development of gene expression systems in
TRANSCRIPT
Development of gene expression systems in
Bifidobacterium bifidum S17 and their
application for tumor therapy
A dissertation submitted to
The Faculty of Natural Science, University of Ulm
for partial fulfillment of the degree of
Doctor of Sciences (Dr. rer. nat.)
presented by
Zhongke Sun
Born in Shaanxi, China
Ulm, July 2014
Amtierender Dekan: Prof. Dr. Steven Jansen
Erstgutachter: Prof. Dr. Bernhard Eikmanns
Zweitgutachter: Prof. Dr. Stefan Binder
Tag der mündlichen Prügung: 30.10.2014
The study was conducted at the Institute of Microbiology and Biotechnology in the Faculty
of Natural Science, University of Ulm.
The work was supported by a PhD fellowship from the German Academic Exchange
Service (DAAD).
1
TABLE OF CONTENTS
TABLE OF CONTENTS .................................................................................................... 1
1 INTRODUCTION ............................................................................................................ 4
1.1 Bifidobacteria: properties and applications.................................................................. 4
1.2 Characteristics of Bifidobacterium bifidum S17 .......................................................... 8
1.3 Expression systems for bifidobacteria ......................................................................... 9
1.3.1 Advantages of bifidobacteria as hosts ................................................................... 9
1.3.2 Plasmids isolated from bifidobacteria ................................................................... 9
1.3.3 Current study of gene expression in bifidobacteria ............................................. 10
1.3.4 Promoters............................................................................................................. 11
1.3.4 Secretion .............................................................................................................. 14
1.4 Bifidobacterial cancer therapy ................................................................................... 15
1.5 The aim of this study.................................................................................................. 17
2 MATERIALS AND METHODS ................................................................................... 18
2.1 Bacterial strains and plasmids.................................................................................... 18
2.1.1 Bacterial strains ................................................................................................... 18
2.1.2 Plasmids and oligonucleotides ............................................................................ 19
2.2 Sample preparation and purification .......................................................................... 19
2.2.1 Preparation of protein samples ............................................................................ 19
2.2.2 Preparation of nucleic acids................................................................................. 20
2.2.3 Purification of nucleic acid samples .................................................................... 22
2.3 Manipulations of nucleic acids .................................................................................. 23
2.3.1 Conventional polymerase chain reaction............................................................. 23
2.3.2 Splicing by overlap extension PCR ..................................................................... 23
2.3.3 Rapid amplification of cDNA ends ..................................................................... 24
2.3.4 Agarose gel electrophoresis................................................................................. 25
2.3.5 DNA restriction and ligation ............................................................................... 26
2.3.6 Transformation of bacteria .................................................................................. 26
2.4 Enzymatic assays ....................................................................................................... 28
2
2.4.1 β-glucuronidase assay.......................................................................................... 28
2.4.2 Phytase assay ....................................................................................................... 28
2.4.3 Nitroreductase assay ............................................................................................ 29
2.5 Growth related assays ................................................................................................ 29
2.5.1 Bacterial growth experiments .............................................................................. 29
2.5.2 Phytate degradation assay.................................................................................... 30
2.5.3 NTR/CB1954 inhibition assay ............................................................................ 30
2.6 Quantification............................................................................................................. 30
2.6.1 BCA protein assay ............................................................................................... 30
2.6.2 2-D Quant protein assay ...................................................................................... 31
2.6.3 DNA/RNA quantification.................................................................................... 31
2.7 Bioinformatic analysis ............................................................................................... 32
3 RESULTS ........................................................................................................................ 33
3.1 Promoter prediction and cloning................................................................................ 33
3.2 Promoter activities in B. bifidum S17 ........................................................................ 35
3.3 Analysis of Pgap .......................................................................................................... 36
3.3.1 Bioinformatic Analysis of Pgap ............................................................................ 36
3.3.2 Experimental determination of TSS .................................................................... 37
3.3.3 Alignment of representatives............................................................................... 38
3.4 Analysis of protein secretion in B. bifidum S17 ........................................................ 40
3.5 Prediction of SPs in B. bifidum S17 ........................................................................... 41
3.7 Construction of a secretion reporter vector................................................................ 44
3.7.1 Clone SP from gene bbif_1734............................................................................ 44
3.7.2 Clone appA as a reporter ..................................................................................... 44
3.7.3 Phytase assay ....................................................................................................... 45
3.8 Comparison of various SPs ........................................................................................ 46
3.9 Secretion of codon adapted ChrR6 in B. bifidum S17 ............................................... 50
4 DISCUSSION .................................................................................................................. 53
4.1 Systems for protein expression and secretion by bifidobacteria ................................ 53
4.1.1 Expression vectors and promoters ....................................................................... 53
4.1.2 Secretion pathway and sorting signal .................................................................. 56
4.1.3 Secretion reporter ................................................................................................ 57
3
4.2 Bifidobacteria as gene delivery vectors in cancer therapy......................................... 60
5 SUMMARY ..................................................................................................................... 63
6 LITERATURES.............................................................................................................. 64
7 APPENDICES ................................................................................................................. 85
7.1 Bacteria strains ........................................................................................................... 85
7.2 Plasmids ..................................................................................................................... 87
7.3 Oligonucleotides ........................................................................................................ 88
7.4 Promoter sequences.................................................................................................... 89
7.5 Putative gap promoters of representative bifidobacteria ........................................... 90
7.6 Reporter gene appA .................................................................................................... 91
7.7 SP sequences .............................................................................................................. 92
7.8 chrR6 sequences......................................................................................................... 93
ABBREVIATIONS ............................................................................................................ 94
PUBLICATIONS ............................................................................................................... 98
ACKNOWLEDGEMENT ................................................................................................ 99
DECLARATION ............................................................................................................. 100
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1 INTRODUCTION
1.1 Bifidobacteria: properties and applications
Bifidobacterium is a genus of Gram-positive, non-sporulating, non-motile
anaerobic bacteria with irregular shapes. They are endosymbiotic inhabitants of the
gastrointestinal tract (GIT), vagina, and mouth of mammals including humans (Picard et al.,
2005). It was reported that bifidobacteria were first isolated in 1899 by Henry Tissier at
the Pasteur Institute in Paris and the present name was proposed late in 1924 by Orla-
Jensen (Matteuzzi and Sgorbati 1980). Since then, a large number of bifidobacterial strains
were isolated from different habitats. Until April 2014, 48 species were assigned to the
genus Bifidobacterium by German Collection of Microorganisms and Cell Cultures
(DSMZ; http://www.dsmz.de/support/bacterial-nomenclature-up-to-date-downloads.html).
Currently, 37 complete genome sequences of bifidobacteria strains are publically available;
with guanine cytosine (GC) contents ranging from 58% to 63% (see details in Table 1-1).
Among these sequenced strains, the majority are commonly observed in the human GIT
including B. adolencent, B. bifidum, B. breve, and B. longum (Turroni et al., 2009). B.
animalis widely presents in a variety of commercial dairy products distributed worldwide
(Briczinski et al., 2009).
Bifidobacteria are characterized by a special metabolic pathway termed bifidus shunt (de
Vries et al., 1967), with the key enzyme fructose-6-phosphate phosphoketolase (F6PPK)
converting hexoses (Scardovi et al., 1971). One main product of bifidobacteria metabolism
is lactate, while they do not belong to traditional Lactic Acid Bacteria (LAB) due to
obvious phylogenetic discriminations (Mattarelli et al., 2014). Most bifidobacteria colonize
and replicate only in anaerobic conditions even their sensitivity to oxygen may vary in
different strains (Shimamura et al., 1992). The characteristic of oxygen sensitive in
bifidobacteria is primarily proposed to be the production of hydrogen peroxide
(Kawasaki et al., 2009).
5
Table 1-1: Bifidobacterium sp. strains for which complete genome sequences are publically available
Gold stamp Organism Size (kb) GC Content (% )
Gc00470 B. adolescentis ATCC 15703 2090, 1709 orfs 59
Gc02229 B. animalis ATCC 25527 1933, 1597 orfs 60
Gc00888 B .animalis AD011 1934, 1587 orfs 60
Gc01215 B. animalis BB-12 1942, 1706 orfs 60
Gc0042985 B. animalis Bi-07 1939, 1661 orfs 60
Gc01943 B. animalis BLC1 1944, 1622 orfs 60
Gc00988 B. animalis Bl-04, ATCC SD5219 1939, 1631 orfs 60
Gc0051871 B. animalis Bl12 1939, 1607 orfs 60
Gc02353 B. animalis B420 1939, 1625 orfs 60
Gc01890 B. animalis CNCM I-2494 1943, 1724 orfs 60
Gc00987 B. animalis DSM 10140 1938, 1629 orfs 60
Gc0025103 B. animalis subsp. lactis ATCC 27673 1963, 1620 orfs 61
Gc01277 B. animalis V9 1944, 1636 orfs 60
Gc009812 B. asteroides PRL2011 2167, 1731 orfs 60
Gc01507 B. bifidum PRL2010 2215, 1767 orfs 63
Gc02297 B. bifidum BGN4 2224, 1903 orfs 63
Gc01417 B. bifidum S17 2187, 1845 orfs 63
Gc01767 B. breve ACS-071-V-Sch8b 2327, 1890 orfs 59
Gc0071448 B. breve JCM 7017 2289 58.7
Gc0071370 B. breve JCM 7019 2359, 1999 orfs 58.6
Gc0071449 B. breve NCFB 2258 2316 58.7
Gc0018521 B. breve S27 2294, 1898 orfs 59
Gc01883 B. breve UCC2003 (NCIMB8807) 2423, 1914 orfs 59
Gc0071444 B. breve 12L 2245 58.9
Gc0071447 B. breve 689b 2332 58.7
Gc01161 B. dentium Bd1 2636, 2197 orfs 59
(Date: 05/05/2014, available from http://www.genomesonline.org/cgi-b in/GOLD/Search.cg i)
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Table 1-1 (continued): Bifidobacterium sp. strains for which complete genome sequences are publically
available
Gold stamp Organism Size (kb) GC Content (% )
Gc00886 B. longum ATCC 15697 2833, 2577 orfs 60
Gc01537 B. longum BBMN68 2266, 1870 orfs 60
Gc00811 B. longum DJO10A 2390, 2074 orfs 60
Gc01485 B. longum F8 2385, 1744 orfs 60
Gc01613 B. longum JCM 1222 2829, 2641 orfs 60
Gc01612 B. longum JCM 1217 2385, 2009 orfs 60
Gc01307 B. longum JDM301 2478, 2022 orfs 60
Gc01871 B. longum KACC 91563 2396, 2050 orfs 60
Gc00108 B. longum NCC2705 2260, 1805 orfs 60
Gc01611 B. longum 157F-NC 2409, 2070 orfs 60
Gc0043015 B. thermophilum RBL67 2292, 1904 orfs 60
(Date: 05/05/2014, available from http://www.genomesonline.org/cgi-b in/GOLD/Search.cg i)
Besides the already mentioned characteristics, probably the most interesting feature of
bifidobacteria is that some strains are proposed to have benefits to human health.
According to the official definition, probiotics are microorganisms, which when
administered in adequate amounts confer a health benefit on the host (FAO/WHO, 2001).
Recently, an expert consensus statement confirmed the term of probiotics with minor
grammatical correction and defined the scope as well as appropriate use of probiotics (Hill
et al., 2014). Therefore, some well-studied bifidobacteria strains are entitled as probiotics
after a series of screens. Considering the long application history of these strains, they are
entered into the International Diary Federation (IDF) and European Food and Feed Culture
Association (EFFCA) joint inventory as Microbial Food Cultures (MFC). Most of these
MFC are used qualified presumption of safety (QPS), which is managed by the European
Food Safety Authority (EFSA) (see the updated authorized lists in Table 1-2).
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Table 1-2: Bifidobacterium sp. authorized by the EFS A used as probiotics in Europe
MFC (IDF-EFFCA, 2012)a QPS (EFS A, 09/2013 updated)
b
B. adolescentis B. adolescentis
B. animalis B. animalis
B. bifidum B. bifidum
B. breve B. breve
B. longum B. longum
B. pseudolongum subsp. pseudolongum
B. thermophilum
MFC, Microbial Food Cultures
IDF-EFFCA, International Diary Federation and European Food and Feed Culture Association
QPS, Qualified Presumption of Safety
EFSA, European Food Safety Authority
a http://www.effca.org/content/inventory-microorganisms
b http://www.efsa.europa.eu/en/efsajournal/doc/3449.pdf
The reported benefits of probiotic bifidobacteria are mainly ameliorations of
gastrointestinal disorders including bacterial or viral induced gastroenteritis, antibiotic-
associated diarrhea, lactose intolerance, constipation, irritable bowel syndrome, and
inflammatory bowel disease (Guyonnet et al., 2007; Guglielmetti et al., 2011; Whelan and
Quigley 2013). Several in vitro or in vivo experimental models provide hints towards
possible explanations for these benefits, such as inhibition of adhesion (Ewaschuk et al.,
2008), immunomodulatory effects (Park et al., 2007; Zhang et al., 2010), production of
antimicrobial substances (Wu et al., 2010), and restoration of the microecology (Relman
2013). Other applications of probiotics are prevention of infectious diseases (Petschow et
al., 2013), obesity (An et al., 2011; Chen et al., 2012), and allergy (Dev et al., 2008).
Despite promising results in animal models, these evidences are not solid enough to
convince related administrative authorities (Katan 2012). For example, the EFSA demands
more systematic studies and well conducted randomized controlled trials to evidence the
efficacy and safety for any health related claim of probiotics (Salminen and van Loveren
2012; Mego et al., 2013; Passariello et al., 2014). Increasing interest of bifidobacteria is
their potential application in cancer gene therapy that will be discussed in one following
part separately.
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1.2 Characteristics of Bifidobacterium bifidum S17
Even there are 48 species of bifidobacteria and many strains belong to different species are
termed as probiotics, specific benefits or characteristics are frequently strain dependent.
One good example is B. bifidum S17, an isolate from feces of a breast-fed infant. This
strain had been sequenced and studied in detail in our lab. The complete genomic
information of B. bifidum S17 is contained on a single circular chromosome of ~2.18 Mb
with an average GC content of 62% (NCBI accession number: CP002220). A total of 1 782
open reading frames (ORFs), 53 tRNA genes and 3 rrn operons were identified (Zhurina et
al., 2011). Completely annotated genome supplied many interesting targets, like a
potential adhesin BopA (encoded by bbif_0636) and the S-ribosylhomocysteinase LuxS
(encoded by bbif_1299). One study suggested that BopA could improve adhesion of B.
bifidum S17 to intestinal epithelial cells (IECs) as a potential adhesin (Gleinser et al.,
2012). However, this result is questioned by another study that concluded that BopA does
not have a major role in adhesion (Kainulainen et al., 2013). LuxS is responsible for the
production of signal molecules, autoinducer-2 (AI-2) in B. bifidum S17 (Sun et al., 2014).
This raises the question whether S17 can perform LuxS/AI-2 dependent quorum sensing
(QS), an important subject for understanding bifidobacteria inter or cross-species signal
communication. Recently, many fluorescent proteins were expressed in B. bifidum S17 that
provides useful tools for in vivo studies (Grimm et al., 2014).
Along with these molecular studies, many physiological studies were conducted to
investigate potential probiotic properties of B. bifidum S17 as well. B. bifidum S17 does
not induce inflammatory events on HT-29 cells in vitro, but almost completely inhibits
LPS-induced NF-κB activation. Meanwhile, pre-treatment with B. bifidum S17
significantly inhibits transcriptional activation of gene IL-8, TNF-α, COX-2, and ICAM-1
in HT-29 cells challenged with LPS in the presence of 50 ml/l human milk (Riedel et al.,
2006a; Riedel et al., 2006b). These results indicate B. bifidum S17 might be a promising
candidate for probiotic intervention of chronic intestinal inflammation. Furthermore, in line
with above in vitro data, a study confirmed the anti- inflammatory capacity of B. bifidum
S17 for amelioration of murine colitis in vivo and this strain can prevent weight loss in
mice (Philippe et al., 2011). Meanwhile, B. bifidum S17 can adhere to three different IECs
9
better than other tested strains (Preising et al., 2010). This property is extremely attractive
when considering the usage of bifidobacteria as mucosal vaccination vehicles.
1.3 Expression systems for bifidobacteria
1.3.1 Advantages of bifidobacteria as hosts
Due to their probiotic properties, bifidobacteria are promising hosts for expression of
foreign genes for both research and application. Since the status of non-pathogenic or even
probiotic, they are highly acceptable for the production of pharmaceutical proteins (Kullen
and Klaenhammer 2000). Furthermore, the strict anaerobic growth makes them attractive
for cancer therapy as they were shown to specifically translocate to and actively replicate
in hypoxic regions of solid tumors (Yazawa et al., 2000; Fujimori 2006; Cronin et al.,
2010). Thus, there are already several studies using recombinant bifidobacteria as gene
delivery vectors for enzyme/pro-drug therapy (Nakamra et al., 2002; Hamaji et al., 2007;
Hidaka et al., 2007). Moreover, bifidobacteria can induce effective mucosal immunity.
Therefore, recombinant bifidobacteria may be used as live oral vaccines for prevention of
infectious diseases (Takata et al., 2006; Ma et al., 2011). There are other reports use
bifidobacteria as hosts for production of cytokines with the aim at enhancing their
functional properties (Reyes Escogido et al., 2007; Shkoporov et al., 2008).
1.3.2 Plasmids isolated from bifidobacteria
Bacterial expression systems are largely based on the isolation and construction of
plasmids. In 1982, the presence of plasmids in the genus Bifidobacterium was first reported
(Sgorbati et al., 1982). The existence and isolation of plasmids paved the way for further
molecular study of bifidobacteria as they are indispensable tools for molecular
manipulation. According to a recent review, 34 plasmids originated from Bifidobacterium
sp. have been completely sequenced (Sun et al., 2012). More than half of them were
isolated from B. longum, with a GC content ranging from 59.0% to 66.2%. Although many
plasmids were structurally characterized in bifidobacteria, their significance remains
largely unknown, as no obvious phenotypic traits associated with their presence. One
10
exception is a B. bifidum plasmid that was reported to be responsible for production of a
bacteriocin termed bifidocin B (Yildirim et al., 1999). Some of the native bifidobacterial
plasmids or their replicons were exploited for the construction of Escherichia coli (E. coli)
- Bifidobacterium sp. shuttle vectors aiming to overcome the lack of molecular tools in
bifidobacteria (Lee and O’Sullivan 2006).
