Cell Size and the Initiation of DNA Replication in Bacteria

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.

Author Summary

DNA replication must be coordinated with growth and division to ensure the viability of cells and organisms. In bacteria, it is believed that cell growth–dependent accumulation of the initiator of DNA replication, DnaA, to critical levels determines the timing of initiation. This view is based primarily on data from the model bacterium E. coli, which initiates replication only upon achieving a particular size. However, recent data from another model organism, B. subtilis, where DnaA is also rate limiting for initiation, suggests that changes in cell size may not impact the timing of DNA replication. This finding prompted us to revisit the relationship between cell size and DNA replication in E. coli. While previous studies examined perturbations in DNA replication on cell size, we instead determined the consequences of cell size defects on DNA replication. This converse approach led to the conclusion that, irrespective of size, DnaA needs to accumulate to a critical amount to trigger initiation in E. coli, as is generally believed to be the case. In contrast, small B. subtilis cells could initiate replication with amounts of DnaA ∼30% less than wild type. Thus, while DnaA is rate limiting for initiation in both organisms, the mechanisms controlling its activity may vary in different bacteria.