The Bacterial Chromosome:

Structure and Function

Organization of the bacterial cellOrganization of the bacterial chromosomeReplication and cell divisionRecombinationDNA repair Gene regulation IGene regulation IIGene regulation IIIGenre regulation IVChaperones and ATP-dependent proteasesSecretion of proteins Adaptation to stress Gene transfer

Time Table

LiteratureLary Snyder and Wendy Champness:

Molecular Genetics of BacteriaASM Press, Washington, D.C., 2003

E.C.C. Lin and A. Simon Lynch:Regulation of Gene Expression in Escherichia coli

Chapman and Hall, 1996

Frederick C. Neidhardt (Editor):Escherichia coli and Salmonella

ASM Press, Washington, D.C., 1996

A.L. Sonenshein, J.A. Hoch and R. Losick:Bacillus subtilis

ASM Press, Washington,D.C., 1993

€ 139

1 Bacterial cell shape

Why bacteria are so small ?Why there are different cell shapes ?Do bacteria have a cytoskeleton ?

Size

Comparison

of Different

Prokaryotes

Average diameter:

0.5 – 2 µm

ER Angert (1993) Nature 362: 239JE Mendell (2008) PNAS 105: 6730

80 x 600 µm

Epulopiscium fishelsonii

Characteristics:1. ~3.8 Mbp genome 2. 50 000 – 120 000

copies of the genome (polyploidy)

3. 85 – 250 pg of DNA (human cells: 6 pg)

4. Viviparity

Light Micrograph of the Terminal Thiomargarita namibiensis Cell in a

ChainDiameter: Up to 750 µm

HN Schulz (1999) Science 284: 493

Why bacteria are so small ?

Typical answer: They require a large surface-to-volume ratio to support their internal biochemistry

The sizes of more typical prokaryotes are not due to the ability to take up nutrients per se but arise from the competition for nutrients

Predation

Predation by protozoa = bacterivory: strong evolutionary pressure to develop means of escape Three basic defensic strategies:1. Escaping capture by being too small or too

fast2. Resisting ingestion by becoming too large or

too long3. Making themselves inaccessible by growing

in agregates or biofilms

Defenses Against Bacterivory

KD Young (2007) Curr. Opin. Microbiol. 10: 596

Diversity of Bacterial Cell Shapes

Borrelia burgdorferi

The causative agent of Lyme disease

Evolution of Bacterial Shapes

Phylogenetic analysis indicate that spherical-shaped bacteria arose periodically during evolution from rod-shaped precursors due to a loss of genes:JL Siefert (1998) Microbiol. 144: 2803

Rod-shaped bacteria can be converted to a spherical morphology by deletion of certain genes:M Doi (1988) J. Bacteriol. 170: 4619

Evolution of Bacterial Shapes,

continued

Other bacteria with more elaborate shapes, such as curved or spiral, have additional genes responsible for their distinctive shape

The Cell Wall (Peptidoglycan) Biosynthesis

Modifiers of the cell wall:Elongation: Requires lateral extension of the

murein sacculus by intercalation of new glycan strands and crosslinking of peptide subunits

Septation: Septal peptidoglycan will form the new pole of each daughter cell

Peptidoglycan Synthesis and Processing

MT Cabeen (2005) Nat. Rev. Microbiol. 3: 601

Peptidoglycan Stability

Lateral murein: Exhibits rapid turnover Polar (septal) murein: Metabolically inert Preseptal murein: Discrete patches of stable murein present in non-septate filaments

The Role of MreB

∆mreB (murein region 'e'): Results in conversion from rod shape to sphere

MreB forms a helical structure extending from pole to pole underlying the cytoplasmic membrane

Comparison of the Crystal Structures

of Eukaryotic Actin and

Bacterial MreB

R Carballido (2006) MMBR 70: 888

Helical Cytoskeletal „Cables“Visualized by Fluorescence Microscopy

of B. subtilis

J Errington (2003) ASM News 69: 608

Schematic View of Cell Shape

Formation

J Errington (2003) ASM News 69: 608

Review Articles

YL Shih (2006) Microbiol. Mol. Biol. Rev. 70:

729

Z Gitai (2005) Cell 120: 577

A Carballido-Lopez (2006) Microbiol. Mol. Biol.

