Identification of a Membrane Protein Required for Lipomannan Maturation and Lipoarabinomannan Synthesis in Corynebacterineae

Abstract

Edited by Dennis R. VoelkerMycobacterium tuberculosis and related Corynebacterineae synthesize a family of lipomannans (LM) and lipoarabinomannans (LAM) that are abundant components of the multilaminate cell wall and essential virulence factors in pathogenic species. Here we describe a new membrane protein, highly conserved in all Corynebacterineae, that is required for synthesis of fulllength LM and LAM. Deletion of the Corynebacterium glutamicum NCgl2760 gene resulted in a complete loss of mature LM/LAM and the appearance of a truncated LM (t-LM). Complementation of the mutant with the NCgl2760 gene fully restored LM/LAM synthesis. Structural studies, including monosaccharide analysis, methylation linkage analysis, and mass spectrometry of native LM species, indicated that the ⌬NCgl2760 t-LM comprised a series of short LM species (8 -27 residues long) containing an ␣1-6-linked mannose backbone with greatly reduced ␣1-2-mannose side chains and no arabinose caps. The structure of the ⌬NCgl2760 t-LM was similar to that of the t-LM produced by a C. glutamicum mutant lacking the mptA gene, encoding a membrane ␣1-6-mannosyltransferase involved in extending the ␣1-6-mannan backbone of LM intermediates. Interestingly, NCgl2760 lacks any motifs or homology to other proteins of known function. Attempts to delete the NCgl2760 orthologue in Mycobacterium smegmatis were unsuccessful, consistent with previous studies indicating that the M. tuberculosis orthologue, Rv0227c, is an essential gene. Together, these data suggest that NCgl2760/ Rv0227c plays a critical role in the elongation of the mannan backbone of mycobacterial and corynebacterial LM, further highlighting the complexity of lipoglycan pathways of Corynebacterineae.

MtrP, a putative methyltransferase in Corynebacteria, is required for optimal membrane transport of trehalose mycolates

“…A similar growth phenotype was observed following loss of tmaT (i.e ΔNCgl2759) but not other genes (i.e. ΔNCgl2760) in this locus (28,31).…”

“…We have previously identified a conserved gene locus in Mycobacteria and Corynebacteria that is required for cell wall assembly (28,29,31). Most of the genes in this locus are essential in M. tuberculosis, hampering their functional characterization in this pathogen (36,37).…”

“…We have previously identified a genetic locus in C. glutamicum that harbors a number of genes involved in regulating cell wall synthesis. This locus is conserved in all Corynebacterineae (15,25,30), and contains NCgl2759 (tmaT, Rv0228) (28), NCgl2760 (Rv0227c) (31) and NCgl2764 (Rv0224c) (Fig. 2).…”

“…Our previous studies on genes of this locus revealed that one of them, NCgl2760, was involved in lipoglycan biosynthesis (31). To determine whether loss of NCgl2764 also impacts the synthesis of other cell wall components, lipomannan (LM) and lipoarabinomannan (LAM) were extracted from delipidated cell pellets and analysed by PAGE and LC-TOF-MS.…”

Cellular and structural basis of synthesis of the unique intermediate dehydro-F420-0 in mycobacteria

“…The 1.6 kb fused insert was then liberated using Xba/SacI and subcloned into Xba/SacI-digested pMSS vector (36), a suicide plasmid for M. smegmatis that contains streptomycin selection and sucrose contra-selection markers. The resultant plasmid, pMSS:ΔMSMEG_5126, was sequenced then electroporated into electrocompetent M. smegmatis mc 2 155 cells, using an ECM 630 electroporator (BTX), selecting for streptomycin-resistant colonies (30 μg/ml), which were then screened for sensitivity to 10% (w/v) sucrose.…”