1887

Abstract

In prior studies, through recombinant expression in , the gene of was shown to encode a rhamnosyltransferase that catalyses the addition of rhamnose (Rha) to the 6-deoxytalose of serovar 2-specific glycopeptidolipid (GPL). Whether RtfA also catalyses the transfer of Rha to the alaninol of the lipopeptide core is unknown. An isogenic mutant of serovar 2 strain TMC724 was derived using a novel allelic exchange mutagenesis system utilizing a multicopy plasmid that contained the gene of and the gene encoding green fluorescent protein (). Overexpression of KatG in resulted in increased susceptibility to isoniazid, thus providing counter-selection by enriching for clones that had lost plasmid DNA. Plasmid loss was confirmed by screening for -negative clones to select putative allelic exchange mutants. Two exchange mutants were created, confirmed by Southern hybridization, and demonstrated loss of serovar 2-specific GPL by thin-layer chromatography (TLC). Gas chromatography of alditol acetate derivatives revealed the loss of Rha and the terminal 2,3--Me-fucose and preservation of 3--Me-Rha and 3,4--Me-Rha substituents at the terminal alaninol of the lipopeptide core. Complementation of through an integrative plasmid restored serovar 2-specific GPL expression identical to wild-type TMC724. This result shows that encodes an enzyme responsible only for the transfer of Rha to the serovar 2-specific oligosaccharide and provides a system of allelic exchange for as a tool for future genetic studies involving this species.

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2003-11-01
2020-04-02
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