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

Abstract

Pathogenic bacteria of the genera Mycobacterium and Corynebacterium cause severe human diseases such as tuberculosis (Mycobacterium tuberculosis) and diphtheria (Corynebacterium diphtheriae). The cells of these species are surrounded by protective cell walls rich in long-chain mycolic acids. These fatty acids are conjugated to the disaccharide trehalose on the cytoplasmic side of the bacterial cell membrane. They are then transported across the membrane to the periplasm where they act as donors for other reactions. We have previously shown that transient acetylation of the glycolipid trehalose monohydroxycorynomycolate (hTMCM) enables its efficient transport to the periplasm in Corynebacterium glutamicum and that acetylation is mediated by the membrane protein TmaT. Here, we show that a putative methyltransferase, encoded at the same genetic locus as TmaT, is also required for optimal hTMCM transport. Deletion of the C. glutamicum gene NCgl2764 (Rv0224c in M. tuberculosis) abolished acetyltrehalose monocorynomycolate (AcTMCM) synthesis, leading to accumulation of hTMCM in the inner membrane and delaying its conversion to trehalose dihydroxycorynomycolate (h2TDCM). Complementation with NCgl2764 normalized turnover of hTMCM to h2TDCM. In contrast, complementation with NCgl2764 derivatives mutated at residues essential for methyltransferase activity failed to rectify the defect, suggesting that NCgl2764/Rv0224c encodes a methyltransferase, designated here as MtrP. Comprehensive analyses of the individual mtrP and tmaT mutants and of a double mutant revealed strikingly similar changes across several lipid classes compared with WT bacteria. These findings indicate that both MtrP and TmaT have nonredundant roles in regulating AcTMCM synthesis, revealing additional complexity in the regulation of trehalose mycolate transport in the Corynebacterineae.

MmpA, a Conserved Membrane Protein Required for Efficient Surface Transport of Trehalose Lipids in Corynebacterineae

“…This hTMCM/h2TDCM phenotype is similar to that originally described for ∆tmaT (NCgl2759) [17,27] and ∆mtrP (NCgl2764) [18] mutants. We previously showed that loss of TmaT and MtrP led to global changes in the lipidomes of mutant lines [18,27].…”

“…This hTMCM/h2TDCM phenotype is similar to that originally described for ∆tmaT (NCgl2759) [17,27] and ∆mtrP (NCgl2764) [18] mutants. We previously showed that loss of TmaT and MtrP led to global changes in the lipidomes of mutant lines [18,27]. To investigate whether similar changes also occurred in the ∆NCgl2761 mutant line, we undertook a detailed analysis of the IM and OM lipid extracts of the WT and mutant strains by LC-ESI-QTOF-MS in positive ionization mode (Figure 6).…”

“…NCgl2759 encodes the acetyltransferase, TmaT, that acetylates the hydroxyl group on the corynomycolic acid chain of hTMCM facilitating the transport of this glycolipid by the membrane transporters CmpL1 and CmpL4 [ 17 ]. NCgl2764 has sequence homology to methyltransferases and is also required for AcTMCM synthesis and corynomycolate transport, although its precise function is unknown [ 18 ]. In contrast, loss of NCgl2760 ( Rv0227c in M. tuberculosis ) had no effect on TMCM/TDCM biosynthesis, but led to a defect in the synthesis of major cell wall components lipomannan (LM) and lipoarabinomannan (LAM) [ 26 ].…”

“…Genetic deletion of tmaT significantly reduced the rate of corynomycolate transport across the IM and synthesis to trehalose dihydroxycorynomycolate (h2TDCM) [ 17 ], indicating that AcTMCM is the major substrate transported by C. glutamicum transporters. A second protein, annotated as a putative methyltransferase, MtrP (NCgl2764), was subsequently shown to be required for AcTMCM synthesis and membrane transport in addition to TmaT [ 18 ]. Here, we provide evidence that a third protein, encoded by the same gene locus, is also required for this process.…”

O-Methylation of the Glycopeptidolipid Acyl Chain Defines Surface Hydrophobicity of Mycobacterium abscessus and Macrophage Invasion

“…To investigate whether fmt encodes a methyltransferase, the amino acid sequence of Fmt was aligned with other validated methyltransferases using CLUSTALW (https://www.genome.jp/tools-bin/clustalw). The aligned sequences include MT0146/CbiT from Methanobacterium thermoautotrophicum 42 , the corynebacterial MtrP protein (NCg12764) assisting the transport of trehalose mycolates 43 , the methyltransferase catalyzing the transfer of a methyl group onto the lipid moiety of phthiotriol and glycosylated phenolphthiotriol dimycocerosates in M. tuberculosis (Rv2952) 44 and the Fmt orthologue in M. smegmatis Mtf2 45 , for which active site residues have been identified. In particular, two glycine residues are conserved in S-adenosyl methionine (SAM)dependent methyltransferase and known to be involved in binding to the methyl donor.…”