Organization of the bacterial cell
Organization of the bacterial chromosome
Replication and cell division
Recombination
DNA repair
Gene regulation I
Gene regulation II
Gene regulation III
Genre regulation IV
Chaperones and ATP-dependent proteases
Secretion of proteins
Adaptation to stress
Gene transfer

Literature
Lary Snyder and Wendy Champness:
Molecular Genetics of Bacteria
ASM 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

Epulopiscium fishelsonii
80 x 600 µm
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
ER Angert (1993) Nature 362: 239
JE Mendell (2008) PNAS 105: 6730

Light Micrograph of the Terminal
Thiomargarita namibiensis Cell in a
Chain
Diameter:
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 fast
2. Resisting ingestion by becoming too large or too long
3. 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

The causative agent of Lyme disease

Evolution of Bacterial Shapes
Phylogenetic analysis indicate that sphericalshaped 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:
ƒ Elongation : Requires lateral extension of the murein sacculus by intercalation of new glycan strands and crosslinking of peptide subunits
ƒ 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 ( m urein r egion ' 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 cell wall membrane
Mycoplasmas
Grampositives membrane cell wall membrane
Gramnegatives

2.1 Cytoplasm
1. The content
2. Microcompartments
3. The cytoskeleton

1. Nucleic acids: chromosome(s), plasmids, prophages = genome unstable RNAs: mRNA = transcriptome stable 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 different proteins
one or more lumen enzymes
TO Yeates (2008) Nature Rev. Mic. 6: 601

Examples
Carboxysomes : CO
2
-fixing enzymes
Ethanolamine microcomp .: degradation of ethanolamine
1,2-propanediol microcomp .: degradation of 1,2propanediol

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 proteins
4 enzymes

Simplified Model of the Carboxysome
6-10 different proteins
RuBisCO:
CO
2
+ ribulose bisphosphate → 3phosphoglycerate

Why microcompartments ?
To retain volatile compounds
Carboxysomes : CO
2
Ethanolamine microcomp .: acetaldehyde
1,2-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

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

What is the function of the cytoplasmic membrane ?
ƒ Boundary
ƒ Selective permeability
ƒ Respiration/photosynthesis
ƒ Cell division
ƒ Cell wall synthesis
ƒ Secretion of proteins
ƒ Anchor flagella

Major Functions of the Cytoplasmic
Membrane

The Three Types of Transport
Systems Across the Membrane
All three systems are energydependent

Mechanisms of Solute Transport

The Phosphotransferase System of
E. coli

What is the advantage of PTS ?
ƒ Molecule less likely to diffuse out of cell
ƒ Molecule ready for glycolysis
ƒ When present primary mode of glucose transport
ƒ PTS sugars preferred by cell over non-PTS sugars

Function of an ATP-Binding Cassette

Endocytosis
ƒ Active transport
ƒ Molecules enclosed in vesicle by movement of
ƒ Found mainly in eukaryotes

Proteins: About 800 different species in E. coli
ƒ Integral membrane proteins with one or more membrane-spanning segments (Triton X-100)
ƒ Peripheral membrane proteins (1 M NaCl)
- permanent
- transient

2.3 Periplasm
ƒ ~10% of the cell volume
ƒ Highly viscous
ƒ Occupied by soluble proteins and the peptidoglycan layers
ƒ Oxidizing environment (formation of disulfide bonds )
ƒ Periplasmic proteins participate in smallmolecule transport or breakdown of polymers

:
1. Murein sacculus
2. Proteins
3. trans -envelope bridges

The Gram-Negative Cell Wall
Lpp

Structure of the E. coli Peptidoglycan

Diagram of the Gram-Positive Cell
Wall

Teichoic Acids and Lipoteichoic
Acids
ƒ Acidic polysaccharides
ƒ Negatively charged: responsible for the negative charge of the cell wall
ƒ Teichoic and lipoteichoic acid synthesized under phosphate repletion conditions
ƒ Teichuronic acid, an anionic polymer without phosphate synthesized under phosphatelimiting conditions

Localization of Periplasm Proteins
Essential protein groups of the periplasm:
ƒ Integral cytoplasmic membrane proteins interacting with the periplasm
- through their periplasmic domains
- their roles in the biogenesis of function of this compartment
ƒ Soluble periplasmic proteins
ƒ Proteins peripherically associated with the periplasmic side of the inner or outer membrane
ƒ Outer 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/transducer
2. An energizing cytoplasmic membranelocalized 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
ƒ PAL = lipoprotein
ƒ Links IM with OM
ƒ Required for OM integrity
H Nikaido (2003) Microbiol. Mol. Biol. Rev. 67: 593

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

Components:
1. Two types of lipids: phospholipids and lipopolysaccharide (LPS)
2. A set of characteristic proteins
3. Unique polysaccharides

Bacterial LPS Layer
MH Saier (2008) Microbe 3: 323

Structure of the LPS
O-Antigen:
ƒ not present in E. coli K12
ƒ responsible for virulence
Core Oligos:
ƒ 6 to 10 core sugars
ƒ bind divalent cations (EDTA)
Lipid A:
ƒ glucosaminyl-(1 → 6)-glucosamine
ƒ substituted 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, PhoE
3. Passive, specific transporters: LamB
(maltose), ScrY (sucrose), Tsx (nucleosides)
4. Channels involved solute efflux: TolC
5. High-affinity receptors
6. 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: OmpX
9. Structural proteins: OmpA
10.Adhesin proteins: NspA, OpcA
11.Channels involved in efflux: TolC
12.Autotransporters

