Dokument

Summary:
Abstract The spatial and temporal regulation of peptidoglycan biosynthesis and its role in cell morphology has been studied intensively in well-characterized model organisms such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus, which divide either by symmetric or asymmetric binary fission. To broaden our knowledge of the mechanisms governing bacterial morphogenesis, we started to investigate the dimorphic marine α-proteobacterium Hyphomonas neptunium as a new model organism. This Gram-negative species is characterized by a unique mode of proliferation, whereby the new offspring is generated by the formation of a bud at the tip of a stalk that emanates from the mother cell body. The main focus of our previous studies was the identification of cell wall biosynthetic enzymes and regulatory factors that are critically involved in stalk and bud biogenesis. These studies revealed that peptidoglycan biosynthesis in H. neptunium is a complex process mediated by an intricate interplay of various factors. Among the open questions, it is still unknown how the generation of the daughter cell is regulated and how the mother cell orchestrates the localization of peptidoglycan remodeling enzymes at specific site of action during the cell cycle. Consequently, that includes the initial localization of enzymes at the stalked pole. At a certain point, they have to diffuse through the stalk into the growing bud. There, they center at the junction between the bud and the stalk to separate the mother cell from the bud. The main goal of the present study our current research is a deeper and more thorough characterization of previously investigated peptidoglycan remodeling enzymes, and especially the lytic enzymes, that cleave the peptidoglycan mesh. We particularly focused on two classes, the M23 metallopeptidases and the amidases. In doing so, we compre-hensively analyzed the six M23 endopeptidases of H. neptunium with localization studies and genetic approaches. Our results revealed a high degree of redundancy among these enzymes, which combined with the absence of a distinct localization pattern, indicated a generalized role in cell wall maintenance. We also investigated the role of the only amidase in H. neptunium in cell separation and bud formation. A deletion of the amidase gene led to an aberrant morphology and a mild chaining phenotype. Importantly, we showed that one of the M23 endopeptidases (LmdE) acts as a regulator of AmiC. Using biochemical approaches, we proved an interaction between AmiC and LmdE, where LmdE stimulates the catalytic activity of AmiC and thus regulates peptidoglycan hydrolysis. A further crucial player in this system is the inner membrane-embedded FtsEX complex. A deletion of the whole complex resulted in cells with very elongated and misshapen stalks. Probably, FtsEX plays a role in the regulation of amidase activity by interacting with LmdE. These results are similar between α- and γ-proteobacteria indicating that the mechanism of amidase regulation is conserved. A further goal of our work was the identification of novel factors that are specifically involved in the regulation of budding in H. neptunium. To this end, we started to establish a transposon mutagenesis system to identify all essential genes in this species. In the future, we will be able to investigate these novel factors and their contribution to cell morphology. Taken together, these results provide insight into the mechanisms of morphogenesis in stalked budding bacteria, thus setting the stage for an in-depth analysis of the regulatory mechanisms that control the spatiotemporal dynamics of the peptidoglycan biosynthetic machinery in these organisms.

Das Dokument ist im Internet frei zugänglich - Hinweise zu den Nutzungsrechten