Analysis of cyclic di-GMP signaling components in "caulobacter crescentus" behavior and cell cycle control

Abstract

Cell cycle progression and polar morphogenesis in Caulobacter crescentus are coordinated by the interplay of multiple proteins in time and space. One major regulatory factor is the second messenger cyclic di-GMP (c-di-GMP) therefore especially the activities of enzymes that are responsible for synthesis and breakdown of this small molecule are tightly regulated. The swarmer cell specific population in the early phase of the cell cycle contains low levels of c-di-GMP due to the action of the phosphodiesterase PdeA. During the course of cell cycle progression, PdeA is degraded and thereby the activity of the diguanylate cyclase (DGC) DgcB is released. At the same time a second DGC, PleD, is activated by a phosphorylation relay, to elevate c-di-GMP levels necessary for cell development. The two proteins DgcB and PleD are the main cyclases in C. crescentus contributing to the intracellular c-di-GMP pool. Cells lacking both DGCs have severe defects affecting cell morphology and cell cycle progression. However, a residual c-di-GMP concentration is still detectable in the pleD dgcB double mutant presumingly due to the activity of other DGCs of C. crescentus. This work addressed the question, which additional GGDEF domain proteins reveal DGC activity and contribute to the c-di-GMP content in C. crescentus cells. This work presented here shows that two additional cyclases, BipB and BipC (bifunctional proteins B and C), are involved in c-di-GMP signaling. Both enzymes belong to the group of so-called composite proteins harboring a GGDEF and EAL domain, encoding for opposing catalytic activities, respectively. Single deletions of either bipB or bipC showed no phenotype. However, in combination with the deletion of pleD and dgcB, no c-di-GMP could be detected. The lack of c-di-GMP resulted in miss-localization of the effector protein PopA that is involved in the degradation of the replication inhibitor CtrA. Therefore, CtrA is stabilized in those cells leading to elongated cell morphology. These phenotypes resemble the phenotypes of a strain lacking all predicted DGCs (gutted strain, GS). To measure specifically low levels of c-di-GMP a strain was used lacking DGCs and in addition all PDEs (really gutted strain, rGS) to avoid immediate degradation in the GS. Introduction of either bipB or bipC in the rGS reverted the strain to a wild-type phenotype, e.g. motility and popA localization, indicating a DGC phenotype in vivo. However, in the presence of different PDEs like in the GS neither bipB nor bipC were able to revert the phenotype to wild-type suggesting weak DGC activity of both enzymes. For BipB bifunctional enzyme activity could be demonstrated in vitro and in vivo, whereas the DGC and the PDE activities were present at the same time. The cyclase activity of BipB is substrate inhibited via c-di-GMP binding to the inhibitory site motif RxxD. Based on these finding we propose that BipB is a bifunctional protein contributing under the applied conditions with BipC, PleD and DgcB to intracellular c-di-GMP levels in C. crescentus. The c-di-GMP signaling circuit involves not only cyclases and phosphodiesterases, which produce c-di-GMP upon an environmental stimulus but also effector proteins that bind c-di-GMP and therefore transmit the signal into an intracellular response. Knowing different c-di-GMP binding proteins would allow understanding c-di-GMP output systems. Therefore, a biochemical screen was carried out using c-di-GMP linked to a capture compound to specifically isolate c-di-GMP binding proteins. Among the novel identified proteins a group clusters next to chemotaxis genes. One of the hits is CmcA (named after its involvement in c-di-GMP dependent motor control), a single domain response regulator lacking the conserved phosphorylation site (aspartate) necessary for the function of a RR. Deletion of cmcA results in an increase in motility. To transmit the chemotactic signal CheY proteins interact directly with the flagellar apparatus. Therefore, the localization pattern of CmcA in different flagellar mutants was determined showing polar localization dependent on the MS-ring forming protein FliF. This localization pattern is missing in c-di-GMP deficient cells. From these results, we concluded that CmcA regulates motility in a c�di-GMP dependent manner.