Auxotrophic mutants of strain ATCC 13059 (parent of AS019, a rifampicin-resistant variant), which were morphologically distinct from the parent and formed protoplasts more readily, had been isolated previously. Mutants MLB130-133 and MLB194 were more sensitive to growth inhibition by isonicotinic acid hydrazide (INH) and glycine, which caused branching and budding. Fatty acid and mycolic acid (MA) profiles were determined after growth in LBG (Luria broth plus glucose), LBG-glycine (LBG- and LBG-INH (LBG-I). The fatty acid profiles of all strains were similar, except that mutant MLB133 showed some increase in stearic acid (C), normally a minor component, late in the growth cycle and oleic acid proportionately decreased. All strains had five major types of MAs (C, C, C, C, C) but the relative proportion of each varied with the strain, age of culture and medium composition. Mutants MLB133 and MLB194 showed slightly higher levels of non-covalentiy bound MAs than the parent and normally showed a higher proportion of longer-chained, unsaturated MAs. The proportion of extracellular MAs increased with culture age for these mutants. Typically, by late stationary phase, mycolic acids in culture fluids increased to 6.5% of the total MAs for MLB194 and 7.9% for MLB133 compared with 3.5% for the parent strain grown in LBG. The main effect of glycine (2%, w/v) addition was to increase the proportion of mycolic acids found in extracellular fluids (16.1 % for AS019 and 31% for MLB133). The most significant effects of INH were seen when strains were cultured in LBG with 8 mg INH ml. When harvested at late stationary phase, strains MLB133 and MLB194 had 18.8% and 21.2% extracellular mycolic acids respectively, with a significant increase in the relative proportion of unsaturated mycolic acids. This effect was not as marked for AS019, which also showed a similar decrease in C relative to increases in the proportion of C and C plus a corresponding increase in the overall proportion of unsaturated mycolic acids and increased extracellular mycolates (8.5%). These results suggest that the mutations in strains MLB133 and MLB194 are associated with synthesis of specific mycolic acids (e.g. C) and attachment of mycolic acids to the cell surface, both of which are likely target sites for glycine and INH action for cell-surface modifications. In addition to previously reported targeting of the peptidoglycan cross-linking, these results show that glycine affects mycolic acid attachment to the cell surface of


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