Data from: Threshold effect of growth rate on population variability of Escherichia coli cell lengths

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.生物学领域长期存在的一个经典问题是生长对细胞大小的影响。本研究通过对微分干涉差（Differential Interference Contrast, DIC）显微镜下固定细胞的图像分析，计算细胞长度变异系数（Coefficient of Variation of Cell Lengths, CVL），以此估算大肠杆菌（Escherichia coli）的生长速率（r）对群体细胞大小分布的影响。研究发现，当生长速率低于每小时1次分裂时，CVL保持恒定；而超过该阈值后，CVL随生长速率提升而升高。我们提出假说：由RecA依赖机制介导的复制停滞引发的细胞分裂随机抑制，结合多叉复制的生长速率阈值（基于Cooper与Helmstetter的研究），或可构成该阈值效应的分子基础。为验证该假说，我们通过羟基脲（Hydroxyurea, HU）处理提升复制叉随机停滞的频率，结果显示仅在生长速率超过该阈值时，细胞长度变异程度显著升高。该群体水平效应在琼脂垫培养及基于“母机”（mother machine）装置实现同步化的单细胞实验中均得到重现。为明确RecA在停滞复制叉修复中的关键作用，我们检测了recA基因缺失大肠杆菌（E. coli ΔrecA）的CVL。结果显示，该突变株的细胞长度变异程度高于野生型菌株，且通过质粒介导的RecA表达可逆转该表型。此外，我们发现当生长速率超过阈值时，RecA-绿色荧光蛋白（RecA-green fluorescent protein, RecA-GFP）融合蛋白向类核（nucleoid）的募集频率更高，且经HU处理后该现象进一步增强。综上，当生长速率超过阈值时，大肠杆菌群体的细胞长度会增加，且该效应至少部分由RecA向类核的募集以及细胞分裂的随机抑制所介导。