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Journal of Bacteriology, May 2008, p. 3690-3699, Vol. 190, No. 10
0021-9193/08/$08.00+0     doi:10.1128/JB.00200-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mutations in pimE Restore Lipoarabinomannan Synthesis and Growth in a Mycobacterium smegmatis lpqW Mutant ^,

Paul K. Crellin,^1,* Svetozar Kovacevic,^1, Kirstee L. Martin,² Rajini Brammananth,¹ Yasu S. Morita,³ Helen Billman-Jacobe,⁴ Malcolm J. McConville,^2, and Ross L. Coppel^1,

Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, and Victorian Bioinformatics Consortium, Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia,¹ Bio21 Institute of Molecular Science and Biotechnology, Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia,² Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University; 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan,³ Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia⁴

Received 8 February 2008/ Accepted 5 March 2008

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.

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* Corresponding author. Mailing address: Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia. Phone: (61 3) 9905 1468. Fax: (61 3) 9905 4811. E-mail: paul.crellin{at}med.monash.edu.au

Published ahead of print on 14 March 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

P.K.C. and S.K. contributed equally to this study.

M.J.M. and R.L.C. contributed equally to this study.

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Journal of Bacteriology, May 2008, p. 3690-3699, Vol. 190, No. 10
0021-9193/08/$08.00+0     doi:10.1128/JB.00200-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.