stressresponssystemen stelt bacteriën in staat te overleven bij

extreme aanvallen van buiten. Verschillende bacteriële moleculaire mechanismen zijn verantwoordelijk voor het waarnemen van een breed scala van veranderingen in het extracellulaire milieu en zetten de cellen aan om zich zo goed mogelijk aan die conditie(s) aan te passen. Een bacteriële cel is in staat zich gelijktijdig aan te passen aan meerdere stresscondities via een gezamenlijk stelsel van regulerende signaaltransductienetwerken. Dit stelsel handhaaft de stabiliteit van het intracellulaire milieu . De meeste bacteriën kunnen worden gekweekt in het laboratorium. In het laboratorium kan onder gecontroleerde condities de beschikbaarheid van voedingsstoffen, een bepalende parameter voor hun groei, worden gevarieerd. Deze variatie kan zo worden ingezet dat ze zo goed mogelijk een natuurlijke situatie benadert. Op deze wijze kan de reactie van de cellen op ‘hongercondities’ bepaald worden. Een dergelijk stressmechanisme is de vorming van sporen.

Bacteriële soorten van het phylum Firmicutes, met name die behorend tot de geslachten Bacillus en Clostridium, hebben de mogelijkheid om dergelijke (endo)sporen te vormen. Sporen zijn ‘slapende’ cellulaire structuren die in staat zijn om onder andere ook de in de voedingsindustrie gebruikelijke conserveringsmethoden met hitte en zuur te overleven. Aangezien sommige van deze soorten sterk pathogeen zijn wint het sporeonderzoek steeds meer belang binnen de biologie. Sporen zijn deeltjes bestaande uit meerdere lagen waarvan de toplagen eiwithoudend zijn id est de sporemantel (‘spore coat’) en het exosporium. De resistentie-eigenschappen van sporen ten aanzien van de verschillende stressvolle condities worden gedeeltelijk toegeschreven aan de eiwitten in de toplagen. Met behulp van de SDS-PAGE 2-DE gel methode zijn in eiwitlokalisatiestudies en genoombrede transcriptoomstudies meer dan zeventig eiwitten geïdentificeerd in de oplosbare fractie van de Bacillus subtilis sporemantel. De sporemantel bevat tevens een onoplosbare eiwitfractie, die wordt gekenmerkt door eiwit-eiwit cross-links. Deze eiwitten zijn moeilijk te analyseren met behulp van gel-gebaseerde methodes voor eiwitextractie.

In Hoofdstuk 2 wordt een "gel-free" protocol gepresenteerd waarmee eiwitten uit de onoplosbare fractie van de B. subtilis sporemantel efficiënt kunnen worden geëxtraheerd en geïdentificeerd. Met behulp van LCMS/MS zijn 55 eiwitten geïdentificeerd in de onoplosbare B. subtilis sporemantelfractie, waarvan 21 mogelijk nieuwe sporemanteleiwitten die nog niet eerder zijn toegewezen aan de sporemantel. Identificatie van sporemanteleiwitten uit een B. subtilis voedselbederfisolaat heeft de algemene toepasbaarheid van ons protocol aangetoond. Ook de gram-positieve Bacillus cereus, verantwoordelijk voor voedselvergiftiging en Clostridium difficile, verwekker van Clostridium difficile-antibiotica geassocieerde diarree (CDAD) zijn pathogene sporenvormende bacteriën die zijn betrokken bij voedselbederf, voedselintoxicatie en andere infecties bij mensen en dieren. Hun exosporium stelt de sporen van deze bacteriën in staat te binden aan oppervlakken hetgeen uiteindelijk kan leiden tot grotere overlevingskansen en als zodanig een extra virulentiefactor vormt. Dus het bestuderen van de mantel, de exosporiumlaag en het vinden van geschikte eiwittargets voor snelle detectie en verwijdering van sporen is van het grootste belang.

In de studie beschreven in Hoofdstuk 3, zijn 100 eiwitten geïdentificeerd in de sporemantel en het exosporium van B. cereus, en 54 eiwitten uit de onoplosbare sporemantelfractie van C. difficile. Bij het definiëren van een universele set van sporetoplaageiwitten zijn 11 gemeenschappelijke superfamiliedomeinen gevonden in de

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geïdentificeerde eiwitten uit twee Bacilli en een Clostridium soort. De onderzochte orthologe relaties van de geïdentificeerde eiwitten tonen een groep van 13 manteleiwitten die zijn geconserveerd binnen de sporevormendebacteriën en 12 geconcerveerde exosporiumeiwitten in de B. cereus groep. Deze eiwitten zijn kandidaten voor eenvoudige detectie bij de screening van sporen of kunnen dienen als targets bij strategieën voor het verwijderen van sporen van oppervlakken. De vorming van de sporemantel is afhankelijk van de morfogenetische sporemanteleiwitten. Eerdere studies hebben aangetoond dat de correcte biogenese van sporemanteleiwitten afhankelijk is van bekende morfogenetische eiwitten zoals SpoIVA, CotE, CotH enz. Voor veel manteleiwitten moet nog worden onderzocht waar en hoe ze zijn ingebouwd in de sporelagen.

