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

• Published in: Microbiology (Society for General Microbiology) 2003-11, Vol.149 (11), p.3193-3202
• Main Authors: Maslow, Joel N, Irani, Vida R, Lee, Sun-Hwa, Eckstein, Torsten M, Inamine, Julia M, Belisle, John T
• Format: Article
• Language: English
• Subjects: 6-deoxytalose; Alleles; Bacterial Proteins - genetics; Biological and medical sciences; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Hexosyltransferases - genetics; Hexosyltransferases - metabolism; Microbiology; Mutagenesis, Site-Directed; Mycobacterium avium; Mycobacterium avium - classification; Mycobacterium avium - enzymology; Mycobacterium avium - genetics; oligosaccharides; Peroxidases - genetics; Plasmids - genetics; Recombinant Proteins - metabolism; rhamnosyltransferase; rtfA gene; Species Specificity; Transformation, Bacterial
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.26565-0

1 Section of Infectious Diseases, VA Medical Center (151), University and Woodland Aves, Philadelphia, PA 19104, USA 2 Division of Infectious Diseases, University of Pennsylvania, Philadelphia, PA, USA 3 New England Regional Primate Center, Southborough, MA, USA 4 Mycobacterial Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA Correspondence Joel N. Maslow joel.maslow{at}med.va.gov 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. Abbreviations: 6-dTal, 6-deoxytalose; GPL, glycopeptidolipid; INH, isoniazid; LP, lipopeptide; nsGPL, non-specific glycopeptidolipid; Rha, rhamnose; ssGPL, serovar-specific glycopeptidolipid