| The bacterium Caulobacter crescentus exhibits a defined program of development at the cell pole. This program requires a number of proteins that exhibit similar subcellular localization patterns and are involved in a complex regulatory network governing the cell cycle and the polar developmental program. This work has focused on one such protein, PodJ. PodJ is an inner-membrane protein required for synthesis of polar structures including pili and the flagellum, and for the proper localization of other regulatory proteins. We conducted a transposon screen to identify additional genes in the development pathway, and identified a mutation in the promoter of the global regulator ctrA. This mutant had reduced levels of CtrA and deficiencies in multiple cell-cycle and developmental characteristics.;Because of the shared localization patterns of proteins required for polar development, and the presence of domains within podJ indicating that it may be involved in protein-protein interactions, we used the yeast two-hybrid system to investigate the interaction of PodJ with itself and other proteins. We showed a PodJ-PodJ interaction, and that this interaction was mediated along the entire cytoplasmic region of the protein. Additionally, a screen to identify other interacting proteins revealed an interaction with the developmental regulator DivL, which has a similar localization pattern to PodJ and requires PodJ for localization.;To gain further insight into the structure-function of PodJ, we undertook a mutagenesis screen to identify functionally deficient mutants of PodJ. The podJ gene and its promoter were randomly mutagenized and introduced into a DeltapodJ background. Mutants were screened for PodJ-dependent polar development phenotypes. Those mutants that expressed detectable levels of PodJ were identified to be mostly frameshift or nonsense mutations, and the mutation sites were largely confined to the periplasmic region of the protein.;Together all these data indicate that, rather than having a specific enzymatic function, PodJ may serve as a central localization hub, coordinating the activities of various regulators of polar development and the cell cycle, and that this process requires the concerted action of multiple protein-protein interaction domains within PodJ. |
