Biochemical disclosure of the mycolate outer membrane of Corynebacterium glutamicum

Abstract

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The cell wall of these bacteria is composed of a heteropolymer of peptidoglycan (PG) linked to arabinogalactan (AG), which in turn is covalently associated with an atypical outer membrane, here called mycomembrane (M). The latter structure has been visualized by cryo-electron microscopy of vitreous sections, but its biochemical composition is still poorly defined, thereby hampering the elucidation of its physiological function. In this report, we show for the first time that the mycomembrane-linked heteropolymer of PG and AG (M-AG-PG) of C. glutamicum can be physically separated from the inner membrane on a flotation density gradient. Analysis of purified M-AG-PG showed that the lipids that composed the mycomembrane consisted almost exclusively of mycolic acid derivatives, with only a tiny amount, if any, of phospholipids and lipomannans, which were found with the characteristic lipoarabinomannans in the plasma membrane. Proteins associated with or inserted in the mycomembrane were extracted from M-AG-PG with lauryl-dimethylamine-oxide (LDAO), loaded on an SDS-PAGE gel, and analyzed by tandem mass spectrometry or by Western blotting. Sixty-eight different proteins were identified, 19 of which were also found in mycomembrane fragments released by the terminal-arabinosyl-transferase-defective ΔAftB strain. Almost all of them are predicted to contain a signal sequence and to adopt the characteristic β-barrel structure of Gram-negative outer membrane proteins. These presumed mycomembrane proteins include the already-known pore-forming proteins (PorA and PorB), 5 mycoloyltransferases (cMytA, cMytB, cMytC, cMytD, and cMytF), several lipoproteins, and unknown proteins typified by a putative C-terminal hydrophobic anchor.

Fig 1

Fractionation of C. glutamicum cell envelope by isopycnic sucrose gradient centrifugation. After cell lysis, membrane-containing fractions were separated according to density on a sucrose step gradient. (A) Centrifugation tubes for the wild-type 13032 strain and for its isogenic Δpks13 mutant. Buoyant densities (d) are indicated in g cm⁻³. (B) Fractions that correspond to F1 and F2, isolated from the 13032 strain, were pooled, treated with 4% SDS, and heated at 100°C for 30 min or left untreated. Insoluble material was collected by ultracentrifugation, and the corresponding pellets are shown. (C) SDS-PAGE protein pattern of F1 and F2 isolated from strain 13032 after Coomassie blue staining. The same amounts of F1 and F2, corresponding to approximately 5 × 10⁸ cells, were loaded on the gel. MW, molecular mass markers. The molecular masses of the bands with the highest intensities are 66 and 27 kDa, respectively.

Fig 2

Visualization of F1 and F2 fractions by freeze fracture electron microscopy. (A) Micrograph of C. glutamicum 13032 cells grown in BHI medium. (B and C) Micrograph of F1 (B) or F2 (C). All images are at the same magnification. Scale bar = 200 nm.

Fig 3

Biochemical analysis of F1 and F2 fractions. (A) Polar lipids were isolated from F1 and F2 and separated on TLC plates. Phospholipids (phosphatidylglycerol [PG] and phosphatidylinositol [PI]) were visualized with the Dittmer reagent. PI derivatives, trehalose monocorynomyclate (TMCM) and trehalose dicorynomyclate (TMDM), were revealed by spraying anthrone. The T lane represents polar lipids extracted from whole cells. (B) NADH oxidase activity was monitored in all fractions collected from the gradient, and the initial velocity was plotted as a percentage of the highest activity (13.2 μmol · min⁻¹). (C) Amino acids, sugars, and peptidoglycan (PG) markers were quantified from purified F1 and F2 fractions as described in Materials and Methods and expressed as nanomoles per milligram of dry material. Bars represent the standard errors of the means for 3 independent experiments. (D) Proteinase K-treated F1 and F2 fractions were loaded on a 15% SDS-polyacrylamide gel, and lipoglycans were revealed as described in Materials and Methods. According to dry weight, equal amounts of material were loaded for F1 and F2. The amount of material loaded in lane “F2 × 10” is 10 times higher than that in the two other lanes.

Fig 4

Effect of PorH-PorA deletion on C. glutamicum cell wall fractionation. Sucrose gradient centrifugation of the C. glutamicum strains. The upper panel shows ultracentrifugation tubes for wild-type ATCC 13032, its derivative PorH-PorA-deficient mutant (ΔporHA), and a complemented strain with pCGL482-porHA. The lower panel shows Western blot analysis of F2 fractions from strain ATCC 13032, the ΔporHA mutant, and the ΔporHA mutant containing pCGL482-PorHA incubated with anti-PorA antibodies. Proteins were visualized by Coomassie blue staining.