Mycolic acid composition of Corynebacterium glutamicum and its cell surface mutants: effects of growth with glycine and isonicotinic acid hydrazide

Auxotrophic mutants of Corynebacterium glutamicum 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 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(18:0)), normally a minor component, late in the growth cycle and oleic acid proportionately decreased. All strains had five major types of MAs (C(32:0), C(34:0), C(34:1), C(36:2)) C(36:2)) 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-covalently 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 far 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 -1 . 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(32:0) relative to increases in the proportion of C(34:1) and C(36:2), 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(32:0)) 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 C. glutamicum.