1. Murein Lipoprotein
ƒ 7,200 Da
ƒ Gene: lpp
ƒ 7 x 10 5 copies per cell
ƒ N-terminal cysteine modified:
- sulfhydryl group substituted with a digylceride
- amino group substituted by a fatty acyl residue
ƒ Anchored into the inner leaflat of the outer membrane
ƒ About one-third of the lipoprotein molecules bound covalently to the murein via a lysine res.
ƒ lpp mutants: unstable outer membrane

2. Classical Porins
ƒ OmpF, OmpC and PhoE
ƒ Trimeric
ƒ Produce nonspecific pores (channels; ~ 1 nm in diameter) that allow the rapid passage of small
(~ 600 Da) hydrophilic molecules
ƒ PhoE is produced only under conditions of phosphate starvation
ƒ Mechanism for opening and closing of the pores

Structure of the OmpF Porin
A: View of the trimer from the top
B: View of the monomeric subunit from the side
C: View of the monomeric subunit from the top showing the constricted region of the channel
H Nikaido (2003) Microbiol. Mol. Biol. Rev. 67: 593

3. The OmpA Protein
ƒ Monomeric porin with a diameter of ~ 0.7 nm
ƒ 10 5 molecules per cell
ƒ ompA mutants are extremely poor recipients in conjugation
ƒ Penetration 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
Cyan: internal cavities
From the top of the membrane
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
A: Side view of the monomeric units
B: View of the monomeric unit from the top
C: View of the greasy slide and its interaction with maltotriose
H Nikaido (2003) Microbiol. Mol. Biol. Rev. 67: 593

5. High-Affinity Receptors
Transport requires the presence of TonB:
anchored in the inner membrane
extends through the periplasmic space
interacts with the receptor
ƒ Btu ( btuB )
- diffusion of vitamin B12
ƒ FadL ( fadL )
- diffusion of long-chain fatty acids

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

How does LPS move to the outer membrane?
ƒ LPS is flipped to the outer leaflat of the IM mediated by MsbA
(ABC-transporter)
ƒ Two models for crossing the periplasm:
- active: LptA
- passive: Bayer‘s bridges
AC McCandish (2007) Microbe 6: 289

Insertion of LPS Into the OM: Role of Imp and RlpB
AC McCandish (2007) Microbe 6: 289

How Proteins Move to the OM
Protein complex required for assembling OM proteins
Skp, DegP and SurA chaperones prevent misfolding and aggregation
Translocation through the
Sec system

3 Extracellular matrices
1. S-layers
2. Capsules and slime layers

ƒ Monomolecular crystalline array of proteinaceous subunits
ƒ S-layers possess pores identical in size and morphology in the 2- to 8-nm range; work as precise molecular sieves
ƒ 40 – 170 kDa
ƒ Some S-layer proteins are glycosylated

S-Layer of the Archaeon
Thermoproteus 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 Layers
ƒ Slimy or gummy material
ƒ Consist mostly of polysaccharide, rarely of proteins
ƒ General term: glycocalyx
ƒ 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

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
ƒ 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 end
ƒ 4-10 flagella per cell
ƒ Consist of three main components:
- basal body: anchors the flagellum in the two membranes
- hook
- filament
ƒ Function: movement and chemotaxis

Arrangements of Flagella in Different
Bacteria

Structure of the Prokaryotic Flagellum and
Attachment to the Cell Wall and Membrane pentameric cap protein HAP2
~ 120 FlgE
C ring:
FliG, FliM,
FliN

Flagella Biosynthesis of Gram-
Negative Bacteria

Manner of Movement in Peritrichously
Flagellated Prokaryotes

Manner of Movement in Polarly
Flagellated 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
ƒ non-covalently in Gram-negative bacteria
ƒ covalently in Gram-positive bacteria
JL Telford (2006) Nature Rev. Mic. 4: 509

Pili (fimbriae)
ƒ Are proteinaceous, hairlike appendages, 2 to
8 nm in diameter, on the surface of bacteria
ƒ Between 3 to 1,000 pili per cell
ƒ Involved in attachment to surfaces

Pili in Gram-Negative Bacteria
Type I pili:
ƒ Rigid rod with flexible tip adhesin
ƒ 1-2 µm long
ƒ 4-5 pilin proteins
Type IV pili:
ƒ flexible rod
ƒ 1-2 µm long
ƒ >2 pilin proteins

Pili in Gram-Negative Bacteria
Curli pili:
ƒ Rigid rod with flexible tip adhesin
ƒ 1-2 µm long
ƒ 2 pilin proteins

Pili in Gram-Positive Bacteria
Fibrils:
ƒ Short, thin rod
ƒ 0.07-0.5 µm long
ƒ 2 pilin proteins
Pili:
ƒ flexible rod
ƒ 0.3-3 µm long
ƒ 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

ƒ Consists of only one protein, the F pilin ( traA )
ƒ The N-terminal amino acid of the pilin (7,000 da) is N-acetylated
ƒ Cells possess one to three pili, 2 to 3 µm in length
ƒ Serve as receptor for some phages

ƒ Produced by many members of the family
Enterobacteriaceae
ƒ Play a major role in
- biofilm development
- pathogenesis during the course of human infections
ƒ 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

ƒ Extracellular 4-6 nm-wide amyloid fibers
ƒ Form a tangled extracellular matrix connecting several neighbouring cells into small groups
ƒ Resist protease digestion, remain insoluble when boiled in 1% SDS
ƒ At least five proteins in E. coli are dedicated to assembling curli on the cell surface
ƒ Major component: 13-kDa CsgA protein

Model of Curli Assembly
A: curli subunit
B: nucleator protein
F, E: required for efficient curli assembly
G: required for secretion
D: transcriptional activator

Interbacterial Complementation
Observation:
ƒ No curli formation in the absence of CsgB
ƒ E. coli csgB
secretes CsgA
ƒ E. coli csgA does not produce curli
ƒ 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