Bacterial Cell Cycle

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February 13, 2009
Bacterial Cell Cycle
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What do cells need to do to reproduce themselves?
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Eukaryotic cell cycle
Image from Purves et al., Life The Science of
Biology, 4th Edition,
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The Bacterial Cell Cycle
initiation of replication
cell division
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1
Nucleoid segregation
Elongation and Origin separation
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2
Termination
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Step 1DNA REPLICATION

• OR
• how to go from 1 copy of your genome to two

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Prokaryotic DNA replication

• Unwind DNA near oriC-topoisomerases
• topoisomerases nick and unwind DNA

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Prokaryotic DNA replication
2. Origin recognition-DnaA
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Prokaryotic DNA replication
DnaA bound to ATP
3. Open complex formation-helicaseSSB
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Prokaryotic DNA replication
4. RNA primer formation-primosome (The primasome
is a complex of 20 proteins in E. coli, one of
which is called Primase-an RNA polymerase)
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Prokaryotic DNA replication
5. Elongation-DNA polymerase III
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Prokaryotic DNA replication
Some proteins involved in DNA replication
Preparing for replication
Gyrase/topoisomerase-unwinds DNA, cuts and
reseals it to relieve supercoils
Helicase-unwinds DNA in front of replication
fork. Doesnt cut DNA.
SSB-single stranded binding protein. Keeps single
stranded DNA at replication fork from reannealing
with complementary DNA.
Primase-an RNA polymerase that uses a DNA
template to lay down RNA primer
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Prokaryotic DNA replication
Some proteins involved in DNA replication
The Polymerases
DNA polymerase III-the replicative polymerase-has
proof reading ability, meaning it can back up
and remove the incorrect nucleotide and then
resume replication. Error rate 1 in 10 million
base pairs.
DNA polymerase I-responsible for removing RNA
primers and replacing them with DNA. Has 5 to 3
exonuclease activity to remove RNA primers and
5to 3 polymerase activity which fills in the
gap left by removal of the primer.
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Prokaryotic DNA replication
Some proteins involved in DNA replication
The Accessory Proteins
The sliding clamp-functions to hold polymerase to
DNA
Tau-holds the two core polymerases together
DNA ligase-seals nicked DNA
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The Sliding Clamp
The Beta subunit of DNA Pol III Functions to
increase the rate and processivity of DNA
replication.
With clamp rate 1000bp/sec processivity up to
50,000bp Without clamp rate 10bp/sec processiv
ity 10 bases
Processivity of DNA polymerase enzymes refers to
their ability to add many hundreds or thousands
of nucleotides to a growing chain without
dissociating from the template.
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How were all these proteins identified?

• Genetic Approach
• Isolate conditional mutants that were temperature
  sensitive-cells grow at 30C but not at 42C.

Two Classes of Mutants quick stop replication
ceases immediately upon shift to restrictive
temperature. slow stop after shifting to
restrictive temperature replication continues but
cells do not initiate another round of
replication.
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How were all these proteins identified?

• Biochemical Approach
• Observation If lysate from wild type cells is
  added to DNA template replication occurs. If
  lysate from dna mutants is added to DNA template
  no replication is observed.

Experimental design In vitro complementation.
Take fractions of extracts from wild type cells
and add it to mutant extracts testing for DNA
polymerase activity. The fraction that restores
DNA replication contains DNA polymerase.
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Prokaryotic DNA replication
Putting it all together
Leading strand
Helicase
Sliding Clamp
Pol III
SSB
t
Primase
t
Clamp Loader
Pol III
Ligase
Lagging strand
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Arthur Kornberg Severo Ochoa 1959 Nobel Prize
in Medicine
"for their discovery of the mechanisms in the
biological synthesis of ribonucleic acid and
deoxyribonucleic acid"
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How fast can E. coli replicate its DNA?
The single molecule of DNA that is the E. coli
genome contains 4.7 x 106 nucleotide pairs.
DNA replication begins at a single, fixed
location in this molecule, the replication
origin, proceeds at about 1000 nucleotides per
second.
How long does it take to replicate the E. coli
genome (remember the fork is bidirectional)?
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Multifork Replication
Slow growing E.coli (40 min or more) each
chromosome undergoes a single round of replication
Rapid growing cells (40 min or less) each
chromosome reinitiates replication before the
first round has terminated.
The ability to undergo multifork replication is a
major difference between the eukaryotic and
prokaryotic cell cycle
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Does DNA move through polymerase or vice versa?
Is polymerase a stationary factory through which
DNA moves? OR Does polymerase move along the
DNA like a train on a track?
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To test if polymerase remains stationary during
replication or if it moves along the DNA,
Katherine Lemon and Alan Grossman examined the
localization pattern of DNA Polymerase III in
live bacterial cells
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The Green Fluorescent Protein
Aequorea victoria
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To visualize DNA Polymerase III in live cells
Lemon and Grossman made a fusion of polIII to a
gene for the green fluorescent protein (GFP) from
the jellyfish Aequorea victoria.
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Green polymerase
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GFP Fluoresces in response to certain wavelengths
of light
home.ncifcrf.gov/ccr/flowcore/ spectra/GFP_WWW.JPG
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GFP Fluoresces in response to certain wavelengths
of light
l510nm
l500nm
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GFP Fluoresces in response to certain wavelengths
of light
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Pol III localization during the cell cycle
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How do you prove that the foci of GFP represent
active polymerase?
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Slow growing cells
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• These data strongly support a model in which DNA
  polymerase remains stationary and the DNA moves
  through the replication machinery.
• This has been dubbed The Factory Model

