| Regulation of cell cycle progression is a complex process in which a cell must analyze information about internal and external conditions, and make decisions on how to respond. Caulobacter crescentus possesses a network of signal transduction molecules that regulate its cell cycle. One central cell cycle protein is the global regulator, CtrA, a transcription factor that is activated by phosphorylation and targets numerous downstream genes. My interest in the signal transduction network regulating the Caulobacter cell cycle led me to investigate whether CtrA is present in other species, described in Chapter 2.; Chapter 3 discusses my work to identify new cell cycle regulators, and describes the cloning, and characterization of a novel gene, divT . The divT mutant exhibits a strong cell division phenotype. DivT is not a two-component system protein, and shows no homology to known proteins, but homologs are present in other α-proteobacteria. DivT has a cell cycle pattern of protein expression, and is regulated transcriptionally by CtrA.; DivK is an essential single-domain response regulator. Previous work identified and cloned divK, but elucidation of its role in Caulobacter cell cycle regulation remained an unanswered question. The main part of my thesis work was to identify DivK's role in Caulobacter . Chapter 4 presents work showing that DivK is present and phosphorylated throughout the cell cycle and exhibits a cell cycle-dependent pattern of protein localization. Chapter 5 describes experiments showing that DivK regulates proteolytic events critical to cell cycle progression. A divK-cs mutant displays phenotypes characteristic of a G1-arrest induced by the presence of active CtrA in the stalked cell. The CtrA protein is stable in a divK-cs mutant at the restrictive temperature, and upon shift to the permissive temperature, CtrA is rapidly degraded. The mutant phenotypes observed in divK-cs are not due to a defect in phosphorylation, and microarray analysis shows that the normal order of gene expression resumes after release from G1-arrest. The divK promoter contains a strong CtrA consensus binding motif where purified CtrA binds. Thus DivK cues the proteolysis of the global regulator, CtrA, allowing cell cycle progression to continue, and is itself regulated by CtrA. |
