The Mycobacterium tuberculosis pks2 gene encodes the synthase for the hepta- and octamethyl-branched fatty acids required for sulfolipid synthesis

Multidrug-resistant tuberculosis is a major global health emergency. Cell w all lipids of Mycobacterium. tuberculosis can play crucial roles in the pat hogenesis, The enzymes involved in their synthesis can be ideal new drug ta rgets against tuberculosis, because many such lipids are unique to this pat hogen, A variety of multiple methyl-branched fatty acids are among such uni que lipids. We have identified seven genes highly homologous to the mas gen e, which is known to be involved in the production of one class of such mul tiple methyl-branched fatty acids. One of these mas-like genes, pks2, was d isrupted using a phage-mediated delivery of the disruption construct. Gene disruption by homologous recombination was confirmed by polymerase chain re action analysis of the flanking regions of the introduced disrupted gene an d by Southern analysis. Thin-layer and radio gas-chromatographic analyses o f lipids derived from [1-C-14]propionic acid and gas chromatography/mass sp ectrometry analysis of the fatty acids and hydroxy fatty acids showed that the pks2 mutant was incapable of producing hepta- and octamethyl phthiocera nic acids and hydroxyphthioceranic acids that are the major acyl constituen ts of sulfolipids. Consequently, pks2 mutant does not produce sulfolipids. Sulfolipid deficiency in pks2 mutant was confirmed by two-dimensional thin- layer chromatographic analysis of lipids derived from [1-C-14]propionic aci d and (SO4-2)-S-35 With this sulfolipid-deficient mutant, it should be poss ible to test for the postulated important roles for sulfolipids in the path ogenesis of M. tuberculosis.