Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) are structurally related lipids noncovalently bound to the outer cell wall layer of Mycobacterium tuberculosis, Mycobacterium leprae, and several opportunistic mycobacterial human pathogens. PDIMs and PGLs are important effectors of virulence. Elucidation of the biosynthesis of these complex lipids will not only expand our understanding of mycobacterial cell wall biosynthesis, but it may also illuminate potential routes to novel therapeutics against mycobacterial infections. We report the construction of an in-frame deletion mutant of tesA (encoding a type II thioesterase) in the opportunistic human pathogen Mycobacterium marinum and the characterization of this mutant and its corresponding complemented strain control in terms of PDIM and PGL production. The growth and antibiotic susceptibility of these strains were also probed and compared with the parental wild-type strain. We show that deletion of tesA leads to a mutant that produces only traces of PDIMs and PGLs, has a slight growth yield increase and displays a substantial hypersusceptibility to several antibiotics. We also provide a robust model for the three-dimensional structure of M. marinum TesA (TesAmm) and demonstrate that a Ser-to-Ala substitution in the predicted catalytic Ser of TesAmm renders a mutant that recapitulates the phenotype of the tesA deletion mutant. Overall, our studies demonstrate a critical role for tesA in mycobacterial biology, advance our understanding of the biosynthesis of an important group of polyketide synthase-derived mycobacterial lipids, and suggest that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections. Mycobacterium tuberculosis (Mtb),3 Mycobacterium leprae, and several opportunistic mycobacterial human pathogens (e.g. M. marinum (Mm)) produce two related groups of diesters of ␤-glycol-containing aliphatic polyketides (e.g. phenolphthiocerols and phthiocerols) and polyketide synthase-derived multimethyl-branched fatty acids (e.g. mycocerosic acids) (Fig. 1). One of these groups is represented by phthiocerol dimycocerosates (PDIMs). The other group is represented by phenolphthiocerol dimycocerosates, which are glycosylated compounds generally known as phenolic glycolipids (PGLs). These complex lipids, which are believed to be noncovalently bound constituents of the outer leaflet of the unique mycobacterial outer membrane, are known important effectors of virulence (for review, see Ref. 1).Cox et al. (3) and Camacho et al.(2) independently demonstrated in 1999 that loss of PDIMs in PGL-deficient Mtb strains correlates with attenuation in animal models of tuberculosis. It has also been documented that production of PGLs confers a hyperlethality phenotype to PDIM-producing Mtb in murine disease models (4). Since these seminal reports, an overwhelming body of evidence has accumulated demonstrating that PDIMs and PGLs play key roles in virulence and host-pathogen interaction ...

The mycobactin siderophore system is present in many Mycobacterium species, including M. tuberculosis and other clinically relevant mycobacteria. This siderophore system is believed to be utilized by both pathogenic and nonpathogenic mycobacteria for iron acquisition in both in vivo and ex vivo iron-limiting environments, respectively. Several M. tuberculosis genes located in a so-called mbt gene cluster have been predicted to be required for the biosynthesis of the core scaffold of mycobactin based on sequence analysis. A systematic and controlled mutational analysis probing the hypothesized essential nature of each of these genes for mycobactin production has been lacking. The degree of conservation of mbt gene cluster orthologs remains to be investigated as well. In this study, we sought to conclusively establish whether each of nine mbt genes was required for mycobactin production and to examine the conservation of gene clusters orthologous to the M. tuberculosis mbt gene cluster in other bacteria. We report a systematic mutational analysis of the mbt gene cluster ortholog found in Mycobacterium smegmatis. This mutational analysis demonstrates that eight of the nine mbt genes investigated are essential for mycobactin production. Our genome mining and phylogenetic analyses reveal the presence of orthologous mbt gene clusters in several bacterial species. These gene clusters display significant organizational differences originating from an intricate evolutionary path that might have included horizontal gene transfers. Altogether, the findings reported herein advance our understanding of the genetic requirements for the biosynthesis of an important mycobacterial secondary metabolite with relevance to virulence.The obligate human pathogen Mycobacterium tuberculosis, most opportunistic mycobacterial human pathogens (e.g., M. avium), and many nonpathogenic saprophytic mycobacteria (e.g., M. smegmatis) produce a structurally complex salicylic acid-derived siderophore known as mycobactin (MBT) (Fig. 1) (5, 33, 35). MBT has a core scaffold of a proposed nonribosomal peptide-polyketide origin consisting of a hydroxyphenylcapped (methyl)oxazoline moiety linked to an N ε -hydroxylysine residue, which is typically connected to a terminal cyclo-N ε -hydroxylysine by a 4-carbon linker. This core scaffold is decorated with a variable fatty acyl substituent on the N ε of the internal N ε -hydroxylysine residue. Structural variants (carboxymycobactins) with acyl substituents terminating in a carboxylate or a methyl ester are also produced. Interestingly, the core scaffold of MBT is remarkably similar to core scaffolds seen in several compounds-some with interesting pharmacological activities-produced by species of the genus Nocardia (Fig. 1). Nocardia is a saprophytic group of actinomycetes closely related to the mycobacteria and includes species that are increasingly recognized as opportunistic human pathogens (2, 25).Studies with cellular and animal models of mycobacterial infection have established the relevance of the M...

BackgroundGlycopeptidolipids (GPLs) are among the major free glycolipid components of the outer membrane of several saprophytic and clinically-relevant Mycobacterium species. The architecture of GPLs is based on a constant tripeptide-amino alcohol core of nonribosomal peptide synthetase origin that is N-acylated with a 3-hydroxy/methoxy acyl chain synthesized by a polyketide synthase and further decorated with variable glycosylation patterns built from methylated and acetylated sugars. GPLs have been implicated in many aspects of mycobacterial biology, thus highlighting the significance of gaining an understanding of their biosynthesis. Our bioinformatics analysis revealed that every GPL biosynthetic gene cluster known to date contains a gene (referred herein to as gplH) encoding a member of the MbtH-like protein family. Herein, we sought to conclusively establish whether gplH was required for GPL production.ResultsDeletion of gplH, a gene clustered with nonribosomal peptide synthetase-encoding genes in the GPL biosynthetic gene cluster of Mycobacterium smegmatis, produced a GPL deficient mutant. Transformation of this mutant with a plasmid expressing gplH restored GPL production. Complementation was also achieved by plasmid-based constitutive expression of mbtH, a paralog of gplH found in the biosynthetic gene cluster for production of the siderophore mycobactin of M. smegmatis. Further characterization of the gplH mutant indicated that it also displayed atypical colony morphology, lack of sliding motility, altered capacity for biofilm formation, and increased drug susceptibility.ConclusionsHerein, we provide evidence formally establishing that gplH is essential for GPL production in M. smegmatis. Inactivation of gplH also leads to a pleiotropic phenotype likely to arise from alterations in the cell envelope due to the lack of GPLs. While genes encoding MbtH-like proteins have been shown to be needed for production of siderophores and antibiotics, our study presents the first case of one such gene proven to be required for production of a cell wall component. Furthermore, our results provide the first example of a mbtH-like gene with confirmed functional role in a member of the Mycobacterium genus. Altogether, our findings demonstrate a critical role of gplH in mycobacterial biology and advance our understanding of the genetic requirements for the biosynthesis of an important group of constituents of the mycobacterial outer membrane.