Temporal and spatial control during Caulobacter cell differentiation

Understanding how cells differentiate is important to further our knowledge of how complex organisms develop and to treat diseases such as cancer.;For the past five years, I have been studying the problem of cell differentiation in the aquatic bacterium, Caulobacter crescentus. Caulobacter crescentus alternates between two phases: a dispersal phase and a non-motile phase. The dispersal stage of Caulobacter is the motile swarmer cell. After one-third of the cell cycle, the swarmer sheds its flagellum, synthesizes a stalk in its place and differentiates into the non-motile stalked cell. The stalked cell initiates DNA replication and cell elongation. Elongation of the stalked cell and synthesis of a new flagellum at the opposite pole gives rise to an asymmetric predivisional cell. The predivisional cell then divides to generate the two different cell types: a swarmer cell and a stalked cell.;The transcription factor Sigma-54, which is encoded by the rpoN gene, is an important regulator of differentiation in Caulobacter . Mutants in rpoN lack flagella and stalks, and are aberrant in cell division. One of the goals of this study was to characterize the role played by the rpoN operon in the cell differentiation of Caulobacter crescentus. To achieve this, the genes in the rpoN operon were isolated and mutational analysis was used to determine whether these genes play a role in modulating Sigma-54 activity. In addition, detailed analysis of rpoN mutants revealed that Sigma-54 is required to activate a developmental checkpoint in C. crescentus.;A second aspect of this study was to study mechanisms that generate asymmetry by looking at the synthesis of the holdfast (a polar organelle) and the regulation of a holdfast attachment gene hfaA. Contrary to accepted dogma, it was determined that the holdfast appears at the pole of cells during the swarmer to stalked cell differentiation. The hfaA gene was shown to be cell cycle regulated, with maximal expression in the swarmer compartment of the predivisional cell.