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Understanding how bacteria coordinate growth with cell cycle events to maintain cell size homeostasis remains a grand challenge in biology. The period of chromosome replication (C period) is a key stage in the bacterial cell cycle. However, the mechanism of in vivo regulation of the C period remains unclear. In this study, we found that titration of the expression of ribonucleotide reductase (RNR), which changes the intracellular deoxynucleoside triphosphate (dNTP) pools, enables significant perturbations of the C period, leading to a substantial change in cell size and DNA content. Our work demonstrates that the intracellular dNTP pool is indeed an important parameter that controls the progression of chromosome replication. Specially, RNR overexpression leads to a shortened C period compared with that of a wild-type strain growing under different nutrient conditions, indicating that the dNTP substrate levels are subsaturated under physiological conditions. In addition, perturbing the C period does not significantly change the D period, indicating that these two processes are largely independent from each other. Overall, titration of ribonucleotide reductase expression can serve as a standard model system for studying the coordination between chromosome replication, cell division, and cell size. IMPORTANCE Bacteria must coordinate growth with cell cycle progression to maintain cell size hemostasis. Cell cycle and cell size regulation is a fundamental concern in biology. The period required for chromosome replication (the C period) is a key stage in the bacterial cell cycle. However, how the C period is controlled in vivo remains largely an open question in this field of bacterial cell cycle regulation. Through introducing a genetic circuit into Escherichia coli for titrating the expression of ribonucleotide reductase, we achieve substantial perturbation of the C period and cell size. Our work demonstrates that the intracellular dNTP pool is an important parameter that controls the progression of chromosome replication. Moreover, our work indicates that bacterial cells manage to maintain subsaturated dNTP levels under different nutrient conditions, leading to a submaximal speed of DNA replication fork movement.

Manipulating the Bacterial Cell Cycle and Cell Size by Titrating the Expression of Ribonucleotide Reductase