The first characterized bifidobacterial plasmid was pMB1 isolated from B. longum. With a
size of 1 847 base pair (bp), it contains repA and repB genes (Matteuzzi et al., 1990; Rossi
et al., 1996). Its replicon was used in a number of shuttle vectors, which were successfully
transformed into B. adolescentis and B. longum strains (Li et al., 2003; Xu et al., 2007).
Another frequently used plasmid is pBC1 isolated from B. catenulatum, which encodes a
repB protein with high similarity to repB of pMB1 (Alvarez-Martin et al., 2007). pBC1
based plasmids were used in expression vectors and were able to replicate in B. breve, B.
pseudocatenulatum, and B. longum subsp. infantis strains (Cronin et al., 2008; Alvarez-
Martinet al., 2008; Ma et al., 2011). Both pMB1 and pBC1 replicate via theta replication
as no accumulation of single strand intermediate and containing corresponding theta type
replicase domain (PF03090) (Alvarez-Martin et al., 2007). Plasmid pTB6, pB44, and
pMG1, all isolated from B. longum strains, are widely used in shuttle vector construction as
well. pTB6 replicates in different strains of B. longum, B. animalis, and B. breve with
highest segregational stability and expression of target genes in B. breve (Hidaka et al.,
2007; Hamaji et al., 2007; Yamamoto et al., 2010). pB44 was used for expression of
secreted fibroblast growth factor (FGF-2) in B. breve UCC2003 and yielded lower
productivity compared to the pB80 replicon (Shkoporov et al., 2008). pMG1 was
exclusively used for expression of various target genes in its native host B. longum MG1
(Moon et al., 2005; Rhim et al., 2006; Park et al., 2008a). These plasmids may replicate by
a rolling circle mechanism (RCM) as single strand intermediates were observed for some
of them with close phylogenetic relationship between their replicases (O'Riordan and
Fitzgerald 1999; Park et al., 1999; Park et al., 2003; Park et al., 2008b).
1.3.3 Current study of gene expression in bifidobacteria
Using the above-mentioned plasmids and their replicons, a variety of E. coli -
Bifidobacterium sp. shuttle and expression vectors have been generated. Based on these
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vectors, heterologous expression of a variety of proteins has been achieved in different
strains of bifidobacteria (Sun et al., 2012). However, the genetic features that determine the
efficiency of expression have not been characterized in detail. The very few reports that
comment on expression levels use indirect measurements of expression by western blot or
functional analysis, suggesting that expression levels are very low.
1.3.4 Promoters
Promoters are DNA elements that constitute binding sites for the RNA polymerase (RNAP)
holoenzyme and transcriptional regulators or other regulatory elements thereby
initiating transcription of downstream genes (Ross et al., 1993). They are usually located
upstream of genes near the transcription start sites (TSS) (Estrem et al., 1999). Due to their
important role in initiation of transcription, promoters are considered as traffic lights in
biotechnology (Patek et al., 2013). Many studies have attempted to identify strong
constitutive promoters driving high- level expression of genes of interest in various
microorganisms including bifidobacteria (Nesvera et al., 2012; Wang et al., 2012).
Constitutive promoters are active in all circumstances and constantly drive transcription
without any regulatory mechanisms. For most biotechnological applications, stable and
efficient production of proteins and simple fermentation processes are considered desirable.
The majority promoters studied for driving gene expression in bifidobacteria are
summarized in table 1-3. The first promoter used for expression of target genes in
bifidobacteria is the promoter region upstream of the hup gene encoding histone- like
DNA-binding protein HU. The cloned DNA fragment containing the hup promoter region
was shown to drive constitutive expression in B. longum (Takeuchi et al., 2002) and was
used for expression of the E. coli cytosine deaminase (CD) for tumors therapy (Nakamura
et al., 2002). The same promoter region was used for expression of Salmonella flagellin
(FliC) in B. animalis ATCC 27536 and B. longum A-105 (Takata et al., 2006; Yamamoto
et al., 2010), and FGF-2 in B. breve UCC2003 (Shkoporov et al., 2008).
12
Table 1-3: Promoters applied for gene expression in bifidobacteria
Promoter Description Target gene Expression host
Phup Promoter of B. longum hup gene cd, fg f-2, IL-10, FliC B. longum,
B. breve,
B. animalis
Pgap Promoter of B. longum gap gene gusA, fgf-2, IL-10,
KatG, SOD
B. longum,
B. breve
PRPL Promoter of bacterial λ phage Endostatin B. adolescentis,
B. longum
P16S rRNA Promoter of B. longum 16S rRNA gusA,
Cholesterol oxidase
Beta-glucosidase
B. breve,
B. longum
B. bifidum
Pamy Promoter of B. longum amylase Pediocin PA -1, CfaB , LTB B. longum (infantis)
Phelp Consensus high expression Listerial
promoter
LuxABCD B. breve
PbetA Promoter of B. longum betA gene, bile
inducible
arabinofuranosidase B. longum,
B. breve,
B. adolescentis,
B. pseudocatenulatum
PbfeUOB Promoter of B. breve bfeUOB operon,
iron inducible
gusA B. breve
ParaBCD Promoter of E. coli araBCD operon,
arabinose inducible
Oxyntmodulin,
IL-10, IL-12
B. longum
Besides the hup promoter region, a number of other bifidobacterial promoters were also
studied. For example, the promoter region together with the signal sequence of the α-
amylase gene of B. adolescentis INT-57 was successfully used for production and secretion
of pediocin PA-1, a class IIa bacteriocin, by B. longum MG1 (Moon et al., 2005). The 16S
rRNA promoter region of B. longum MG1 was used for expression of the E. coli gusA in B.
breve NCIMB 8807 (Sangrador-Vegas et al., 2007), a cholesterol oxidase in B. longum
MG1 (Park et al., 2008a) and three potential beta-glucosidases in B. bifidum BGN4 (Kim
et al., 2012).
Another promoter region widely used in bifidobacteria is the DNA sequence upstream of
the glyceraldehyde-3-phosphate dehydrogenase gene (gap). Microarray data suggests that
13
gap is a housekeeping gene highly expressed in B. longum NCC2705 and thus its promoter
can drive high- level expression. The corresponding DNA sequence was used to drive the
gusA reporter in pMDY23 as a suitable promoter test system (Klijn et al., 2006). Using the
same promoter, levels of FGF-2 expression were achieved comparable to the hup promoter
(Shkoporov et al., 2008). Moreover, a recent paper used this promoter for driving
overexpression of two genes resulting in improved resistance to oxidative stress (Zuo et al.,
2014).
Besides all these originated from bifidobacteria themselves, other promoters (cloned from
other microorganisms or artificially designed) may break the restriction of species and
strength limit of self-driving. At present, two examples can be tracked (do not include
inducible promoter). One is the high expression listerial promoter (help), which was
constructed for high level expression of the luxABCDE operon in Listeria monocytogenes
(Riedel et al., 2007) and subsequently used for the same purpose in B. breve UCC2003
(Cronin et al., 2008). The other is λPRPL, a phage hybrid promoter, which was shown to
drive expression of endostatin in B. adolescentis (Li et al., 2003; Xu et al., 2007).
Promoters for regulated/inducible expression have been successfully established in E. coli
and a wide range of other bacteria, such as the T7 promoter (Tabor and Richardson 1985),
the Lac promoter (Duong et al., 2011), and the Tet promoter (Debowski et al., 2013).
However, there is no good system for regulated expression in bifidobacteria so far. In
many cases, promoters from bifidobacteria are recognized for driving recombinant protein
expression in E. coli (Rhim et al., 2006; Gleinser et al., 2012). This suggests that the
structural consensus of some promoters and their binding sites are consistent between
Bifidobacterium sp. and E. coli. However, most regulated promoters developed in E. coli
have not been effectively used in Gram-positive bacteria in general and bifidobacteria in
particular (Patek et al., 2013; Sun et al., 2012). One exception is the E. coli araBAD
system which was used for arabinose- inducible expression of several targets including
oxyntmodulin, interleukin 10 (IL-10), and IL-12 in B. longum (Long et al., 2010; Yao et al.,
2011; Yu et al., 2011). However, the use of this system in bifidobacteria is limited since
various species of bifidobacteria including B. bifidum S17 do not harbor genes for
arabinose transporters.
14
A starting point for the generation of inducible systems for bifidobacteria is the
identification of bile- and iron- inducible genes or operons. The first inducible gene
characterized in bifidobacteria was betA of B. longum NCC2705, which is highly up
regulated in the presence of bile (Ruiz et al., 2011). A DNA fragment of 469 bp
immediately upstream of betA was found to be the smallest unit that retains bile- inducible
expression. A second study, assayed the promoter region upstream of bfeUOB, a presumed
ferrous iron uptake system of B. breve UCC2003 and found a 2-5 folds increase of reporter
activity with multi-copy plasmid (Cronin et al., 2012). These promoters might be useful for
physiological study but be of limited potential on regulating heterologous gene expression
as they can be strongly affected by other environment factors and their inducible capacity
is very limited.
In summary, there are a number of promoters for expression in bifidobacteria. However, to
date none of these promoters was experimentally characterized and limited information is
available on conserved promoter elements (-10, -35 regions, and ribosome binding site
(RBS)) of bifidobacteria.
1.3.4 Secretion
To perform the intended function properly and to achieve maximal efficacy, it is essential
to ensure that recombinant proteins are expressed at the correct location of a bacterial cell.
In case of extracellular proteins, secretion is necessary for correct folding. Certain
extracellular components produced by probiotic bacteria may be responsible for some
benefits that they exerted on host (Sanchez et al., 2010). Moreover, secretion might be
advantageous in high- level production of recombinant proteins to avoid inclusion body
formation and facilitate purification from the supernatant omitting harvesting strategies
that involve cell disruption (Georgiou and Segatori 2005). Meanwhile, recombinant
bifidobacteria may increase the commercial value of functional foods by secreted
substances, e.g. secondary metabolites or primary proteins/enzymes (Guglielmetti et al.,
2013). For bifidobacteria tumor therapy, specifically, live cells with safe vectors can
actively translocate to hypoxia and necrotic regions. Therefore, the secretion of protein
drugs in these areas may significantly improve the therapeutic effect.
15
In an early study, a genomic library of B. breve UCC2003 strain was screened for
secretion signals using an export-specific nuclease reporter approach and a few putative
signal peptides were identified (MacConail et al., 2001). These identified bifidobacteria
signal peptides contain the AxA consensus cleavage site. Analysis of several
bifidobacterial genomes indicates that these bifidobacteria strains have most
components of the general secretary pathway (Sec) commonly found in Gram-positive
bacteria (Pallen et al., 2003). In contrast, components of the twin arginine translocation
(Tat) secretion machinery are absent in most of the analyzed strains
(http://www.genome.jp/kegg-bin/show_pathway).
Secretion of recombinant proteins by bifidobacteria was first described for fusion of the α-
amylase signal peptide of B. adolescentis INT-57 to the Pediococcus sp. class II
bacteriocin pediocin PA-1. The resulting construct was successfully expressed in B.
longum and found in the culture supernatant (Moon et al., 2005). Other examples are
pB44- and pB80-derived vectors carrying a Sec2 signal peptide-encoding sequence of B.
breve. Functionality of these constructs was tested using human fgf-2 gene (Shkoporov et
al., 2008). Secretion of FGF-2 was verified by Western blot using precipitated supernatants
of the B. breve engineered strains. A series of vectors were constructed using the signal
peptides of protein AmyB of B. breve and ApuB of B. adolescentis for expression and
secretion of recombinant human IL-10 (Khokhlova et al., 2010). Interestingly, the ApuB
signal peptide increased IL-10 secretion in culture medium compared to the Sec2 signal
peptide. Nevertheless, knowledge on protein secretion and secretion signal sequences in
bifidobacteria is still limited.
1.4 Bifidobacterial cancer therapy
A wide range of bacterial species/strains was tested for their antitumor potential, including
Caulobacter, Listeria, Clostridium, Salmonella, Escherichia, Bifidobacterium, Proteus,
and Streptococcus (Forbes 2010). Among them, Salmonella and Clostridium are the most
well studied bacteria for tumor therapy in different mouse models including breast,
pancreatic, prostate, and colon cancer. In theory, bacteria can be utilized for cancer therapy
by either their intrinsic toxicity or by production of antitumor reagents. Spores of
Clostridium sp. have been studied extensively as tumor targeting vectors with more or less
16
encouraging results (Fox et al., 1996; Agrawal et al., 2004; Maletzki et al., 2010). The
major problem of establishing bacteria as tumor therapeutics is safety issues. For some
strains of Salmonella typhimurium, high doses of bacteria are required for therapeutic
efficacy, which is associated with a high risk of systemic toxicity (Dang et al., 2001;
Palumbo et al., 2013). Thus, the use of genetically engineered, non-pathogenic bacteria as
delivery vehicles has come into focus as this approach can overcome the adverse effect.
One representative strain is a virulence attenuated Salmonella typhimurium VNP20009
(Luo et al., 2001). This strain had already been tested in clinical trials but shown limited
efficiency (Toso et al., 2002). Another group of bacteria that has been considered for
cancer therapy is bifidobacteria. These microorganisms have a very good safety record and
were shown can specifically target the hypoxic region of solid tumors in mice (Tangney
2010). Native bifidobacteria have a limited potential to induce tumor regression or inhibit
tumor growth (Kohwi et al., 1978). In consequence, genetic modification of bifidobacteria
is critical for potential tumor therapy (Baban et al., 2010).
One widely used approach for cancer therapy using bacteria as gene delivery vectors is
gene directed enzyme pro-drug therapy (GDEPT, Cheng et al., 2008). In this concept,
therapy is based on expression of an enzyme by a bacterial of viral delivery system
specifically in tumor tissue. This ensures that conversion of a pro-drug that is administered
to the patient systemically is confined to its intended site of action thereby limiting side
effects and increasing efficacy (Chen et al., 2004; Djeha et al., 2005; Mitchell and Minchin
2008; Warmann et al., 2009). Several enzyme/pro-drug systems were frequently reported.
These include cytosine deaminase/5-fluorocytosine (CD/5-FC, Huber et al., 1994), purine
nucleoside phosphorylase/6-Methylpurine-deoxyriboside (MePdR/PNP, Hughes et al.,
1995; Stritzker et al., 2008), β-glucuronidase/9-aminocamptothecin glucuronide (βG/9-
ACG, Cheng et al., 2008), nitroreductase/5-aziridin- l-yl-2,4-dinitrobenzamide
(NTR/CB1954, Barak et al., 2006; Vass et al., 2009); and herpes simplex virus thymidine
kinase/ganciclovir (HSV-TK/GCV, Immonen et al., 2004). Some of these systems have
been used for generation of recombinant bifidobacteria for tumor therapy (Yi et al., 2005;
Tang et al., 2009; Yin et al. , 2013). However, most of these genes are expressed at unknown
level and intracellularly. Thus, there is a need for further development of more efficient
bifidobacterial expression and secretion systems to enhance the therapeutic effect.
17
1.5 The aim of this study
The aim of this study was to establish a system for efficient expression and secretion of a
pro-drug converting enzyme and to use this system engineering a recombinant
Bifidobacterium sp. strain as a gene delivery vector for cancer therapy. This goal was
approached in three consecutive parts: (i) identification and characterization of a strong
constitutive promoter for high- level expression, (ii) screening of a sorting signal for
efficient secretion of proteins, and (iii) construction of a recombinant strain of B. bifidum
S17 that produces and secretes a pro-drug converting enzyme.
18
2 MATERIALS AND METHODS
2.1 Bacterial strains and plasmids
2.1.1 Bacterial strains
All bacterial strains used in this study were listed in appendices (Table 7-1). Storage and
propagation of these strains were conducted according to standard operation procedures.
For long term storage, 10 ml of a culture incubated for 16 h were harvested by
centrifugation and the bacterial pellet was resuspended in 0.5 ml fresh sterile medium,
mixed with an equal volume of 30% sterilized glycerol and store at -80°C. For short-term
storage, 0.5 ml of an overnight culture was mixed with 0.5 ml 30% glycerol and store at -
20°C for up to 3 months.
E. coli strains used in this study are E. coli DH5 and E. coli DH10B. Both strains were
routinely cultivated in Luria-Bertani (LB) broth (10 g/l peptone, 5 g/l yeast extract, 5 g/l
sodium chloride) or LB agar (additionally 12 g/l agar) at 37 °C. Medium was supplemented
with 100 μg/ml spectinomycin (Spc) when necessary. For liquid culture, glass tubes
containing 5 ml LB were agitated at 170 revolutions per minute (rpm); for solid culture, 9
cm agar plates were placed in an incubator.
Bifidobacteria were routinely grown by inoculating 100 μl of a glycerol stock into 10 ml
pre-boiled MRSc (Lactobacilli MRS medium (BD DifcoTM, USA) supplemented with 0.5
g/l L-cysteine HCl) and grown at 37°C overnight (16-24 h) under anaerobic conditions.
Anaerobic conditions were created by placing CO2 producing AnaeroGenTM sachets (Oxoid,
Germany) in a sealed jar (Anaerocult, Merck, Germany). For growth experiments,
overnight pre-cultures were used to inoculate fresh medium to an initial optical density at
600nm (OD600) of 0.1. Medium was supplemented with 100 μg/ml Spc where appropriate.
For solid media, 12 g/l agar was added.
19
2.1.2 Plasmids and oligonucleotides
All plasmids used in this study were listed in appendices (Table 7-2). Plasmids were
routinely maintained in and isolated from E. coli DH10B grown in LB broth with 100
μg/ml Spc. Pure plasmid DNA was dissolved in 10 mM Tris-HCl, pH8.5, and was stored at
-20°C.
All oligonucleotides used in this study were listed in appendices (Table 7-3). All primers
were designed with primer3plus (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi)
using public available genome sequences (www.ncbi.nlm.nih.gov), and synthesized a t high
purity salt free (HPSF) grade by a commercial supplier (Eurofins MWG, Germany). With
the exception of the GSPR primers, which were dissolved in nuclease- free water, all other
primers were dissolved in double distilled water (ddH2O) at a concentration of 100 μM
(stocks) and stored at -20°C. For frequently used primers, stocks were mixed to
corresponding primer pairs at a final concentration of 10 μM (each oligonucleotide) and
stored at 4°C.
2.2 Sample preparation and purification
2.2.1 Preparation of protein samples
Sample preparation for β-glucuronidase assays
Bacterial cells were harvested at different time points by centrifugation (4 226 relative
centrifuge force (rcf), 10 min, 4°C) of 10 ml cultures grown in MRSc containing 100 μg/ml
Spc under anaerobic condition. Cell pellets were washed with ddH2O twice and then
resuspended in 1 ml GUS buffer (50 mM phosphate sodium, pH7.0; 1 mM
ethylenediaminetetraacetic acid (EDTA); 5 mM dithiothreitol (DTT), 0.1% Triton X-100).