Rev. 70: 888

MT Cabeen (2005) Nature Rev. Microbiol. 3:

601

2 Structure of the bacterial cell

1. Cytoplasm

2. Cytoplasmic membrane

3. Cell wall

4. Outer membrane

5. Periplasm

6. Extracellular matrices

7. Appendages

The Bacterial Envelopes

membrane

Mycoplasmas

cell wallmembrane

Gram-positives

membranecell wall

membrane

Gram-negatives

2.1 Cytoplasm

1. The content

2. Microcompartments

3. The cytoskeleton

Content of the cytoplasm:

1. Nucleic acids: chromosome(s), plasmids, prophages = genomeunstable RNAs: mRNA = transcriptomestable RNAs: tRNAs, rRNAs, small RNAs

2. Proteins = proteome: machines (ribosomes, replisome, molecular chaperones, ATP-dependent proteases), structural and functional proteins

3. Metabolites = metabolome

Microcompartments

Definition:Primitive organelles composed entirely of protein subunits ranging in size from 100 to 200 nm

Consist of - a protein shell composed of 5-10 differentproteins

- one or more lumen enzymes

TO Yeates (2008) Nature Rev. Mic. 6: 601

Examples

Carboxysomes: CO2-fixing enzymes Ethanolamine microcomp.: degradation of ethanolamine1,2-propanediol microcomp.: degradation of 1,2-

propanediol

Shell Proteins Contain a Conserved Sequence Referred to as the Bacterial

Microcompartment (BMC) Domain

CA Kerfeld (2005) Science 309: 936

Electron Micrograph of Polyhedral

Microcompartments

a The carboxysomes of Helicobacter neapolitanus

b Microcompartments of Salmonella enterica

TA Bobik (2007) Microbes 2: 25

Purified Bacterial Microcompartments from S. enterica Grown on 1,2-

Propanediol

Composition:

7 different putative shell proteins4 enzymes

Simplified Model of the Carboxysome

6-10 different proteins

RuBisCO:CO2 + ribulosebisphosphate → 3-phosphoglycerate

Why microcompartments ?

To retain volatile compoundsCarboxysomes: CO2

Ethanolamine microcomp.: acetaldehyde1,2-propanediol microcomp.: propionaldehyde

How widespread are

microcompartments ?

About 25% or 85 of 337 bacterial genomes sequenced contain genes coding for putative shell proteins

These genes are absent from Archaea and Eucarya

2.2 Cytoplasmic (inner) membrane

General Structure of the E. coli Cell Envelope

N Ruiz (2005) Nature Rev. Microbiol. 4: 57

Structure of a Phospholipid Bilayer

~ 50% Phospholipids: E. coli

70-80% phosphatidylethanolamine

15-20% phosphatidylglycerol

5% cardiolipin

~ 50% Proteins

Composition

1. Energy generation and conservation

2. Regulated transport of nutrients and metabolic products

3. Translocation of proteins→ Secretion

4. Transmembrane signaling → Two-component signal transduction systems

The cytoplasmic membrane carries out a number and variety of important cellular functions:

What is the function of the cytoplasmic membrane ?

Boundary Selective permeability Respiration/photosynthesis Cell division Cell wall synthesis Secretion of proteins Anchor flagella

Major Functions of the Cytoplasmic Membrane

The Three Types of Transport Systems Across the Membrane

All three systems are energy-dependent

Mechanisms of Solute Transport

The Phosphotransferase System of E. coli

Molecule less likely to diffuse out of cellMolecule ready for glycolysisWhen present primary mode of glucose transportPTS sugars preferred by cell over non-PTS sugars

What is the advantage of PTS ?

Function of an ATP-Binding Cassette

Active transportActive transportMolecules enclosed in vesicle by movement of Molecules enclosed in vesicle by movement of plasma membraneplasma membraneFound mainly in eukaryotesFound mainly in eukaryotes

Endocytosis

Integral membrane proteins with one or more membrane-spanning segments (Triton X-100)

Peripheral membrane proteins (1 M NaCl)- permanent- transient

Proteins: About 800 different species in E. coli

2.3 Periplasm

~10% of the cell volume Highly viscous Occupied by soluble proteins and the peptidoglycan layersOxidizing environment (formation of disulfide bonds) Periplasmic proteins participate in small-molecule transport or breakdown of polymers

