@article {689,
	title = {Controlled expression of branch-forming mannosyltransferase is critical for mycobacterial lipoarabinomannan biosynthesis.},
	journal = {J Biol Chem},
	volume = {285},
	year = {2010},
	month = {2010 Apr 30},
	pages = {13326-36},
	abstract = {Lipomannan (LM) and lipoarabinomannan (LAM) are phosphatidylinositol-anchored glycans present in the mycobacterial cell wall. In Mycobacterium smegmatis, the mannan core of LM/LAM constitutes a linear chain of 20-25 alpha1,6-mannoses elaborated by 8-9 alpha1,2-monomannose side branches. At least two alpha1,6-mannosyltransferases mediate the linear mannose chain elongation, and one branching alpha1,2-mannosyltransferase (encoded by MSMEG_4247) transfers monomannose branches. An MSMEG_4247 deletion mutant accumulates branchless LAM and interestingly fails to accumulate LM, suggesting an unexpected role of mannose branching for LM synthesis or maintenance. To understand the roles of MSMEG_4247-mediated branching more clearly, we analyzed the MSMEG_4247 deletion mutant in detail. Our study showed that the deletion mutant restored the synthesis of wild-type LM and LAM upon the expression of MSMEG_4247 at wild-type levels. In striking contrast, overexpression of MSMEG_4247 resulted in the accumulation of dwarfed LM/LAM, although monomannose branching was restored. The dwarfed LAM carried a mannan chain less than half the length of wild-type LAM and was elaborated by an arabinan that was about 4 times smaller. Induced overexpression of an elongating alpha1,6-mannosyltransferase competed with the overexpressed branching enzyme, alleviating the dwarfing effect of the branching enzyme. In wild-type cells, LM and LAM decreased in quantity in the stationary phase, and the expression levels of branching and elongating mannosyltransferases were reduced in concert, presumably to avoid producing abnormal LM/LAM. These data suggest that the coordinated expressions of branching and elongating mannosyltransferases are critical for mannan backbone elongation.},
	keywords = {Bacterial Proteins, Cell Wall, Gene Deletion, Lipopolysaccharides, Mannose, Mannosyltransferases, Mycobacterium smegmatis},
	issn = {1083-351X},
	doi = {10.1074/jbc.M109.077297},
	author = {Sena, Chubert B C and Fukuda, Takeshi and Miyanagi, Kana and Matsumoto, Sohkichi and Kobayashi, Kazuo and Murakami, Yoshiko and Maeda, Yusuke and Kinoshita, Taroh and Morita, Yasu S}
}
@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 {694,
	title = {Identification of a novel protein with a role in lipoarabinomannan biosynthesis in mycobacteria.},
	journal = {J Biol Chem},
	volume = {281},
	year = {2006},
	month = {2006 Apr 7},
	pages = {9011-7},
	abstract = {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{\textquoteright} 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.},
	keywords = {Cell Membrane, Gene Expression Regulation, Bacterial, Genes, Bacterial, Lipopolysaccharides, Mutation, Mycobacterium smegmatis, Phenotype, Phosphatidylinositols, Virulence},
	issn = {0021-9258},
	doi = {10.1074/jbc.M511709200},
	author = {Kovacevic, Svetozar and Anderson, Dianne and Morita, Yasu S and Patterson, John and Haites, Ruth and McMillan, Benjamin N I and Coppel, Ross and McConville, Malcolm J and Billman-Jacobe, Helen}
}
@article {697,
	title = {Function of phosphatidylinositol in mycobacteria.},
	journal = {J Biol Chem},
	volume = {280},
	year = {2005},
	month = {2005 Mar 25},
	pages = {10981-7},
	abstract = {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.},
	keywords = {Inositol, Lipopolysaccharides, Mannose, Mycobacterium smegmatis, Phosphatidylinositols},
	issn = {0021-9258},
	doi = {10.1074/jbc.M413443200},
	author = {Haites, Ruth E and Morita, Yasu S and McConville, Malcolm J and Billman-Jacobe, Helen}
}