Hoofdstuk 4 beschrijft een studie die is opgezet om de sporemanteleiwitten in kaart te brengen voor de ΔcotA , Δ cotE en Δtgl sporen. Een subset van sporemanteleiwitten is zo geïdentificeerd welke afhankelijk is en/of beïnvloed wordt door het ontbreken van de genen cotE en tgl. De tests van de thermische weerstand van deze sporen toont aan dat Δtgl sporen beter bestand zijn tegen thermische stress dan ΔcotE sporen. CotA is een laccase en aangezien laccasen kunnen deelnemen in de vorming van cross-links tussen eiwitten is het het effect van de verwijdering van het cotA gen op het coat eiwit profiel onderzocht. Er werd evenwel geen aanpassing van het manteleiwitprofiel gevonden bij de afwezigheid van het CotA eiwit.

Natte hitteresistentie van sporen is ontwikkeld in de latere stadia van sporevorming en tijdens de verdere rijping van de vrijgekomen sporen. Tijdens die rijping is de efficiëntie van de tryptische digestie van de eiwitten in de sporemantel gevolgd gedurende 10 dagen. Dit is gedaan met behulp van onze gel-vrije methode gecombineerd met kwantitatieve LC-FT-ICR-MS/MS analyses van de tryptische peptiden met de tryptische peptiden van de 15N metabool gelabelde volwassen sporen als kwantificeer-referentie. De resultaten, gepresenteerd en besproken in Hoofdstuk 5, laten zien dat tijdens het sporerijpingsproces verlies optreedt van de digestie-efficiëntie voor de buitenste mantellaag en korst eiwitten. Dit efficiëntieverlies is gesynchroniseerd met de toename in hitteresistentie. Dit verband impliceert dat het rijpingsproces van de sporen gepaard gaat met het vormen van chemische cross-links tussen de eiwitten in de buitenste mantel- en korstlaag. Voor eiwitten in de diepere coatlagen blijft de digestie efficiëntie ongewijzigd . Het blijkt dat de efficiëntie van de digestie van sporencoat eiwitten is gekoppeld aan hun locatie binnen de mantel- en korstlagen. Een mogelijk verband tussen de rijping van sporen en de heterogeniteit in het tempo van ontkieming van de sporen is ook bestudeerd met behulp van een single-spore live imaging microscopie techniek. Analyse van bacteriële proteomen van de buitenlagen van sporen vraagt om functionele karakterisering van de nieuw geïdentificeerde vermeende sporemanteleiwitten. Hoewel een paar van deze eiwitten al zijn gebruikt voor biotechnologische toepassingen, blijven de duurzaamheid en het vermogen van de sporen te ontkomen aan het immuun systeem, bedreigingen voor voedselveiligheid en medische behandelingen.

Hoofdstuk 6 bespreekt de specifieke moleculaire eigenschappen van sporeoppervlakte-eiwitten, die kunnen worden gebruikt als targets voor de ontwikkeling van betrouwbare sporen detectie en verwijderingssystemen. Met behulp van bioinformatisch software programma’s is voor deze eiwitten de rol van specifieke aminozuurdistributies, molecuulmassa’s, pI en GRAVY hydrophobiciteitsdistributies in kaart gebracht met betrekking tot de hechting van sporen aan oppervlakken. Ten slotte zijn met behulp van in silico algoritmen, de bruikbaarheid van de peptidesequenties van de geïdentificeerde eiwitten onderzocht waar het, het ontwikkelen van geneesmiddelen tegen resistente pathogenen betreft.

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Studies beschreven in dit proefschrift laten zien dat kwantitatieve analyses van geidentificeerde eiwitten die betrokken zijn bij de rijping van sporen hebben bijgedragen aan nieuwe inzichten met betrekking tot de functies van deze eiwitten en de vorming van intermoleculaire eiwit cross-links. Vervolgonderzoek is nodig om meer gedetailleerde moleculaire informatie te verkrijgen over de tijdopgeloste koppeling tussen de sporulatie van bacteriën, de assemblage van manteleiwitten, de vorming van cross-links tussen sporemanteleiwitten, en de kiemingsefficiëntie van sporen.

