The antimycobacterial compound ethambutol [Emb; dextro-2,2'-(ethylenediimino)-di-1-

The biosynthesis of lipoarabinomannan (LAM), a key mycobacterial lipoglycan that has been implicated in numerous immunoregulatory functions, was examined utilizing D-mannosamine (ManN) as a tool to identify mannosyltransferase genes involved in LAM synthesis. Cell-free reactions utilizing cellular membranes of mycobacteria as the enzyme source indicated that ManN inhibited the synthesis of phosphatidylinositol mannosides, early precursors to LAM. A selection strategy was devised to screen a Mycobacterium tuberculosis genomic library in Mycobacterium smegmatis for clones conferring conditional resistance to ManN, with the rationale that overexpression of the gene(s) encoding a target of ManN would impart a ManN-resistant phenotype under these conditions. This strategy led to the identification of pimB, whose deduced amino acid sequence shows similarity to mannosyltransferases and other glycosyltransferases. Partially purified recombinant PimB protein from Escherichia coli or membranes from M. smegmatis overexpressing the pimB gene were used in cell-free assays to show that PimB catalyzes the formation of triacylphosphatidylinositol dimannoside from GDP-mannose and triacylphosphatidylinositol monomannoside.

(9) was compared with that of the AAD(9) encoded by staphylococcal transposon Tn554. The two proteins shared approximately 39% amino acid identity, which was expanded to 53% when conservative amino acid changes were included. When the streptococcal protein was compared with an AAD(3')(9) protein of E. coli, the degrees of identity were 27 and 47%, on the basis of actual amino acids and conservative replacements, respectively. The cloning and nucleotide base sequence analyses of the spectinomycin AAD(9) determinant from E. faecalis that results in high-level Spr when transferred to S.sanguis or E. coli are presented.

Molecular cloning and sequencing showed that Mycoplasma gallisepticum, like Mycoplasma capricolum, contains both tRNAUCA and tRNACCA genes, while Mycoplasma pneumoniae and Mycoplasma genitalium each appear to have only a tRNAUCA gene. Therefore, these mycoplasma species contain a tRNA with the anticodon UCA that can translate both UGA and UGG codons.Although UGA is a stop (opal) (Fig. 1A) were employed as probes, and they gave identical results (not shown). The hybridizing DNA fragments from M. genitalium (AluI, DraI, and EcoRI fragments of 390 bp, 622 bp, and 5 kbp, respectively) and M. gallisepticum (410-and 850-bp AluI and 5.45-and 5.75-kbp EcoRI fragments) were cloned in E. coli. These regions were mapped, and relevant portions of both strands were sequenced.The cloned M. genitalium DNA was found to contain a tRNA gene with the anticodon UCA (Fig. 2B), while the M. gallisepticum clones contained both a tRNA gene with the anticodon UCA (Fig. 2C) and one with the anticodon CCA 504 on June 7, 2019 by guest

Bacteria within the Mycobacterium avium complex are prominent in the environment and are a source of serious disseminated infections in patients with AIDS. Serovars of the M. avium complex are distinguished from all other mycobacteria and from one another by the presence of highly antigenic glycolipids, the glycopeptidolipids, on their surfaces. A genomic library of DNA from serovar 2 of the M. avium complex was constructed in the Escherichia coli-Mycobacterium shuttle cosmid, pYUB18, and used to clone and express in Mycobacterium smegmatis the genes responsible for the biosynthesis of the oligosaccharide segment of the M. avium serovar 2-specific glycopeptidolipid. The responsible gene cluster was mapped to a 22-to 27-kb functional region of the M. avium genome. The recombinant glycolipid was also isolated by high-pressure liquid chromatography and chemically characterized, by gas chromatography-mass spectrometry and fast atom bombardment-mass spectrometry, to demonstrate that the lipopeptide core originated in M. smegmatis, whereas the oligosaccharide segment arose from the cloned M. avium genes. This first-time demonstration of the cloning and expression, in a nonpathogenic mycobacterium, of the genes encoding complex cell wall glycoconjugates from a pathogenic mycobacterium presents a new approach for studying the role of such products in disease processes.