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Step 2Chromosome Segregation
OR how to separate your newly replicated genome
into two separate entities
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Eukaryotic Chromosome Segregation
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How do bacteria segregate their chromosomes?
initiation of replication
cell division
Chromosome separation
Origin separation
Chromosome separation is a two step process
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Chromosome Separation
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Chromosome Separation
Mutations in bacterial smc lead to decondensed
chromosomes, and guillotining (when the septum
bisects the nucleoid) as well.
Wild Type
smc mutants
smc mutants are significantly sicker when grown
in rich media (media that supports rapid growth)
but not in minimal media.
Why?
Photomicrographs from Britton and Grossman, 1999
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Origin Separation
Visualizing the origin of replication in live
cells
To view the localization dynamics of the
chromosomal origin in live cells Chris Webb and
colleagues tagged a region of the B. subtilis
chromosome with a tandem array of lac operator
cassettes and expressed a version of the Lac
repressor (LacI) fused to GFP
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Origin Separation
Webb et al, 1998
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Chromosome Separation
Since bacteria do not have any obvious
cytoskeletal machinery the mechanism by which
they separate their origins of replication and
their chromosomes remains a mystery.
What do you think could be driving chromosome
separation in bacteria?
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Replication initiates at midcell from a
stationary replisome factory
Lemon and Grossman, 1998
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Chromosome Separation A potential model

• The extrusion-capture model for bacterial
  chromosome partitioning
• (Lemon and Grossman, 2001)
• After the origin region is replicated, the two
  sister origins (light gray circles) are extruded
  from (arrows pointing toward cell poles) the
  centrally located replisome (overlapping
  triangles)
• The origins are then captured on opposite halves
  of the cell at or near the cell quarters.
• The terminus region (dark gray square) remains at
  mid cell until it is duplicated.

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Step 3Cell Division
OR how to separate yourself into two separate
entities
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Cell Division The end or the beginning of the
cycle?
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Cell division is a beautiful thing.....
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FtsZ and other division proteins were identified
as conditional mutations that blocked cell
division. These mutants were called fts for
filamentous temperature sensitive. DNA
replication and partitioning is not affected in
fts mutants.
B. subtilis ftsZ mutant
Wild Type B. subtilis cells
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One of the proteins identified as an fts mutation
was FtsZ
Essential cell division protein
Conserved in bacteria, archaea, and plants
GTPase with similar crystal structure to
tubulin
Assembles into a ring at the nascent division
site
J. Lutkenhaus
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During exponential growth FtsZ forms a ring at
midcell
FtsZ
Cell wall and FtsZ
DNA and FtsZ
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In bacteria, FtsZ establishes the location of the
division site
Most well conserved Fts protein
Required for localization of other Fts proteins
to the septum
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FtsZ localization is coupled to the cell cycle
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Mechanistically, cell division is extraordinarily
well conserved
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Whether FtsZ provides the force necessary for
cytokinesis is still an open question
Two models for how FtsZ might drive constriction
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The spatial regulation of cytokinesis
While we still dont know what establishes the
division site at midcell, we have a few clues
about what prevents aberrant FtsZ ring formation
and division at cell poles.
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Division inhibitors that function in tandem to
prevent FtsZ ring formation and septation at cell
poles
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The MinCD complex is concentrated at the cell
poles
FtsZ
MinCD
MinCD
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Mutations in minCD result in anucleate minicells
Wild type
min-
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-

minCD causes the formation of polar rings of FtsZ
DAPI and FtsZ
Cell wall and FtsZ
FtsZ
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In E. coli (but not B. subtilis) the MinCD is
kept away from midcell by the presence of a
third protein, MinE
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MinE prevents MinCD from localizing to midcell in
E. coli
FtsZ
MinCD
MinCD
MinE
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The MinCD complex oscillates from pole to pole in
a MinE dependent manner.
FtsZ
MinE
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The MinCD complex oscillates from pole to pole in
a MinE dependent manner.
FtsZ
MinE
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The MinCD complex oscillates from pole to pole in
a MinE dependent manner.
FtsZ
MinE
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The MinCD complex oscillates from pole to pole in
a MinE dependent manner.
FtsZ
MinE
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The MinCD complex oscillates from pole to pole in
a MinE dependent manner.
FtsZ
MinE
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The MinCD complex oscillates from pole to pole in
a MinE dependent manner.
FtsZ
MinE
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The MinCD complex oscillates from pole to pole in
a MinE dependent manner.
FtsZ
MinE
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The MinCD complex oscillates from pole to pole in
a MinE dependent manner.
FtsZ
MinE
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(Raskin DM de Boer PA, 1999)
MinD flipping in E. coli cells
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The Bacterial Cell Cycle
initiation of replication
cell division
3
1
Nucleoid segregation
Elongation and Origin separation
2
2
Termination
2
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Questions

• What factors are responsible for localizing oriC
  and FtsZ to midcell?
• What drives origin separation and chromosome
  segregation?
• How is FtsZ ring formation and constriction
  coupled to the bacterial cell cycle?
• Does FtsZ play a mechanistic role in cytokinesis?