Inactivation of Corynebacterium glutamicum NCgl0452 and the Role of MgtA in the Biosynthesis of a Novel Mannosylated Glycolipid Involved in Lipomannan Biosynthesis^*

Abstract

Mycobacterium tuberculosis PimB has been demonstrated to catalyze the addition of a mannose residue from GDP-mannose to a monoacylated phosphatidyl-myo-inositol mannoside (Ac₁PIM₁) to generate Ac₁PIM₂. 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-C₁₆/C_18:1-(α-d-mannopyranosyl)-(1→4)-(α-d-glucopyranosyluronic acid)-(1→3)-glycerol (ManGlcAGroAc₂) and Gl-A as the precursor, GlcAGroAc₂. In addition, C. glutamicum::pimB was still able to produce Ac₁PIM₂, suggesting that Cg-PimB catalyzes the synthesis of ManGlcAGroAc₂ from GlcAGroAc₂. 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-GroAc₂ in C. glutamicum::pimB led to a severe reduction in LM. These results suggested that ManGlcAGroAc₂ 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 α-mannosyltransferase from our in vitro analyses and is involved in the biosynthesis of ManGlcAGroAc₂.