Abstract
A long-standing question in biology is the effect of growth on cell size. Here, we estimate the effect of Escherichia coli growth rate (r) on population cell size distributions by estimating the coefficient of variation of cell lengths (CVL) from image analysis of fixed cells in DIC microscopy. We find that the CVL is constant at growth rates less than one division per hour, whereas above this threshold, CVL increases with an increase in the growth rate. We hypothesize that stochastic inhibition of cell division owing to replication stalling by a RecA-dependent mechanism, combined with the growth rate threshold of multi-fork replication (according to Cooper and Helmstetter), could form the basis of such a threshold effect. We proceed to test our hypothesis by increasing the frequency of stochastic stalling of replication forks with hydroxyurea (HU) treatment and find that cell length variability increases only when the growth rate exceeds this threshold. The population effect is also reproduced in single-cell studies using agar-pad cultures and ‘mother machine’-based experiments to achieve synchrony. To test the role of RecA, critical for the repair of stalled replication forks, we examine the CVL of E. coli ΔrecA cells. We find cell length variability in the mutant to be greater than wild-type, a phenotype that is rescued by plasmid-based RecA expression. Additionally, we find that RecA-GFP protein recruitment to nucleoids is more frequent at growth rates exceeding the growth rate threshold and is further enhanced on HU treatment. Thus, we find growth rates greater than a threshold result in increased E. coli cell lengths in the population, and this effect is, at least in part, mediated by RecA recruitment to the nucleoid and stochastic inhibition of division.
Highlights
By increasing replication fork stalling with hydroxyurea (HU) in multiple mutant strains, we demonstrate that DNA replication fork dynamics can affect population cell size distributions in a RecA-dependent manner
With the aim of measuring the effect of growth rate, r, on cell size, E. coli MG1655 cells were grown in LB, yeast extract broth (YEB), tryptone broth (TB) and M9 supplemented with glucose, succinate and acetate
Because the genome content dependence of CVL does not, reproduce a biphasic, threshold dependence in cell length variability, we examined whether perturbing replication dynamics below and above the presumptive growth rate threshold could be used as a test of replication stochasticity as the underlying mechanism
Summary
Many genetic factors that link nutrient sensing to cell size regulation have been identified [9,10,11] These pathways, link growth rate via pathways independent of replication to cell size. Combined with fluorescence microscopy of subcellular components [20,21], it has become possible to address single-cell dynamics of the bacterial cell division cycle. These advances allow us to address the effect of population sizes and physical factors and probe the mechanisms that control cell sizes and cell size variability. The effect of molecular regulatory networks on cell size and the correlation of cell size variability with growth rate remain unclear
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