A bacterial scaffolding protein keeps the cell cycle and differentiation in check by regulating histidine kinase activity

One protein involved in regulating the differentiation of Caulobacter crescentus is the polar development protein PodJ, which is required for efficient dispersal in low density agar, flagellum release, and the synthesis of the holdfast and pili. To investigate the role of PodJ, co-immunoprecipitation followed by mass spectroscopy were performed to identify proteins that interact with PodJ. SpmX and DivJ co-immunoprecipitated with PodJ. SpmX is a lysozyme homologue that regulates the localization to the stalked pole and activation of the histidine kinase DivJ, which recruits and phosphorylates the single domain response regulator DivK to the stalked pole. Microscopy of functional, fluorescently tagged PodJ, SpmX and DivJ showed that all three proteins colocalize at the flagellar pole in swarmer cells. In vitro phosphorylation assays showed that PodJ promotes the dephosphorylation of DivJ. DivJ-mediated phosphorylation of the DivK response regulator stimulates swarmer cell differentiation and initiation of DNA replication. As expected, a DeltapodJ deletion mutant had more phosphorylated DivK (DivK~P), less CtrA~P, and prematurely initiated DNA replication. In contrast, overexpression of PodJ inhibited polar development. We conclude that PodJ prevents premature cell differentiation and initiation of DNA replication by inhibiting DivJ at the swarmer pole. The kinase activity of DivJ was previously thought to exclusively fulfill the role of DivJ during the C. crescentus cell cycle, but the role of its phosphatase activity was not known. To investigate the role of DivJ phosphatase activity, we uncoupled kinase and phosphatase activities of DivJ by site-directed mutagenesis based on previous studies of PleC and EnvZ. Unexpectedly, the kinase active phosphatase inactive (K+P-) DivJ T342Y mutant was unable to completely perform DivJ developmental functions, and the kinase inactive phosphatase active (K-P+) DivJY502L mutant was able to partially perform DivJ developmental functions. We conclude that both phosphatase and kinase activities of DivJ are involved in developmental regulation. Furthermore, in vitro phosphorylation and epistasis analysis revealed that DivJ may regulate stalk biogenesis by acting as a phosphatase for the transcriptional factor TacA, which controls stalk biogenesis with sigma54. We propose that DivJ controls stalk elongation and forms a feedback loop with TacA to modulate its own localization and activation.