title: Identification and recombinant expression of a Mycobacterium avium rhamnosyltransferase gene (rtfA) involved in glycopeptidolipid biosynthesis.
abstract: The Mycobacterium avium complex is a source of disseminated infections in patients with advanced AIDS. This group of mycobacteria is distinguished by the presence of highly antigenic, surface-exposed glycopeptidolipids, and these glycolipids possess variant oligosaccharide structures that are the chemical basis of the 28 distinct serovars of the M. avium complex. We previously described the ser2 gene cluster, encoding the synthesis of the haptenic oligosaccharide (2, 3-dimethylfucose-rhamnose-6-deoxytalose-) of the serovar 2-specific glycopeptidolipid, and revealed a locus (ser2A) encoding a putative rhamnosyltransferase. Sequencing of the ser2A locus demonstrated the presence of three open reading frames, two of which yielded significant homology to several glycosyltransferases, and the deduced amino acid sequences of these two putative glycosyltransferases had 63% identity. These two genes were expressed in Mycobacterium smegmatis, and the resulting recombinant glycopeptidolipids were characterized by thin-layer chromatography and gas chromatography-mass spectrometry. These analyses demonstrated that only one of these genes, termed rtfA, encoded the rhamnosyltransferase responsible for the transfer of rhamnose to 6-deoxytalose. The identification of rtfA will permit further evaluation of glycopeptidolipid biosynthesis and the construction of isogenic mutants of multiple M. avium complex serovars. Moreover, such mutants will help define the role of glycopeptidolipids in the intracellular survival of these bacteria.
authors.affiliation: Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523-1677, USA.
authors.forename: T M
authors.initials: TM
authors.lastname: Eckstein
authors.forename: F S
authors.initials: FS
authors.lastname: Silbaq
authors.forename: D
authors.initials: D
authors.lastname: Chatterjee
authors.forename: N J
authors.initials: NJ
authors.lastname: Kelly
authors.forename: P J
authors.initials: PJ
authors.lastname: Brennan
authors.forename: J T
authors.initials: JT
authors.lastname: Belisle
mesh_terms: D001426:Bacterial Proteins; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial; D004187:Disaccharides / Q000032:analysis; D015870:Gene Expression; D006017:Glycolipids / Q000096:biosynthesis*; D006020:Glycopeptides / Q000096:biosynthesis*; D006602:Hexosyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009162:Mycobacterium avium / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004187:Disaccharides; D006017:Glycolipids; D006020:Glycopeptides; D011993:Recombinant Fusion Proteins; D006602:Hexosyltransferases; C116032:RtfA protein, Mycobacterium avium; D004262:DNA Restriction Enzymes