Prokaryotic Cell Biology

The Bacterial Cell Cycle (Escherichia coli)

B period: before chromosome replication

C period: replication of chrom, starts at initiation, ends at termination, constant length (40min), analogous to S (eukaryotes)

D period: after replication, starts at termination, ends at completion of cell division, constant length (20min), analogous to M/G2 (eukaryotes)

Replication of the Bacterial Cell

Chromosome Replication

contain single chrom – large circular DNA molec

E. Coli: 4,639,221 bp long (~4,600 kbp) and contains ~4,300 genes

Initiation of replication occurs at oriC

Initiator protein DnaA binds to oriC, forcing the double helix to open up (at a A-T rich region), allowing access to the DNA polymerase complex

From oriC, replication is bi-directional via 2 replication forks (1 CW, 1 CCW)

Both sides replicate ~1000 bp/sec Þ 40min total; and meet at dif (terminus region)

Chromosome segregation

physical unlinking of the 2 sister chromatids

linked non-covalently (catenation)

decatenation by type 2 topoisomerases: Topo II (DNA gyrase) and Topo IV

molecular targets for antibiotics called quinolones (cells grow, don’t segregate, die)

linked covalently

result of homologous recombination between 2 replicated portions of a replicating chrom
uneven # of recombination events takes place, product of replication = chrom dimer
dimer = large circular molec with the sister chrom lined head to tail (2 oriC and 2 dif)
dimers resolved into 2 separable molec by site-specific recombination at dif or by RecA

lateral movement of chrom to opposite ends of the mother cell

microtubule spindles absent in prokaryotes
the origin in young cells is located near the oldest cell pole
after duplication of the origin, one of the sister origins rapidly moves to the opposite pole
chrom segregation appears to start well before chromosomal replication is finished
the terminus stays in place in the middle and replicates

GFP-LACI = repressor of Lac Operon so Insert a string of 236 lacO into oriC and use GFP to trace the events that occur during segregation

Cell Division (cytokinesis)

last stage of cell cycle when mother cell separates into 2 daughter cells

division septum: structure formed by the invaginating envelope; usually placed at midpt of long axis

In E. coli, 10 genes are dedicated to the cell constriction process

Cytokinesis driven by a septal ring; major component = FtsZ

FtsZ (filament temp sens): 40kD, well conserved, progenitor of tubulin, binds GTP

Earliest event is assembly of cytoplasmic FtsZ into a ring at the prospective division site

The FtsZ ring attracts other division proteins which together assemble the septal ring organelle

penicillin-binding protein 3 (PBP3 or FtsI): target of penicillin, required to synthesize the murein (peptidoglycan) component of the septum

Placement of the division septum

division septum can form near one of the cell poles in mutant forms
polar septation in wildtype cells is prevented by endogenous division inhibitors (MinC and MinD)
MinCD blocks FtsZ ring formation and, hence, septation
A 3^rd protein (MinE) prevents MinCD-mediated division inhibition at midcell so normal division

Overlapping cell cycles

durations of C and D periods together take 60min, yet doubling time of E. coli is 20min so E. coli must start a cell cycle before the previous one has ended
Thus, cells are born with an already half-replicated chrom