| The gram-negative aquatic bacterium Caulobacter crescentus displays two distinct cell types: the swarmer cell and the stalked cell. Swarmer cells spend a variable amount of time in the swarmer phase before differentiating into stalked cells, and this interval depends critically on the cell's nutrient environment. Low carbon or nitrogen promote long swarmer phases, while low phosphorus promotes a short swarmer phase. Electron microscopy of Caulobacter isolates reveals prominent polyphosphate (polyP) inclusions, present as one or two granules within cells. The so-called "phosphorus clock" hypothesis of C. crescentus development unites these observations by asserting that this inherited polyP, in the form of polyP granules, dictates how long a swarmer will remain in the swarmer state: if carbon or nitrogen is exhausted, polyP is accumulated in predivisional cells, and the resultant newborn swarmers can spend a long time in that state before drawing down their polyP reserves. In contrast, if phosphorus is exhausted, polyP accumulation halts, and newborn swarmers do not receive a store of polyP and quickly differentiate into stalked cells. This hypothesis had remained untested until the present. Moreover, no molecular mechanism had previously been attributed to the timing of swarmer differentiation, and though polyP granules have been known to occupy characteristic positions within bacteria since 1895, no studies concerning the molecular determinants of granule localization had been conducted in any species. This dissertation sheds light on both phenomena. Chapter 2 describes our discovery that polyP, as well as the stringent response effector ppGpp, are important for the timing of swarmer cell differentiation. Our findings represent the first molecular clues to how these cells modulate their cell cycle to respond to changing nutrient conditions. Furthermore, we partially confirm the "phosphorus clock" hypothesis, demonstrating that without polyP, swarmers do not accumulate in carbon starvation. Subsequently, Chapter 3 details our analysis of polyP as a subcellular entity. Struck by the consistent localization of polyP granules within nascent C. crescentus mother and daughter cells, we explore the molecular mechanism underpinning this organization, determining that it is intimately related to the cell's chromosomal state. Finally in Chapter 4, we present unpublished phenotypic and transcriptional analysis of a ppk1 null strain in an attempt to understand the link between polyP and C. crescentus metabolism, which remains mysterious, as it has in other species. As an Appendix, we present a significant literature review of a class of ligand-binding protein domains; though thematically unrelated, this review contains significant primary research and represents a contribution to the field of microbiology generally. |
