Phosphatidylinositol mannosides (PIMs) are a major class of glycolipids in all mycobacteria. AcPIM2, a dimannosyl PIM, is both an end product and a precursor for polar PIMs, such as hexamannosyl PIM (AcPIM6) and the major cell wall lipoglycan, lipoarabinomannan (LAM). The mannosyltransferases that convert AcPIM2 to AcPIM6 or LAM are dependent on polyprenol-phosphate-mannose (PPM), but have not yet been characterized. Here, we identified a gene, termed pimE that is present in all mycobacteria, and is required for AcPIM6 biosynthesis. PimE was initially identified based on homology with eukaryotic PIG-M mannosyltransferases. PimE-deleted Mycobacterium smegmatis was defective in AcPIM6 synthesis, and accumulated the tetramannosyl PIM, AcPIM4. Loss of PimE had no affect on cell growth or viability, or the biosynthesis of other intracellular and cell wall glycans. However, changes in cell wall hydrophobicity and plasma membrane organization were detected, suggesting a role for AcPIM6 in the structural integrity of the cell wall and plasma membrane. These defects were corrected by ectopic expression of the pimE gene. Metabolic pulse-chase radiolabeling and cell-free PIM biosynthesis assays indicated that PimE catalyzes the ␣1,2-mannosyl transfer for the AcPIM5 synthesis. Mutation of an Asp residue in PimE that is conserved in and required for the activity of human PIG-M resulted in loss of PIM-biosynthetic activity, indicating that PimE is the catalytic component. Finally, PimE was localized to a distinct membrane fraction enriched in AcPIM4 -6 biosynthesis. Taken together, PimE represents the first PPM-dependent mannosyltransferase shown to be involved in PIM biosynthesis, where it mediates the fifth mannose transfer.

Five rough colony mutants of Mycobacterium smegmatis mc2155 were produced by transposon mutagenesis. The mutants were unable to synthesize glycopeptidolipids that are normally abundant in the cell wall of wild‐type M. smegmatis. The glycopeptidolipids have a lipopeptide core comprising a fatty acid amide linked to a tetrapeptide that is modified with O‐methylated rhamnose and O‐acylated 6‐deoxy talose. Compositional analysis of lipids extracted from the mutants indicated that the defect in glycopeptidolipid synthesis occurred in the assembly of the lipopeptide core. No other defects or compensatory changes in cell wall structure were detected in the mutants. All five mutants had transposon insertions in a gene encoding an enzyme belonging to the peptide synthetase family. Targeted disruption of the gene in the wild‐type strain gave a phenotype identical to that of the five transposon mutants. The M. smegmatis peptide synthetase gene is predicted to encode four modules that each contain domains for cofactor binding and for amino acid recognition and adenylation. Three modules also have amino acid racemase domains. These data suggest that the common lipopeptide core of these important cell wall glycolipids is synthesized by a peptide synthetase.

All species of Mycobacteria synthesize distinctive cell walls that are rich in phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). PIM glycolipids, having 2-4 mannose residues, can either be channeled into polar PIM species (with 6 Man residues) or hypermannosylated to form LM and LAM. In this study, we have identified a Mycobacterium smegmatis gene, termed lpqW, that is required for the conversion of PIMs to LAM and is highly conserved in all mycobacteria. A transposon mutant, Myco481, containing an insertion near the 3 end of lpqW exhibited altered colony morphology on complex agar medium. This mutant was unstable and was consistently overgrown by a second mutant, represented by Myco481.1, that had normal growth and colony characteristics. Biochemical analysis and metabolic labeling studies showed that Myco481 synthesized the complete spectrum of apolar and polar PIMs but was unable to make LAM. LAM biosynthesis was restored to near wild type levels in Myco481.1. However, this mutant was unable to synthesize the major polar PIM (AcPIM6) and accumulated a smaller intermediate, AcPIM4. Targeted disruption of the lpqW gene and complementation of the initial Myco481 mutant with the wild type gene confirmed that the phenotype of this mutant was due to loss of LpqW. These studies suggest that LpqW has a role in regulating the flux of early PIM intermediates into polar PIM or LAM biosynthesis. They also suggest that AcPIM4 is the likely branch point intermediate in polar PIM and LAM biosynthesis.Members of the genus Mycobacterium cause important diseases in humans, including tuberculosis and leprosy. Mycobacterium tuberculosis is thought to infect nearly one-third of the world population and to cause two to three million deaths each year (1). This threat to global health is growing as drug-resistant strains emerge and coinfections with human immunodeficiency virus increase the number of individuals with active tuberculosis. All species of mycobacteria synthesize a highly distinctive cell wall that contributes to the ability of pathogenic mycobacteria to survive within the endosomal network of human macrophages and to their innate resistance to many antibiotics. The mycobacterial cell wall has a multilaminate structure, comprising an asymmetric outer membrane and an inner layer of arabinogalactan polysaccharide and peptidoglycan (2, 3). The asymmetric outer membrane has an inner leaflet of tightly packed, long chain (C70 -C90) mycolic acids and an outer leaflet of free (glyco)lipids. This asymmetric outer membrane is responsible for the low permeability properties of the cell wall and also contains lipids that play key roles in the pathogenesis of these organisms.Although the (glyco)lipid composition of the mycobacterial cell wall can vary among mycobacterial species, all species synthesize an abundant class of phosphatidylinositol mannosides (PIMs) 7 and the hypermannosylated PIMs, lipomannan (LM) and lipoarabinomannan (LAM) (4, 5). The PIMs, LM and LAM, may be located in the plasm...