The suspensions were transferred to 2 ml screw cap tubes containing 250 μl glass beads
(diameter 0.1 mm) and bacteria were disrupted in a Precellys24 (Peqlab, Germany) during
2 cycles of 35 s at 6 500 rpm). Cell crude extract was prepared by collection of the
supernatant after centrifugation (17 949 rcf, 15 min, 4°C). The cell crude extract containing
glucuronidase was stored at 4°C and enzymatic assays were conducted within three days.
20
Total protein in the cell crude extract was measured by a 2-D Quant protein assay kit (see
2.6.2).
Sample preparation for phytase assays
For phytase assays, special attention is required to deplete potential phosphate
contaminations. All reagents and media were prepared in ultrapure water (18 MΩ·cm H2O,
Millipore, USA). Supernatants and cell pellets containing phytase protein were collected at
different time points by centrifugation (17 949 rcf, 5 min, 4°C) of recombinant B. bifidum
S17 strains grown in reinforced clostridia medium (RCM, Oxoid) containing 100 μg/ml
Spc under standard conditions. Bacterial pellets were rinsed twice in 0.2 M sodium citrate
buffer, pH5.5, and then resuspended in 500 μl of the same buffer for disruption in
Precellys24 as described above. Both supernatant (containing extracellular phytase) and
cell lysate (containing intracellular phytase) were stored at 4°C and phytase assays were
conducted within one week. Total protein in cell lysate was quantified by a BCA protein
assay kit (see 2.6.1).
Sample preparation for nitroreductase assays
For nitroreductase assays, supernatants and bacterial pellets of recombinant B. bifidum S17
strains were collected by centrifugation (17 949 rcf, 5 min, 4°C) after growth for 16 h in 10
ml RCM containing 100 μg/ml Spc under standard condition. Pellets were rinsed twice in
10 mM Tris-HCl buffer, pH7.0, and then resuspended in 500 μl same buffer disrupted as
described above. Both supernatant (containing extracellular nitroreductase) and cell lysate
(containing intracellular nitroreductase) were stored at 4°C and enzymatic assays were
conducted within three days. Total protein in cell lysate was quantified by a BCA protein
assay kit (see 2.6.2).
2.2.2 Preparation of nucleic acids
Preparation of genomic DNA
Template DNA for PCR cloning of target DNA fragments, such as promoter, reporter, and
SP, was prepared by centrifugation (14 549 rcf, 5 min) of 1 ml culture fluid. The pellet was
rinsed twice in 1 ml ddH2O, and then resuspended in 100 μl ddH2O. After heat treatment at
21
98°C for 10 min, cell debris was spin down (14 549 rcf for 5 min). Supernatant containing
genomic DNA was stored at -20°C for up to 3 month and used as template DNA for PCR.
Preparation of plasmid DNA
Plasmid DNA was prepared based on the principle of alkaline-sodium dodecyl sulfate
(SDS) lysis and ethanol precipitation, using E.Z.N.A. Plasmid Mini Kit I according to
manufacturer’s guideline (Omega, USA). 2 ml overnight culture of recombinant E. coli
DH10B was collected for plasmid isolation. Elution of DNA from the columns of the kit
was generally performed in 40 μl elution buffer (10 mM Tris-HCl, pH 8.5). After elution, 2
μl plasmids were resolved on 1% agarose to estimate its size and another 2 μl were used for
quantification (see 2.6.3). Plasmids were stored at -20°C for further application.
Preparation of total RNA
Total RNA of bifidobacteria was prepared as described previously after phenol-chloroform
extraction of proteins from crude extracts obtained by mechanical disruption as described
above (Gleinser et al., 2012). 10 ml of an overnight culture were mixed with 40 ml
quenching buffer (mixture of 16 ml 66.7 mM HEPES pH 6.5 and 24 ml 100% methanol) in
a 50 ml falcon tube and stored at -80°C for 30 min. Bacteria were harvested by
centrifugation (4 226 rcf, 20 min, 4°C) and resuspended in 200 μ l ddH2O. The suspension
was transferred into screw cap tubes filled with 0.5 ml glass beads (diameter 0.1 mm), 30
µl sodium acetate (3 M, pH 5.2), and 30 µl SDS (10% weight/volume (w/v)). Then, 400 µl
phenol and 100 µl chloroform were added. Cells were disrupted by spinning the tubes in a
Precellys24 for 2 cycles at 6500 rpm for 35 s. Aqueous and organic phases were formed by
centrifugation (17 949 rcf, 15min, 4°C). The aqueous phase was carefully transferred to a
sterile 1.5 ml Eppendorf (EP) tube and mixed with 1.5 volumes of 100% ethanol. The
mixture was incubated at -20°C over night for precipitation. The next day, precipitated
samples were centrifuged and washed with 70% ethanol (17 949 rcf, 30 min, 4°C). RNA
pellets were air dried by placing the tube under the airflow in a clean cabinet for 15-20 min.
Dried pellets were dissolved in 200 μ l 65°C pre-warmed nuclease-free water. Then,
dissolved RNA samples were treated with 200 U DNase I in 1 × DNase I reaction buffer at
37°C for 60 min (Thermo Scientific, Germany). DNase I was denatured by adding 15 µl 50
mM EDTA and incubation at 65°C for 10 min. 8 μ l of DNAse I-treated sample were
analyzed by agarose gel electrophoresis (see 2.3.4) and RNA concentration was quantified
22
(see 2.6.3). RNA samples were used immediately for downstream applications or stored at
-80°C until use.
2.2.3 Purification of nucleic acid samples
DNA fragments produced by PCR or enzymatic restriction were purified by using
NucleoSpin Extract II Kit according to the user manual (Macherey-Nagel, Germany). For
isolation of vector DNA or PCR products from agarose gels, the corresponding bands were
excised from the gel after electrophoresis, weighted, dissolved in two volumes (w/v) NTI
buffer at 50°C for 5 min, and then purified according to the Gel Extract protocol. To verify
successful purification of target DNA, samples were analyzed by agarose gel
electrophoresis (see 2.3.4) and quantified spectrophotometrically (see 2.6.3).
Ligation products
To achieve efficient transformation, ligation mixes were concentrated by ethanol
precipitation. Briefly, 10-20 μl of a ligation reaction were mixed with 10%
(volume/volume, v/v) 3 M sodium acetate, pH5.2 and precipitated by 2.5 volumes 98%
ethanol at -20°C for 1 h. Precipitated DNA was collected by centrifugation (14 549 rcf, 10
min) and washed twice with 70% ethanol. Then, DNA pellet was air dried by incubation at
37°C for 15 min and the dried pellet was dissolved in 2-5 μl 65°C pre-warmed ddH2O.
RNA
RNA samples were purified using RNeasy Mini Kit (Qiagen, Germany) according the
manufacturer’s instructions. Briefly, samples were mixed with an equal volume of 70%
ethanol and loaded onto an RNeasy Mini spin column by centrifugation. Columns were
rinsed twice with RPE buffer and RNA eluted in 30 µl of RNase-free H2O with incubation
for 10 min before centrifugation.
23
2.3 Manipulations of nucleic acids
2.3.1 Conventional polymerase chain reaction
Polymerase chain reaction (PCR) is a basic technique used for amplification of specific
DNA fragments (Bartlett et al., 2003). In this study, PCR was employed for general
cloning of target genes or screening of transformants for cloned inserts. Phusion DNA
polymerase was used for amplification according to supplier’s instruction (Thermo
Scientific, Germany). The composition of a typical PCR reaction was:
Template DNA
1-100 ng
Primer mixture 50-500 nM
dNTPs 200 nM
5 × HF buffer (containing 7.5mM MgCl2) 1 ×
Phusion DNA polymerase
0.02 U/μl
*DMSO 0-10%
*optional, DMSO was added only for amplification of complex templates
Thermo cycling was performed on a FlexCycler (Analytik Jena, Germany) by pre-
denaturation at 98°C for 2 min followed by 25-35 cycles of denaturation (95°C for 10 s),
annealing (50-70°C for 10 s), and elongation (68-72°C for 30-60 s), and a final elongation
(72°C for 5 min). PCR products were stored at 4°C until use. Annealing temperature was
optimized for each primer pair.
2.3.2 Splicing by overlap extension PCR
Splicing by overlap extension PCR (SOE-PCR) is used to fuse two (or more) PCR
products without the use of restriction enzymes and DNA ligase. In a first round, PCR is
performed to generate two gene segments with overlapping ends introduced by internal
primers with complementary 5’ ends. Then, these two PCR products are used together as
template DNA for the second round PCR to create a full- length chimerical product. A
standard procedure for SOE-PCR was described (Heckman and Pease 2007). In both
24
rounds of PCR, composition of the SOE-PCR reaction and parameters for cycling was
identical to conventional PCR (see 2.3.1) with optimization of annealing temperature.
2.3.3 Rapid amplification of cDNA ends
The protocol used for rapid amplification of cDNA 5’-end (5’-RACE) was described
previously (Scotto-Lavino, et al., 2006). The 2-Generation RACE Kit (Roche, Switzerland),
which is based on this principle as shown in figure 2-1 (Fig. 2-1) was used. Briefly, first-
strand cDNA was synthesized from 2 μg total RNA using a gene-specific primer (GSPR1).
Synthesized single strand cDNA was purified with a High-Pure PCR Clean-up Kit (Roche,
Switzerland) before appending a homopolymeric tail using terminal deoxynucleotide
transferase (TdT) and deoxyadenosine triphosphate (dATP). The tailed cDNA was directly
amplified by PCR using the oligo dT-anchor primer and the target-specific primer GSPR3.
A second round of PCR was conducted with anchor primer and the target-specific primer
GSPR4 to increase specificity. The PCR products were resolved on 1.5 % (w/v) agarose
gel by electrophoresis. Specific target band was cut out of the ge l and purified from the
agarose gel as described above. Isolated DNA fragments were cloned into pJET1.2
(Thermo Scientific, Germany) and sequenced with pJet1_PF by Sanger sequencing (GACT
biotech, Germany).
25
Fig. 2-1: Schematic representation of the procedure for identification of transcriptional start sites by
5’-RACE including reverse transcription, cDNA purification, poly A appending, two rounds of PCR,
and blunt end cloning.
2.3.4 Agarose gel electrophoresis
Agarose gel electrophoresis is used to analyze DNA molecules for detection, size
determination, or purification. In this study, agarose concentrations of 1-3 % (w/v) were
used for resolving nucleic acid fragments with different sizes. Briefly, 1-3% agarose was
dissolved in 1× TAE buffer (40 mM Tris-HCl, 10 mM acetic acid, 1 mM EDTA, pH 8.0)
by heating in microwave. Gels were casted using standard electrophoresis apparatus
(Peqlab, Germany). Samples were mixed with 6 × loading dye (Thermo Scientific,
Germany) and loaded to the slots on gel. Electrophoresis was carried out at constant
voltage (100 V) for 30-60 min. When bromophenol blue in loading dye had migrated to
approximate 0.5 mm from the bottom of the gel, electrophoresis was terminated and
agarose gel was stained for 5-10 min in ethidium bromide staining solution (1 µg/ml in
dH2O). Gels were rinsed with distilled water (dH2O) to remove excess ethidium bromide
and captured under exposure to UV light (302 nm) with a photo documentation system
(MWG-Biotech, Germany). Under these conditions, DNA stained by ethidium bromide
26
produces bright orange fluorescence. For determination of the size of DNA fragments,
GeneRulerTM 1 kb DNA Ladder or 50 bp DNA Ladder (Thermo Scientific, Germany) was
run in a separate lane on every gel.
2.3.5 DNA restriction and ligation
DNA restriction is a method to produce complementary DNA ends between that
effectively ligation can be achieved. All restriction enzymes were purchased from Thermo
Scientific, Germany. In this study, DNA restriction was performed using two different
restriction enzymes simultaneously. For each combination of enzymes, optimal buffers
were selected according to the recommendation of the supplier
(http://www.thermoscientificbio.com/webtools/doubledigest/; Thermo Scientific,
Germany). Digestion was carried out at 37°C for 3-20 h in a total volume of 40 µl in sterile
PCR tubes. Typical digestion reaction was composed of:
DNA sample (vector or insert)
1-2 µg
Restriction enzyme 1 10 U
Restriction enzyme 2 10 U
Recommended compatible buffer
1 ×
Digested insert DNA fragments were purified using NucleoSpin II columns (Machery-
Nagel, Germany) according to the PCR Extract protocol. Digested Vector DNA was
purified from agarose gels. All recovered DNA was analyzed by agarose gel
electrophoresis. For ligation, vector and insert DNA were mixed at molar ratios between 1 :
3 and 1 : 10 in a total volume of 10-20 µl containing 5 U T4 DNA ligase and 1 × T4 DNA
ligase buffer (Thermo Scientific, Germany). For successful ligation, a minimum of 0.5
pmol insert (calculated, 660 pg/pmol) was always used. Ligation reaction was carried out
by incubation at 22°C for 15-60 min.
2.3.6 Transformation of bacteria
Transformation of E. coli DH10B
27
Competent cell of E. coli DH10B was prepared as described (Gonzales et al., 2013). E. coli
DH10B was transformed by electroporation of 50 µl competent cells and 2 µl ligation
mixtures in an ice-cold electroporation cuvette (electrode distance 1mm; Peqlab, Germany).
After incubation for 5 min on ice, electroporation was performed in a Gene Pulser XcellTM
system (Bio-Rad, Germany) with following settings: 1 500 V, 200 Ω, and 25 µF.
Immediately after the pulse, 950 µl ice cold LB broth were added followed by incubation
on ice for 2 min. Then, cell suspension was transferred into a sterile 1.5 ml EP tube and
incubated for 60 min at 37°C with agitation (150 rpm). Finally, bacteria were collected by
centrifugation and spread on two LB agar (corresponding to 100 and 900 µl of regenerated
bacterial suspension) supplemented with the appropriate antibiotic and incubated overnight
at 37°C aerobically.
Transformation of B. bifidum S17
For efficient transformation, B. bifidum S17 competent cells were always prepared freshly
as described elsewhere (Serafini et al., 2012). Briefly, an overnight pre-culture was used
to inoculate 100 ml pre-warmed MRSc supplemented with 16 % (w/v) Actilight® fructo-
oligosaccharides (Beghin Meiji, France) to an initial OD600 of 0.1. Cultures were grown
under standard conditions (anaerobic, 37°C) for 6-8 h to an OD600 of ~ 0.5. Bacteria were
harvested by centrifugation (4°C, 4 226 rcf, 10 min) and washed three times with ice cold
citrate sucrose buffer (CSB, 1 mM citric acid, 0.5 M sucrose, pH 5.8). After the final
washing step, the pellet was resuspended in 1 ml CSB and used immediately as competent
cells. An aliquot of 100 µl of competent cells were mixed with 2 µl plasmid (100-500 ng)
in an ice-cold electroporation cuvette (1 mm electrode distance; Peqlab, Germany) and
incubated on ice for 5-10 min for transformation. Bacteria and plasmid mixture were
pulsed in a Gene Pulser XcellTM system with the following settings: 1 750 V, 200 Ω, 25
µF. After the pulse, bacteria was resuspended in 900 µl ice cold RCM broth immediately
and kept on ice for 2 min. The bacterial suspension was transferred to a sterile 1.5 ml EP
tube and incubated for 2.5-3 h at 37°C under anaerobic condition. Regenerated bacteria
were centrifuged (14 549 rcf, 2 min), pellets resuspended in two aliquots corresponding to
100 and 900 µl of the regenerated suspension were spread on two RCM agar plates
supplemented with 100 µg/ml Spc and incubated for 48-72 h at 37 °C under anaerobic
conditions.
28
2.4 Enzymatic assays
2.4.1 β-glucuronidase assay
Promoter activities were assayed using the gusA reporter gene of pMDY23 (Klijn et al.,
2006). The method for determination of GusA β-glucuronidase activity is described
elsewhere (Grimm et al., 2014). Briefly, bacterial crude extract corresponding to 1-5 μg
total protein were mixed with 25 μL 4-Methylumbelliferyl-β-D-glucuronide (4-MUG)
substrate solution (5 mM stock) and brought to a total volume of 100 μl GUS buffer ( 50
mM disodium hydrogen phosphate pH 7, 1 mM EDTA, 0.1% Triton X-100, 5 mM
dithiothreitol (DTT)) in the wells on a 96-well plate. After incubation for 30 min at 37°C,
the reaction was stopped and 5 μl transferred into another 96 well plate containing 195 μl
stop buffer (0.2 M Sodium carbonate in ddH2O, pH 9.5). Fluorescence of the product 4-
methylumbelliferone (4-MU) was measured in triplicate using an Infinite M200 multimode
microplate reader (Tecan, Switzerland) with excitation at 388 nm and emission at 480 nm.
The β-glucuronidase activity was defined as fluorescence units per microgram protein
(FU/μg). B. bifidum S17 harboring the promoter- less pMDY23 plasmid was included as a
negative control.
2.4.2 Phytase assay
Phytase assay was adapted from the Phytex method described elsewhere (Kim and Lei
2005). Phytase can catalyze the release of inorganic phosphate from phytate. Free
phosphate reacts with ammonium molybdate and forms stable blue complex, which has a
specific absorbance. Briefly, 100 μl sample (supernatant or crude extract) were incubated
at 37°C for 5 min and then mixed with 100 μl 10.8 mM sodium phytate (50% phytic acid
diluted in 0.2 M sodium citrate buffer, Sigma, Switzerland). The reaction was carried out at
37°C for 15 min, and then stopped by adding 200 μl 15% tricholoroacetic acid (TCA).
After centrifugation at 14549 rcf for 2min, an aliquot of 20 μl was mixed with 480 μl 18
MΩ·cm H2O and 500 μl color solution (mix of 1M sulfuric acid, 2.5% (w/v) ammonium
molybdate in ddH2O, and 10% (w/v) ascorbic acid in ddH2O at a ratio of 3:1:1). The
mixture was incubated at 50°C for 15 min, and 100 μl were transferred to a transparent 96-
29
well plate (Thermo Scientific, Germany). Absorbance at 820 nm was measured in triplicate
using an Infinite M200 multimode microplate reader (Tecan, Switzerland). Phytase activity
was calculated according to a standard curve produced by a serial dilution of 9 mM
potassium dihydrogen phosphate in aqueous solution. One phytase unit (FTU) was defined
as the amount of enzyme that catalyzes the release of 1 μmol of inorganic phosphate per
minute from 5.4 mM sodium phytate under above conditions at 37°C. Enzyme activities
were expressed as FTU/ml in supernatant and FTU/mg protein in cell lysate.
