Second messenger mediated spatiotemporal control of cell cycle and development

Abstract

During the biphasic life cycle of Caulobacter crescentus motile, free-living swarmer cells differentiate into sessile, surface attached stalked cells. The swarmer cell is replication inert and is unable to divide. During the swarmer-to-stalked cell differentiation, degradation of CtrA, a master regulator that blocks replication initiation, leads the onset of chromosome replication. After this obligate cell differentiation step, which is mainly regulated by the degradation of the master cell cycle regulator CtrA, stalked cells immediately initiate their chromosome replication. Recently, dynamic colocalization of CtrA and its protease ClpXP to cell pole was proposed as a timing mechanism for cell cycle-dependent CtrA degradation. We have identified the response regulator PopA as an essential regulator for CtrA sequestration to the incipient stalked cell pole and for subsequent CtrA degradation by the nearby ClpXP protease complex. Time laps fluorescence microscopy of PopA-GFP showed that PopA itself dynamically sequesters to the cell poles during the C. crescentus cell cycle. While PopA sequestration to the flagellated pole depends on PodJ, a swarmer pole specificity factor, localization to the incipient stalked pole depends on the C-terminal GGDEF output domain of PopA. We demonstrate that in contrast to most GGDEF domain proteins, PopA lacks diguanylate cyclase activity. Instead, PopA functions as cyclic di-GMP effector protein, which specifically binds the bacterial second messenger at a conserved binding site (I-site) within the GGDEF domain. An intact PopA I-site is required for PopA sequestration to the incipient stalked pole as well as for CtrA degradation during the cell cycle. PopA directs CtrA to the ClpXP occupied cell pole via a direct interaction with an adaptor protein, RcdA. Based on this we postulate that c-di-GMP bound PopA facilitates the dynamic distribution of CtrA to the cell pole where it s degraded by ClpXP. This is the first report that links cdi- GMP to protein dynamics and cell cycle control in bacteria. In addition to its prominent role in cell cycle control, PopA was identified as novel component of the complex regulatory network that orchestrates polar development in C. crescentus. PopA, together with PleD and DgcB, two active diguanylate cyclases, controls cell motility, holdfast formation and surface attachment. Our data suggest that PopA interferes with PleD and DgcB to coordinate cell motility, stalk biogenesis, holdfast formation and finally surface attachment. Based on this, we propose that PopA is a bifunctional protein, involved in control and coordination of C. crescentus cell cycle and development.