Analysis of the c-di-GMP mediated cell fate determination in Caulobacter crescentus

Abstract

Cyclic-di-GMP (c-di-GMP) is a ubiquitous second messenger in bacteria, which has been recognized as a key regulator, antagonistically controlling the transition between motile, planktonic cells and surface attached, multicellular communities. The biosynthesis and degradation of c-di-GMP are mediated by the opposing enzymatic activities of di-guanylate cyclases (DGCs) and phosphodiesterases (PDEs), generally in response to internal and environmental signals. These activities reside in GGDEF and EAL domains respectively, which represent two large families of output domains often found in bacterial one- and twocomponent systems. In this work, the cell cycle-embedded differentiation from a free-living, motile swarmer cell into a sessile stalked cell in the model organism Caulobacter crescentus, and the role of c-di-GMP in this process was investigated. A systematic analysis was used to identify key regulatory enzymes involved in c-di-GMP metabolism that influence this developmental process. The function and regulation of these genes was then examined. One component that has already been implicated in this process, the DGC PleD, was investigated in more detail, with special emphasis on the mechanisms underlying its timed activation and cell cycle specific subcellular localisation. In the first part of this work, a systematic functional analysis of all GGDEF, EAL and GGDEF/EAL composite proteins from C. crescentus with a focus on motility and surface attachment is described. In this screen, the phosphodiesterase PdeA was identified as a gatekeeper that prevents premature paralysis of the flagellum and holdfast synthesis in the C. crescentus swarmer cell. It is shown that PdeA, together with its antagonistic DGCs DgcB and PleD, are components of converging pathways and orchestrate polar development during the swarmer-to-stalked cell transition. Furthermore, evidence is presented for a proteolytic regulation mechanism for PdeA. Secondly, the PleD localisation factor CC1064 is analysed. This transmembrane protein has pleiotropic effects on motility, surface attachment and polar localisation of PleD. It is shown that the motility and PleD localisation phenotypes of a Δcc1064 strain are conditional and depend on environmental factors such as oxygen and temperature stress. Moreover, evidence is presented that the impaired motility of a Δcc1064 mutant is caused by an assembly defect of the motor proteins MotA and MotB, leading to paralysis of the flagellum. A model is suggested that links altered membrane composition under environmental stress conditions to the Δcc1064 phenotypes. In Paul, Abel et al. (2007), insights were gained into the regulation of PleD. In addition to the well characterised non-competitive feedback inhibition, a second independent layer of activity control via dimerisation was investigated. The response regulator PleD is activated by phosphorylation of the N-terminal receiver domain. Here we show that the phospho-mimetic chemical beryllium fluoride specifically activates the enzymatic activity of PleD in vitro and in addition leads to dimerisation. Fractionation experiments showed that the DGC activity exclusively resides within the dimer fraction. Finally, evidence is provided that dimerisation of PleD is not only required for catalytic activity, but also leads to sequestration to the differentiating stalked pole of the C. crescentus cell, thereby providing an elegant way of restricting PleD activity to a subcellular region of the cell. In Paul, Jaeger & Abel et al. (2008), a network of proteins belonging to the two component system that regulates PleD activation and thereby leads to its localisation were investigated in detail. The single domain response regulator DivK is controlled by the phosphatase activity of PleC and the kinase DivJ. It is shown that DivK allosterically activates the kinase activities of PleC and DivJ and thereby switches PleC from a phosphatase into a kinase state. Increased DivJ activity further activates DivK in a feed-forward loop, while PleC and DivJ together stimulate PleD activity and localisation. Evidence is provided that DivJ, PleC, and DivK colocalise in a short time window during the cell cycle, directly prior to PleD activation, suggesting a role for the spatial distribution of these proteins. At last, the wider role of single domain response regulators in the interconnection of two-component signal transduction circuits is discussed. Finally, in Dürig, Folcher, Abel et al. (2008), a role for c-di-GMP in the cell cycle of C. crescentus via regulation of targeted proteolysis of the regulator CtrA is shown. During the swarmer-to-stalked cell transition CtrA is recruited to the incipient stalked pole, where it is degraded by its protease ClpXP. This recruitment and subsequent degradation is dependent on the enzymatically inactive GGDEF domain protein PopA. PopA itself localises to the cell pole and can bind c-di-GMP. It is shown that mutants in the c-di-GMP binding site fail to localise to the developing stalked pole and consequently fail to promote CtrA degradation. Finally, evidence is provided that interconnects PopA with the pathway responsible for substrate inactivation and protease localisation in a cell cycle dependent manner