Identification of a peptide synthetase involved in the biosynthesis of glycopeptidolipids of Mycobacterium smegmatis

Five rough colony mutants of Mycobacterium smegmatis mc(2)155 were produced by transposon mutagenesis. The mutants were unable to synthesize glycopept idolipids that are normally abundant in the cell wall of wild-type M. smegm atis. The glycopeptidolipids have a lipopeptide core comprising a fatty aci d amide linked to a tetrapeptide that is modified with O-methylated rhamnos e and O-acylated 6-deoxy talose. Compositional analysis of lipids extracted from the mutants indicated that the defect in glycopeptidolipid synthesis occurred in the assembly of the lipopeptide core. No other defects or compe nsatory changes in cell wall structure were detected in the mutants. All fi ve mutants had transposon insertions in a gene encoding an enzyme belonging to the peptide synthetase family. Targeted disruption of the gene in the w ild-type strain gave a phenotype identical to that of the five transposon m utants. The M. smegmatis peptide synthetase gene is predicted to encode fou r modules that each contain domains for cofactor binding and for amino acid recognition and adenylation. Three modules also have amino acid racemase d omains. These data suggest that the common lipopeptide core of these import ant cell wall glycolipids is synthesized by a peptide synthetase.