Regulation of a bacterial histidine kinase by a phase separating scaffolding protein

Scaffolding proteins customize the response of signaling networks to support cell development and behaviors. We investigated how the bacterial scaffolding protein PodJ regulates the histidine kinase PleC involved in the asymmetric cell division of Caulobacter crescentus . We reconstituted the PleC-PodJ signaling complex through both heterologous expression in E. coli and in vitro studies. In vitro PodJ phase separates as a biomolecular condensate that recruits and inhibits PleC kinase activity. By constructing an in vivo PleC-CcaS chimeric histidine kinase reporter assay, we have demonstrated how PodJ leverages its intrinsically disordered region (IDR) to bind and regulate PleC-CcaS signaling. Moreover, we observed that full-length PodJ L regulates PleC-CcaS signaling, while a truncated PodJ s could not regulate signaling activity. These results support a model where PodJ biomolecular condensate formation regulates the localization and activity of the cell fate determining kinase PleC.