Inactivation of Corynebacterium glutamicum NCgl0452 and the role of MgtA in the biosynthesis of a novel mannosylated glycolipid involved in lipomannan biosynthesis.
Mycobacterium tuberculosis PimB has been demonstrated to catalyze the addition of a mannose residue from GDP-mannose to a monoacylated phosphatidyl-myo-inositol mannoside (Ac(1)PIM(1)) to generate Ac(1)PIM(2). Herein, we describe the disruption of its probable orthologue Cg-pimB and the chemical analysis of glycolipids and lipoglycans isolated from wild type Corynebacterium glutamicum and the C. glutamicum::pimB mutant. Following a careful analysis, two related glycolipids, Gl-A and Gl-X, were found in the parent strain, but Gl-X was absent from the mutant. The biosynthesis of Gl-X was restored in the mutant by complementation with either Cg-pimB or Mt-pimB. Subsequent chemical analyses established Gl-X as 1,2-di-O-C(16)/C(18:1)-(alpha-d-mannopyranosyl)-(1-->4)-(alpha-d-glucopyranosyluronic acid)-(1-->3)-glycerol (ManGlcAGroAc(2)) and Gl-A as the precursor, GlcAGroAc(2). In addition, C. glutamicum::pimB was still able to produce Ac(1)PIM(2), suggesting that Cg-PimB catalyzes the synthesis of ManGlcAGroAc(2) from GlcAGroAc(2). Isolation of lipoglycans from C. glutamicum led to the identification of two related lipoglycans. The larger lipoglycan possessed a lipoarabinomannan-like structure, whereas the smaller lipoglycan was similar to lipomannan (LM). The absence of ManGlcA-GroAc(2) in C. glutamicum::pimB led to a severe reduction in LM. These results suggested that ManGlcAGroAc(2) was further extended to an LM-like molecule. Complementation of C. glutamicum::pimB with Cg-pimB and Mt-pimB led to the restoration of LM biosynthesis. As a result, Cg-PimB, which we have assigned as MgtA, is now clearly defined as a GDP-mannose-dependent alpha-mannosyltransferase from our in vitro analyses and is involved in the biosynthesis of ManGlcAGroAc(2).
Tatituri
Raju V V
RVV
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
Illarionov
Petr A
PA
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
Dover
Lynn G
LG
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
Nigou
Jerome
J
Institut de Pharmacologie et de Biologie Structurale, UMR CNRS 5089, Toulouse, France.
Gilleron
Martine
M
Institut de Pharmacologie et de Biologie Structurale, UMR CNRS 5089, Toulouse, France.
Hitchen
Paul
P
Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College, London SW7 2AZ, United Kingdom.
Krumbach
Karin
K
Institute for Biotechnology 1, Research Centre Juelich, D-52425 Juelich, Germany.
Morris
Howard R
HR
Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College, London SW7 2AZ, United Kingdom; M-SCAN Mass Spectrometry Research and Training Centre, Wokingham, Berks RG41 2TZ, United Kingdom, and the.
Spencer
Neil
N
School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
Dell
Anne
A
Institute for Biotechnology 1, Research Centre Juelich, D-52425 Juelich, Germany.
Eggeling
Lothar
L
Institute for Biotechnology 1, Research Centre Juelich, D-52425 Juelich, Germany.
Besra
Gurdyal S
GS
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. Electronic address: g.besra@bham.ac.uk.
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics; D017353:Gene Deletion; D005816:Genetic Complementation Test; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics; D010716:Phosphatidylinositols / Q000096:biosynthesis* / Q000235:genetics
D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D026901:Membrane Transport Proteins; D010716:Phosphatidylinositols; C064351:lipomannan; C034154:phosphatidylinositol mannoside; C093299:GDP-mannosyltransferase; D008364:Mannosyltransferases; C400532:PimB protein, Mycobacterium tuberculosis; D000251:Adenosine Triphosphatases; C093585:MgtA protein, bacteria