GENE KNOCKOUT REVEALS A NOVEL GENE-CLUSTER FOR THE SYNTHESIS OF A CLASS OF CELL-WALL LIPIDS UNIQUE TO PATHOGENIC MYCOBACTERIA

Surface-exposed unusual lipids containing phthiocerol and phenolphthio cerol are found only in the cell wall of slow-growing pathogenic mycob acteria and are thought to play important roles in host-pathogen inter action, The enzymology and molecular genetics of biosynthesis of phthi ocerol and phenolphthiocerol are unknown, We postulate the domain orga nization of a set of multifunctional enzymes and a cluster of genes (p ps) that would encode these enzymes for the biosynthesis of phthiocero l and phenolphthiocerol, A cosmid containing the postulated pps gene c luster was identified by screening a genomic library of Mycobacterium bovis BCG with the postulated homologous domains from mycocerosic acid synthase and fatty acid synthase genes as probes, Homologous cosmids were also identified in the genomic libraries of Mycobacterium tubercu losis and Mycobacterium leprae, M. bovis BCG was transformed with a pp s disruption construct, made from the BCG cosmid by introducing the hy gromycin resistance gene as the positive-selectable marker and the sac B gene as the counter-selectable marker, Gene disruption by homologous recombination with double crossover was confirmed by polymerase chain reaction and Southern hybridization, Chromatographic analysis showed that the phenolphthiocerol derivative, mycoside B, and phthiocerol dim ycocerosates were not produced by the gene knockout mutants, This resu lt confirms the identity of the pps genes, With the identification of the pps gene clusters in both M. tuberculosis and M. leprae, it should be possible to test the postulated roles of these unique lipids in tu berculosis and leprosy.