Glycopeptidolipids (GPLs) are major components of the cell walls of several species of mycobacteria. We have isolated a transposon mutant of Mycobacterium smegmatis that is unable to synthesize mature GPLs and that displays a rough colony morphology. The disrupted gene, mtf1, shares a high degree of homology with several S-adenosylmethionine-dependent methyltransferases. The enzyme encoded by mtf1 is required for the methylation of a single rhamnose residue that forms part of the conserved GPL core structure. This conclusion is supported by the finding that (a) the mutant synthesized only GPLs with undermethylated (either mono-or nonmethylated instead of di-or trimethylated) rhamnose residues; (b) complementation of the mutant with a wild-type copy of mtf1 restored high levels of synthesis of GPLs containing di-and trimethylated rhamnose; and (c) S-adenosylmethionine-dependent methylation of rhamnosylated GPLs could be detected in cell lysates of wild-type cells and mtf1-complemented mutant cells, but not in mutant cells lacking intact mtf1. Structural analysis of wild-type and mutant GPLs suggests that disruption of mtf1 specifically inhibits addition of O-methyl groups to the 3 (or 2)-position of the rhamnose. In the absence of 3-O-methylation, further methylation of GPL rhamnose is apparently inhibited, and overall GPL synthesis is down-regulated by 90%.Several species of mycobacteria cause important human diseases. For example, Mycobacterium tuberculosis is the causative agent of tuberculosis, the leading cause of death from a single bacterial infection, whereas species of the Mycobacterium avium complex cause intractable infections in immunocompromised individuals (1). Many features of mycobacteria, including their ability to proliferate within phagolysosomes of host macrophages and their general resistance to a wide range of antibiotics, have been attributed to the fact that all these organisms synthesize distinctive lipid-rich cell walls (1-3). In addition to forming a highly effective permeability barrier, specific components in this wall have been shown to contribute to pathogenesis and/or to mediate specific host-bacterial interactions (4). The mycobacterial cell wall is composed of a core peptidoglycan-arabinogalactan layer surrounded by an outer lipid bilayer. The inner leaflet of the lipid bilayer is composed of mycolic acids, whereas several distinct classes of glycolipids and phospholipids form the outer leaflet (2, 3). The outer layer glycolipids are thought to contribute to the distinct surface properties of the different mycobacterial species and are also important surface antigens. The predominant outer layer glycolipids in members of the M. avium complex are glycopeptidolipids (GPLs), 1 which characteristically contain a tripeptideamino alcohol core that is modified with an amide-linked fatty acid, a 6-dTal residue, and a variably O-methylated Rha residue ( Fig. 1) (5-7). GPLs having this core structure are termed non-serovar-specific GPLs (nsGPLs) and are found in most isolates of the M. avi...

Phosphatidylinositol (PI) is an abundant phospholipid in the cytoplasmic membrane of mycobacteria and the precursor for more complex glycolipids, such as the PI mannosides (PIMs) and lipoarabinomannan (LAM). To investigate whether the large steady-state pools of PI and apolar PIMs are required for mycobacterial growth, we have generated a Mycobacterium smegmatis inositol auxotroph by disruption of the ino1 gene. The ino1 mutant displayed wild-type growth rates and steady-state levels of PI, PIM, and LAM when grown in the presence of 1 mM inositol. The non-dividing ino1 mutant was highly resistant to inositol starvation, reflecting the slow turnover of inositol lipids in this stage. In contrast, dilution of growing or stationary-phase ino1 mutant in inositol-free medium resulted in the rapid depletion of PI and apolar PIMs. Whereas depletion of these lipids was not associated with loss of viability, subsequent depletion of polar PIMs coincided with loss of major cell wall components and cell viability. Metabolic labeling experiments confirmed that the large pools of PI and apolar PIMs were used to sustain polar PIM and LAM biosynthesis during inositol limitation. They also showed that under non-limiting conditions, PI is catabolized via lyso-PI. These data suggest that large pools of PI and apolar PIMs are not essential for membrane integrity but are required to sustain polar PIM biosynthesis, which is essential for mycobacterial growth.Mycobacterium tuberculosis, the causative agent of tuberculosis, infects nearly one third of the world population and causes active disease in an estimated 16 million people worldwide (1). The distinctive cell wall of M. tuberculosis and other pathogenic mycobacteria (M. leprae, M. avium-M. intracellulare complex, and M. ulcerans) confers protection against a range of microbicidal processes and many classes of antibiotics and undoubtedly contributes to the success of these organisms as pathogens. The mycobacterial cell wall contains a number of unusual features, including the presence of a highly structured peptidoglycan-arabinogalactan (AG) 1 -mycolic acid macromolecule and a diverse array of glycolipids that form an asymmetric outer bilayer with the mycolic acids (2-4). Mycobacteria and other members of the Actinomycetales also differ from other eubacteria in synthesizing phosphatidylinositol (PI) and the biosynthetically related lipoglycans, PI mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM) (5-7). Whereas there is accumulating evidence that the PIMs and LM/LAM have potent immuno-modulatory activities that may be important for the pathogenesis of M. tuberculosis (7-10), the presence of structurally related PIMs and LAMs in saprophytic mycobacterial species suggests that these lipoglycans have a more fundamental role(s) in mycobacterial physiology. This conclusion is supported by the finding that both phosphatidylinositol synthase and PimA, the first mannosyltransferase in the PIM/LM/LAM pathway (Fig. 1), are essential for growth and viability of the saprophytic...