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

Abstract

In prior studies, through recombinant expression in Mycobacterium smegmatis, the rtfA gene of Mycobacterium avium 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 rtfA mutant of M. avium serovar 2 strain TMC724 was derived using a novel allelic exchange mutagenesis system utilizing a multicopy plasmid that contained the katG gene of Mycobacterium bovis and the gene encoding green fluorescent protein (gfp). Overexpression of KatG in M. avium 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 gfp-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 of rtfA in trans through an integrative plasmid restored serovar 2-specific GPL expression identical to wild-type TMC724. This result shows that rtfA encodes an enzyme responsible only for the transfer of Rha to the serovar 2-specific oligosaccharide and provides a system of allelic exchange for M. avium as a tool for future genetic studies involving this species.

Structural Characterization of a Specific Glycopeptidolipid Containing a Novel N -Acyl-Deoxy Sugar from Mycobacterium intracellulare Serotype 7 and Genetic Analysis of Its Glycosylation Pathway

“…rtfA functions to catalyze only the addition of Rha to 6-d-Tal (26), and which gene cluster transfers additional sugars to L-Rha elongated from 6-d-Tal is unclear. ORFs 1, 3, and 9 have high homology to the glycosyltransferases GtfA, Rv1516c, and Rv1518, respectively.…”

“…PCR was used to isolate cosmid clones carrying the rhamnosyltransferase (rtfA) gene with primers rtfA-F (5Ј-TTTTGGAGCGACGAGTTCATC-3Ј) and rtfA-R (5Ј-GTGTAGTTGACCACGCCGAC-3Ј). rtfA encodes an enzyme responsible for the transfer of Rha to 6-d-Tal in OSE (11,26). The insert of cosmid clone 49 was sequenced using a BigDye Terminator, version 3.1, Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and an ABI Prism 310 gene analyzer (Applied Biosystems).…”

“…The standard technique for classification of MAC strains has been serologic typing based on the oligosaccharide (OSE) residue of the GPL. More recently, advanced chemical synthesis of various haptenic OSEs was demonstrated, and the genes encoding the glycosylation pathways in biosynthesis of GPL were identified and characterized (10,17,26).…”

Phylogeny of Mycobacterium avium strains inferred from glycopeptidolipid biosynthesis pathway genes

“…However, the exact function of the genes potentially involved remains unknown, with one exception. It has been experimentally demonstrated that the rtfA gene encodes rhamnosyltransferase that catalyses the addition of rhamnose to 6-dTal (Eckstein et al, 1998;Maslow et al, 2003). Glycosylation performed by rhamnosyltransferase is assumed to be the first step in the conversion of the non-specific GPLs to serotype-specific GPLs.…”

Identification and Characterization of Two Novel Methyltransferase Genes That Determine the Serotype 12-Specific Structure of Glycopeptidolipids of Mycobacterium intracellulare

“…Modification of the GPL structure might play an important role not only in antigenicity but also in host immune responses and bacterial physiology (18). Recently, chemical synthesis of various haptenic OSEs was demonstrated, and the genes encoding glycosylation pathway enzymes for the biosynthesis of GPLs were identified and characterized (8,12,19,21). However, genes responsible for serotype-specific glycosylation have yet to be analyzed for most of the serotypes.…”