Inactivation of two genes located in the GPL biosynthetic locus leads to morphological changes in Mycobacterium smegmatis

Glycopeptidolipids (GPLs) are surface exposed molecules found either in saprophytic or in clinically-relevant non-tuberculous mycobacteria. These molecules, which may represent more than 70% of total surface lipids, are required for cell aggregation, sliding motility and biofilm formation and seem to act as surface antigens stimulating host macrophages response. The GPLs structure is conserved among mycobacteria and is made by a common glycosylated lpopeptide core that is variably modified by O-methylation and O-acetylation. In the fast growing Mycobacterium smegmatis, all the genes necessary for GPL biosynthesis are clustered in a single region of 65 kb and most of them have been identified experimentally or by in silico prediction. Here we report the isolation of two mutant strains of M. smegmatis, carrying null mutations in genes coding for two hypothetical proteins. The two genes (MSMEG_0412; MSMEG_0394) belong to the GPL gene cluster. Preliminary experiments show that inactivation of both genes has drastic impact on bacterial surface properties. Both mutants display rough phenotype, lack of sliding motility, altered biofilm formation and increased antimicrobial susceptibility. GPLs analysis by TLC and MALDI-TOF is in progress to assess whether the observed phenotypes arise from GPLs deficiency and/or structural modification.