SignificanceWhen and at what size to divide are critical decisions, requiring cells to integrate internal and external cues. While it is known that the 24-h circadian clock and the environment modulate division timings across organisms, how these signals combine to set the size at which cells divide is not understood. Iterating between modeling and experiments, we show that, in both constant and light−dark conditions, the cyanobacterial clock produces distinctly sized and timed subpopulations. These arise from continuous coupling of the clock to the cell cycle, which, in light−dark cycles, steers cell divisions away from dawn and dusk. Stochastic modeling allows us to predict how these effects emerge from the complex interactions between the environment, clock, and cell size control.

How cells maintain their size has been extensively studied under constant condi ons. In the wild, however, cells rarely experience constant environments. Here, we examine how the 24-hour circadian clock and environmental cycles modulate cell size control and division mings in the cyanobacterium Synechococcus elongatus using single-cell me-lapse microscopy. Under constant light, wild type cells follow an apparent sizer-like principle. Closer inspec on reveals that the clock generates two subpopula ons, with 1 . CC-BY-NC 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/183558 doi: bioRxiv preprint first posted online Sep. 1, 2017; cells born in the subjec ve day following different division rules from cells born in subjec ve night. A stochas c model explains how this behaviour emerges from the interac on of cell size control with the clock. We demonstrate that the clock con nuously modulates the probability of cell division throughout day and night, rather than solely applying an on-off gate to division as previously proposed. Itera ng between modelling and experiments, we go on to show that the combined effects of the environment and the clock on cell division are explained by an effec ve coupling func on. Under naturally graded light-dark cycles, this coupling shi s cell division away from dusk and dawn, when light levels are low and cell growth is reduced. Our analysis allows us to disentangle, and predict the effects of, the complex interac ons between the environment, clock, and cell size control.

Bacterial cell wall peptidoglycan is essential, maintaining both cellular integrity and morphology, in the face of internal turgor pressure. Peptidoglycan synthesis is important, as it is targeted by cell wall antibiotics, including methicillin and vancomycin. Here, we have used the major human pathogen Staphylococcus aureus to elucidate both the cell wall dynamic processes essential for growth (life) and the bactericidal effects of cell wall antibiotics (death) based on the principle of coordinated peptidoglycan synthesis and hydrolysis. The death of S. aureus due to depletion of the essential, two-component and positive regulatory system for peptidoglycan hydrolase activity (WalKR) is prevented by addition of otherwise bactericidal cell wall antibiotics, resulting in stasis. In contrast, cell wall antibiotics kill via the activity of peptidoglycan hydrolases in the absence of concomitant synthesis. Both methicillin and vancomycin treatment lead to the appearance of perforating holes throughout the cell wall due to peptidoglycan hydrolases. Methicillin alone also results in plasmolysis and misshapen septa with the involvement of the major peptidoglycan hydrolase Atl, a process that is inhibited by vancomycin. The bactericidal effect of vancomycin involves the peptidoglycan hydrolase SagB. In the presence of cell wall antibiotics, the inhibition of peptidoglycan hydrolase activity using the inhibitor complestatin results in reduced killing, while, conversely, the deregulation of hydrolase activity via loss of wall teichoic acids increases the death rate. For S. aureus, the independent regulation of cell wall synthesis and hydrolysis can lead to cell growth, death, or stasis, with implications for the development of new control regimes for this important pathogen.