The role of two novel proteins YvcJ/YhbJ and YvcK/YbhK in cell wall biosynthesis in presumable connection to the PTS in Bacillus subtilis and Escherichia coli

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The bacterial phosphotransferase System (PTS) catalyzes the simultaneous transport and phosphorylation of sugars using a phosphorylation cascade composed of several proteins. The phosphorylation state of the PTS regulates a number of signal transduction chains in the cell implicated in catabolite repression, in chemotaxis and in regulation of numerous genes. The PTS can therefore be regarded as the bacterial brain . We identified two novel genes named yvcJ and yvcK in Bacillus subtilis with a fundamental role in biosynthesis of the cell wall or its regulation. These two genes exist in many evolutionary divergent bacteria and often cluster on the genome together with genes related to the PTS, suggesting a functional connection. In Bacilli they are present within the yvd-N-operon encoding also Crh, a homologue of the phosphocarrier protein HPr of the PTS. We found that B. subtilis mutants defective for yvcK acquire an L-form-like cell shape and finally lyse when carbon sources are utilized that require gluconeogenesis for their metabolism. Our data suggest that this mutant is unable to provide a sufficient level of glycolytic intermediates required for the synthesis of cell wall precursor molecules. In Escherichia coli gene yhbJ (homologous to yvcJ) is present within the rpoN operon that also encodes the alternative ¿54-factor and the proteins IIANtr and NPr which are orthologues of PTS-enzymes and of unknown function. We found that deletion of yhbJ leads to huge overproduction of the glucosamine synthase GlmS that catalyzes the formation of glucosamine-6-phosphate, a key reaction in cell wall synthesis. An abnormal high GlmS level would also perfectly explain the effects of an yvcK deletion in B. subtilis suggesting that both gene products are interconnected in the control of the GlmS amount. In E. coli, ybhK (homologous to yvcK) is separately encoded and probably expressed from a ¿54-dependent Promoter. The data point to a model in which YhbJ controls the ¿54-dependent expression of YbhK which in turn regulates the GlmS level in E. coli. Here we propose to investigate this signal transduction pathway in the two model bacteria B. subtilis and E. coli. First, we want to explore whether glmS transcription or translation or degradation of its product is affected by YhbJ (YvcJ). Second, we will study whether YhbJ acts directly on GlmS or indirectly perhaps by interaction with ¿54 thereby modulating the expression of YbhK which in turn could regulate the GlmS amount in E. coli. Finally, we want to address the role of the PTS-orthologues co-expressed together with yhbJ (yvcJ) and whether they modulate this signal transduction pathway towards GlmS and thereby adjust cell wall biosynthesis to the metabolic state of the cell.