2.4.3 Nitroreductase assay
Nitroredutase assay was performed with CB1954 as substrate and β-nicotinamide adenine
dinucleotide reduced (NADH) as cofactor (Sigma, Switzerland), as described elsewhere
(Race et al., 2007) with minor modifications. In brief, NADH was dissolved in 0.01N
sodium hydroxide to give a stock concentration of 5 mM and prevented from light.
CB1954 was dissolved in pure dimethylsulfoxide (DMSO, Merck, Germany) at a stock
concentration of 10 mM. Cofactor and substrate stocks were stored at 4°C and used within
one week. In a 1 ml reaction, 100 μl NADH stock (final 500 μM) and 10μl CB1954 stock
(final 100 μM) were mixed with 100 μl 100 mM Tris-HCl pH7.0 (final 10 mM)). Reaction
was started by adding 100 μl crude extract or 500 μl spent culture supernatant. Volumes
were brought to 1 ml with ddH2O and reactions were carried out for 10 min at room
temperature. Then absorbance was measured at 340 nm in a plastic cuvette with 10 mm
light path (Thermo Scientific, Germany) using a Nanophotometer (Implen, Germany). One
NTR unit (U) was defined as the amount of enzyme that catalyzes the conversion of 1
μmol NADH to NAD per minute under the described conditions. Enzyme activities were
expressed as mU/ml in culture supernatants or mU/mg protein in crude extracts.
2.5 Growth related assays
2.5.1 Bacterial growth experiments
Cell growth was monitored by reading OD600 spectrophotometrically using either an
Infinite M200 multimode microplate reader (Tecan, Switzerland) with 200 μl culture fluids
30
in a transparent 96-well plates (Nuc, Thermo Scientific, Germany) or a Nanophotometer
(Implen, Germany) with 1mL 1:10 diluted cultures in a plastic cuvette with 10 mm light
path (Thermo Scientific, Germany).
2.5.2 Phytate degradation assay
As a first indication for phytase secretion of recombinant B. bifidum S17 strains, a phytate
degradation assay was employed. Strains were cultured on a solid phytase screen medium
(PSM) using calcium phytate as substrate (Quan et al., 2001). For the PSM medium, RCM
agar containing 100 μg/ml Spc was supplemented with 0.5% calcium phytate. A defined
volume of 2 μl of the strains to be tested was spotted on the PSM agar and incubated
anaerobically at 37°C for 72 h in a sealed jar. Ability of recombinant strains to hydrolyze
calcium phytate is indicated by clear zones on the agar surrounding bacterial colonies.
2.5.3 NTR/CB1954 inhibition assay
An initial screen for expression of ChrR6 by recombinant B. bifidum S17 was performed
by a growth inhibition assay. Strains expressing NTR can convert CB1954 into the
cytotoxic drug, which inhibits cell growth. Briefly, recombinant bifidobacteria were grown
in 10 ml RCM broth containing 100 μg/ml Spc and different concentrations of CB1954.
After 24 h anaerobic incubation at 37°C, OD600 was assayed and compared. NTR
expressing strains showed significantly lower final OD600.
2.6 Quantification
2.6.1 BCA protein assay
For quantification of the protein in crude extracts used for phytase and NTR assays, BCA
Protein Assay Kit (Thermo Scientific, Germany) was used according to the manufacturer’s
instructions. The assay is based on bicinchoninic acid (BCA) for the colorimetric
quantification of total protein. It combines the reduction of Cu+2 by protein in alkaline
31
solution with colorimetric detection of Cu+1 using a reagent containing BCA. The purple
reaction product of this assay is formed by chelating of two molecules of BCA with one
Cu+1 ion. The absorbance of this water-soluble product directly correlates to protein
concentrations over a broad working range (linear range 20-2000 μg/ml). The method was
adapted small sample volumes (10-25 μl). Absorbance at 562 nm was measured for each
sample in triplicate using an Infinite M200 multimode microplate reader (Tecan,
Switzerland).
2.6.2 2-D Quant protein assay
For quantification of the protein in crude extracts used for glucuronidase assay, the 2-D
Quant Kit (GE Healthcare, Germany) was used according to the manufacturer’s
instructions. This kit is developed primarily to accurately quantify protein samples
prepared for two-dimensional electrophoresis as well as any sample that contains
substances interfering with common protein quantification. The procedure uses a
combination of 500 μl patented precipitant and 500 μl co-precipitant to precipitate up to 50
μg in a total volume of 1-50 μl protein sample while leaving interfering contaminants in
solution. Precipitated protein is then collected by centrifugation and resuspended in 100 μl
alkaline copper solution. 1 ml colorimetric agent that reacts with unbound cupric ions is
added. The color density is inversely related to the concentration of protein in the sample.
Since cupric ions bind to the polypeptide backbones of any protein without dependence on
amino acid composition, protein concentration can be accurately estimated by comparison
to a bovine serum albumin (BSA) standard curve. Absorbance was measured for each
sample in duplicate using an Infinite M200 multimode microplate reader at 480 nm (Tecan,
Switzerland).
2.6.3 DNA/RNA quantification
Nucleic acids were quantified by measuring absorbance at 230, 260, and 280 nm of 2 μl
samples spectrophotometrically using a quartz-based NanoQuant Plate and an Infinite
M200 multimode microplate reader (Tecan, Switzerland) and the DNA or RNA mode of
the Tecan i-control™ Software. Wells on the plate were rinsed for three times with ddH2O
32
every time before loading new samples or blanks. Blanking was always conducted for each
well individually twice.
2.7 Bioinformatic analysis
The prediction of core promoter regions of hup (bl_1798), 16S rRNA (blr_01), gap
(bbif_0612) and luxS (bbif_1299) was predicted using the BPROM online software tool
(http://linux1.softberry.com/BPROM) and Neural Network Promoter Prediction (NNPP,
www.fruitfly .org/seq_tools /promoter .html ) using complete inter-region sequences
upstream of the respective genes (for full sequences see appendices 7.4). Potential SPs
were identified in the N terminal 60 amino acid residues of deduced protein sequences
using SignalP4.0 (www.cbs.dtu.dk/services/SignalP/). Corresponding DNA sequences of
predicted SPs were listed in appendices 7.7. Proteins with predicted SPs were searched in
ePSORTb database for their annotation to check whether they are experimentally
confirmed as extracellular proteins. The 8 signal peptides cloned in this study were also
analyzed by TatP (http://www.cbs.dtu.dk/services/TatP/) for potential twin arginine
translocation motifs. To confirm correct open reading frames and to rule out mutations all
cloned constructs were sequenced and obtained sequences aligned online with the Basic
Local Alignment Search Tool (BLAST, www.ncbi.nlm.nih.gov/BLAST/). CLC workbench
(CLC Bio, Denmark) was used for local alignment of 5’-RACE sequences and
representative Pgap sequences (see appendices 7.5). Codon optimization for gene synthesis
was performed according to B. bifidum S17 codon usage table provided by a commercial
service provider (Eurofins MWG, Germany, see appendices 7.8).
33
3 RESULTS
3.1 Promoter prediction and cloning
The range of promoters tested included the promoter regions upstream of the gap gene
(bbif_0612) encoding glyceraldehyde-3-phosphate dehydrogenase (Pgap) and the luxS
(bbif_1299) gene for the methylthioadenosine/S-adenosyl-homocysteine (MTA/SAH)
nucleosidase LuxS (PluxS) of B. bifidum S17. Furthermore, the sequence upstream of the
gene bl_1798 for histone-like HU protein (Phup) and the sequence upstream (P16S rRNA) of
16S rRNA gene (blr_01) of B. longum NCC2705 were included. The intergenic regions
upstream of these four genes (for complete sequences see appendices 7.4) were used to
predict potential bacterial promoters using BPROM, which was developed for bacterial
promoter prediction based on the recognition of E. coli σ70 type sigma factors. This
analysis yielded single promoters with relatively high linear discrimination function (LDF)
in front of gap, hup, and 16S rRNA when the default unchangeable threshold value equals
0.2. By contrast, no predicted promoter was found in front of luxS.
Table 3-1: Predicted promoters core regions by BPROM
Gene *Core sequence (50bp, 5’3’) LDF Potential TF
gap GTACAGACATATTTGTTAGCGTTAACGAAATA
TGGCCGTTTTATGCTCAA
3.37 rpoS17,
soxS
hup ACCCTTATAAAACGCGGGTTTTCGCAGAAACA
TGCGCTAGTATCATTGAT
5.18 rpoD16,
rpoD15
16S rRNA TCGTCAATTTTTGTTTTGAGA GTCATCTATTCG
GATGCTTTTCATGAAGT
2.40 rpoD15,
rpoD17
*Red nucleotides are proposed -35 regions; blue nucleotides are proposed -10 regions. LDF: linear
discrimination function; TF, transcription factor
The promoter elements of the gap and hup promoters predicted by BPROM are not ideal
because the spacer length between predicted -10 and -35 sequences was considerably
shorter (11 and 13 bp, respectively) than in consensus promoter (17 ± 1 bp; Lisser and
34
Margalit 1993). Since expression of LuxS has been reported in different bifidobacteria
(Yuan et al., 2008; Sun et al., 2014), further in silico promoter predictions were performed
using Neural Network Promoter Prediction (NNPP). This prediction produced several
possible core promoter regions with potential TSS for gap, hup, and luxS (Table 3-2).
Table 3-2: Some predicted core regions of promoters by NNPP
Gene Core promoter sequences (50bp, 5’3’) Scores
gap TATTTGTTAGCGTTAACGAAATATGGCCGTTTTATGCTCAAAGCAAGCGC
TCGCATCGAATCGCCGCAGGCTGTACAGACATATTTGTTA GCGTTAACGA
ACACCGTTGCTCTAGTACAGACGGCGCATTACAGTAGACACTGTTGGTAA
0.92
0.46
0.22
luxS TATAGCCAAGAATGCGGCGAGACGCGCGCGATAGTGAGACAATGGTCGAT
ACCGGACTCGCCTCGTGCCGTATATGCCGTATATGAGGAA CGCCTATAGC
AGAATGCGGCGAGACGCGCGCGATAGTGAGACAATGGTCGATGGAATGCC
0.55
0.23
0.20
hup CATGAAGTGGCTTGACAAGCATAATCTTGTCTGATTCGTCTATTTTCATA
CGTCTATTTTCATACCCCCTTCGGGGAAATAGATGTGAAAACCCTTATAA
GCTTGACAAGCATAATCTTGTCTGATTCGTCTATTTTCATA CCCCCTTCG
0.89
0.78
0.73
Potential TSS is underlined
In order to probe the functionality of these possible promoters, the reporter vector
pMDY23 was used (Fig. 3-1). PluxS and Pgap were amplified from the genome of B. bifidum
S17 and Phup and P16S rRNA were amplified from the genome of B. longum NCC2705 by
PCR introducing XhoI and BglII restriction sites. These restriction sites were used for
directional cloning of the promoter fragments in front of the gusA reporter gene in
pMDY23 as exact transcriptional fusions. Resulting plasmids were verified for absence of
any mutations and transformed into B. bifidum S17.
35
Fig. 3-1: Schematic representation of promoter fragments cloned into pMDY23. Four promoter
regions are amplified py PCR and cloned using BglII and XhoI restriction sites into the reporter vector
pMDY23 in front of gusA (repA and repB: bi fidobacterial origin of replication; spc: s pectinomycin
resistance gene).
3.2 Promoter activities in B. bifidum S17
The transcriptional activities of the four putative promoters were assayed by β-
glucuronidase activities in crude extracts of cloned B. bifidum S17 derivatives containing
the respective plasmids. Preliminary time course experiments reveal measurable β-
glucuronidase activity in B. bifidum S17/pMDY23-Pgap and B. bifidum S17/pMDY23-Phup
grown in MRSc with the highest level in early exponential phases, i.e. 8 h growth (Fig. 3-
2). Transcriptional activity of Pgap was significantly higher than Phup. Unexpectedly, no β-
glucuronidase activity above background levels (i.e. those observed with the empty
pMDY23 plasmid) could be detected with the luxS and 16S rRNA promoters. Monitor of
OD600 shown there is no difference in growth of all recombinant B. bifidum S17 strains
excluding any effect of growth on β-glucuronidase activity (data unshown).
36
Fig. 3-2: β-glucuronidase activity of crude extracts of recombinant B. bifidum S17 strains containing
di fferent promoter probe constructs derived from pMDY23. Samples were prepared from bacteria
grown 8 h in MRSc, i.e. in exponential growth phase. Values are fluorescence units per µg protein
(FU/µg) and are mean ± standard deviation of three independent cultures each measured in triplicate.
Since the promoter region of the gap gene of B. bifidum S17 yielded highest transcriptional
activity among these promoters tested. This region was used in all further experiments and
analysis.
3.3 Analysis of Pgap
3.3.1 Bioinformatic Analysis of Pgap
Complete sequence upstream of gap gene was shown in Fig. 3-3. As predicted in Table 3-1
and 3-2, and marked in this figure, potential -35 region, -10 region, and TSS were proposed
by online software. In silico, analysis suggested a putative bacterial promoter in the cloned
gap promoter region with a predicted TSS 114 bp upstream of the gap ATG start codon.
The 5’-untranslated region upstream of the ATG start codon of gap contains a putative
RBS as well.
37
Fig. 3-3 : Sequence of the DNA fragment upstream of gap of B. bifidum S17 cloned for reporter analysis.
The sequence contains a putative bacterial promoter predicted independently with two software tools.
The predicted transcription start site (position 1), -10, and -35 regions, as well as the putative
ribosome-binding site are indicated by black boxes.
During initiation of translation, the RBS interacts with a sequence motif in the 3’-end of
the 16S rRNA termed anti-Shine-Dalgarno sequence (anti-SD). The anti-SD of B. bifidum
S17 is highly complementary with only one mismatched base pair to the anti-SD (Fig. 3-4)
indicative of efficient translation initiation.
Fig. 3-4: Base pairing of the putative RBS of the Pgap promoter (RBS Pgap) with the anti-Shine-Dalgarno
sequence (Anti -SD) at the 3’-end of the 16S rRNA of B. bifidum S17
3.3.2 Experimental determination of TSS
To confirm the TSS of the gap gene of B. bifidum S17 experimentally, 5’-RACE was
conducted using RNA samples prepared from two independent cultures of B. bifidum S17
grown on MRSc to late exponential growth phase. For each RNA sample, inserts of two
randomly selected, positive E. coli clones containing pJet1.2-derivatives were sequenced.
Alignment of all four inserts equivocally indicated a thymidine (T) residue 62 bp upstream
of the ATG start codon of the gap open reading frame as TSS (Fig. 3-5), which is
considerable different to the predicted TSS (Table 3-2 and Fig. 3-3).
38
Fig. 3-5: Sequences of four inserts obtained from 5’-RACE experiments performed on two independent
RNA samples were aligned to the genomic region of the putative Pgap promoter. The experimentally
determined transcriptional start site (TSS), presumable ribosome binding site (RBS), and the ATG
start codon (Start) of the gap mRNA are indicated by grey boxes. The gene s pecific primer used for the
amplification of the gap transcripts is marked by a black box. Sequences of the oligo dT16 anchor
primer upstream of the TSS and vector sequences up and downstream of the two primers were clipped.
3.3.3 Alignment of representatives
Upon visual analysis, no sequences motifs with obvious homology to the consensus -10
(TATAAT) or -35 (TTGACA) sequences of σA-dependent promoters of Bacillus subtilis,
i.e. the prototype housekeeping promoter of Gram-positive bacteria, could be identified. In
order to identify potential -35 and -10 regions, the Pgap sequence of B. bifidum S17 was
aligned to the DNA sequences upstream of the gap genes of five other Bifidobacterium sp..
The results suggest a -35 region with the sequence TTGCTC, a spacer of 17 bases, and a -
10 region consisting of the nucleotides TACAGT for Pgap of B. bifidum S17 (Fig. 3-6).
39
Fig. 3-6: Alignment of the DNA sequences immediately upstream of the gap genes from 5
representative strains of Bifidobacterium. The degree of conservation is indicated on a grey scale with
black indicating highest conservation and conserved residues are shown as sequence logo. Presumable
or determined transcription start sites (TSS), corres ponding -10 and -35 regions are indicated by black
boxes. For orientation, the predicted TSS of bbif_0612 is also indicated. B. longum E18 (blong_1334), B.
breve UCC2003 (bbr_1233), B. adolescentis ATCC 15703 (bad_1079), B. dentium Bd1 (bdp_1514), B.
lactis ATCC 27673 (blac_0592), B. bifidum S17 (bbif_0612)
The consensus -35 and -10 sequences of the analyzed gap promoters of bifidobacteria are
TTGCCN and TANAGT, respectively, with a spacer of 17-19 bases (Fig. 3-6). Compared
to the canonical -35 (TTGACA) and -10 (TATAAT) regions of σ70- and σA-dependent
promoters of E. coli (Lisser and Margalit 1993) and Bacillus subtilis (Helmann 1995), the
bifidobacterial Pgap consensus -35 and -10 sequences each differ in two nucleotides (Fig. 3-
7).
Fig. 3-7: Alignment of the consensus sequences of bifidobacterial gap promoters and σA
-dependent
housekeeping promoters of Gram-positive bacteria.
40
3.4 Analysis of protein secretion in B. bifidum S17
As a basis for protein secretion in bifidobacteria, the genome sequences of two strains
frequently used in our labs were analyzed for genes of protein export systems. A number of
homologues of the Sec-dependent protein secretion are present in both B. bifidum S17 and
B. longum E18 (Table 3-3). An integral membrane complex composed by SecY, SecE and
SecG is the core component of Sec translocon. YajC plays a part in stabilizing and
regulating secretion through the SecYEG via SecA, which is an ATPase supplying energy.
This pathway is responsible for the secretion of the majority of extracellular proteins in
both Gram-negative and Gram-positive bacteria (Pugsley 1993; van Roosmalen et al.,
2004). Additionally, a complete twin arginine translocon operon was found in the genome
of B. longum E18 (BLONG_0088: TatB; BLONG_0089: TatC; BLONG_0090: TatA),
suggesting that this strain can secrete folded protein by the twin-arginine translocation (Tat)
pathway.
