Quantitative lipid composition of cell envelopes of Corynebacterium glutamicum elucidated through reverse micelle extraction

Abstract

Cells of the Corynebacterium-Nocardia-Mycobacterium group of bacteria are surrounded by an outer membrane (OM) containing mycolic acids that are covalently linked to the underlying arabinogalactan-peptidoglycan complex. This OM presumably acts as a permeability barrier that imparts high levels of intrinsic drug resistance to some members of this group, such as Mycobacterium tuberculosis, and its component lipids have been studied intensively in a qualitative manner over the years. However, the quantitative lipid composition of this membrane has remained obscure, mainly because of difficulties in isolating it without contamination from the inner cytoplasmic membrane. Here we use the extraction, with reverse surfactant micelles, of intact cells of Corynebacterium glutamicum and show that this method extracts the free OM lipids quantitatively with no contamination from lipids of the cytoplasmic membrane, such as phosphatidylglycerol. Although only small amounts of corynomycolate were esterified to arabinogalactan, a large amount of cardiolipin was present in a nonextractable form, tightly associated, possibly covalently, with the peptidoglycan-arabinogalactan complex. Furthermore, we show that the OM contains just enough lipid hydrocarbons to produce a bilayer covering the cell surface, with its inner leaflet composed mainly of the aforementioned nonextractable cardiolipin and its outer leaflet composed of trehalose dimycolates, phosphatidylinositol mannosides, and highly apolar lipids, similar to the Minnikin model of 1982. The reverse micelle extraction method is also useful for extracting proteins associated with the OM, such as porins.

Fig. 1.

Dioctylsulfosuccinate sodium (AOT).

Fig. 2.

TLC profiles of lipids recovered by CMW extraction of whole cells (A), RMS extraction of whole cells (B), CMW extraction of residues after RMS extraction (C), and reextraction with CMW after lysozyme treatment of CMW-extracted cells (D). Thin-layer plates were developed with CMW 30:8:1, followed by visualization of different classes of lipids. Thus, phospholipids (PLs) were detected by spraying with phosphospray and amino-group containing lipids with ninhydrin reagent. Anthrone spray, which produces a characteristic blue color, was used to detect glycolipids (GLs; brown-colored spots are not GLs). Lipids were quantified by radioactivity through phosphorimaging. PIMs, phosphatidylinositol mannosides, PG, phosphatidylglycerol, CL, cardiolipin.

Fig. 3.

Apolar and polar lipids resolved by TLC using different solvent systems. (A) Apolar lipids resolved using hexane-diethyl ether-acetic acid (70:30:1). (B) Phospholipids resolved using CMW (65:25:1). (C) PI and PIMs resolved using chloroform–methanol–13 M ammonia–1 M ammonium acetate–water (180:140:9:9:23, vol/vol) (16). Lipids in the extracts were detected with a phosphorimager. Lane 1, CMW extract of whole cells; lane 2, RMS extract of whole cells; lane 3, CMW extract of RMS-treated cells (IM); lane 4, CMW reextract of lysozyme-treated, CMW-extracted cells (post-lyso CMW). Standards: MAG, monoacylglycerol (1-olein); DAG, diacylglycerol; TAG, triacylglycerol (triolein); FFA, oleic acid; PG, phosphatidylglycerol, CL, cardiolipin. UI lipid, unidentified lipid.

Fig. 4.

Cardiolipin becomes extractable in the isolated cell envelope or after lysozyme treatment of cells. (A) Lipid composition of the CMW extract of intact cells (lane 1) and the CMW extract of the isolated cell envelope (lane 2) as detected by TLC with CMW (65:25:1 vol/vol). Lanes 3–5 contain phospholipid standards. PG, phosphatidylglycerol; CL, cardiolipin. Phosphomolybdotungstate was used for detection. (B) ESI-MS profile of the major lipid band, migrating like CL, isolated from the TLC plate of CMW reextract of lysozyme-treated delipidated cells.

Fig. 5.

Proteins extracted with RMS analyzed by 12% SDS/PAGE. Lane 1, aqueous wash after RMS treatment of C. glutamicum. Lane 2, aqueous wash after RMS treatment of Mycobacterim smegmatis mc²-155. The leftmost lane shows molecular weight standards (in kDa).