Gap, a mycobacterial specific integral membrane protein, is required for glycolipid transport to the cell surface

Abstract

SummaryThe cell envelope of mycobacteria is a complex multilaminar structure that protects the cell from stresses encountered in the environment, and plays an important role against the bactericidal activity of immune system cells. The outermost layer of the mycobacterial envelope typically contains species-specific glycolipids. Depending on the mycobacterial species, the major glycolipid localized at the surface can be either a phenolglycolipid or a peptidoglycolipid (GPL). Currently, the mechanism of how these glycolipids are addressed to the cell surface is not understood. In this study, by using a transposon library of Mycobacterium smegmatis and a simple dye assay, six genes involved in GPLs synthesis have been characterized. All of these genes are clustered in a single genomic region of approximately 60 kb. We show by biochemical analyses that two non-ribosomal peptide synthetases, a polyketide synthase, a methyltransferase and a member of the MmpL family are required for the biosynthesis of the GPLs backbone. Furthermore, we demonstrate that a small integral membrane protein of 272 amino acids named Gap ( gap : GPL addressing protein) is specifically required for the transport of the GPLs to the cell surface. This protein is predicted to contain six transmembrane segments and possesses homologues across the mycobacterial genus, thus delineating a new protein family. This Gap family represents a new paradigm for the transport of small molecules across the mycobacterial envelope, a critical determinant of mycobacterial virulence.

Direct Visualization of the Outer Membrane of Mycobacteria and Corynebacteria in Their Native State

“…M. smegmatis mc 2 155 (ATCC 700084) and M. smegmatis tmptB (49) were cultured in LB (Luria-Bertani broth; Difco, Basel, Switzerland) at 37°C with aeration. M. bovis BCG Pasteur (ATCC 35734) was cultured in 0.05% Tween 80, oleic acid-albumin-dextrose-catalase-enriched (oleic acid dextrose complex) Middlebrook 7H9 broth (Difco) at 37°C with aeration.…”

Clonal Diversification and Changes in Lipid Traits and Colony Morphology in Mycobacterium abscessus Clinical Isolates

“…We analyzed 8 genes in the GPL locus, including the genes in which spontaneous insertions/deletions (indels) were previously identified in rough M. abscessus clinical strains (mps1 and mmpL4b) (22) as well as those which when experimentally rendered nonfunctional resulted in a switch from smooth to rough in M. abscessus or M. smegmatis (gap, mps2, mbtH, mmpL4b, and mmpS4) (17,(23)(24)(25). fmt and mmpL4a were included because of their adjacency to the above genes (Fig.…”

Identification of the Polyketide Synthase Involved in the Biosynthesis of the Surface-Exposed Lipooligosaccharides in Mycobacteria

“…Accordingly, we postulated that the MmpL protein putatively encoded by MSMEG_4741 would have such a function in LOS synthesis. The last stage of the envelope biogenesis would be the addressing of the glycolipids to the outer layers, a function probably carried out by Gap (for GPLaddressing protein) in GPL synthesis (45) and possibly performed by the paralogous MSMEG_4733 (45) in the case of the LOS. It is noteworthy that paralogues of these five genes (pks, pap, fadD, mmpL, and gap) are conserved in the GPL (41), and M. avium (14) but also in the LOS biosynthetic gene cluster of M. marinum (40) and in the postulated LOS cluster of M. tuberculosis (40).…”

“…Lipids were analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) as previously described (28,45 Phenotypic tests. Sliding motility, Congo red binding, hydrophobic index, and adhesion on PVC were assessed as previously described (16,17,39).…”

“…We have previously assigned the functions of two of them, namely, MSMEG_0408 (Pks1), which is involved in GPL biosynthesis (45), and MSMEG_6392 (Pks13), which catalyzes the last condensation step of mycolic acid biosynthesis (36). MSMEG_4512 (MbtD) and MSMEG_4513 are postulated to participate in the synthesis of the mycobactin siderophore.…”