PMID:22396664

Citation	Hill, NS, Kadoya, R, Chattoraj, DK and Levin, PA (2012) Cell size and the initiation of DNA replication in bacteria. PLoS Genet. 8:e1002549
Abstract	In eukaryotes, DNA replication is coupled to the cell cycle through the actions of cyclin-dependent kinases and associated factors. In bacteria, the prevailing view, based primarily from work in Escherichia coli, is that growth-dependent accumulation of the highly conserved initiator, DnaA, triggers initiation. However, the timing of initiation is unchanged in Bacillus subtilis mutants that are ~30% smaller than wild-type cells, indicating that achievement of a particular cell size is not obligatory for initiation. Prompted by this finding, we re-examined the link between cell size and initiation in both E. coli and B. subtilis. Although changes in DNA replication have been shown to alter both E. coli and B. subtilis cell size, the converse (the effect of cell size on DNA replication) has not been explored. Here, we report that the mechanisms responsible for coordinating DNA replication with cell size vary between these two model organisms. In contrast to B. subtilis, small E. coli mutants delayed replication initiation until they achieved the size at which wild-type cells initiate. Modest increases in DnaA alleviated the delay, supporting the view that growth-dependent accumulation of DnaA is the trigger for replication initiation in E. coli. Significantly, although small E. coli and B. subtilis cells both maintained wild-type concentration of DnaA, only the E. coli mutants failed to initiate on time. Thus, rather than the concentration, the total amount of DnaA appears to be more important for initiation timing in E. coli. The difference in behavior of the two bacteria appears to lie in the mechanisms that control the activity of DnaA.
Links	PubMed PMC3291569 Online version:10.1371/journal.pgen.1002549
Keywords	Bacillus subtilis/genetics; Bacterial Proteins/genetics; Cell Size; DNA Replication/genetics; DNA Replication Timing; DNA-Binding Proteins/genetics; Escherichia coli/genetics; Mutation
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Significance

This paper highlights that the mechanisms for coordination of cell growth and DNA replication are different in E. coli 

B. subtilis and consequently, not conserved in Bacteria.

The questions that are posed and solved through experimental data are:

1. Will reduction in cell size lead to a delay in DNA replication? Approaches:

- Two mutants of E. coli (pgm::kan & ftsA*) and one mutant of Bacillus (pgcA::Tn10).

- For E. coli, both WT and mutants were cultured in LB + glucose, LB, or AB minimal media. For Bacillus subtilis, both WT and mutants were cultured in LB or 750 minimal media.

- The cultures were started from overnight grown cells, diluted to an OD of 0.005, grown to an OD of 0.2-0.6, diluted again to an OD of 0.005, grown to an OD of 0.3

- Added antibiotics and performed flow cytometry

- Calculated origins per cell using the Cell Quest Pro software

Results and Conclusions: Fig. 2. Cell size delays DNA replication in E.coli but not B.subtilis

2. Are DNA replication rates increased in E. coli mutants?

Approaches: - Cells were grown to OD 0.3, treated with 300 ug/ml sodium azide and lysed

- DNA proximal to the origin or terminus was amplified by qPCR and results analyzed using the Pfaffl method.

- Marker ratios were normalized to the ori/ter ratio of either E. coli cells treated with 300 ug/ml rifampicin and 36 ug/ml cephalexin or DNA prepared from Bacillus spores

Results and Conclusions: Fig. 3A-3D. C period is smaller in cell size mutants suggesting faster replication rates.

3. Are origin and replication fork numbers reduced in E. coli mutants but not in Bacillus subtilis mutants?

Approaches: - Markers of active replication forks to examine the origin and replication fork frequency: "E. coli: YPet fusion to single-stranded DNA binding protein (SSB-YPet) "B.subtilis": GFP-fusion to the replication protein Tau (DnaX-GFP)

- Grown to OD 0.25-0.35, stained with FM4-64 and placed on an agarose pad

- Scored for number of distinct foci

Results and Conclusions: Fig. 3E-3F. Due to a replication delay in E. coli mutants, the number of origins and replication forks are smaller. B. subtilis does not have change in number of origins and replication forks

4. How are DnaA levels in E. coli mutants versus B.subtilis mutant? Approaches:

- Grown to OD 0.25-0.35, normalized lysates from cultures to either OD to determine the relative concentration of DnaA, or to cell number to determine the relative levels of DnaA per cell

- Subjected to SDS-PAGE.

- Using either E. coli rabbit anti-DnaA antibody or "B. subtilis" chicken anti-DnaA antibody to perform immunoblots

- Cognate goat anti-rabbit or donkey anti-chicken secondary antibody conjugated to horseradish peroxidase

- DnaA levels were determined relative to FtsZ in individual strains using ImageQuant software and plotted in Excel

Results and Conclusions: Fig. 4A-4D. There is no difference in DnaA per mass for both species when compared to the respective wild type. However the DnaA per cell was less in the smaller size mutants of both species.

5. Increasing DnaA levels by a modest amount could restore normal replication timing in E. coli?

Approaches: - Low-copy plasmid with an arabinose inducible copy of dnaA: pDS596

- Cultured in LB and back-diluted with/without arabinose without ampicillin

- Evaluated by flow cytometry, marker frequency analysis and quantitative immunoblotting as described above

Results and Conclusions: Fig. 4E and table 2. Yes. DnaA increased levels restores wild type replication time

6. Do the origins per cell increase in larger cells?

Approaches: - Increased cell size by using inducible-repressible promoter constructs that permit depletion of FtsZ

- By titrating levels of inducer (sialic acid for E. coli and IPTG for Bacillus) under steady-state conditions, they generated cells ranging in size from near wild type to two-fold larger

- In E. coli, they also generate slightly smaller cells.

- Determined cell origin numbers by examining DNA content by flow cytometry

Results and Conclusions: Fig. 5. Number of origins per cell increase in larger cells

Annotations

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References

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