Identification of a methyltransferase from Mycobacterium smegmatis involved in glycopeptidolipid synthesis

Glycopeptidolipids (GPLs) are major components of the cell walls of several species of mycobacteria. We have isolated a transposon mutant of Mycobacte rium smegmatis that is unable to synthesize mature GPLs and that displays a rough colony morphology. The disrupted gene, mtf1, shares a high degree of homology with several S-adenoslmethionine-dependent methyltransferases. Th e enzyme encoded by mtf1 is required for the methylation of a single rhamno se residue that forms part of the conserved GPL core structure. This conclu sion is supported by the finding that (a) the mutant synthesized only GPLs with undermethylated (either mono- or nonmethylated instead of di- or trime thylated) rhamnose residues; (b) complementation of the mutant with a wild- type copy of mtf1 restored high levels of synthesis of GPLs containing di- and trimethylated rhamnose; and (c) S-adenosylmethionine-dependent methylat ion of rhamnosylated GPLs could be detected in cell lysates of wild-type ce lls and mtf1-complemented mutant cells, but not in mutant cells lacking int act mtf1. Structural analysis of wild-type and mutant GPLs suggests that di sruption of mtf1 specifically inhibits addition of O-methyl groups to the 3 (or 2)-position of the rhamnose, In the absence of 3-O-methylation, furthe r methylation of GPL rhamnose is apparently inhibited, and overall GPL synt hesis is down-regulated by 90%.