@article {691,
	title = {Mutations in pimE restore lipoarabinomannan synthesis and growth in a Mycobacterium smegmatis lpqW mutant.},
	journal = {J Bacteriol},
	volume = {190},
	year = {2008},
	month = {2008 May},
	pages = {3690-9},
	abstract = {Lipoarabinomannans (LAMs) and phosphatidylinositol mannosides (PIMs) are abundant glycolipids in the cell walls of all corynebacteria and mycobacteria, including the devastating human pathogen Mycobacterium tuberculosis. We have recently shown that M. smegmatis mutants of the lipoprotein-encoding lpqW gene have a profound defect in LAM biosynthesis. When these mutants are cultured in complex medium, spontaneous bypass mutants consistently evolve in which LAM biosynthesis is restored at the expense of polar PIM synthesis. Here we show that restoration of LAM biosynthesis in the lpqW mutant results from secondary mutations in the pimE gene. PimE is a mannosyltransferase involved in converting AcPIM4, a proposed branch point intermediate in the PIM and LAM biosynthetic pathways, to more polar PIMs. Mutations in pimE arose due to insertion of the mobile genetic element ISMsm1 and independent point mutations that were clustered in predicted extracytoplasmic loops of this polytopic membrane protein. Our findings provide the first strong evidence that LpqW is required to channel intermediates such as AcPIM4 into LAM synthesis and that loss of PimE function results in the accumulation of AcPIM4, bypassing the need for LpqW. These data highlight new mechanisms regulating the biosynthetic pathways of these essential cell wall components.},
	keywords = {Cell Wall, Lipopolysaccharides, Lipoproteins, Mannosyltransferases, Mutation, Mycobacterium smegmatis, Phosphatidylinositols},
	issn = {1098-5530},
	doi = {10.1128/JB.00200-08},
	author = {Crellin, Paul K and Kovacevic, Svetozar and Martin, Kirstee L and Brammananth, Rajini and Morita, Yasu S and Billman-Jacobe, Helen and McConville, Malcolm J and Coppel, Ross L}
}
@article {692,
	title = {PimE is a polyprenol-phosphate-mannose-dependent mannosyltransferase that transfers the fifth mannose of phosphatidylinositol mannoside in mycobacteria.},
	journal = {J Biol Chem},
	volume = {281},
	year = {2006},
	month = {2006 Sep 1},
	pages = {25143-55},
	abstract = {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 alpha1,2-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 mannosyl-transferase shown to be involved in PIM biosynthesis, where it mediates the fifth mannose transfer.},
	keywords = {Amino Acid Sequence, Cell Proliferation, Cell Wall, Cell-Free System, Genome, Bacterial, Humans, Mannose, Mannosides, Mannosyltransferases, Molecular Sequence Data, Mycobacterium, Mycobacterium smegmatis, Phosphates, Phosphatidylinositols, Sequence Homology, Amino Acid},
	issn = {0021-9258},
	doi = {10.1074/jbc.M604214200},
	author = {Morita, Yasu S and Sena, Chubert B C and Waller, Ross F and Kurokawa, Ken and Sernee, M Fleur and Nakatani, Fumiki and Haites, Ruth E and Billman-Jacobe, Helen and McConville, Malcolm J and Maeda, Yusuke and Kinoshita, Taroh}
}
@article {696,
	title = {Compartmentalization of lipid biosynthesis in mycobacteria.},
	journal = {J Biol Chem},
	volume = {280},
	year = {2005},
	month = {2005 Jun 3},
	pages = {21645-52},
	abstract = {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 PMf, 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 PMf and PM-CW fractions. The PMf 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 PMf marker enzymes to non-PM-CW fractions. The formation of the PMf 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.},
	keywords = {Bacterial Proteins, Biochemistry, Cell Membrane, Cell Wall, Hemagglutinins, Lipid Metabolism, Lipids, Mannosides, Mannosyltransferases, Microscopy, Electron, Models, Biological, Mycobacterium smegmatis, Phosphatidylethanolamines, Phosphatidylinositols, Phospholipids, Protein Structure, Tertiary, Subcellular Fractions},
	issn = {0021-9258},
	doi = {10.1074/jbc.M414181200},
	author = {Morita, Yasu S and Velasquez, Ren{\'e} and Taig, Ellen and Waller, Ross F and Patterson, John H and Tull, Dedreia and Williams, Spencer J and Billman-Jacobe, Helen and McConville, Malcolm J}
}
