Cell envelope of bacteria producing glycopeptide antibiotics

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Glycopeptide antibiotics inhibit the synthesis of the bacterial cell wall by blocking the transpeptidation and transglycosylation. The resistance mechanism in most pathogens against glycopeptides involves the synthesis of alternative cell wall precursors. Due to the complexity of the resistance mechanism which comprises of at least three essential enzymes, it was expected that glycopeptide producers are the evolutionary origin of the resistance genes. However, none of the essential resistance genes was found in the antibiotic biosynthesis gene clusters of the producing Amycolatopsis species. The only functions which are localised in the cluster and may be related to resistance are a putative ABC-transporter, as well as a two component regulatory system and a carboxypeptidase. The latter have counterparts on vancomycin resistance transposons, but are dispensable for expressing resistance. The proposed research should answer the following questions: * How does the producer protect its cell wall from the action of its antibiotic? * How are the accessory resistance genes involved in resistance? * Are different cell wall precursors synthesized during exponential and stationary (=production) phase and how is their synthesis regulated? This will be analysed by mutation and overexpression studies of the putative resistance genes. The genes encoding the synthesis of the "normal" cell wall will be isolated and characterised. The regulation of the genes as well as the synthesis of the proteins will be investigated, with respect to co-regulation of resistance and antibiotic production. Furthermore, we plan to study the chemical structure of the murein precursors at different time points and in the mutants (cooperation with chemistry).