1. Murein sacculus

2. Proteins

3. trans-envelope bridges

Components:

The Gram-Negative Cell Wall

Lpp

Structure of the E. coli Peptidoglycan

Diagram of the Gram-Positive Cell Wall

Teichoic Acids and Lipoteichoic Acids

Acidic polysaccharidesNegatively charged: responsible for the negative charge of the cell wall Teichoic and lipoteichoic acid synthesized under phosphate repletion conditionsTeichuronic acid, an anionic polymer without phosphate synthesized under phosphate-limiting conditions

Localization of Periplasm Proteins

Essential protein groups of the periplasm:Integral cytoplasmic membrane proteins inter-acting with the periplasm - through their periplasmic domains - their roles in the biogenesis of function of this compartment

Soluble periplasmic proteins Proteins peripherically associated with the periplasmic side of the inner or outer membraneOuter membrane proteins that protrude into the periplasmic space

Trans-Envelope Signal Transduction

1. TonB-dependent regulatory system

2. The Pal – Tol system

What happens with molecules to big to diffuse through porins ?

There are uptake systems consisting of two or four different components:1. An outer membrane receptor/transducer2. An energizing cytoplasmic membrane-

localized protein complex, where a TonB domain contacts the receptor/transducer

3. An inner membrane-anchored anti-sigma factor

4. An ECF sigma factor

Structural Organization of

TonB-Dependent Regulatory

Systems

R Koebnik (2005) Trends Microbiol. 13: 343

The PAL – Tol System

H Nikaido (2003) Microbiol. Mol. Biol. Rev. 67: 593

PAL = lipoproteinLinks IM with OMRequired for OM integrity

2.4 Outer membrane

Serves as permeability barrier to the outside milieu Is highly asymmetric: - inner leaflat composed of phospholipids - outer leaflat composed of LPS Contains lipoproteins and β-barrel proteins

1. Two types of lipids: phospholipids and lipopolysaccharide (LPS)

2. A set of characteristic proteins

3. Unique polysaccharides

Components:

Bacterial LPS Layer

MH Saier (2008) Microbe 3: 323

Structure of the LPS

O-Antigen:not present in E. coli K12responsible for virulence

Core Oligos:6 to 10 core sugarsbind divalent cations (EDTA)

Lipid A:glucosaminyl-(1→6)-glucosaminesubstituted with 6 or 7 saturated fatty acids

The Mycobacterial Cell Envelope

MH Saier (2008) Microbe 3: 323

The Protein Pattern of the Outer Membrane

1. Murein Lipoprotein: Lpp (homotrimer) 2. General nonspecific diffusion pore (porins):

OmpC, OmpF, PhoE3. Passive, specific transporters: LamB

(maltose), ScrY (sucrose), Tsx (nucleosides)4. Channels involved solute efflux: TolC5. High-affinity receptors6. Active transporters for iron complexes (Fhu,

FepA, FecA) and cobalamin (BtuB)

The Protein Pattern of the Outer Membrane, continued

7. Enzymes such as proteases (OmpT), lipases (OmPIA), acyltransferase (PagP)

8. Toxin binding defense proteins: OmpX9. Structural proteins: OmpA 10.Adhesin proteins: NspA, OpcA11.Channels involved in efflux: TolC 12.Autotransporters

1. Murein Lipoprotein

7,200 DaGene: lpp7 x 105 copies per cellN-terminal cysteine modified:- sulfhydryl group substituted with a digylceride- amino group substituted by a fatty acyl residueAnchored into the inner leaflat of the outer membraneAbout one-third of the lipoprotein molecules bound covalently to the murein via a lysine res.lpp mutants: unstable outer membrane

2. Classical Porins

OmpF, OmpC and PhoETrimericProduce nonspecific pores (channels; ~ 1 nm in diameter) that allow the rapid passage of small (~ 600 Da) hydrophilic moleculesPhoE is produced only under conditions of phosphate starvationMechanism for opening and closing of the pores

Structure of the OmpF Porin

H Nikaido (2003) Microbiol. Mol. Biol. Rev. 67: 593

A: View of the trimer from the topB: View of the monomeric subunit from the sideC: View of the monomeric subunit from the top

showing the constricted region of the channel

3. The OmpA Protein

Monomeric porin with a diameter of ~ 0.7 nm105 molecules per cellompA mutants are extremely poor recipients in conjugationPenetration of solutes is about two orders of magnitude slower than through the OmpF channel

