3 The Cell Cycle: Replication of the

Bacterial Chromosome,
Partitioning and Cell Division

1
Questions:

1. Which genes are involved ?

2. What is the function of the gene products ?
3. How are the different processes regulated at
both the genetic and the biochemical level ?
4. If there are pertubations, how does the cell
react ?

2
Schematic Representation of the
Bacterial Life Cycle

3
 Schematic
Description of
the Bacterial
Cell Cycle

Chromosome arms =
replichores

F Touzain (2011)
Nature Rev Microbiol
9: 15 4
3.1 Replication

5
 Nomenclature

oriC

terC

dif

Replisome

Continuous – discontinuous synthesis

Leading – lagging strand

6
3.1.1 Components Involved in
Replication

7
 Subunit Composition of DNA
Polymerase III Holoenzyme

Three functional complexes are distinguished:

1. Sliding clamp (β subunit): Forms a ring around
the DNA
2. Clamp loader (γ complex): γ3δδ’χψ
3. Pol III core: α-ε-θ

8
Schematic Diagram of the β Subunit
Dimer

9
The Pol III* Subassembly of DNA
Polymerase III Holoenzyme
= clamp loader

= sliding clamp

T Katayama (1999)
10
Biochemie 81: 835
DnaA:
ƒ Key protein for initiation of replication
ƒ Is active as DnaA-ATP
ƒ Three major functions:
1. Acts as a pacemaker of initiation through
ordered binding to five 9mers called DnaA
boxes
2. Promotes local unwinding of the AT-rich region
in the left part of oriC (formation of the open
complex)
3. Acts as a preprimosome by guiding the DnaB
11
helicase to its entry site
 Important Additional Proteins
Involved in DNA Replication
DnaB: Replicative helicase; hexamer
DnaC: Loader for DnaB; hexamer
DnaG: Primase; length of primers: 11 +/- 1 nucl.
SSB: Covers the single-stranded DNA
DNA gyrase: Relaxation of DNA; acts as a swivel
DNA polymerase I: Removes primers and
polymerizes the gaps at the same time
DNA ligase: Seals the nicks between the replaced
primers and the adjacent Okazaki fragments
12
Rep helicase: Removes proteins from the DNA
3.1.2 Initiation of Replication

13
 Structural Organization of the
Escherichia coli oriC region

Aim: Strand separation at the AT-rich 13mers

W Messer (2002) FEMS Microbiol. Rev. 26: 355

14
Minimal oriC: 245 bp

Contains:
1. Five DnaA boxes
2. Binding sites for FIS, IHF and H-NS: involved in
the organization of the correct three-
dimensional structure of oriC
3. IciA: Inhibitor of chromosomal initiation A; may
block opening of the DNA duplex
4. 11 GATC sites

How to identify a minimal oriC ?

15
Model for the Initiation Complex at
the Escherichia coli oriC

20 - 40 DnaA-ATP
monomers
16
Steps of Initiation of DNA Replication
at oriC
1. Binding of DnaA-ATP: Formation of the open complex
2. Coating of the single-stranded DNA by SSB
3. Binding of DnaC-DnaB complex to the two single
strands: Formation of the prepriming complex I
4. DnaB translocates from the entry to the start site,
dissociation of DnaC: formation of the prepriming
complex II
5. DnaG interacts with DnaB and synthesizes the primer:
Priming complex
6. DNA polymerase III elongates the primer: Formation of
the replisome 17
 Schematic Representation of the
Orientation of Two DnaB Hexamers
bound to the Two Arms of the
Replication Fork

W Bujalowski (2003)
TIBS 28: 116
18
Initiation of Replication at
the Escherichia coli oriC

W Messer (2002) 26: 355

19
Two types of primosomes:

1. Replicative primosome

2. Replication restart primosome

20
The DNA Replication Fork

21
B Albers (2003) Nature 421: 431
Leading and Lagging Strand DNA
Synthesis

B Albers (2003) Nature 421: 431

22
Proteins at the Replication Fork

DNA gyrase

SSB

B Albers (2003) Nature 421: 431

23
The Lagging and Leading Strand
DNA Polymerases Remain Tied

B Albers (2003) Nature 421: 431 24

 The Replisome Complex

CS McHenry (2003) Mol. Microbiol. 49: 115725

3.1.3 Elongation of Replication

26
Model of SOS Translesion Replication
by DNA Polymerase V

DNA V polymerase
does not have a
proof-reading activity

error-prone

EC Friedberg (2005)
Nature Rev. Mol. Cell
Biol. 6: 943
27
3.1.4 Termination of Replication

Two components:

1. Termination sites (ter elements)

2. Protein binding to the ter elements

28
3.1.4.1 Termination of Replication in
Escherichia coli

29
 Map of the E. coli Chromosome
Showing Locations of Termination
Sites
ter sites:
- 22 bp
- polarity
- bind Tus (Terminus
utilization substance)
- not essential

