The plasma membrane of Mycobacterium sp. is the site of synthesis of several distinct classes of lipids that are either retained in the membrane or exported to the overlying cell envelope. Here, we provide evidence that enzymes involved in the biosynthesis of two major lipid classes, the phosphatidylinositol mannosides (PIMs) and aminophospholipids, are compartmentalized within the plasma membrane. Enzymes involved in the synthesis of early PIM intermediates were localized to a membrane subdomain termed PM f , that was clearly resolved from the cell wall by isopyknic density centrifugation and amplified in rapidly dividing Mycobacterium smegmatis. In contrast, the major pool of apolar PIMs and enzymes involved in polar PIM biosynthesis were localized to a denser fraction that contained both plasma membrane and cell wall markers (PM-CW). Based on the resistance of the PIMs to solvent extraction in live but not lysed cells, we propose that polar PIM biosynthesis occurs in the plasma membrane rather than the cell wall component of the PM-CW. Enzymes involved in phosphatidylethanolamine biosynthesis also displayed a highly polarized distribution between the PM f and PM-CW fractions. The PM f was greatly reduced in non-dividing cells, concomitant with a reduction in the synthesis and steady-state levels of PIMs and amino-phospholipids and the redistribution of PM f marker enzymes to non-PM-CW fractions. The formation of the PM f and recruitment of enzymes to this domain may thus play a role in regulating growth-specific changes in the biosynthesis of membrane and cell wall lipids.

Mycobacteria are acid-fast bacteria that have characteristic lipid-rich cell envelopes that afford the cells protection from desiccation, chemical disinfectants, and some antibiotics. The cell envelopes of pathogenic mycobacteria are one of the major virulence factors that assist the bacteria to live within host macrophages and withstand the bactericidal defenses of such cells. The mycobacterial cell envelope has a multilaminar structure, comprising an inner layer of peptidoglycan and arabinogalactan polysaccharides and mycolic acids and an outer layer made of species-specific glycolipids and phospholipids. Among the lipids of the outer layer are the glycopeptidolipids (GPLs) that are characteristic of some nontuberculosis mycobacteria such as Mycobacterium avium and Mycobacterium smegmatis. The core structure of most GPLs comprises a long-chain fatty acid (3-hydroxy and 3-methoxy C 26-34 ) amidated with a tripeptideamino alcohol (D-Phe-D-allo-Thr-D-Ala-L-alaninol). In most cases, the hydroxyl groups of the allo-Thr and/or alaninol are modified with 6-deoxytalose (dTal) or rhamnose (Rha), respectively. In M. smegmatis, the dTal is variably acetylated, while the Rha residue can be modified with up to three methyl groups. Heterogeneity in the methylation of both amide-linked fatty acids and the Rha residue results in the appearance of four main GPL species in M. smegmatis. GPL-1 and GPL-2 have 3-methoxy C 26-34 fatty acids but differ from each other in the degree of methylation of the terminal Rha, in that GPL-1 has 2,3,4-tri-O-Me-Rha and GPL-2 has 3,4-di-O-Me-Rha. GPL-1a and GPL-2a have tri-and di-Me-Rha as above; however, these GPLs have 3-hydroxy C 26-34 fatty acids. We have simplified our nomenclature of the GPLs from that used in Patterson et al. (9). M. avium GPLs have the same lipopeptide core as M. smegmatisGPLs, but the alaninol is glycosylated with 3-O-Me-Rha or 3,4-di-O-Me-Rha, and the dTal can be extended with additional sugars. A locus designated ser2 contains some of the genes encoding enzymes required for synthesis of the haptenic disaccharide of GPLs of serotype 2 M. avium. Eckstein et al. showed that the simpler GPLs naturally found in M. smegmatis could serve as intermediates in the biosynthesis of a ser2-like locus encoding GPL in recombinant M. smegmatis (3). We have identified an equivalent region in M. smegmatis, shown that the mps gene encodes a peptide synthetase that makes the GPL peptide, and assigned the roles to two of the four potential methyltransferases encoded in the GPL biosynthetic locus (1, 7, 9). Mtf1 is a rhamnosyl 3-O-methyltransferase, and mtf2 encodes a fatty acid O-methyltransferase that modifies the hydroxyl at C 3 of the GPL fatty acid (7,9). For the purposes of clarity, we propose that the M. smegmatis genes formerly designated mtf1, mtf2, mtf3, and mtf4 (GenBank accession no. AY138899) be renamed rmt3 for rhamnosyl 3-O-methyltransferase, fmt for fatty acid O-methyltransferase, rmt4 for rhamnosyl 4-O-methyltransferase, and rmt2 for rhamnosyl 2-Omethyltransferase, respect...