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

Abstract

Cell walls of bacteria of the genera Mycobacterium and Corynebacterium contain high levels of (coryno)mycolic acids. These very long chain fatty acids are synthesized on the cytoplasmic leaflet of the inner membrane (IM) prior to conjugation to the disaccharide, trehalose, and transport to the periplasm. Recent studies on Corynebacterium glutamicum have shown that acetylation of trehalose monohydroxycorynomycolate (hTMCM) promotes its transport across the inner membrane. Acetylation is mediated by the membrane acetyltransferase, TmaT, and is dependent on the presence of a putative methyltransferase, MtrP. Here, we identify a third protein that is required for the acetylation and membrane transport of hTMCM. Deletion of the C. glutamicum gene NCgl2761 (Rv0226c in Mycobacterium tuberculosis) abolished synthesis of acetylated hTMCM (AcTMCM), resulting in an accumulation of hTMCM in the inner membrane and reduced synthesis of trehalose dihydroxycorynomycolate (h2TDCM), a major outer membrane glycolipid. Complementation with the NCgl2761 gene, designated here as mmpA, restored the hTMCM:h2TDCM ratio. Comprehensive lipidomic analysis of the ΔtmaT, ΔmtrP and ΔmmpA mutants revealed strikingly similar global changes in overall membrane lipid composition. Our findings suggest that the acetylation and membrane transport of hTMCM is regulated by multiple proteins: MmpA, MtrP and TmaT, and that defects in this process lead to global, potentially compensatory changes in the composition of inner and outer membranes.

Keywords: Corynebacterium glutamicum; Mycobacterium tuberculosis; cell wall; glycolipid; lipid bilayer; trehalose monoacetylcorynomycolate (AcTMCM); trehalose monohydroxycorynomycolate (hTMCM).

Figure 1

NCgl2761 is a predicted integral membrane protein encoded within a conserved genetic locus highly conserved in Corynebacterineae. (A) The tmaT locus of Corynebacterineae. Likely orthologous genes in the four species are shown using the same colour. The focus of the current study NCgl2761 and its mycobacterial orthologs are in bold. Previously studied genes are NCgl2759 (tmaT; [17]), NCgl2764 (mtrP; [18]) and NCgl2760 [26] while the remaining genes are uncharacterized. (B) Protter prediction [20] of the membrane topology of NCgl2761. Numbers indicate predicted transmembrane helices while the putative N-terminal signal sequence is red. (C) Structural model of NCgl2761 generated using the Alphafold2 AI system [21] reveals a multi-pass membrane protein with a single non-membrane domain near the C-terminus. Colors represent predicted IDDT confidence values. The N-terminus (-N), C-terminus (-C) and putative transmembrane helices are labelled.

Figure 2

RT-PCR analysis reveals co-transcription of NCgl2760 and NCgl2761. (A) The genetic locus with binding sites and orientations of the two RT-PCR primers within the two genes indicated by thin horizontal arrows. (B) Agarose gel electrophoresis of RT-PCR reaction products. RNA was prepared from WT C. glutamicum and incubated with reverse transcriptase (RT) in the presence of random hexamers or in their absence (negative control). The samples were then subjected to a PCR using primers RT1 and RT2 to detect complementary DNA (cDNA) that spans both genes. Purified C. glutamicum genomic DNA (gDNA), PCR amplified using the same primer pair, was used as a positive control. M, λHindIII/EcoRI DNA size markers (indicated in kb).

Figure 3

Disruption strategy and growth assessment of C. glutamicum ΔNCgl2761. (A) Diagram showing the arrangement of genes in the NCgl2761 region of WT C. glutamicum (above) and ΔNCgl2761 mutant (below). XhoI restriction sites, sizes of expected bands on Southern blot and position of the probe used are shown. Small horizontal arrows indicate the binding sites for the four primers used to construct the ΔNCgl2761 mutant (a, NCgl2761-left_F; b, NCgl2761-left_R; c, NCgl2761-right_F; d, NCgl2761-right_R). (B) Southern blot analysis of XhoI-digested DNA of C. glutamicum WT (lane 1), a single cross-over strain (lane 2) and the ΔNCgl2761 mutant (lane 3). Positions of DIG-labelled λDNA standards digested with HindIII are indicated in kilobase pairs (kb). (C) Growth curves of WT C. glutamicum, the ΔNCgl2761 mutant and complementation strains in liquid BHI medium. Each strain was grown to saturation, then diluted 1:100 in BHI medium. Triplicate cultures were sampled at the times indicated to determine the optical density (OD) at a wavelength of 600 nm. Growth curves were plotted as mean ± SD.

Figure 4

Cell wall free glycolipid analysis of a C. glutamicum ΔNCgl2761 mutant. Lipids were extracted from WT C. glutamicum, ΔNCgl2761, ΔNCgl2761 containing empty pSM22 and ΔNCgl2761 containing pSM22:NCgl2761, then analysed by HPTLC. Glycolipids were visualized by orcinol-sulfuric acid staining. The identities of glycolipids indicated were based on previously published reports. CL, cardiolipin; PG, phosphatidylglycerol; Gl-X, Man.GlcA-DAG; AcPIM2, Man₂-acyl-PI.

Figure 5

A C. glutamicum ΔNCgl2761 mutant has a defect in TMCM surface transport. Wild-type and the ΔNCgl2761 mutant were metabolically labelled with [¹⁴C]-acetate and sequentially extracted in 1-butanol (BuOH) and chloroform/methanol (C/M; 2:1 v/v). Parallel cultures were directly extracted in chloroform/methanol/water (Total lipid; 1:3:1 v/v). The three fractions from each bacterial line were analysed by HPTLC (A) and labelled species detected by fluorography and quantitated (B).

Figure 6

Comparison of the relative abundances of IM and OM lipids in C. glutamicum WT and a ΔNCgl2761 mutant. The relative abundance of 144 species, identified by LC-MS/MS profiling, in the IM and OM fractions. Lipid abundances (based on ion intensities) represent the mean value of four replicates. The relative abundance of DAG and TAG based lipid classes (A), PG and CL-based lipid classes (B), PI/PIM and Gl glycolipid classes (C), hTMCM species (D), other trehalose and glucose corynomycolates (E) and h2TDCM species (F) are shown.

Figure 7

Proposed role of C. glutamicum MmpA in trehalose corynomycolate synthesis and transport. Corynomycolic acids, synthesized by the FAS-I and FAS-II pathways and Pks13, are linked to trehalose to form keto-TMCM and the keto group is subsequently reduced by CmrA reductase to form hTMCM. The hydroxylated corynomycolic acid in hTMCM is acetylated by the membrane acetyltransferase, TmaT, and AcTMCM transported across the IM by CmpL1 or CmpL4. AcTMCM is deacetylated by an unknown enzyme in the periplasmic space (dashed arrows) and hTMCM used as a corynomycolic acid donor by corynomycolyltransferases (CMTs) to synthesize h2TDCM or mycolated AG. Released trehalose is recycled by the LpqY-SucABC importer complex [32]. Additional acylation variants exist of hTMCM, AcTMCM and hTDCM [27]. Results of the current study place NCgl2761 (MmpA, orange) at the same step of the pathway as NCgl2759 (TmaT) [17] and NCgl2764 (MtrP) [18].