Timing the process of cell differentiation during the Caulobacter cell cycle
Keywords: Medicine.
Developmental Biology.

While cell differentiation is considered a hallmark process in the development of multicellular organisms, many unicellular bacteria undergo the process of cell differentiation. One such bacterium, Caulobacter crescentus, undergoes differentiation from a motile swarmer cell into a stationary, replication competent stalked cell with each cell cycle. The process of swarmer to stalked cell differentiation entails shedding of the polar flagellum, retraction of the sensory pili, initiation of chromosome replication and biogenesis of the polar stalk appendage. After differentiation, the stalked cell develops into a pre-divisional cell, forming a flagellum and pili at the pole opposite the stalk. The pre-divisional cell then undergoes asymmetric cell division, giving rise to a daughter swarmer cell that will disperse, and a daughter stalked cell that will begin a new round of replication. This bi-phasic cell cycle allows Caulobacter to exist as planktonic cells, capable of colonizing new habitats, and stationary cells, capable of proliferation and biofilm formation. The process of swarmer to stalked cell differentiation is obligate to Caulobacter's cell cycle and requires activation of a signaling pathway. Therefore, timing of this differentiation event dictates the ratio of swarmer versus stalked and pre-divisional cells, ultimately affecting Caulobacter's population-level fitness. The process of swarmer to stalked cell differentiation occurs in the absence of an environmental stimulus and is initiated upon cell-cycle regulated activation of an internal signaling pathway. Key to activation of this signaling pathway is the bifunctional histidine kinase, PleC, that acts as either a kinase or a phosphatase at distinct points in the cell cycle. Here, we present evidence that PleC enzymatic activity and subcellular localization are controlled by the polar scaffold protein, PodJ. PodJ recruits PleC to the new flagellar pole, through interaction with PleC's PAS domains, simultaneously inhibiting PleC kinase activity on PleD. Upon disruption of PodJ's interaction with PleC, freed PleC molecules exit the pole, and switch to their kinase form, allowing phosphorylation of the diguanylate cyclase, PleD. When phosphorylated, PleD synthesizes the small molecule, cyclic-di-GMP, initiating the signaling pathway responsible for cell differentiation. Therefore, PodJ interaction with PleC's PAS domains is critical in regulating the timing of this differentiation event. In this work, we demonstrate that a mutant lacking PleC PAS domains, that is free from PodJ inhibition, undergoes premature swarmer to stalked cell differentiation. Additionally, we provide evidence that PodJ interaction with PleC PAS domains does not impact PleC phosphatase activity, which is required for development of the new flagellar pole prior to asymmetric cell division. We show that mutant cells lacking PleC PAS domains are able to form pili and rotate their flagella, suggesting that new pole development is not perturbed in this mutant. These results suggest that PleC's PAS domains enable independent regulation of PleC kinase and phosphatase activities, allowing separate governance of cell differentiation and new pole development. In this way, PAS domains allow precise regulation of a single histidine kinase, acting on diverse substrates, to result in independent regulation of developmental events.