β-Barrel Membrane

Protein OmpA

From the plane of the membrane

From the top of the membrane

Cyan: internal cavities

R Koebnik (20000) Mol. Microbiol. 37: 239

4. The Specific Channels

• LamB (lamB)- porin-like trimeric protein- allows the passage of maltose and maltodextrins

- receptor for phage λ• T6 receptor (tsx)

- specific diffusion of nucleosides

X-Ray Crystallographic Structure of LamB

H Nikaido (2003) Microbiol. Mol. Biol. Rev. 67: 593

A: Side view of the monomeric unitsB: View of the monomeric unit from the topC: View of the greasy slide and its interaction

with maltotriose

5. High-Affinity Receptors

Btu (btuB)- diffusion of vitamin B12FadL (fadL)- diffusion of long-chain fatty acids

Transport requires the presence of TonB:- anchored in the inner membrane- extends through the periplasmic space- interacts with the receptor

6. Proteins Involved in Direct Import/Export of Proteins and Drugs

TolC- Involved in the entry of some colicins- Serves as a channel for the export of hemolysinPapC- Recognizes specifically the various subunits of the Pap pilus

PulD- Many proteins are secreted through this pore, e.g., filamentous phage protein IV

- Involved in phage export

Outer Membrane Biogenesis

N Ruiz (2005) Nature Rev. Microbiol. 4: 57AC McCandish (2007) Microbe 6: 289

1. Movement of LPS from the cytoplasm into

the outer leaflat of the OM

2. Movement of β-barrel proteins from the

cytoplasm into the OM

How does LPS move to the outer membrane?

AC McCandish (2007) Microbe 6: 289

LPS is flipped to the outer leaflat of the IM mediated by MsbA(ABC-transporter) Two models for

crossing the periplasm: - active: LptA- passive: Bayer‘s

bridges

Insertion of LPS Into the OM: Role of Imp and RlpB

AC McCandish (2007) Microbe 6: 289

How Proteins Move to the OM

Translocation through the Sec system

Skp, DegP and SurAchaperones prevent misfolding and aggregation

Protein complex required for assembling OM proteins

3 Extracellular matrices

1. S-layers

2. Capsules and slime layers

Monomolecular crystalline array of proteinaceous subunitsS-layers possess pores identical in size and morphology in the 2- to 8-nm range; work as precise molecular sieves40 – 170 kDaSome S-layer proteins are glycosylated

2. S-layers

S-Layer of the ArchaeonThermoproteus tenax

Electron Micrograph of a Freeze-Etched Preparation

Architecture of Cell Envelopes

Containing S-Layers

Gram-positive Gram-negative

UB Sleytr (1999) Trends Microbiol. 7: 253

3. Capsules and Slime LayersSlimy or gummy materialConsist mostly of polysaccharide, rarely of proteinsGeneral term: glycocalyxFunctions:

- Attachment of certain pathogenic bacteria to their hosts

- Encapsulated bacteria are more difficult for phagocytic cells of the immune system (Pneumococcus)

- binds a significant amount of water: plays some role in dessication

3. Capsules and Slime Layers

Functions:- Attachment of certain pathogenic bacteria to their hosts

- Encapsulated bacteria are more difficult for phagocytic cells of the immune system (Pneumococcus)

- binds a significant amount of water: plays some role in dessication

→ biofilms

Bacterial Capsules

Acinetobacter Rhizobium trifolii

A Model for Assembly of the K5 Capsule

4 Appendages

1. Flagellum (flagella)

2. Pilus (pili) = fimbrium (fimbriae)

3. Curli

4.1 Flagellum (Flagella)

GS Chilcott (2000) MMBR 64: 694

OA Soutourina (2003) FEMS Microbiol. Rev. 27: 505

Flagella = nanomotor

Are long, thin, up to 15 µm long (10x the length of the bacterium) appendages free at one end and attached to the cell at the other end4-10 flagella per cell Consist of three main components:- basal body: anchors the flagellum in the two membranes

- hook- filamentFunction: movement and chemotaxis

Arrangements of Flagella in Different Bacteria

Structure of the Prokaryotic Flagellum and Attachment to the Cell Wall and Membrane

