Download The Bacterial Chromosome: Structure and Function

The Bacterial Chromosome:
Structure and Function
Time Table
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
Borrelia burgdorferi
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 (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
membrane
Mycoplasmas
cell wall
cell wall
membrane
membrane
Gram-
Gram-
positives
negatives
2.1
Cytoplasm
1. The content
2. Microcompartments
3. The cytoskeleton
Content of the cytoplasm:
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: CO2-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 1,2Propanediol
Composition:
7 different putative
shell proteins
4 enzymes
Simplified Model of the Carboxysome
6-10 different proteins
RuBisCO:
CO2 + ribulose
bisphosphate → 3phosphoglycerate
Why microcompartments ?
To retain volatile compounds
Carboxysomes: CO2
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
Composition
~ 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
plasma membrane
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
Components:
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
7,200 Da
Gene: lpp
7 x 105 copies per cell
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
ƒ
ƒ
ƒ
ƒ
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 105 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
2. S-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 FreezeEtched 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 GramNegative 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
Examples:
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
The F Pilus
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
The Type I Pili
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 TPilus
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
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 csgAstrain 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