Table 3-3: Some components of Sec translocon in B. bifidum S17 and B. longum E18
Sec components B. bifidumS17 B. longum E18
SecA BBIF_1223 BLONG_1273
SecY BBIF_1484 BLONG_1793
SecE BBIF_0279 BLONG_2073
SecG BBIF_0980 BLONG_1190
YajC BBIF_0957 BLONG_1111
On the genomes of B. bifidum S17 and B. longum E18, the type I SPases (LepB) are
encoded by bbif_0379 and blong_1040 that recognize type I SP. BBIF_0514 and
BLONG_1429 are type II SPase (LspA) that response for cleavage of lipoprotein. The type
III SPs are prepilin- like SPs, that differ from the former two SPs as they do not contain any
hydrophobic regions. They also called type IV prepilin SP because they are recognized by
type IV prepilin peptidases. Type IV pili structure and function had been studied in B.
breve UCC2003 (Turroni et al., 2013), and the corresponding SPase annotated in the
genomes of many bifidobacteria strains. BBIF_0835 (annotated as hypothetical protein in
B. bifidum S17) has 58% identity to B. longum JDM301 type IV prepilin peptidase
(BLJ_0873).
41
3.5 Prediction of SPs in B. bifidum S17
In theory, proteins with SPs can be composed by three main groups: extracellular proteins,
cell wall proteins, and some proteins may have multiple localization sites. All proteins
containing SPs are potential candidates for cloning of a SP used for sorting protein
secretion. To screen reliable SPs, a complete list of proteins with SPs of B. bifidum S17
was compiled by in silico analysis using cPSORTb database (Table 3-4). Meanwhile, the
most N terminal 60 amino acids of these proteins were analyzed one by one with SignalP,
which yielded either positive SPs with potential cleavage sites or negative results.
Furthermore, both cPSORTb and SignalP positive proteins were searched in ePSORTb
database. Proteins existed in ePSORTb were underlined in this table, which had been
confirmed as extracellular proteins in E. coli.
42
Table 3-4: List of proteins with signal peptide in B. bifidum S17 predicted by cPSORTb
Gene tag Annotation E-Score SignalP 4.1 (cleavage site) D-score
extracellular proteins
BBIF_0022 Alpha-L-arabinofuranosidase 9.26 pos. 36 - 37: AYA-AD 0.718
BBIF_0048 1,4-beta-N-acetylmuramidase 9.98 pos. 30 - 31: AYA-LQ 0.732
BBIF_0246 peptidylprolylisomerase, FKBP-type 9.26 pos. 36 - 37: SGS-SS 0.556
BBIF_0285 hypothetical protein containing multip le
sugar recognition domains
9.76 pos. 27 - 28: ASA-AP 0.886
BBIF_0483 conserved protein with the pectin lyase
fold domain
9.13 pos. 30 - 31: AAA-ST 0.723
BBIF_0507 beta-galactosidaseBbgIII 7.74 pos. 32 - 33: AWA-VE 0.665
BBIF_1193 serine/cysteine peptidase 9.13 pos. 30 - 31: ASA-SP 0.836
BBIF_1317 alpha-L-fucosidase 9.97 pos. 37 - 38: AVA-AN 0.714
BBIF_1380 hypothetical protein BBIF_1380 9.13 pos. 27 - 28: ANA-AD 0.831
BBIF_1426 hypothetical protein with NlpC/P60
domain
9.73 pos. 25 - 26: AAA-AV 0.633
BBIF_1461 beta-N-acetylglucosamin idase 9.76 pos. 29 - 30: AYA-AG 0.76
BBIF_1576 beta-N-acetylglucosamin idase 9.98 pos. 34 - 35: AMA-AS 0.68
BBIF_1732 sialidase 9.97 pos. 39 - 40: ANA-AD 0.792
BBIF_1734 sialidase 9.98 pos. 35 - 36: ASA-AS 0.862
BBIF_1740 Alkaline phosphatase 9.73 pos. 29 - 30: AFA-AS 0.776
Cell wall proteins
BBIF_0215 cell wall protein containing Ig-like
domains (group2 - 3)
0.61 pos. 41 - 42: AAA-SD 0.681
BBIF_0301 hypothetical protein containing von
Willebr- factor type A domain
0 pos. 26 - 27: AIA-AN 0.884
BBIF_0302 hypothetical protein with Cna B-type
domain
0 pos. 29 - 30: ANA-AE 0.815
E-score, extracellu lar score, is calculated by cPSORTb; Pos., position of amino acid residues. Potential
cleavage sites are predicted by SignalP 4.1; D-Score, discrimination score. Proteins existed in ePSORTb are
underlined.
43
Table 3-4 (continued): List of proteins with signal peptide in B. bifidum S17 predicted by cPSORTb
Gene tag Annotation E-Score SignalP 4.1 (cleavage site) D-score
Cell wall proteins
BBIF_0522 hypothetical protein with CHAP domain 0.78 pos. 36 - 37: EQA-AA 0.716
BBIF_0592 peptide/nickel transport system,
substrate-binding protein
0.78 pos. 28 - 29: VKD-AN 0.612
BBIF_1382 hypothetical protein containing bacterial
Ig-like domain (group 2)
0.63 pos. 34 - 35: ASA-AD 0.768
BBIF_1452 hypothetical protein with the 5'-
nucleotidase domain
0.61 pos. 27 - 28: VTA-AY 0.837
BBIF_1607 hypothetical protein BBIF_1607 0.78 pos. 34 - 35: AGA-NP 0.652
BBIF_1648 hypothetical protein containing CnaB
domain - LPXTG-anchor
0.06 pos. 29 - 30: ANA-QV 0.729
BBIF_1761 Cell surface protein with gram positive
anchor - Cna protein B-type domains
0 pos. 31 - 32: ANA-AD 0.879
Proteins may have multiple localization sites
BBIF_0449 ATPase associated with various cellular
activities, AAA3
5.15 No SP 0.374
BBIF_0636 peptide/nickel transport system,
extracellular solute-binding protein
4.87 pos. 34 - 35: GSA-GN 0.644
BBIF_1059 hypothetical protein BBIF_1059 0.02 pos. 19 - 20: ALA-AV 0.473
BBIF_1257 hypothetical protein containing bacterial
Ig-like domain (group 2)
4.87 pos. 37 - 38: AQA-AG 0.641
BBIF_1681 subtilysin family peptidase (lactocepin) 4.87 pos. 28 - 29: ALA-AP 0.798
BBIF_1769 hypothetical protein with collagen-
binding domain
5.15 pos. 36 - 37: GVA-AR 0.54
E-score, extracellu lar score, is calculated by cPSORTb; Pos., position of amino acid residues. Potential
cleavage sites are predicted by SignalP 4.1; D-Score, discrimination score. Proteins existed in ePSORTb are
underlined.
44
3.7 Construction of a secretion reporter vector
3.7.1 Clone SP from gene bbif_1734
Amongst all SPs detected in silico, the SP of protein BBIF_1734 (S0 in short) has the
highest D-score. BBIF_1734 is annotated as a putative extracellular sialidase. In addition,
the ePSORTb database indicates that the E. coli sialidase is expressed as extracellular
protein. The BBIF_1734 SP was further confirmed by SignalP (Fig. 3-8) and TatP
prediction (data unshown). To test its potential as a sorting signal on heterologous protein
secretion, it was cloned by PCR amplification with primer S0F/S0R. To preserve the
predicted cleavage site between A35 and A36, two extra amino acids were included (A36S37).
Fig. 3-8: Prediction of the SP sequence of BBIF_1734 by SignalP4.1 using the N-terminal 60 amino
acids. The violet line is the default cutoff (score = 0.5 ). SignalP produces three scores: C-score (raw
cleavage site score), S-score (signal peptide score), and Y-score, which is a combination of the C-score
and the slope of the S-score.
3.7.2 Clone appA as a reporter
To test the utility of S0 as a sorting signal, a suitable reporter is needed. appA gene
encoding a phytase was amplified from the genome of E. coli DH10B by PCR and fused
with S0 by SOE-PCR using primers S0F/S0R, P0F/PhyR, and S0F/PhyR. The SOE-PCR
45
product (S0P) was ligated into pMDY23-Pgap to replace the gusA gene and the resulting
vector named pMgapS0P (Fig. 3-9, panel A). Meanwhile, appA was amplified using
primers PhyF and PhyR and cloned into pMDY23-Pgap to replace gusA by appA. The
resulting vector named pMgapP was used as a control to express phytase intracellularly
(Fig. 3-9, panel B). Positive constructs were confirmed by PCR with primers PMF/PhyR
and Sanger sequencing. Then, they were introduced into B. bifidum S17 by electroporation
for heterologous phytase expression.
Fig. 3-9: Plas mid map of pMgapS0P (A) and pMgapP (B). On pMgapS0P, gusA was replaced by S0
fused mature appA between Xho I and Hind III. S0 is standing for the predicted SP of gene bbif_1734.
Pgap is gap promoter. On pMgapP, gusA was replaced by mature appA between Xho I and Hind III
with an extra ATG start codon.
3.7.3 Phytase assay
Both plasmids were transformed into B. bifidum S17 and phytase activity was measured at
various time points during growth in crude extracts (intracellular) and culture supernatants
(extracellular). Both strains displayed almost identical growth ruling (data unshown).
Phytase activity markedly increased over time in supernatants of B. bifidum
S17/pMgapS0P whereas little to no activity was measured for the control strain without S0
before 12 h (B. bifidum S17/pMgapP, Fig. 3-10, panel A). By contrast, throughout growth
phytase activities were higher in crude extracts of B. bifidum S17/pMgapP. The relative
levels of extra- and intracellular phytase activity in both strains suggests efficient yet not
complete secretion mediated by the S0 (Fig. 3-10, panel B)
46
Fig. 3-10: Phytase activi ty in the supernatants (A) and crude extracts (B) of B. bifidum S17
recombinant constructs. B. bifidum S17/pMgapS0P and the SP-less control strain B. bifidum
S17/pMgapP were grown in RCM containing 100 μg/ml Spc. Supernatants and crude extracts were
collected at indicated time points. FTU is the standard unit of phytase activi ty that was defined as the
amount of enzyme needed for catalyzing the release of 1 μmol phosphate from phytate per minute.
Values are FTU per ml culture supernatant (A) or FTU per mg protein (B) and are mean ± standard
deviation of three independent cultures each measured in triplicate.
3.8 Comparison of various SPs
Following the successful establishment of an appA-based reporter vector and assay for S0
in B. bifidum S17, this system was used to test various other SPs of bifidobacteria selected
from three strains, exactly, B. bifidum S17, B. longum E18, and B. breve S27 (Table 3-5).
For B. longum E18 and B. breve S27, key words “secreted” and “secreted protein” were
searched from the annotation of genomic datasets. This yielded several candidates that
might be secreted proteins. Further prediction of all these candidates by SiganlP excluded
some of them as they are SP negative (Gram-positive model, D-score cutoff = 0.5). Some
representatives of them with relative high D-scores were selected as they might secreted
through different pathways according to functional annotation or report in literatures. For
example, BLONG_0223 and BLONG_1620 were annotated as Tat secreted proteins.
47
Table 3-5: 7 selected proteins and their signal peptides
Gene function Gene tag (NCBI) D-score Signal peptide
Tat-secreted glycosidase blong_0223 0.865 S1, 34 aa
Putative tat-secreted pectin lyase-like protein blong_1620 0.772 S2, 32 aa
Hypothetical secreted protein with NlpC/P60 domain blong_1728 0.891 S3, 31 aa
Conserved hypothetical secreted protein with CHAP
domain
blong_0476 0.838 S4, 36 aa
subtilisin family peptidase (lactocepin) bbif_1681 0.798 S5, 30 aa
surface protein with gram positive anchor and Cna
protein B-type domains
bbif_1761 0.879 S6, 33 aa
Conserved hypothetical secreted protein with CHAP
domain
S27_0508
0.823 S7, 36 aa
Analysis of the amino acids composition of these predicted SPs indicates they are type I SP
as possessing the typical AxA cleavage site (Table 3-6). Although BLONG_0223 (S1) and
BLONG_1620 (S2) were annotated as Tat secreted proteins on the genome B. longum E18,
the Tat signal motif (RRxFLK) was not detected by TatP. It seems no type II SPs among
them as the conserved feature structure is absent. These potential SPs (S1-S7) range from
30 to 36 amino acids (aa), which is typical in Gram-positive bacteria (Paetzel et al., 2002).
Table 3-6: Amino acid sequences of the seven predicted signal peptides
Signal peptide Sequence
S1 MHQSTRKRW LASIGAVAAVATLATGGAVTAQA*AD
S2 MTSRQGRQAIAATAAMGVAVALALPTAAFA*QS
S3 MKTKTVASSIVAAIVSCACLFMFVPTATA*AE
S4 MTNVRVIKPALAALVAAAACVGGLAFSSAQPAQA*DT
S5 MANKQWPRW VAAVAALAMAGSLPGTALA*AP
S6 MKSLMKKVFAAAAAIATVFGLAATTVATANA*AD
S7 MTKAKTMRPVLAACAASAICVAGLLASPVQPARA*NT
*indicates predicted cleavage sites; amino acids of conserved AxA motifs are shown in red.
48
To test the functionality of these predicted SPs, they were fused to the appA reporter gene
by SOE-PCR, cloned into the pMDY23-Pgap plasmid replacing the gusA gene and
introduced into B. bifidum S17 by electroporation. Monitoring of growth on RCM
indicated that all recombinant strains show the same growth characteristics (data not
shown). Moreover, preliminary temporal analysis indicated that for all strains phytase
mainly accumulated in supernatants in stationary phase. Thus in all further experiments,
samples were collected from cultures grown for 16 h for phytase assays. Phytase activity
was detected in the supernatants of all recombinant strains of B. bifidum S17 harboring
predicted SPs. B. bifidum S17 strains introduced plasmids pMgapS1P, pMgapS4P,
pMgapS6P, or pMgapS7P shown comparable or higher activities than B. bifidum
S17/pMgapS0P. In contrast, strains harboring pMgapS2P, pMgapS3P, and pMgapS5P
show lower activity than B. bifidum S17/pMgapS0P. B. bifidum S17/pMgapP, which does
not encode an SP, has no obvious activity in supernatant (Fig. 3-11).
Fig. 3-11: Phytase activi ties in the supernatants of different B. bifidum S17 constructs. Samples were
prepared from RCM derived s pent medium containing 100 μg/ml S pc after inoculation for 16 h. FTU
is the standard unit of phytase activity that was defined as the amount of enzyme needed for catalyzing
the release of 1 μmol phos phate from phytate per minute. Values are FTU per ml supernatant (FTU/ml)
and are mean ± standard deviation of three independent cultures each measured in triplicate.
In cell crude extracts, B. bifidum S17/pMgapP, which expressing appA intracellularly,
showed highest phytase activity. All strains with SP-containing constructs showed
markedly lower activity than B. bifidum S17/pMgapP as expected (Fig. 3-12).
49
Fig. 3-12: Phytase activi ties in the cell lysate of different B. bifidum S17 constructs. Samples were
prepared from 16 h incubated cells grown in RCM containing 100 μg/ml S pc. FTU is the standard unit
of phytase activity that was defined as the amount of enzyme needed for catalyzing the release of 1
μmol phos phate from phytate per minute. Total protein is quantified with a BCA kit. Values are FTU
per mg protein (FTU/mg) and are mean ± standard deviation of three independent cultures each
measured in triplicate.
To confirm the expression and secretion of phytase in B. bifidum S17 visually, a
phenotypic assay based on degradation of insoluble calcium phytate by extracellular
phytase in solid medium was used. Clear zones of degradation were observed for B.
bifidum S17 strains harboring pMgapS0P, pMgapS1P, pMgapS4P, pMgapS6P, and
pMgapS7P whereas strains carrying pMgapS2P, pMgapS3P, and pMgapS5P respectively
showed low or only weak calcium phytate degradation (Fig. 3-13). The degradation
potentials of these constructs are consistent with their enzymatic assay results. Collectively,
this data suggests that S6 is the most effective protein secretion signal.
Fig. 3-13: Calcium phytate degradation by recombinant B. bifidum S17 strains. P, B. bifidum
S17/pMgapP; S0-S7, B. bifidum S17/pMgapS0P-pMgapS7P; N, B. bifidum S17/pMDY23-Pgap. One
representative of three independent experiments is shown.
50
3.9 Secretion of codon adapted ChrR6 in B. bifidum S17
The ultimate goal of this thesis was the generation of a recombinant bifidobacterium
expression a prodrug-converting enzyme for cancer therapy using a suitable promoter
combined with protein secretion. As a therapeutic approach, nitroreductase (NTR)
converting the non-toxic pro-drug 5-aziridin- l-yl-2,4-dinitrobenzamide (CB1954) to the
tumor therapeutic drug 4-hydroxylamine (4-HX), which intercalates with DNA thereby
inhibiting proliferation, was selected. To construct a recombinant B. bifidum S17 for
conversion of CB1954, the gene encoding ChrR6 was synthesized codon-optimized for B.
bifidum S17. The ChrR6 is an artificial NTR created by mutation of the E. coli YieF and
shows faster enzyme kinetics than its native form (Thorne et al., 2009). The codon-
optimized chrR6 gene was sub-cloned under control of Pgap and containing the S6 secretion
signal thereby creating pMgapS6C for secretion (Fig. 3-14).
Fig. 3-14: Plasmid map of pMgapS6C. Pgap, in red, the gap promoter; S6, in green, SP of gene
bbif_1671; chrR6, codon optimized chromate reductase gene, also a nitroreductase gene.
Expression and secretion of ChrR6 in recombinant B. bifidum S17 was measured by NTR
enzymatic assay of culture supernatant and crude extracts. No NTR activity was observed
in empty controls (fresh medium or Tris-HCl buffer, data not shown). In crude extracts of
B. bifidum S17/pMgapS6C, about 240 mU/mg NTR activity were measured (Fig. 3-15,
panel A). In the negative vector control strain B. bifidum S17/pMDY23-Pgap, NTR activity
was markedly lower yet still detectable (~50 mU/mg), possibly due to the intrinsic NTR of
51
the strain. Similarly, NTR levels were by far higher in culture supernatants of B. bifidum
S17/pMgapS6C compared to B. bifidum S17/pMDY23-Pgap (Fig. 3-15, panel B).
Fig. 3-15: NTR activity in crude extracts (A) and culture supernatant (B) of B. bifidum S17/pMgapS6C
or B. bifidum S17/pMDY23-Pgap (empty vector control) grown for 16 h in RCM containing 100 μg/ml
Spc. 1U is the enzyme required for oxidizing 1 μmol NADH per minute. Values are NTR activity in
mU/ml supernatant (A) or mU/mg protein in crude extracts and are mean ± standard deviation of
three independent experiments each measured in triplicate.