S Muluga (2001)
Proc. Natl. Acad. Sci
USA 98: 9569 30
Termination of Replication

R Rothstein (2000) Genes Dev. 14: 1 31

Model: Polar Antihelicase Activity of
Tus

32
3.1.4.2 Termination of Replication
in Bacillus subtilis

33
The Replication Termini of Bacillus
subtilis

Structure of the ter Sites

34
 The Replication Termini of Bacillus
subtilis

35
DE Bussière et al. (1999) Mol. Microbiol. 31: 1611
3.1.5 Regulation of Chromosomal
Replication

36
Three negative regulation
mechanisms identified:

1. By SeqA

2. Titration of DnaA

3. DnaA-ATP ⇔ DnaA-ADP

37
Model of the Sequestration of the oriC
Region of the E. coli Chromosome
After Initiation of Replication

38
Titration of DnaA:

datA (DnaA titration A)

ƒ datA = chromosomal region of about 1 kb

ƒ Contains five 9- mer sequences (DnaA boxes)
ƒ in vitro, datA binds 370 DnaA molecules
ƒ DnaA is present in about 1000 molecules per
cell

39
Regulatory Inactivation of DnaA (RIDA):
DnaA-P ⇔ DnaA-ADP: hdaA (homologous to dnaA)

T Katayama (1999) Biochemie 81: 835

40
3.1.6 Decatenation

41
Decatenation of Terminated
Daughter Molecules

KP Lemon (2001) Genes Dev. 15: 2031 42

Resolution of a Chromosome Dimer
Into two Monomers by Resolvases
XerCD

KP Lemon (2001) Genes Dev. 15: 2031 43

How FtsK Might Promote Synapsis of
Directly Repeated dif Sites

FtsK interacts
with short
polarized
recombination
activating gene
(RAG) sequences

SC Ip (2003) EMBO J. 22: 6399

44
The sequence-directed DNA
translocase FtsK is involved in:

1. Chromosome dimer resolution

2. Segregation
3. Decatenation

45
 The Effector Sequence of FtsK

GNGNAGGG and its complement

O Levy (2005) PNAS 102: 17618 46

3.1.7 Replication of Linear
Chromosomes and Plasmids

47
The problem:

Replication of the 5' ends (telomers)

5'-----------------------------3'
3'-----------------------------5'
⇓ --- = RNA primer
5'-----------------------------3'
←------------←-----------

--------→---------------→
3'-----------------------------5' RNA primase cannot
start at the ends of
DNA 48
Unusual primers:

1. Tyrosine residue of a protein

covalently linked to the 5' ends

2. tRNA

3. „covalently closed hairpin ends“

49
 Replication of Linear DNA with
Hairpin Ends (Borrelia)

L, R: left,
right inverted
repeat

50
G Chaconas (2010) Ann Rev Microbiol 64: 185
Telomer Resolution
by ResT

G Chaconas (2010) Ann Rev

Microbiol 64: 185

51
3.2 Partitioning (Segregation) of
the Daughter Chromosomes

Three models:
1. The replicon model (Jacob, 1963)
2. The tracking model
3. The spooling model

52
 The Replicon Model

The two replicating oriC‘s are bound to the

membrane and separated during elongation of
the cell

53
 The Tracking Model

The sister replisomes migrate away from each other

D Bates (2008) Mol Microbiol 69: 1341 54

 The Spooling Model

The DNA moves, spooling through a stationary

replisome
55
D Bates (2008) Mol Microbiol 69: 1341
How Do Origin Regions Migrate to
the Cell Poles ?
E. coli:
ƒ Does not depend on the presence of oriC per se
ƒ Potential centromer: migS (25-bp sequence;
imperfect stem-loop)

B. subtilis:
ƒ RacA binds near oriC during sporulation and
interacts with DivVA, a membrane protein
ƒ RacA-binding motif: 14-bp hairpin
56
Model for the E. coli 'Centromere'

57
Y Yamaichi (2004) EMBO J. 23: 221
Model for the B. subtilis ‚Centromere'

RacA

S Ben-Yehuda (2003) Science 299: 532 58

 Condensins

Proteins involved in the compaction of the

bacterial chromosome:

ƒ MukBEF

ƒ SMC (Structural Maintenance of Chromosomes)

59
Formation, Extension and Reconden-
sation of the Condensed Filament

MukBEF

RB Case (2004) Science 305: 222 60

A Speculative Model for Condensed
DNA in vivo

61
 Structure of SMC Proteins

PL Graumann (2001) Biochimie 83: 53 62

Model for the Function of SMC
During the Bacterial Cell Cycle

PL Graumann (2001) Biochimie 83: 53 63

3.3 Cell Division

Mechanisms of cell division:

1. Binary fission
2. Production of multiple endospores:
Metabacterium polyspora
Segmented filamentous bacteria
3. Viviparity in Epulopiscium
4. Multiple offspring by multiple fission
64
 1. Binary fission