C ring: FliG, FliM, FliN

~ 120 FlgE

pentameric capprotein HAP2

Flagella Biosynthesis of Gram-Negative Bacteria

Manner of Movement in Peritrichously Flagellated Prokaryotes

Manner of Movement in PolarlyFlagellated Prokaryotes

Electron

Micrograph of

Vibrio

paraheamolyticus

SL Brady (2003)

Microbiol. 149: 295

4.2 Pilus (Pili) = Fimbrium (Fimbriae)

Pilin subunits are attached to each other

non-covalently in Gram-negative bacteriacovalently in Gram-positive bacteria

JL Telford (2006) Nature Rev. Mic. 4: 509

Are proteinaceous, hairlike appendages, 2 to 8 nm in diameter, on the surface of bacteriaBetween 3 to 1,000 pili per cellInvolved in attachment to surfaces

Pili (fimbriae)

Pili in Gram-Negative Bacteria

Type I pili:Rigid rod with flexible tip adhesin 1-2 µm long 4-5 pilin proteins

Type IV pili:flexible rod 1-2 µm long >2 pilin proteins

Pili in Gram-Negative Bacteria

Curli pili:Rigid rod with flexible tip adhesin 1-2 µm long 2 pilin proteins

Pili in Gram-Positive Bacteria

Fibrils:Short, thin rod 0.07-0.5 µm long 2 pilin proteins

Pili:flexible rod 0.3-3 µm long 2-3 pilin proteins

Pili are assembled by at least four different pathways:

1. The chaperone-usher pathway

2. The secretin pathway

3. The curli pathway

4. The sortase pathway

1. The F-pilus

2. The type I pili

3. The T-pilus

4. The Pap-Pilus

5. Curli

6. The pilus of Corynebacterium

diphtheriae

Examples:

The F Pilus

Consists of only one protein, the F pilin (traA)

The N-terminal amino acid of the pilin (7,000 da)

is N-acetylated

Cells possess one to three pili, 2 to 3 µm in

length

Serve as receptor for some phages

The Type I Pili

Produced by many members of the family

Enterobacteriaceae

Play a major role in

- biofilm development

- pathogenesis during the course of human

infections

E. coli cells can switch from a completely

piliated state to a completely nonpiliated state =

phase variation

Model of the

Biogenesis

of the T-

Pilus

E.-M. Lai (2000) Trends Microbiol. 8:

361

Formation of the Cyclic T-Pilin

E-M Lai (2000) Trends Microbiol. 8: 361

Genetic Organization of the pap

Gene Cluster

DG Thanassi (2000) Methods 20: 111

Model of Pap Pilus Assembly

FG Sauer (2000) Curr. Opin. Struct. Biol. 10: 548

Curli Belong to the „Functional“

Amyloids

What are amyloids ?

Amyloidogenic proteins (amyloids) are found in several medically related disorders such as- Alzheimer disease - Huntington disease - Parkinson disease - Transmissible spongiform encephalopathies

Amyloid Formation

Uncontrolled conversion of soluble proteins into biochemically and structurally related fibers 4-12 nm wide

Amyloidogenic proteins are mostly unstructured or contain mixtures of β-sheets and α-helices in their native structure

Electron Micrographs of Curli

a Curlis present

b Curlis absent

c Purified fibers

Curli Fibers

Extracellular 4-6 nm-wide amyloid fibersForm a tangled extracellular matrix connecting several neighbouring cells into small groupsResist protease digestion, remain insoluble when boiled in 1% SDSAt least five proteins in E. coli are dedicated to assembling curli on the cell surfaceMajor component: 13-kDa CsgA protein

Model of Curli Assembly

A: curli subunitB: nucleator

protein F, E: required for

efficient curli assembly

G: required for secretion

D: transcriptional activator

Interbacterial Complementation

Observation:No curli formation in the absence of CsgBE. coli csgB- secretes CsgAE. coli csgA- does not produce curli If both strains are grown together the csgA-

strain will form curli

Pilus Assembly in Corynebacterium diphtheriae: Polymerization

A Mandlik (2008) PNAS 105: 14152

Pilus Assembly in Corynebacterium diphtheriae: Anchoring

A Mandlik (2008) PNAS 105: 14152