Additionally, the enzymatic activity of Chr6R on CB1954 conversion was indirectly
demonstrated by growth inhibition experiment. For this purpose, OD600 was measured of B.
bifidum S17 derivatives grown for 24 h in the presence of different concentrations of
CB1954. The control strain B. bifidum S17/pMDY23-Pgap was highly resistant to CB1954
with supplementation of as much as 500 μM CB1954 only reduced the final OD600 to about
90%. By contrast, ChrR6 expressing B. bifidum S17/pMgapS6C was by far more sensitive
to CB1954 and growth of B. bifidum S17/pMgapS6C was inhibited by CD1954 in a dose-
dependent pattern (Fig. 3-16) indicating effective conversion of CB1954 to cytotoxic 4-HX.
52
Fig. 3-16: Growth inhibition of recombinant B. bifidum S17 strains by adding CB1954. B. bifidum
S17/pMgapS6C and B. bifidum S17/pMDY23 -Pgap (empty vector control) were grown for 24 h in RCM
broth containing 100 μg/ml S pc and different concentrations CB1954. Values are final OD600 relative to
the culture without CB1954 and are mean ± standard deviation of three independent experiments
assayed in triplicate.
53
4 DISCUSSION
4.1 Systems for protein expression and secretion by
bifidobacteria
4.1.1 Expression vectors and promoters
In this study, a series of E. coli - Bifidobacterium sp. shuttle vectors were constructed
based on the reporter vector pMDY23, which is based on the cryptic plasmid pNCC293
(Klijn et al., 2006). pNCC293 has 98.8% identity to the 1 847 bp plasmid pMB1, which is
believed to replicate by a theta mechanism. The plasmid pMDY23 harbors an E. coli gusA
gene as transcriptional reporter , is 100% stable in B. longum NCC2705 for at least 10
generations without antibiotic pressure, and has a medium copy number around 10 (Klijn,
et al., 2006). This plasmid was shown to stable replicate in B. bifidum S17 (Gleinser et al.,
2012), and was used previously by our group as a promoter probe vector in this strain
(Grimm et al., 2014).
In silico prediction by BPROM of putative promoters using complete inter-region
sequences yielded potential promoters for all promoter regions except for the fragment
upstream of luxS. Moreover, in the fragments with a positive hit for a bacterial promoter,
potential binding sites for RpoS and RpoD were detected. This indicates that these
promoters may dependent on σA (encoded by rpoD) consistent with a role as promoters of
housekeeping genes (Nesvera et al., 2012; Patek et al., 2013). Using BLAST analysis, a
gene (bbif_1216) was identified in the genome of B. bifidum S17. The deduced amino acid
sequence of BBIF_1216 shows high homology to the prototype σA of Bacillus subtilis
(Haldenwang 1995). The protein contains Sigma-70 factor regions 1.2, 2, 3, and 4
(residues 205-240, 272-342, 351-427 and 440-493, respectively) with a helix-turn-helix
motif for DNA binding and a motif for interaction with the RNA polymerase (RNAP) β’
core subunit in region 2. Together with the high conservation of the -35 and -10 promoter
elements, this suggests that the gene product of bbif_1216 is likely to be the housekeeping
sigma factor σA of B. bifidum S17 directing the RNAP to Pgap to initiate transcription.
54
Using pMDY23, a number of DNA fragments containing potential bifidobacterial
promoters were analyzed for transcriptional activity. Pgap and Phup were already used for
protein expression in bifidobacteria by different groups (Sun et al., 2012). PluxS was
assumed to bear transcriptional activity based on the detection of autoinducer-2 activity in
culture supernatants of B. longum NCC2705 (Sun et al., 2014).
Among the promoter fragments tested, the gap promoter of B. bifidum S17 and the hup
promoter of B. longum NCC2705 yielded detectable GusA reporter. This is in line with the
suspected role of gap and hup as housekeeping genes. The results also consistent with
previous study by our group showing higher Pgap-driven reporter activity in B. bifidum S17
compared to other promoters in all growth phases tested (Grimm et al., 2014). Furthermore,
several other groups also report on high transcriptional activity of bifidobacterial gap
promoters and these promoters have been used successfully for expression in bifidobacteria
(Klijn et al., 2006; Khokhlova et al., 2010; Sun et al., 2014). The gap genes encode the
enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12), which
catalyses the conversion of glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate
(Nagradova 2001). Although GAPDH is primarily know for its role in glycolysis, it is also
required for formation of lactate from glyceraldehyde 3-phosphate and, in consequence,
efficient energy conservation from substrates that are fermented via the bifidus shunt. Thus,
it seems reasonable to hypothesize that the gap gene is constitutively expressed at high
levels in a wide range of bacteria including bifidobacteria.
Histone- like HU proteins are ubiquitous in the bacterial kingdom (Chouayekh et al., 2009).
In E. coli, HU proteins are encoded by two genes, hupA and hupB (Claret and Rouviere-
Yaniv, 1997). In most other bacteria including bifidobacteria, only hupB was annotated.
HU proteins are able to bend and condense DNA and play an important role in bacterial
nucleotide organization (Czapla et al., 2008) and are involved in numerous cellular
processes, such as replication, transcription and gene regulation (Kamashev et al., 2008;
Mukherjee et al., 2009). Again, this indicates that the hup genes are constitutively
expressed.
The results indicate that Pgap has higher transcriptional activity under the conditions tested
than Phup. This contradicts previous studies showing comparable expression of FGF-2 in B.
breve using the Pgap and Phup sequences of B. longum (Shkoporov et al., 2008). The
55
difference may due to better recognition of Pgap by the RNAP holoenzyme based on
differences in the sequences (Fig. 3-6). The luxS and 16S rRNA promoters did not show
any transcriptional activity under the conditions tested although other studies have reported
considerable activity of the 16S rRNA promoter of B. longum (Park et al., 2008a).
Although several putative promoters have been successfully applied in bifidobacteria to
facilitate gene expression, none of these promoters’ elements has been experimentally
confirmed. Two important features that have an impact on promoter activity are the -10
and -35 regions. AT rich elements in the -10 region are involved in formation of
transcription bubbles (Feklistov and Darst 2011). Using 5’-RACE experiments, the
transcription start site (TSS) of Pgap was shown to be a thymidine (T) residue 62 bp
upstream of the translation start site. This is different from the TSS prediction by NNPP.
This suggests that bifidobacterial housekeeping promoters may be somewhat different to
those used for software-based promoter prediction.
Following the identification of the TSS, the -35 and -10 regions of Pgap can be localized by
alignment of other putative gap promoters of bifidobacteria. Alignment of five
representative potential Pgap sequences originated from different species/strains using the
core sequences from the ATG start codon to 50 bp upstream of the TSS suggested
conserved -10 and -35 regions (Fig. 3-6). Notably, nucleotides near the TSS are highly
conserved. This indicates that Pgap of B. bifidum S17 is likely to show transcriptional
activity in other bifidobacteria as well. In fact, this has been demonstrated by successful
expression of different proteins in B. bifidum S17, B. breve S27 and B. longum E18
(Grimm et al., 2014, Gleinser et al., 2012). In addition, Pgap is able to drive high- level gusA
reporter activity in E. coli (data not shown).
The 5’-untranslated region upstream of the ATG start codon of gap contains a putative
ribosome-binding site (RBS, Fig. 3-3). During initiation of translation, the RBS interacts
with a sequence motif in the 3’-end of the 16S rRNA termed anti-Shine-Dalgarno sequence
(anti-SD) (Laursen et al., 2005). The anti-SD of B. bifidum S17 is identical to that of other
Gram-positive bacteria including Bacillus subtilis and Lactococcus lactis (Wegmann et al.,
2013). Moreover, the RBS of Pgap and anti-SD of B. bifidum S17 are highly complementary
with only one mismatched base pair to the anti-SD (Fig. 3-4) indicative of efficient
translation initiation (Makrides 1996).
56
4.1.2 Secretion pathway and sorting signal
Generally, bacteria possess three different pathways for protein secretion: the Sec-
dependent (Sec), Sec-independent, and the twin arginine translocation (Tat) pathway with
the Sec pathway being the most common secretion machinery (Dautin and Bernstein 2007;
Tseng et al., 2009; Jongbloed et al., 2002). Proteins secreted via the Sec-dependent
pathway utilize a common machinery consisting of the integral membrane complex
SecYEG and other accessory and regulatory proteins including SecA, SecB, YajC
(Kostakioti et al., 2005). Tat pathway is responsible for secretion of readily folded proteins
(Jongbloed et al., 2004). Search of corresponding components in Bifidobacterium (NCBI
taxid: 1678) indicates that a large number of bifidobacteria possess a Sec-dependent
pathway, whereas Tat systems are only presented in a few strains of B. longum. Sec-
independent pathways allow direct export from the cytoplasm to the extracellular
environment in one-step. These pathways may include ABC (ATP-binding cassette)
exporters (Holland et al., 2005; Schneewind and Missiakas 2012) and the type III secretion
systems (T3SS, Hueck 1998). However, there is no study of Sec- independent proteins
secretion in bifidobacteria at present.
In biotechnology, protein secretion is required for a number of applications, when the
protein exerts its function outside the bacterial cell, e.g. in recombinant vaccine strains or
for downstream purification (Quax 1997). Secretion of recombinant protein by
bifidobacteria was first achieved by testing various signal peptides (SPs) with a nuclease
reporter using B. breve UCC2003 as host (MacConaill et al., 2003). Later, fusion of the α-
amylase SP to pediocin PA-1 leads to active protein secretion in B. longum (Moon et al.,
2005). Other examples are pB44- and pB80-derived vectors carrying a sec2 SP, cloned
from B. breve for secretion of FGF-2 (Shkoporov et al., 2008).
In this thesis, eight SPs were cloned from three strains of bifidobacteria. In a first set of
experiments, The SP of sialidase (encoded by bbif_1734) termed S0 was used to establish a
reporter system for protein secretion in B. bifidum S17 since this SP has the highest D
score and the corresponding protein was experimentally confirmed to be secreted in other
organisms (Corfield 1992; Crennell et al., 1993). The E. coli appA gene encoding phytase
57
was selected as reporter. This reporter gene was successfully expressed in bacteria
(Shouldice et al., 2010), plant (Zimmermann et al., 2003), and animal (Golovan et al.,
2001). The S0 SP was fused to the phytase gene and cloned downstream of Pgap in the
pMDY23 backbone lacking gusA. Following transformation of this construct into B.
bifidum S17, phytase activity was measurable in culture supernatants of the recombinant
strain and this activity increased whereas specific phytase activity in crude extracts
decreased during growth. These results demonstrate that S0 is a functional SP and appA
can be used in combination with the pMDY23 backbone and Pgap as a reporter for protein
secretion in bifidobacteria.
In a second step, several selected SPs that may direct protein secretion through different
pathways were cloned into the reporter plasmid and assayed for protein secretion. S1 and
S2 are thought to be Tat type secretion signals as corresponding proteins were annotated as
Tat secreted proteins. However, S1 mediates phytase secretion in B. bifidum S17 that lacks
Tat secretion system indicating it is in fact not a Tat SP. Of the other SPs used, only S4, S6
and S7 result in marked protein secretion. S4 and S7 are signal peptides of two
homologues (conserved hypothetical protein with CHAP domain) of B. longum E18 and B.
breve S27. Their functionality in B. bifidum S17 suggests that SP maybe recognized by
different strains/species within the genus Bifidobacterium. S5 and S6 are SPs of B. bifidum
S17 proteins with S5 being predicted to mediate secretion by the Sec-dependent and S6 by
the Sec-independent pathway. Interestingly S6, SP of a surface protein with Gram-positive
anchor and Cna protein B-type domains was the most efficient SP amongst those tested. In
all, comparison of these SPs in the same host highlights the importance of choosing sorting
signal in protein secretion. Finally, the relatively efficient S6 was selected for targeting
prodrug converting enzymes in following part as its obvious supper performance on sorting
phytase (Fig. 3-10).
4.1.3 Secretion reporter
Reporter systems are widely used in molecular biology. The most frequently used reporters
are enzymes, e.g. β-galactosidase (lacZ), chloramphenicol acetyltransferase (Cat), and β-
glucuronidase (gusA) (Poyart and Trieu-Cuot 1997; Palmano et al., 2001; Jefferson et al.,
1987). Another group of reporters is fluorescent proteins like green fluorescent protein
58
(GFP, Chary et al., 2005) or luciferases such as the Photorhabdus luminescence
luxCDABE (Hakkila et al., 2002; Waidmann et al., 2011). All these reporter systems are
convenient tools for assessment of promoter activities. However, naturally occurring
fluorescence can lead to high background levels during measurements (Greer and Szalay
2002). Moreover, enzymatic assays that involve cell disruption and addition of specific
substrates are usually more accurate (Kim et al., 2006).
A number of factors determine successful application of a reporter. Two important criteria
are the absence of similar activities in the host and the availability of simple and sensitive
methods for quantitative assays. In the extracellular environment, there are many
substances including proteases, acids and ions that might have an impact on the activity of
a secreted protein reporter. For bifidobacteria at laboratory scale, some specific
characteristics must be considered, e.g. producing large amount acid and growth under
anaerobic conditions in complex medium. Many common reporters designed for
intracellular expression in neutral pH are inactive in the complex and ac idic extracellular
environment and are thus undetectable.
In the presented thesis, the gusA reporter was used for promoter probing experiments and
its enzymatic activity is assayed with bacteria cell lysate at near neutral pH as it was widely
used a reporter (Jefferson et al., 1987). The Michaelis constant (Km) for one substrate, p-
nitrophenyl-beta-D-glucuronide (PNPG) was found to be 0.22 mM (Kim et al., 1995). The
cytosolic enzyme has an optimal pH 6-7 and culture supernatants of bifidobacteria easily
reach pH below 4.5 (Sun et al., 2014, supplementary material). Moreover, the active form
of this enzyme is composed of four subunits with a molecular weight of approximately
290-kilo Dalton (KD) (Kim et al., 1995). Thus, other authors have concluded that the (E.
coli) β-glucuronidase is not a good reporter enzyme to assay protein secretion (Kim et al.,
2006; Kavita and Burma 2008). This is confirmed by results of the present study showing
that β-glucuronidase activity cannot be detected in culture supernatants of recombinant B.
bifidum S17 containing constructs with S0 fused to the gusA reporter under the control of
Pgap (data unshown).
A number of genes were studied as secretion reporters. One is the secreted alkaline
phosphatase of E. coli, which was successfully used in bacteria and yeast (Bina et al., 1997;
Nallaseth and Anderson 2013). In eukaryotic organisms a similar protein, secreted
59
embryonic alkaline phosphatase (SEAP) is widely used as secreted reporter (Berger et al.,
1988; Christou and Parks 2011). Another example is the non-specific, class C family acid
phosphatase of Staphylococcus aureus, which is used as a secretion reporter in both Gram-
positive and Gram-negative bacteria (du Plessis et al., 2007). However, these phosphatases
are limited in their application due to presence of both alkaline phosphatase and non-
specific acid phosphatase in a wide range of bacteria resulting in high background levels. B.
bifidum S17 harbors two acid phosphatases (gene bbif_1161 and bbif_1680) and one
alkaline phophatase (bbif_1740). Moreover, the protein encoded by bbif_1740 contains a
type I SP with extracellular sub- location and the same protein in E. coli was experimentally
confirmed as extracellular protein (see Table 3-4). Thus, this protein is expected to
interfere with a phosphatase reporter.
Other secretion reporters include the luciferase from the marine copepod Gaussia princeps
(Gluc) and nuclease (Nuc) from Staphylococcus aureus. Gluc was used in Salmonella
enteric (Wille et al., 2012) and Mycobacterium smegmatis (Andreu et al., 2010). However,
high luminescence was detected in supernatants of Salmonella and Mycobacterium
smegmatis regardless of the presence or absence of a secretion signal. Nuc, a heat stable
phosphodiesterase is secreted as an active small polypeptide that undergoes proteolytic
cleavage to produce its active form (Shortle 1983, Poquet et al., 1998). Nuc is efficiently
secreted by numerous Gram-positive bacteria when its native signal sequence is replaced
by the SP of exported proteins (Downing et al., 1999; MacConaill et al., 2003; Chouayekh
et al., 2009; Karlskas et al., 2014). Of note, Nuc is the first and the only secretion reporter
used in bifidobacteria (MacConaill et al., 2003). However, similar to Gluc, considerable
nuclease activity is also observed in the absence of a functional SP. It has been suggested
that the release of the staphylococcal nuclease to the extracellular environment was not due
to cell lysis but by an unknown SP-independent pathway (Recchi et al., 2002).
Considering all advantages and disadvantages, these reporters might be unsuitable to
investigate SPs in bifidobacteria. Thus, the E. coli appA gene encoding a histidine acid
phytase (myo-inositol hexakisphosphate phosphohydrolase) was selected as a secretion
reporter. This enzyme catalyzes the stepwise removal of phosphates from phytic acid
(inositol hexakisphosphate, IP6) or its salt phytate and other phosphate donors. It is active
in a broad of pH profile and resistant to protease (Lei et al., 2013). Ideally, most
bifidobacteria including B. bifidum S17 do not harbor a phytase homologue. Exceptions are
60
B. pseudocatenulatum ATCC 27919 and B. longum subsp. infantis ATCC 15697, which
were reported to harbor phytase genes (Haros et al., 2007; Haros et al., 2009). Moreover,
this single subunit enzyme can be easily assayed with high specific activity in both cell
lysate and culture supernatants. For E. coli appA encoded phytase, the Km = 0.13 μM, and
Kcat/Km = 4.476 × 107 (Greiner et al., 1993). In fact, phytase were successfully secreted by
different host strains including Lactobacillus plantarum (Kerovuo and Tynkkynen 2000), B.
longum (Park et al., 2005), Pichiapastoris (Xiong et al., 2003) and Arxula adeninivorans
(Boer et al., 2009).
There are several methods for determining phytase activity based on similar chemical
principles. In this work, the Phytex procedure was employed, which was developed
specifically for E. coli derived phytase (Kim and Lei 2005; Weaver et al., 2009). In all
experiments, RCM other than MRS medium was used for cultivation of bacteria prior to
phytase assays since MRS contains 2 g/l sodium phosphate, which interfere the assay
(Olstorpe et al., 2009).
