Loci of Mycobacterium avium ser2 gene cluster and their functions

Abstract

The highly antigenic glycopeptidolipids present on the surface of members of the Mycobacterium avium complex serve to distinguish these bacteria from all others and to define the various serovars that compose this complex. Previously, the genes responsible for the biosynthesis of the disaccharide hapten [2,3-di-0-methyl-CL-L-fucopyranosyl-(1--*3)-cK.L-rhamnopyranose] of serovar 2 of the M. avium complex were isolated, localized to a contiguous 22-to 27-kb fragment of the M. avium genome, and designated the ser2 gene cluster (J. T. Belisle, L. Pascopella, J. M. Inamine, P. J. Brennan, and W. R. Jacobs, Jr., J. Bacteriol. 173:6991-6997, 1991). In the present study, transposon saturation mutagenesis was used to map the specific genetic loci within the ser2 gene cluster required for expression of this disaccharide. Four essential loci, termed ser2A, -B, -C, and -D, constituting a total of 5.7 kb within the ser2 gene cluster, were defined. The ser2B and ser2D loci encode the methyltransferases required to methylate the fucose at the 3 and 2 positions, respectively. The rhamnosyltransferase was encoded by ser2A, whereas either ser2C or ser2D encoded the fucosyltransferase. The ser2C and ser2D loci are also apparently involved in the de novo synthesis of fucose. Isolation of the truncated versions of the hapten induced by the transposon insertions provides genetic evidence that the glycopeptidolipids ofM. avium serovar 2 are synthesized by an initial transfer of the rhamnose unit to the peptide core followed by fucose and finally 0 methylation of the fucosyl unit.Knowledge of the composition and biosynthesis of components of the cell envelope of mycobacteria is paramount in addressing issues such as mechanisms of pathogenesis and drug sensitivity and resistance, all pertinent to diseases such as tuberculosis, leprosy, and atypical mycobacterioses. The fundamental architecture of the cell envelope common to all mycobacteria has been elucidated through concurrent chemical (26) and ultrastructural (18) analyses. Mycolic acids form the outermost layer and are attached to the centrally located polysaccharide, arabinogalactan, which, in turn, is linked to the innermost peptidoglycan. In addition to these conserved cell envelope components, mycobacteria contain a range of complex free lipids that are probably associated with the mycolic acid matrix and have different molecular identities in the various Mycobacterium species (18,26). In members of the Mycobacterium avium complex, i.e., M avium and Mycobactenum intracellulare (32), the copious amounts of glycopeptidolipids (GPLs) present on the surface of the bacteria not only serve to distinguish members from other mycobacteria but also differentiate one serovar from another (9). The GPLs that confer serological specificity are multiglycosylated with various serovar-specific sugars at the threonine substituent on an otherwise invariant lipopeptide core (9, 11).Biosynthetic pathways for assembling these complex structures have been proposed (6,15,26,31), but the isolation of bi...

Characterization of the Fucosylation Pathway in the Biosynthesis of Glycopeptidolipids fromMycobacterium aviumComplex

“…AF125999.1). This region contains five genes: mdhtA and merA, whose deduced amino acid sequences show a high level of similarity to those of enzymes involved in Fuc synthesis; mtfF, previously identified as the fucosyl 2-O-methyltransferase gene; and gtfC and gtfD, putative glycosyltransferase genes whose functions remain unknown (13,18). Since M. smegmatis only produces core GPLs, we introduced the chromosomal integrating vector pYMrtfA-int possessing the M. avium gene rtfA, whose gene product transfers the Rha residue to 6-d-Tal of core GPLs, and obtained the recombinant strain rtfA-int, which produces GPL with a terminal Rha residue (termed GPL-S1) that could be a substrate for the synthesis of serovar 2 GPL.…”

Mycobacterium aviumGenes Associated with the Ability To Form a Biofilm

“…The nsGPLs are further elaborated with oligosaccharide structures to produce the antigenically important serovar-specific GPLs (5). M. smegmatis, which produces only nsGPL, has been used to identify the genes for the glycosyltransferase and methyltransferase involved in elaborating the nsGPL with the hepatenic oligosaccharides of M. avium serovar 2 (11,21). Furthermore, the M. avium A5 transposon mutant of 4B2 obtained in the present study had the pstB gene inactivated, which was encoded in the sequences of the GPL biosynthesis gene cluster and daunorubicine gene in M. avium 2151 (GenBank accession no.…”

Modification of glycopeptidolipids by an O-methyltransferase of Mycobacterium smegmatis a aThe GenBank accession number for the sequence determined in this work is AY138899.

“…The GPLs of M. smegmatis, which are not glycosylated at 6-dTal, are non-serovar-specific GPLs and represent the biosynthetic precursor of the ssGPLs of M. avium (Eckstein et al, 1998). The M. avium genomic region encoding glycosylation of serovar-2-specific GPLs has been identified (Belisle et al, 1991(Belisle et al, , 1993Mills et al, 1994). Deletion of the ser2 gene cluster results in GPL-deficient mutants that have rough colony morphology (Eckstein et al, 2000 D. Jeevarajah and others and a potential transporter protein (TmptC) (BillmanJacobe et al, 1999 ;Patterson et al, 2000 ;Recht & Kolter, 2001 ;Recht et al, 2000).…”