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 (p arent of AS019, a rifampicin-resistant variant), which were morphologi cally distinct from the parent and formed protoplasts more readily, ha d been isolated previously. Mutants MLB130-133 and MLB194 were more se nsitive to growth inhibition by isonicotinic acid hydrazide (INH) and glycine, which caused branching and budding. Fatty acid and mycolic ac id (MA) profiles were determined after growth in LBG (Luria broth plus glucose), LBG-glycine (LBG-G) and LBG-INH (LBG-I). The fatty acid pro files of all strains were similar, except that mutant MLB133 showed so me increase in stearic acid (C-18:0), normally a minor component, late in the growth cycle and oleic acid proportionately decreased. All str ains had five major types of MAs (C-32:0, C-34:0, C-34:1, C-36:1, C-36 :2) but the relative proportion of each varied with the strain, age of culture and medium composition. Mutants MLB133 and MLB194 showed slig htly higher levels of non-covalently bound MAs than the parent and nor mally showed a higher proportion of longer-chained, unsaturated MAs. T he proportion of extracellular MAs increased with culture age for thes e mutants. Typically, by late stationary phase, mycolic acids in cultu re fluids increased to 6.5% of the total MAs for MLB194 and 7.9% for M LB133 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 strain s were cultured in LBG with 8 mg INH ml(-1). When harvested at late st ationary phase, strains MLB133 and MLB194 had 18.8% and 21.2% extracel lular mycolic acids respectively, with a significant increase in the r elative proportion of unsaturated mycolic acids. This effect was not a s marked for AS019, which also showed a similar decrease in C-32:0, re lative to increases in the proportion of C-34:1, and C-36:2, plus a co rresponding increase in the overall proportion of unsaturated mycolic acids and increased extracellular mycolates (8.5%). These results sugg est that the mutations in strains MLB133 and MLB194 are associated wit h 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 sit es for glycine and INH action for cell-surface modifications. In addit ion to previously reported targeting of the peptidoglycan cross-linkin g, these results show that glycine affects mycolic acid attachment to the cell surface of C. glutamicum.