4.2 Bifidobacteria as gene delivery vectors in cancer
therapy
Cancer is one of the most challenging diseases worldwidely. Despite many approaches for
treatment, resistance to chemotherapy and radiotherapy is still a major obstacle limiting
therapeutic success (de Groot et al., 2011; Quispe-Tintaya et al., 2013). Alternative and
supplementary therapies such as bacterial tumor targeting have been studied for many
years (Hoption Cann et al., 2003). However, this approach only recently has attracted
increasing interest owing to advances in genetic engineering of promising bacterial strains
that so far have been difficult to access, e.g. clostridia and bifidobacteria (Forbes 2006).
The most frequently used facultative or anaerobic bacterial vectors for delivery of
therapeutic genes are Salmonella, Clostridium, and Bifidobacterium (Forbes 2010). The
therapeutic approaches include expression of toxins, cytokines, ligands and pro-drug
converting enzymes by these bacteria (see reviews, Patyar et al., 2011; Zu and Wang 2014).
Solid tumors are characterized by hypoxic and necrotic regions (Dewhirst et al., 2008), and
this microenvironment provides suitable conditions for the colonization of facultative and
61
obligate anaerobes (Fujimori et al., 2002). Anaerobic bacteria including bifidobacteria and
clostridia were shown to localize to these regions in solid tumors in mouse models
selectively (Cronin et al., 2008; Cronin et al., 2010; Lambin et al., 1998). The main
advantage of therapeutic proteins produced in situ in tumors by live bacteria is high local
concentrations while at the same time reducing toxicity at other sites. To date, two classes
of therapeutic proteins have been successfully expressed in bifidobacteria. One class of
proteins have direct cytotoxic effects such as toxins, cytokines, antibodies and antigens (Li
et al., 2003; Zhu et al., 2009; Li et al., 2012; Yin et al., 2012). The other class is enzymes
that convert non-toxic prodrugs into their active derivatives (Hamaji et al., 2007; Tang, et
al., 2009; Yin et al., 2013).
Gene directed enzyme prodrug therapy (GDEPT) is considered as one of the most
promising strategies since prodrug activation and the associated cytotoxicity are co nfined
to certain cells or regions (Palmer et al., 2003; Sasaki et al., 2006). In bifidobacteria,
several GDEPT approaches have been pursued. Herpes simplex virus thymidine kinase
(HSV-TK) expression by B. infantis was used to activate ganciclovir (GCV) and was
shown to result in antitumor activity in a rat model of bladder cancer (Tang et al., 2009).
However, the highly-polar, phosphorylated GCV derivative, which is the active drug,
shows limited diffusion across cell membranes (Mesnil et al., 2000) and two phase III
clinical trials using this approach had contradictory results (Rainov 2000; Immonen et al.,
2004).
At present, the mostly well-studied prodrug-converting enzyme system in bifidobacteria is
cytosine deaminase/5-Fluorocytosine (CD/5-FC, Hidaka et al., 2007; Hamaji et al., 2007).
Mammalian cells are CD negative and thus CD expressed by bacterial vectors can be used
to convert 5-FC into the toxic nucleotide analogue 5-fluorouracil (5-FU) (Kuriyama et al.,
1999; Longley et al., 2003). In contrast to HSV-TK/GSV system, the CD/5-FC system has
much stronger bystander effect, i.e. diffusion of the active compound to neighboring cells
without direct cell-cell contact (Ichikawa et al., 2004). However, the efficiency of 5-FC
penetration into bacterial cells is limited and hepatic metabolism of 5-FC is associated with
moderate systemic toxicity (Hooiveld et al., 2004; van Kuilenburg 2004).
Nitroreductase (NTR) combined with nitro aromatic pro-drugs is attractive as these drugs
are toxic to both replicative and non-replicative cells (Blackwood et al., 2001), and the
62
prodrug CB1954 can freely diffuse across membranes (Christofferson and Wilkie 2009).
NTR converts CB1954 into the tumor drug 4-hydroxylamine (4-HX), which alkylates
DNA (Vass et al., 2009). To date, this system has not been tested in bifidobacteria. The
efficiency of enzyme/pro-drug therapy is largely confined by the actual activity of the
converting enzyme in vivo. Of note, activities of wild type NTRs are quiet low, e.g. E. coli
ChrR Km = 260 μM (Ackerley et al., 2004). To test this approach for bifidobacterial tumor
targeting, a codon-optimized nitroreductase gene (chrR6) was cloned under Pgap for high-
level heterologous expression in B. bifidum S17. ChrR6, also named as Y6, is a mutant
version of ChrR with much higher enzyme kinetics to CB1954 (Barak et al., 2006).
Moreover, the S6 SP was fused to the ChrR6 coding sequence for efficient secretion to
enhance efficiency as well as decrease cytotoxity to the expression host. The presented
preliminary results indicate the secretion of active ChrR6 into medium as shown by
transformation of substrate CB1954 and NADH by culture supernatants of the recombinant
B. bifidum S17 strain. The potential of ChrR6 secretion by recombinant bifidobacteria for
cancer therapy will be tested in tumor models in vitro and in vivo in the future.
63
5 SUMMARY
Based on their beneficial properties and their generally recognized as safe status,
bifidobacteria are used as probiotics in food and pharmaceutical industries. In the future,
recombinant strains with enhanced and/or new beneficial properties may become an option
for alternative or supplementary treatment of various diseases including inflammatory
bowel disease or cancer. The aim of this thesis was to establish an efficient system for
production of secreted proteins with therapeutic potential using recombinant bifidobacteria.
This was achieved by following work.
1. Four putative promoter sequences were analyzed in silico and in vitro using a
glucuronidase reporter system in B. bifidum S17. This revealed that the DNA
sequences upstream of the gap gene yielded highest levels of GusA reporter activity
amongst the promoters tested.
2. The transcription start site of the gap promoter (Pgap) was determined by 5’-RACE and
was found to be a thymidine residue 62 bp upstream of the translational start site.
Using bioinformatics comparison to other putative gap promoters of bifidobacteria, the
-10, -35 and ribosome-binding site of Pgap were identified. The Pgap core regions are
highly conserved in all five Bifidobacterium sp. analyzed indicating that Pgap of B.
bifidum S17 is functional across these species.
3. Using the E. coli appA gene and the predicted signal peptide of a sialidase
(BBIF_1734), a reporter vector for analysis of signal peptides in B. bifidum S17 was
generated. This system was used to analyze the efficacy of other seven predicted signal
peptides (S1-S7) to mediate protein secretion. One of these signal peptides showing
best performance was chosen in further experiments.
4. The most efficient signal peptide were fused to a codon adapted gene encoding for a
nitroreductase, which is a prodrug converting enzyme (ChrR6), and cloned under
control of Pgap. The resulting vector was transformed into B. bifidum S17 and this
recombinant strain exhibited high nitroreductase activity in culture supernatants. The
engineered strain can efficiently convert the prodrug CB1954 to its active derivative.
64
6 LITERATURES
1. Ackerley DF, Gonzalez CF, Keyhan M, Blake R 2nd, Matin A (2004) Mechanism of
chromate reduction by the Escherichia coli protein, NfsA, and the role of different
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7 APPENDICES
7.1 Bacteria strains
All bacteria strains, with short description and their sources were listed in table 7-1.
Table 7-1: Bacteria strains used in this study
Strain Relevant genotype or description Source
E. coli DH5a Type strain Invitrogen
E. coli DH10B Type strain Invitrogen
E. coli DH10B/pMDY23 E. coli DH10B harboring plasmid pMDY23 This study
E. coli DH10B/pMDY23-PluxS E. coli DH10B harboring plasmid pMDY23-PluxS This study
E. coli DH10B/pMDY23-P16S
rRNA
E. coli DH10B harboring plasmid pMDY23-P16S rRNA This study
E. coli DH10B/pMDY23-Phup E. coli DH10B harboring plasmid pMDY23-Phup This study
E. coli DH10B/pMDY23-Pgap E. coli DH10B harboring plasmid pMDY23-Pgap This study
E. coli DH10B/pMgapP E. coli DH10B harboring plasmid pMgapP This study
E. coli DH10B/pMgapS0P E. coli DH10B harboring plasmid pMgapS0P This study
E. coli DH10B/pMgapS1P E. coli DH10B harboring plasmid pMgapS1P This study
E. coli DH10B/pMgapS2P E. coli DH10B harboring plasmid pMgapS2P This study
E. coli DH10B/pMgapS3P E. coli DH10B harboring plasmid pMgapS3P This study
E. coli DH10B/pMgapS4P E. coli DH10B harboring plasmid pMgapS4P This study
E. coli DH10B/pMgapS5P E. coli DH10B harboring plasmid pMgapS5P This study
E. coli DH10B/pMgapS6P E. coli DH10B harboring plasmid pMgapS6P This study
E. coli DH10B/pMgapS7P E. coli DH10B harboring plasmid pMgapS7P This study
E. coli DH10B/pMgapS6C E. coli DH10B harboring plasmid pMgapS6C This study
B. longum NCC2705 Sequenced type strain (Schell et al.,
2002)
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Table 7-1 (continued): Bacteria strains used in this study
Strain Relevant genotype or description Source
B. bifidum S17 Sequenced type strain (Zhurina et al.,
2011)
B. longum E18 Sequenced infants isolate (Zhurina et al.,
2013)
B. breve S27 Infants isolate Our lab
B. bifidum S17/pMDY23 B. bifidum S17 harboring plasmid pMDY23 This study
B. bifidum S17/pMDY23-PluxS B. bifidumS17 harboring plas mid pMDY23-PluxS This study
B. bifidum S17/pMDY23-P16S
rRNA
B. bifidumS17 harboring plas mid pMDY23-P16S
rRNA
This study
B. bifidum S17 /pMDY23-Phup B. bifidum S17 harboring plasmid pMDY23-Phup This study
B. bifidum S17/pMDY23-Pgap B. bifidum S17 harboring plasmid pMDY23-Pgap This study
B. bifidum S17/pMgapP B. bifidum S17 harboring plasmid pMgapP This study
B. bifidum S17/pMgapS0P B. bifidum S17 harboring plasmid pMgapS0P This study
B. bifidum S17/pMgapS1P B. bifidum S17 harboring plasmid pMgapS1P This study
B. bifidum S17/pMgapS2P B. bifidum S17 harboring plasmid pMgapS2P This study
B. bifidum S17/pMgapS3P B. bifidum S17 harboring plasmid pMgapS3P This study
B. bifidum S17/pMgapS4P B. bifidum S17 harboring plasmid pMgapS4P This study
B. bifidum S17/pMgapS5P B. bifidum S17 harboring plasmid pMgapS5P This study
B. bifidum S17/pMgapS6P B. bifidum S17 harboring plasmid pMgapS6P This study
B. bifidum S17/pMgapS7P B. bifidum S17 harboring plasmid pMgapS7P This study
B. bifidum S17/pMgapS6C B. bifidum S17 harboring plasmid pMgapS6C This study
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7.2 Plasmids
All plasmids with short description and their sources were listed in table 7-2.
Table 7-2: Plasmids used in this study
Plasmid Description Source
pJET1.2 Linear b lunt end cloning vector Life Science
pMDY23 E. coli – Bifidobacterium sp. shuttle vector, promoter-less, gusA
reporter
Nestle
pMDY23-PluxS pMDY23 inserted 208 bp potential LuxS promoter region This study
pMDY23-P16S rRNA pMDY23 inserted 569 bp 16S rRNA promoter This study
pMDY23-Phup pMDY23 inserted 241 bp hup promoter region This study
pMDY23-Pgap pMDY23 inserted 209 bp gap promoter region This study
pMgapP gusA was replaced by appA in pMDY23-Pgap, for phytase
overexpression
This study
pMgapS0P S0 was fused in pMgapP, for secretion of phytase This study
pMgapS1P S1 was fused in pMgapP, for secretion of phytase This study
pMgapS2P S2 was fused in pMgapP, for secretion of phytase This study
pMgapS3P S3 was fused in pMgapP, for secretion of phytase This study
pMgapS4P S4 was fused in pMgapP, for secretion of phytase This study
pMgapS5P S5 was fused in pMgapP, for secretion of phytase This study
pMgapS6P S6 was fused in pMgapP, for secretion of phytase This study
pMgapS7P S7 was fused in pMgapP, for secretion of phytase This study
pZA-SchrR cloning vector containing synthetic codon adapted ChrR6 This study
pMgapS6C gusA was replaced by S6 fused ChrR6 in pMgap, for secretion of
ChrR6
This study
S0: pred icted signal peptide (SP) of sialidase from B.bifidum S17 encoded by gene bbif_1734
S1: pred icted SP of Tat-secreted glycosidase from B. longum E18 encoded by gene blong_0223
S2: pred icted SP of putative Tat-secreted pectin lyase-like protein from B. longum E18 encoded by gene
blong_1620
S3: pred icted SP of hypothetical secreted protein with NlpC/P60 domain from B. longum E18 encoded by
gene blong_1728
88
S4: pred icted SP of conserved hypothetical secreted protein with CHAP domain from B. longum E18
encoded by gene blong_0476
S5: pred icted SP of subtilisin family peptidase (lactocepin) from B. bifidum S17 encoded by gene bbif_1681
S6: pred icted SP of surface protein with Gram-positive anchor and Cna protein B-type domains from B.
bifidum S17 encoded by gene bbif_1761
S7: pred icted SP of conserved hypothetical secreted protein with CHAP domain from B. breve S27 encoded
by gene S27_0508
7.3 Oligonucleotides
Oligonucleotides were designed based on the DNA sequences available from the National
Center for Biotechnology Information (NCBI) and listed in table 7-3 with short description.
Table 7-3: Primers used in this study
Name Sequence Description
GSPR1 GGAGTGAGGGATGGTGTTGA Reverse transcription
GSPR3 GTTCTTGACCCACGGGATGT First round PCR
GSPR4 CATCCTTCTCGGCGTAGACC Second round PCR
PMF GCATATGACTAGTGAGCTC Colonies screen
PMR AGGACGTAAGTCGACATG Colonies screen
PgapF GAAGATCTGCGGAATGCCTCGCATCGAATC For. primer of Pgap
PgapR GGCCTCGAGCTCCCTTTGTAGGGTAGA Rev. primer of Pgap
PhupF GAAGATCTACGAGGAGTACCGGTGGATCG For. primer of Phup
PhupR GGCCTCGAGCTCCTGTACTCCTGCACAG Rev. primer of Phup
PluxF GAAGATCTTCGCTACCAATGCGTTCGG For. primer of PluxS
PluxR GGCCTCGAGCGCTCGTTGTCGTTGTCCAT Rev. primer of PluxS
P16SF GAAGATCTCGGTGTACTGGATCAATGATGATTCCG For. primer o f P16S rRNA
P16SR GGCCTCGAGA CCTCCTTACGCCGCCAGCGTTCATCC Rev. primer of P16S rRNA
PhyF GGCCTCGAGATG CAGAGTGAGCCGGAGCTGA For. primer of appA
PhyR CCCAAGCTTAGCCTCAGAGCATTCAGGTAAC Rev. primer of appA
S0F GGCCTCGAGATGGTTCGTTCGACCAAGCCATCGCT For. primer of S0
S1F GGCCTCGAGATGCATCAATCAACACGAAAGCGGTG For. primer of S1
S2F GGCCTCGAGATGACATCCCGTCAGGGCAGA For. primer of S2
S3F GGCCTCGAGATGAAGACCAAAACTGTAGCTTCT For. primer of S3
S4F GGCCTCGAGATGACGAACGTACGTGTGATCAA For. primer of S4
S5F GGCCTCGAGATGGCCAATAAGCAATGGCCTCGCTG For. primer of S5
89
Table 7-3 (continued): Primers used in this study
Name Sequence Description
S6F GGCCTCGAGATGAAATCACTGATGAAAAAGGTTTTCGC For. primer of S6
S7F GGCCTCGAGATGACTAAGGCGAAAACGATGAGGCCAG For. primer of S7
S0R TCAGCTCCGGCTCACTCTGGCTGGCCGCACTCGCGGTGGATA Rev. primer of S0
S1R TCAGCTCCGGCTCACTCTGATCGGCTGCCTGCGCGGT Rev. primer of S1
S2R TCAGCTCCGGCTCACTCTGTGACTGCGCGAACGCAGC Rev. primer of S2
S3R TCAGCTCCGGCTCACTCTGCTCAGCTGCAGTTGCTGTAG Rev. primer of S3
S4R TCAGCTCCGGCTCACTCTGGGTGTCTGCCTGGGCAGG Rev. primer of S4
S5R TCAGCTCCGGCTCACTCTGCGGAGCCGCGAGCGCGGTA Rev. primer of S5
S6R TCAGCTCCGGCTCACTCTGATCCGCTGCGTTGGCCGT Rev. primer of S6
S7R TCAGCTCCGGCTCACTCTGGGTGTTTGCCCGCGCCGG Rev. primer of S7
P0F TATCCACCGCGAGTGCGGCCAGCCAGAGTGAGCCGGAGCTGA For. primer of P0
P1F ACCGCGCAGGCAGCCGATCAGAGTGAGCCGGAGCTGA For. primer of P1
P2F GCTGCGTTCGCGCAGTCACAGAGTGAGCCGGAGCTGA For. primer of P2
P3F CTACAGCAACTGCAGCTGAGCAGAGTGAGCCGGAGCTGA For. primer of P3
P4F CCTGCCCAGGCAGACACCCAGAGTGAGCCGGAGCTGA For. primer of P4
P5F TACCGCGCTCGCGGCTCCGCAGAGTGAGCCGGAGCTGA For. primer of P5
P6F ACGGCCAACGCAGCGGATCAGAGTGAGCCGGAGCTGA For. primer of P6
P7F CCGGCGCGGGCAAACACCCAGAGTGAGCCGGAGCTGA For. primer of P7
Restriction sites are underlined. For., forward; Rev., reverse
7.4 Promoter sequences
DNA sequences for potential promoters of gene gap, luxS in B. bifidum S17, hup and 16S
rRNA in B. longum NCC2705 were cloned and sequenced as following.