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List of Publications Abhyankar, W., Ter Beek, A., Dekker, H.L., Kort, R., Brul, S. and de Koster, C. G. (2011), Gel-free proteomic identification of the Bacillus subtilis insoluble spore coat protein fraction. PROTEOMICS, 11: 4541–4550. Abhyankar, W., Hossain, A.H., Djajasaputra, A., Permpoonpattana, P., Ter Beek, A., Dekker, H.L., Cutting, S.M., Brul, S., de Koning, L.J., de Koster, C.G. (2013), In Pursuit of Protein Targets: Proteomic characterization of Bacterial spore outer layers. J. Proteome Res., 2013, 12 (10), pp 4507–4521. Abhyankar, W., Pandey, R., Ter Beek, A., Brul, S., de Koning, L. J., de Koster, C. G. (2013) Monitoring the progress in cross-linking of spore coat proteins during maturation of Bacillus subtilis spores. Food Microbiology (Submitted)

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Acknowledgements

No achievement can be cherished without acknowledging the helpers and well-wishers and same was the case with me during my PhD work. Therefore, before I put my pen down, I would like to thank all those countless people who made my personal and professional life enjoyable during these last 4 years.

First and foremost, I express my sincere gratitude to my promoters Prof. Dr. Chris de Koster and Prof. Dr. Stanley Brul for giving me an opportunity to conduct my research in, what I would say, a fascinating field of endospores. Both of you have been very kind and supportive when it came to knowledge, research, patience, success and personal as well professional life. I will always be thankful to you for the same.

I would like to extend my gratitude to my co-promoter Dr. Leo de Koning, who always challenged my thoughts when it came to making sense out of the FTMS data. Thank you so much Leo for stimulating my thoughts and motivating me to do those little extra efforts especially in the last few months. It made a lot of positive changes and created interesting opportunities in my research. I agree with you and “I love it when the plans come together”.

I would like to extend my sincerest thanks and appreciation to the members of my doctoral committee Prof. Dr. Adam Driks, Prof. Dr. Ezio Ricca, Prof. Dr. Mike Peck, Prof. Dr. Remco Kort and Prof. Dr. Peter Schoenmakers for investing their precious time to review this thesis and for their valuable comments.

Jasper Faber, Niels Molenaar and Inés Crespo - thank you so much because without you it wouldn’t have been possible for me to come to the Netherlands. Thank you Erasmus Mundus programme (EMECW15) for the financial grants. Jasper and Niels you made my life much easier especially during the initial period. I will always cherish those small coffee sessions and chats with you two. Inés, thank you so much for your help throughout the first two years and I wish you all the success with your career and life.

I would like to take this opportunity to thank my collaborators Prof. Dr. Simon Cutting and Dr. Patima Permpoonpattana. Thank you for showing your interest in my work and trust in me.

Though, the Department of Mass spectrometry was small, about 7-8 people, the amount of fun, noise, creativity that emerged through this department was much more than a cricket team! Thanks to none other than Henk and Winfried! Henk, you have always been the source of “positiveness”. Though I struggled in my second year, you never let me think of quitting, you always kept on fighting the problem with me and eventually we got through it. Thank you so much for all the help, information and knowledge shared, the jokes, the fun, the Driehuis trip etc. - in short for everything! Winfried, you were also not far behind. Your technical skills always have impressed me; in fact sometimes the ease with which you tackled the technical problems has left me awestruck. Thank you for all

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the fun, the encouraging chats, the jokes, pranks and your help especially to make the batch reactor system “functional”! Thank you Hans Peters for that small time when we were neighbours in the office and all the other times when we just saw each other in the corridors and labs. The simple “Hi” with a smile always helped relaxing the nerves. Luitzen, I especially thank you as you always made me think of research as a jigsaw puzzle - the more pieces you collect the more easy it gets to solve the puzzle. Your keen observations, expertise in biochemistry, immense patience, research attitude and at the same time your efforts and interest to keep the quality of life on the priority, have all been special to me. Thank you for the wonderful times that I spent at your house as well as during the ice skating in the freezing cold winters.

