Dokument

Summary:
The peptidoglycan (PG) sacculus is a large macromolecule enclosing most bacterial cells. During progression of the cell cycle, it needs to be continuously remodelled to enable elongation of the cell body and, finally, cell division. This process requires a delicate balance between synthetic and hydrolytic reactions, which are executed by an array of different enzymes. However, the roles of individual components in this complex machinery and the mechanisms underlying their temporal and spatial regulation are still incompletely understood. In particular, the functional significance of many PG hydrolases is still unclear. While acting at all stages of cell wall biogenesis, PG hydrolases have a particularly important role during cell division, facilitating the coordinated invagination of the PG layer as constriction proceeds. Previous work has identified the putative PG hydrolase DipM as a key component of the Caulobacter crescentus divisome. However, the extent to which other hydrolases contribute to PG remodelling during the constriction process in this species has remained unknown. To identify the major players and elucidate their function, I have analysed the full set of putative amidases, lytic transglycosylases (LTs), endopeptidases (EPases) and LD-transpeptidases (LD-TPases) encoded in the C. crescentus genome. For this purpose, I generated a variety of fluorescent fusions and deletion mutants and characterized them using microscopic and biochemical approaches. The results obtained indicate that the PG hydrolases of C. crescentus have highly redundant functions. Based on the observation of changes in cell morphology, localization dynamics, stress and antibiotic resistance I have identified a particularly important role of EPases in maintaining cell wall integrity. Deletion of the amiC and chap genes encoding proteins with PG amidase activity had no detectable effect. In contrast, mutants lacking multiple EPases of the NlpC/P60 or LytM subgroups formed either long smooth filaments or occasionally cell chains. Furthermore, the presence of either NlpC/P60 domain containing EPase NlpA, or the Chap amidase is essential to maintain proper growth. Inactivation of all soluble lytic transglycosylases (SLTs), led to cell filamentation accompanied by outer-membrane blebbing. Depletion of DipM, an EPase homologue lacking catalytic activity in strains lacking either all SLTs or all of the remaining LytM factors (a subgroup of EPases), led to a complete block in cell division and finally to cell death. Interestingly, a similar morphological defect was observed upon depletion of a DipM in a strain lacking another LytM factor with degenerated active site, LdpF. Cultivation in stress conditions revealed critical functions of LdpF, SdpA (falling into the SLTs group) and Chap in cell envelope biogenesis, since single deletions of the respective genes led to either osmo- or antibiotic sensitivity. Moreover, SdpA displayed a dynamic localization pattern, characteristic for the divisome components implicated in the final stages of cell wall remodelling, indication a role in the cell division. Collectively, this work provides the first comprehensive analysis of PG hydrolases in C. crescentus and underscores the key role of the catalytically inactive EPase homologues DipM and LdpF in the autolytic system of this species.

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