Bacterial cell cycle dynamics: size regulation during exponential growth and role of polyphosphate during starvation response

Bacteria are the most diverse and abundant kingdom of life and have adapted to survive and thrive in habitats around the globe. When provided with ample nutrients they grow and divide at staggering rates, increasing their population exponentially. Upon nutrient depletion on the other hand, they quickly adapt by drastically altering their metabolism, halting growth to survive for a very long time. Since bacteria are tiny -about a few micrometers-, visualizing these processes requires microscopy. To measure the dynamics of their shape and inner structures precisely, one needs to choose a technique that balances spatial resolution, temporal resolution and photo-toxicity. In this thesis, I present two projects using advanced dynamic microscopy, first to study cell size regulation during exponential growth in the abundance of nutrients and then to elucidate the role and positioning of polyphosphate granules during cell cycle exit in response to nutrient starvation. During exponential growth, bacteria balance growth and division to regulate their size, resulting in a narrow size distribution, referred to as cell size homeostasis. Recent work has tried to uncover what cells sense to decide to divide in order to achieve size homeostasis: time, size, growth or a combination of those. Control of cell division is often equated to control of constriction onset; however, the constriction period still accounts for up to 40% of cell growth and could thus contribute significantly to cell size regulation. We used SIM microscopy to measure constriction kinetics and their impact on cell size regulation in Caulobacter crescentus. We found that constriction rate regulation can determine cell size. Moreover, constriction rate modulation compensates for variability in elongation before constriction, allowing a higher fidelity cell size homeostasis. We suggest a parsimonious model where excess cell wall precursors accumulate proportionally to elongation before constriction and set the rate of constriction. This is the first direct demonstration that constriction rate can contribute to cell size control and homeostasis in bacteria. Upon nutrient starvation on the other hand, bacteria exit their cell cycle to preserve energy and nutrients. In many bacteria, such as Pseudomonas aeruginosa, this is associated with the accumulation of polyphosphate (polyP) in intracellular granules. PolyP is created by polyphosphate kinases (Ppk’s), which are required for successful cell cycle exit and survival of and recovery from longterm starvation. Interestingly, these polyP granules are regularly spaced within the nucleoid. To date, it is not known during which stage polyP is required for cell cycle exit, and what causes the spacing of the granules. Here, we use fluorescence microscopy to probe the cell cycle stage of Δppk cells arrested during nutrient starvation as well as the localization and dynamics of Ppk’s. We show that a majority of Δppk cells are arrested with open replication forks. Furthermore, we find that Ppk’s localize in distinct patterns, already prior to starvation and polyP granule production, which could be responsible for the positioning of polyP granules. To this end, we developed a background subtraction algorithm to remove cytoplasmic fluorescence, improving accuracy of spot detection and localization.