A cell grows to twice its size and than splits in

two

Important:
ƒ The cell must divide at the appropriate time
and at the correct location in the cell
ƒ The cell must ensure that each progeny cell
receives a complete complement of genes
with high fidelity 65
 The Life Cycle of Bacillus subtilis

Endospore formation = an exceptional adaptive

advantage
66
EA Angert (2005) Nature Rev. Microbiol. 3: 214
2. Production of Multiple Endospores:
Metabacterium polyspora

Metabacterium polyspora:
ƒ Inhabits the gastrointestinal tract of guinea
pigs
ƒ Produces up to nine endospores per mother
cell

67
Formation of Multiple Endospores

a: Cell divides at both

poles
b: Forespore are
engulfed by the
mother cell
c: Forespores are
capable of binary
fission
d: Forespores
continue to divide
and grow
e: Forespores mature
68
into endospores
 2. Production of Multiple Endospores:
Segmented filamentous bacteria (SFB)

SFB:
ƒ Have been found in the intestinal tracts of
various animals
ƒ Develop as a multicellular filament that is
anchored to the epithelial lining of the distal
ileum

69
Segmented Filamentous
Bacteria
Up to 1 mm long

Intracellular offspring
Holdfast ƒ vegetative cells
ƒ spores

70
 3. Viviparity in Epulopiscium:
Intracellular offspring formation

Up to 12 offspring per mother cell have been

observed 71
4. Multiple Offspring by Multiple
Fission:

72
4. Multiple Offspring by Multiple
Fission: Pleurocapsalean
cyanobacteria

ƒ Cell enlarges, undergoes

multiple fissions to produce
offspring known as baeocytes
(„small cells“; 1-2 µm in
diameter; 4 to more than 1000)
per mother cell
ƒ The extracellular matrix tears
open, releasing the baeocytes
73
4. Multiple Offspring by Multiple
Fission: Pleurocapsa

ƒ Grow and divide by

binary fission forming
filamentous aggregates
ƒ Some of the cells
produce baeocytes

74
4. Multiple Offspring by Multiple
Fission: Dermocarpelia

ƒ The cell enlarges

asymmetrically
ƒ The cell divides
asymmetrically
ƒ The larger reproductive
cell undergoes multiple
fission and releases
baeocytes

75
4. Multiple Offspring by Multiple
Fission: Bdellovibrio

ƒ Forms a multinucleoid
filamentous cell in the
periplasm
ƒ When the host‘s
resources are
exhausted, multiple
fissions
76
4. Multiple Offspring by Multiple
Fission: Pedomicrobium

77
4. Multiple Offspring by Multiple
Fission: Ancalomicrobium

78
4. Multiple Offspring by Multiple
Fission: Planctomyces

79
 Proteins Involved in Cell Division

1. FtsZ: Forms the Z ring at midcell

2. MinCDE: Accomplish the placement of the

division site at midcell

3. FtsA, ZipA, FtsK, FtsQ, FtsL, FtsW, FtsI and

FtsN are recruited to the septal ring and form
the divisome

80
The Role of FtsZ in E. coli Cell Division

Polymerization
of FtsZ is
inhibited by
SfiA (= SulA;
SOS response)

81
Model of MinCDE Action

one new - two old

septation sides

82
Assembly and Disassembly of the
MinD Tube

Time lapse [sec]

83
 Model of Min
Oscillation

J Lutkenhaus (2003)
Mol. Microbiol. 48: 295
84
 Model for MinD Binding to the
Membrane

Amphipathic α-helix:
- mainly hydrophobic
amino acid residues
on one side
- mainly hydrophilic once
on the other side

J Lutkenhaus (2003) Mol. Microbiol. 48: 29585

 Complex Cycle of MinD ATPase

J Lutkenhaus (2003) Mol. Microbiol. 48: 295

86
 Organization of the Min Proteins
Within E. coli

87
Z Gitai (2003) Proc. Natl. Acad. Sci. USA 100: 7423
 Schematic Representation of the Ten
Known E. coli Cell Division Proteins

N Buddelmeijer (2002) Curr. Opin.Microbiol. 5:88553

Recruitment of Division Proteins to
the Septal Ring of E. coli

KLN Mercer (2002) J. Bacteriol. 184: 904 89

3.4 Plasmid- and Chromosome-
Encoded Toxin-Antitoxin
Modules

3.4.1 Postsegregational killing of plasmid-free

cells

3.4.2 Chromosomal toxins-antitoxins

90
 Schematic
Representation of
Cell Death Induced
by Plasmid-
Located TA
Modules
red = toxin
blue = antitoxin
green = protease

F Hayes (2003) Science 301: 1496

91
Location of Known Type I and Type II
TA Modules
red = type I
yellow = type II

F Hayes (2003) Science 301: 1496

92
 E. coli mazEF-Mediated Cell Death

93
H Engelberg-Kulka (2006) PLoS Genetics 2: e135