AN ACYL-COA SYNTHASE (ACOAS) GENE ADJACENT TO THE MYCOCEROSIC ACID SYNTHASE (MAS) LOCUS IS NECESSARY FOR MYCOCEROSYL LIPID-SYNTHESIS IN MYCOBACTERIUM-TUBERCULOSIS VAR. BOVIS BCG

An open reading frame, ORF3, first identified adjacent to the mycocero sic acid synthase gene in Mycobacterium bovis BCG encodes a protein wi th acyl-CoA synthase (ACoAS) activity. Genes homologous to acoas are f ound adjacent to other multifunctional polyketide synthase genes in th e mycobacterial genome. To test whether these gene products are necess ary to esterify the fatty acids generated by the adjacent polyketide s ynthase gene products, the acoas gene was disrupted in M. bovis BCG us ing a suicide vector containing the acoas gene with an internal deleti on and the hygromycin-resistant gene as selection marker. Allelic exch ange at the acoas locus was confirmed by Southern hybridization and po lymerase chain reaction amplification of both nanking regions expected hom homologous recombination, Immunoblot analysis indicated that the 65-kDa ACoAS protein product was absent in the mutant. Chromatographic analysis of lipids derived from [1-C-14]propionate showed that the mu tant did not produce mycocerosyl lipids, although it produced normal l evels of mycocerosic acid synthase. These results suggest that ACoAS i s involved in the synthesis of mycocerosyl Lipids of the mycobacterial cell wall.