Polar development and cell division in Caulobacter crescentus are controlled and coordinated by multiple signal transduction proteins. divJ encodes a histidine kinase. A null mutation in divJ results in a reduced growth rate, cell filamentation, and mislocalized stalks. Suppressor analysis of divJ identified mutations in genes encoding the tyrosine kinase (divL) and the histidine kinase (cckA). The divL and cckA suppressor alleles all have single amino acid substitutions, some of which confer a temperature-sensitive phenotype, particularly in a wild-type background. Analysis of transcription levels from several positively regulated CtrA-dependent promoters reveals high expression in the divJ mutant, suggesting that DivJ normally serves to reduce CtrA activity. The divL and cckA suppressors reduce the amount of transcription from promoters positively regulated by CtrA, indicating that the mutations in divL and cckA are suppressing the defects of the divJ mutant by reducing the abnormally high level of CtrA activity. Immunoblotting showed no major perturbations in the CtrA protein level in any of these strains, suggesting that the high amount of CtrA activity seen in the divJ mutant and the reduced amount of activity in the suppressors are regulated at the level of activation and not transcription, translation, or degradation. In vivo phosphorylation assays confirmed that divJ mutants have elevated levels of CtrA phosphorylation and that this level is reduced in the suppressors with mutations in divL.The aquatic gram-negative bacterium Caulobacter crescentus has a dimorphic life cycle, beginning as a motile, piliated swarmer cell incapable of DNA replication. The swarmer cell differentiates into a stalked cell by ejecting its flagellum, retracting the pili, and synthesizing a stalk with an adhesive holdfast at the same pole that contained the flagellum. The stalked cell initiates DNA replication and cell division and synthesizes a new flagellum at the pole opposite the stalk, producing a new motile swarmer cell during each cell cycle. Multiple signal transduction proteins are involved in coordinating polar development and cell division in C. crescentus. The global response regulator CtrA controls the expression of at least 144 genes (25) involved in cell division, DNA methylation, holdfast synthesis, flagellum biogenesis, and pilus biogenesis (22,25,34,43). CtrA prevents the initiation of DNA replication in swarmer cells by binding to the origin of replication (35) and inhibits cell division by repressing transcription of ftsZ, which encodes the first cell division protein to localize to the site of division (22). Late in the cell cycle, CtrA activates transcription from the P QA promoter, which cotranscribes ftsQ and ftsA, ensuring ordered expression of ftsZ, ftsQ, and ftsA (22,38,50

Summary Subcellular protein localization is thought to promote protein-protein interaction by increasing the effective concentration and enabling spatial coordination and proper segregation of proteins. We found that protein overexpression allowed the assembly of a productive polysaccharide biosynthesis-export-anchoring complex in the absence of polar localization in Caulobacter crescentus. Polar localization of the holdfast export protein, HfsD, depends on the presence of the other export proteins, HfsA, and HfsB, and on the polar scaffold protein PodJ. The holdfast deficiency of hfsB and podJ mutants is suppressed by the overexpression of export proteins. Restored holdfasts are randomly positioned and co-localize with a holdfast anchor protein in these strains, indicating that functional complexes can form at non-polar sites. Therefore, overexpression of export proteins surpasses a concentration threshold necessary for holdfast synthesis. Restoration of holdfast synthesis at non-polar sites reduces surface adhesion, consistent with the need to spatially coordinate the holdfast synthesis machinery with the flagellum and pili. These strains lack the cell-specific segregation of the holdfast, resulting in the presence of holdfasts in motile daughter cells. Our results highlight the fact that multiple facets of subcellular localization can be coupled to improve the phenotypic outcome of a protein assembly.