Biosynthetic specificity of the rhamnosyltransferase gene of Mycobacterium avium serovar 2 as determined by allelic exchange mutagenesis

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Abstract
In prior studies, through recombinant expression inMycobacterium smegmatis, thertfAgene ofMycobacterium aviumwas 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 isogenicrtfAmutant ofM. aviumserovar 2 strain TMC724 was derived using a novel allelic exchange mutagenesis system utilizing a multicopy plasmid that contained thekatGgene ofMycobacterium bovisand the gene encoding green fluorescent protein (gfp). Overexpression of KatG inM. aviumresulted in increased susceptibility to isoniazid, thus providing counter-selection by enriching for clones that had lost plasmid DNA. Plasmid loss was confirmed by screening forgfp-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-O-Me-fucose and preservation of 3-O-Me-Rha and 3,4-O-Me-Rha substituents at the terminal alaninol of the lipopeptide core. Complementation ofrtfAin transthrough an integrative plasmid restored serovar 2-specific GPL expression identical to wild-type TMC724. This result shows thatrtfAencodes an enzyme responsible only for the transfer of Rha to the serovar 2-specific oligosaccharide and provides a system of allelic exchange forM. aviumas a tool for future genetic studies involving this species.