Pgap (bbif_0612)
AGATCTGCGGAATGCCTCGCATCGAATCGCCGCAGGCTGTACAGACATATTTGTTAGCGTTAAC
GAAATATGGCCGTTTTATGCTCAAAGCAAGCGCGACACCGTTGCTCTAGTACAGACGGCGCATT
ACAGTAGACACTGTTGGTAAACAAAGGCCATAGCGCATCCATGCGCAAACGGTCTACCCTACA
AAGGGAG CTCGAG
PluxS (bbif_1299)
AGATCTGCAGTTCCTCGCCGTCAACGGCGTGGGTGACAGCAAGCTGGCCAAATACGGCAAGCG
ATTCATGGAGGCCATCGCCGGCTTCGACGATTGACAGCGCCGTTACAGCGCACCGGACTCGCCT
CGTGCCGTATATGCCGTATATGAGGAACGCCTATAGCCAAGAATGCGGCGAGACGCGCGCGAT
90
AGTGAGACAATGGTCGATGGAATGCCGGCTAACGGTCGTACGCGCCGGCGTCATATCGAGCAA
AAGGAGTCTGCG CTCGAG
Phup (bl_1798)
AGATCTCCGCCACTTTGCTGCACCGCGTGGTGTTCTACTGGCTGCGCATTCCGCTGGGCGCGGCG
GCCATGAAGTGGCTTGACAAGCATAATCTTGTCTGATTCGTCTATTTTCATACCCCCTTCGGGGA
AATAGATGTGAAAACCCTTATAAAACGCGGGTTTTCGCAGAAACATGCGCTAGTATCATTGATG
ACAACATGGACTAAGCAAAAGTGCTTGTCCCCTGACCCAAGAAGGATGCTTT CTCGAG
P16S rRNA (blr_01)
AGATCTACACGCCGAGCGAAGCCTGTCGTTTCAACGATTTGGGCTTATCTTCGTGCGCCCGATTT
GCGCGAGTCCGTGAATGCGTGTAAGTTATTCCCTTGCTGCCGCTCAGCGAGTGGTTCTCCTCCGG
GGAGCTGCGAGGTGGGTGAGTGGTGGTGGTTTGAGAACTCAAGAGCGTGTTTGTACTACTTCTT
TATAGTCAATGATTGCCAGTTCATTCCTCGCCTGATTGCCTGTCGTGGTGGTTGGGTACCCGGGA
GGGTTTGATGAGGGGTGAGGTTTTTTGAGGGCGTCCTTCCTTAAGGACGTGCTCGTCAATTTTTG
TTTTGAGAGTCATCTATTCGGATGCTTTTCATGAAGTTTTTTTGTGGAGGGTTCGATTCTGGCTCA
GGATGAACGCTGGCGGCGtaaggaggCTCGAG
7.5 Putative gap promoters of representative bifidobacteria
Sequences of putative gap promoters were extracted from the genomes of corresponding
bifidobacteria strains publicly in NCBI database.
B. bifidum S17, promoter of bbif_0612
>gi|310286520:c752203-751998
GGCCGCGGTGCGGAATGCCTCGCATCGAATCGCCGCAGGCTGTACAGACATA TTTGTTAGCGTT
AACGAAATATGGCCGTTTTATGCTCAAAGCAAGCGCGACACCGTTGCTCTAGTACAGACGGCGC
ATTACAGTAGACACTGTTGGTAAACAAAGGCCATAGCGCATCCATGCGCAAACGGTCTACCCTA
CAAAGGGAGAACAC
B. dentium Bd1, promoter of bdp_1514
>gi|283454959:1693570-1693730
AAACTCATTGTGAGCGTTCACGGAAACGACGTCGTTTTCTGAAACACACGACCGCCATACGTTG
CCCTAAGTACAGGTGACGCACTAAAGTGGGCGGTGTTGGTAAACAATGGCTTCATTGCGCCTCG
CGCATGTGTGGCCTACCCTAAAGGGAGAATTAC
91
B. adolescentis ATCC 15703, promoter of bad_1079
>gi|119025018:1338696-1338866
ACGCATGCGGGACATGCATGTGAGCGTTCACTGAAAATCGCACTGCATTTTGGAACACGCGCCT
CGGAACGTTGCCCTAAGTACAGGTGACGCACTACAGTGGACGGTGTTGGTAAACAATGGCTTCA
GTGTGCCTGGCACACGCGTGGCCTACCCTAAAGGGAGAATTAC
B. breve UCC2003, promoter of bbr_1233
>gi|476417161:1536384-1536616
CCGGACGTGAGCCCCAACTTCCGTGGATTCACGCCGTCGGTCCAATACGGGAAACCGCATATCG
GTTGCCGGCGATCCACAATGTGAGCGCTCACAAAAAAACGACGTTTTTGCTCAAAACGCACGCT
CAGACATTGCCATGTGTACAGAGTCGGCATTACAGTAGCAACTGTTGGTAAACAATGGCCCGGT
GTGCCAGAGCGCACCACGGGCACCCTACAAGGGAGAATTAC
B. longum E18, promoter of blong_1334
>gi|58036264:c266275-266034
CTTGCATGCCGCGCGCTTGCTCATGATGATTCGAGACATTCCTCCAAAGGAGAGGAAAAATCAC
ATGTCGGTTGCGGGCAATCCGAATTGTGAGCGCTCACAGAAAATACGGCATTTTTGCCCAAAAC
GCACGCTGAAACGTTGCCATGTGTACAGAGTCGGCATTACAGTAGCAACTGTTGGTAAACAATG
GCCCGGTGTGCCAAAGCGCGCCAAGGCCACCCTACAAGGGAGAATTACAT
7.6 Reporter gene appA
DNA sequence of mature form appA in Escherichia coli DH10B (GenBank: CP000948.1)
was used as a reference for cloning of reporter.
CAGAGTGAGCCGGAGCTGAAGCTGGAAAGTGTGGTGATTGTCAGTCGTCATGGTGTGCGTGCTC
CAACCAAGGCCACGCAACTGATGCAGGATGTCACCCCAGACGCATGGCCAACCTGGCCGGTAA
AACTGGGTTGGCTGACACCGCGNGGTGGTGAGCTAATCGCCTATCTCGGACATTACCAACGCCA
GCGTCTGGTAGCCGACGGATTGCTGGCGAAAAAGGGCTGCCCGCAGTCTGGTCAGGTCGCGATT
ATTGCTGATGTCGACGAGCGTACCCGTAAAACAGGCGAAGCCTTCGCCGCCGGGCTGGCACCTG
ACTGTGCAATAACCGTACATACCCAGGCAGATACGTCCAGTCCCGATCCGTTATTTAATCCTCTA
AAAACTGGCGTTTGCCAACTGGATAACGCGAACGTGACTGACGCGATCCTCAGCAGGGCAGGA
GGGTCAATTGCTGACTTTACCGGGCATCGGCAAACGGCGTTTCGCGAACTGGAACGGGTGCTTA
ATTTTCCGCAATCAAACTTGTGCCTTAAACGTGAGAAACAGGACGAAAGCTGTTCATTAACGCA
GGCATTACCATCGGAACTCAAGGTGAGCGCCGACAATGTCTCATTAACCGGTGCGGTAAGCCTC
GCATCAATGCTGACGGAGATATTTCTCCTGCAACAAGCACAGGGAATGCCGGAGCCGGGGTGG
GGAAGGATCACCGATTCACACCAGTGGAACACCTTGCTAAGTTTGCATAACGCGCAATTTTATT
92
TGCTACAACGCACGCCAGAGGTTGCCCGCAGCCGCGCCACCCCGTTATTAGATTTGATCAAGAC
AGCGTTGACGCCCCATCCACCGCAAAAACAGGCGTATGGTGTGACATTACCCACTTCAGTGCTG
TTTATCGCCGGACACGATACTAATCTGGCAAATCTCGGCGGCGCACTGGAGCTCAACTGGACGC
TTCCCGGTCAGCCGGATAACACGCCGCCAGGTGGTGAACTGGTGTTTGAACGCTGGCGTCGGCT
AAGCGATAACAGCCAGTGGATTCAGGTTTCGCTGGTCTTCCAGACTTTACAGCAGATGCGTGAT
AAAACGCCGCTGTCATTAAATACGCCGCCCGGAGAGGTGAAACTGACCCTGGCAGGATGTGAA
GAGCGAAATGCGCAGGGCATGTGTTCGTTGGCAGGTTTTACGCAAATCGTGAATGAAGCACGC
ATACCGGCGTGCAGTTTGTAA
7.7 SP sequences
SPs were predicted by SignalP and corresponding DNA sequences were listed as following
including 6 nucleotides after predicted cleavage sites.
S0 (bbif_1734)
ATGGTTCGTTCGACCAAGCCATCGCTGCTGCGCCGTCTTGGCGCGCTGGTGGCGGCCGCTGCCA
TGCTGGTGGTACTGCCCGCAGGGGTATCCACCGCGAGTGCGGCCAGC
S1 (blong_0223)
ATGCATCAATCAACACGAAAGCGGTGGCTTGCGTCAATCGGCGCGGTTGCAGCGGTCGCCACAC
TGGCCACCGGCGGTGCAGTCACCGCGCAGGCAGCCGAT
S2 (blong_1620)
ATGACATCCCGTCAGGGCAGACAAGCCATAGCCGCGACGGCCGCAATGGGCGTGGCCGTCGCG
CTGGCGTTGCCGACCGCTGCGTTCGCGCAGTCA
S3 (blong_1728)
ATGAAGACCAAAACTGTAGCTTCTTCCATTGTTGCCGCGATTGTCTCCTGTGCCTGCTTGTTTAT
GTTTGTTCCTACAGCAACTGCAGCTGAG
S4 (blong_0476)
ATGACGAACGTACGTGTGATCAAGCCCGCACTGGCGGCACTGGTGGCCGCCGCAGCCTGTGTGG
GAGGTCTGGCGTTCAGCTCGGCACAGCCTGCCCAGGCAGACACC
S5 (bbif_1681)
ATGGCCAATAAGCAATGGCCTCGCTGGGTTGCCGCTGTAGCGGCCCTGGCGATGGCAGGCAGCC
TGCCCGGTACCGCGCTCGCGGCTCCG
93
S6 (bbif_1761)
ATGAAATCACTGATGAAAAAGGTTTTCGCTGCCGCCGCGGCGATTGCCACCGTATTTGGATTGG
CTGCGACGACAGTCGCCACGGCCAACGCAGCGGAT
S7 (S27_0508)
ATGACTAAGGCGAAAACGATGAGGCCAGTGTTGGCCGCATGCGCGGCCAGCGCCATATGCGTG
GCCGGGCTGCTGGCCAGCCCTGTTCAGCCGGCGCGGGCAAACACC
7.8 chrR6 sequences
DNA sequences of E. coli and B. bifidum S17 codon adapted chrR6 were supplied as
following.
E. coli chrR6, GenBank: DQ987901.1
ATGTCTGAAAAATTGCAGGTGGTTACGTTACTGGGGAGCCTGCGCAAAGGCTCATTTAATGGCA
TGGTTGCACGTACCCTGCCGAAAATTGCTCCGGCGAGCATGGAAGTCAATGCGTTACCATCCAT
TGCCGACATTCCCTTGTATGACGCTGACGTACAGCAGGAAGAAGGTTTTCCAGCAACGGTTGAA
GCTCTGGCGGAACAGATCCGTCAGGCTGACGGTGTGGTGATCGTCACGCCGGAATATAACTACT
CGGTACCGGGTGGGCTGAAAAATGCCATCGACTGGCTTTCCCGCCTGCCGGATCAACCGCTGGC
AGGTAAACCGGTGTTGATTCAGACCAGCTCAATGGGCGCGATTGGCGGCGCGCGCTGTCAGAAT
CACCTGCGCCAGATTCTGGTCTTCCTCGATGCAATGGTGATGAACAAGCCGGAATTTATGGGCG
GCGTGATTCAGAACAAAGTTGATCCGCAAAACGGAGAAGTGATTGATCAGGGTACGCTGGACC
ACCTGACCGGGCTATTGACCGCATTTGGTGAGTTTATTCAGCGAGTTAAGATCTAA
B. bifidum S17 codon adapted chrR6
ATGTCGGAAAAGCTCCAGGTGGTCACGTTGCTCGGCAGTCTGCGCAAAGGGAGCTTCAACGGC
ATGGTCGCCCGTACCCTTCCGAAGATTGCACCGGCGAGCATGGAGGTGAATGCCCTGCCTTCGA
TAGCGGACATACCCCTGTATGACGCGGATGTGCAGCA GGAAGAAGGCTTTCCGGCTACCGTCGA
AGCCTTGGCCGAACAGATACGCCAAGCCGATGGAGTGGTCATCGTTACCCCGGAGTACAACTAC
TCCGTACCAGGCGGACTGAAGAACGCGATCGACTGGCTCTCCAGACTGCCGGATCAACCCCTTG
CTGGTAAGCCGGTCCTGATCCAGACCTCCTCCATGGGCGCGATTGGCGGTGCAAGGTGCCAGAA
CCACTTGCGGCAGATCCTGGTGTTCCTGGACGCCATGGTCATGAACAAACCCGAGTTCATGGGT
GGCGTGATCCAGAACAAGGTAGACCCGCAGAATGGCGAGGTTATCGACCAGGGAACGCTGGAC
CATCTCACTGGGCTGCTGACAGCCTTTGGCGAGTTCATCCAACGCGTGAAAATTTAA
94
ABBREVIATIONS
% percent
Ω Ohm
σ sigma
μ micro
℃ degree centigrade
4-HX 4-hydroxylamine
4-MU 4-methylumbelliferone
4-MUG 4-methylumbelliferxl-β-D-glucuronide
5-FC 5-fluorocytosine
5-FU 5-fluorouracil
5’-RACE Rapid amplification of cDNA 5’-end
AT adenine and thymine
ATP adenosine triphosphate
B. Bifidobacterium
BCA bicinchoninic acid
BLAST basic local alignment search tool
bp base pair
BSA bovine serum albumin
CB1954 5-aziridin- l-yl-2,4-dinitrobenzamide
CD cytosine deaminase
cDNA complementary deoxyribonucleic acid
CSB citrate sucrose buffer
95
dATP deoxyadenosine triphosphate
ddH2O double distilled water
dH2O distilled water
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
DNase deoxyribonuclease
DTT dithiothreitol
E. Escherichia
EDTA Ethylenediaminetetraacetatic acid
EFSA theEuropean Food Safety Authority
EP Eppendorf
FGF-2 basic fibroblast growth factor
Fig. figure
g gram
GAPDH glyceraldehyde 3-phosphate dehydrogenase
GC guanine and cytosine
GCV ganciclovir
GDEPT gene directed enzyme prodrug therapy
GFP green fluorescent protein
GIT gastrointestinal tract
GRAS generally recognized as safe
GUS β-glucuronidase
h hour
HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid
HPSF high purity salt free
96
HSV-TK herpes simplex virus thymidine kinase
IBD inflammatory bowel disease
ICAM-1 intercellular adhesion molecule 1
IECs intestinal epithelial cells
IFN Interferon
l liter
IL interleukin
LB Luria Bertani
LDF linear discrimination function
LPS lipopolysaccharides
m mili
min minute
mRNA messenger ribonucleic acid
MRS de Man-Rogosa-Sharpe medium
NADH nicotinamide adenine dinucleotide (reduced)
NCBI National Center for Biotechnology Information
NF-κB nuclear factor kappa-light-chain-enhancer of activated B
cells
NNPP neural network promoter prediction
NTR nitroreductase
OD600 optical density at wavelength 600nm
OE-PCR overlap extension polymerase chain reaction
ORF open reading frame
PCR polymerase chain reaction
PSM phytase screening medium
97
RBS ribosome binding site
rcf relative centrifuge force
RCM reinforced clostridial medium
rpm resolutions per minute
RNA ribonucleic acid
RNAP RNA polymerase
s second
SDS sodium dodecyl sulphate
SOD superoxide dismutase
SP signal peptide
SPase signal peptidase
Spc spectinomycin
TAE tris acetate EDTA buffer
Tat two arginine translocation
TCA trichloroacetic acid
TdT terminal deoxynucleotidyl transferase
TNF tumor necrotic factor
tRNA transfer RNA
TSS transcription start site
U unit
UV ultraviolet
V volt
v/v volume per volume
w/v weight per volume
98
PUBLICATIONS
1. Sun Z, Baur A, Riedel CU. A secretion reporter system and heterologous secretion of
nitroreductase in Bifidobacterium bifidum S17 for tumor therapy. Manuscript in
preparation
2. Sun Z, Westermann C, Yuan J, Riedel CU. Experimental determination and
characterization of the gap promoter of Bifidobacterium bifidum S17. Bioengineered.
2014. 5(6):1-7.
3. Sun Z, He X, Brancaccio VF, Yuan J, Riedel CU. Bifidobacteria exhibit LuxS-
dependent autoinducer 2 activity and biofilm formation. PLoS ONE. 2014. 9(2):
e88260.
4. Sun Z, Baur A, Zhurina D, Yuan J, Riedel CU. Accessing the inaccessible: molecular
tools for bifidobacteria. Appl Environ Microbiol. 2012. 78(15):5035-42.
99
ACKNOWLEDGEMENT
During the last four years, many of you contributed in a certain way to the completion of
this thesis. Without those help and support from you, this thesis cannot be done timely. I
would like to express my sincere gratitude to all of you listed below.
Prof. Dr. Bernhard Eikmanns, thank you for giving me the opportunity to be a PhD student
under your official supervision. Your kind help and supportive comments in the institute
seminars are much appreciated. Prof. Dr. Peter Dürre also supplied valuable suggestions.
Dr. Christian Riedel, you supervised my study from writing proposa l to finalizing results.
Your contribution on the completion of the thesis is highly acknowledged. Thank you very
much for your patience on my work, and spending your limited time on correct every
pieces of my paper carefully. I cannot manage to conduct the project smoothly without
your firm support and constructive suggestions.
Much appreciation goes to Prof. Dr. Stefan Binder. Thank you for spending time to review
my thesis. Gratitude to Prof. Dr. Steven Jansen, Prof. Dr. Marion Kazda, and Dr. Frank
Rosenau as well, for you support as examiners in my defense.
I would also like to thank all former and present members of AG Riedel, especially Dr.
Daria, Dr. Marita, Dr. Mark, Verena, Marion, Annika, and Christina for their support in lab
and tolerance to share a crowded office.
This project would not have been possible without the financial support provided by the
German Academic Exchange Service (DAAD). I am very grateful to DAAD former staff
Mr. David Hildebrand. Thank you very much for your kind support in the early phase
when I reached Germany.
At last, I want to express deep gratitude to my wife and my father. Without their support
and sacrifice, I will be impossible to pursue higher education abroad for years. I dedicate
this thesis to them.
100
DECLARATION
I hereby declare that this thesis presented herein was composed by me independently, and
its content was originated entirely from my own work during the PhD study in Ulm
University. Information from the published and unpublished work of others has been fully
acknowledged in the text and cited in references.
I certify that this thesis has not been submitted in any form for acquiring another degree at
any university or institute. I also declare that the work presented in the thesis is original
and I was the person primarily involved in those related publications.
Ulm, July 2014
Zhongke Sun