Apart from all the senior members of the department, I would like to thank all the junior members starting with Clemens, Alice, Sacha, Linli and none other than the great Hansuk! I assure all of you that you have been an invaluable part of the past 4 years. I have learnt a lot from all of you and I wish you all a great future! I would like to thank Clemens for guiding me in the initial period for the proteomics work. Sacha you are a great pal and an excellent encyclopaedia of beers. I will cherish each and every moment when you, me and Hansuk went for drinks! Linli thanks a bunch for that small period when we worked together. I am sure your visit to India must have strengthened our friendship. All the best for your future endeavours. Both Sacha and Linli thanks for being my paranymphs. Hansuk, what can I say! You have been really special. You were my friend, philosopher and guide. Always! I enjoyed each and every moment that we spent as flatmates, as colleagues and as photographers I will never forget this time. Once during our struggling period I had said that you will be promoted before me and you actually took it seriously! Amazing! Thank you for all the encouragements, discussions, food, pranks and just everything! I salute your commitment towards research and I wish you a great success in your life. Also with Hansuk, I would like to extend my thanks to Weina and Inambao for being a part of my life. You both have been kind, supportive.

All the members, of Molecular Biology and Microbial Food safety group, I thank you for critical discussions, helping tips, guidance, and continuous encouragement. Alex and Frans, I especially thank you for taking a keen interest in all my works and criticizing me when needed. Alex I will always cherish the conference times that we spent together. It was really amazing! Thanks and all the best! Daniel, you have been a good friend. I wish you all the good luck and strength in the future. Rachna, thanks a lot for everything. All the parties, all the travelling journeys, all the fun, jokes, encouragement, support and everything that helped me understand how things work in general. Thank you for that tasty food that you cooked for me especially when you yourself had a fast! I will never forget these moments since I have captured them in my camera. I wish you all the best, and happy and a successful future! Other friends and colleagues - Johan, Jan, Nadine, Reuben, Marco, Richard, Janfang, Soraya, Azmat, Laura, Gertien, Hans, Yelena, Orawan, Jeroen, Pascal, Andreas, Que, Wei, Rosanne, Kornel and each and every one including the master and bachelor students form the MBMFS and MMP groups, I thank you all. I always enjoyed your company. Andreas and Nadine, it was great to be a part of the

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temporary valley ball team. I wish all of you a great success in the future. Dennis, Jos and Jolanda, special thanks to you for timely help and management of my lab requirements. Without your help it wasn’t possible! Ethayaraja, Venkatesh, Vijay-Antina, Poonam-Jop, Nakul-Anisha, Nanda-Sutanuva, Maithili, Aatish, Monalisa, Vinod, Mihir, Kandan, Suraj, Andjoe, Odile and all other Erasmus Mundus grantees from India whom I met are all thanked form deep of my heart. Abdul, Rashid, Hanan, Ronny and Siraje - the borders will never come in between our friendship. Thank you all for those enjoyable moments.

All my students, Abeer, André, Tijmen, Khadra and Rokus - you all were wonderful and have made a big contribution to my PhD. So a lot of credit goes to you. Thanks for your help and thanks for being such nice friends. Abeer, I will especially thank you for all the fun time, movies and together with Hansuk those great times at Mesken and Rancho! I wish you all a great success.

Outside the university, the entire Indian friend circle from Amstelveen and the members of Maharashtra Mandal as well as the “Rashmin” group, Netherlands, thank you for your support and all the fun and the precious moments. Thank you for giving me a space in your world where I could relax and take out some of the frustrations at times. I was lucky to be a part of this big family.

Hub and Anju mawshi, I can’t forget to thank you! Whether it was festival or it was some other help, you were always there. Even if lately we could not meet I always had someone to look to. Thank you and I really appreciate your help and support.

The list will not be complete without all my teachers who showed me the correct path, all the relatives, friends and visitors who visited me in these years. Spending time with you all was a different but still a special experience. So thank you all for giving me an opportunity to be your host. Our meetings always refreshed me. I hope the feeling is mutual.

Last but not the least, my parents and all other family members, all friends back in India, Imran, Rishikaysh, Yamini and my love Bhagyashree, you all have been the main support for me. Thank you all for your patience and your trust in me. I am blessed to have you all in my world. Bhagyashree, I have troubled you the most but I salute your courage, patience and the endless love. The 4 years of PhD work were very difficult staying far away from you.

लहानपणापासून आजी, आजोबा, काका, आ ण इतर ये ठ, व र ठ मंडळींनी मा या नकळतच

मा यावर जे सं कार केले याचंच फळ हणून आज मी या ठकाणी पोहोचू शकलो आहे. आज ते

न क च मा यावर खूष असतील आ ण मलाह यांचं व न पूण के याचं समाधान आज मळत

आहे. तुकोबा हणतात तसं, ' याचसाठ केला होता अ ाहास, शेवटचा द स गोड हावा.'

Wishwas Abhyankar

Amsterdam, 2014

uva-dare (digital academic repository) the proteome of spore … · sporulation is an irreversible...

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