Membrane interaction of the glycosyltransferase MurG: a special role for cardiolipin

Abstract

MurG is a peripheral membrane protein that is one of the key enzymes in peptidoglycan biosynthesis. The crystal structure of Escherichia coli MurG (S. Ha, D. Walker, Y. Shi, and S. Walker, Protein Sci. 9:1045-1052, 2000) contains a hydrophobic patch surrounded by basic residues that may represent a membrane association site. To allow investigation of the membrane interaction of MurG on a molecular level, we expressed and purified MurG from E. coli in the absence of detergent. Surprisingly, we found that lipid vesicles copurify with MurG. Freeze fracture electron microscopy of whole cells and lysates suggested that these vesicles are derived from vesicular intracellular membranes that are formed during overexpression. This is the first study which shows that overexpression of a peripheral membrane protein results in formation of additional membranes within the cell. The cardiolipin content of cells overexpressing MurG was increased from 1 +/- 1 to 7 +/- 1 mol% compared to nonoverexpressing cells. The lipids that copurify with MurG were even further enriched in cardiolipin (13 +/- 4 mol%). MurG activity measurements of lipid I, its natural substrate, incorporated in pure lipid vesicles showed that the MurG activity is higher for vesicles containing cardiolipin than for vesicles with phosphatidylglycerol. These findings support the suggestion that MurG interacts with phospholipids of the bacterial membrane. In addition, the results show a special role for cardiolipin in the MurG-membrane interaction.

FIG. 1.

Schematic representation of the role of MurG within the peptidoglycan biosynthesis pathway. The substrate for MurG is lipid I, which consists of undecaprenylpyrophosphate and the sugar N-acetylmuramic acid (MurNAc) pentapeptide (l-Ala-γd-Glu-l-Lys-d-Ala-d-Ala). MurG adds the sugar GlcNAc to lipid I to form lipid II. Subsequently, lipid II is translocated across the cell membrane, after which the disaccharide-pentapeptide is transferred to the peptidoglycan network.

FIG. 2.

Expression and purification of His-tagged MurG. Lane 1, E. coli BL21(DE3)/plysS cells containing the pet21b+MurG vector before IPTG induction; lane 2, E. coli BL21(DE3)/plysS cells containing the pet21b+MurG vector 3 h after IPTG induction, just before harvesting; lane 3, purified MurG. A protein size marker is shown on the left.

FIG. 3.

Freeze fracture electron micrograph of purified MurG with copurified lipids.

FIG. 4.

Freeze fracture electron micrographs of cells. (A) E. coli BL21(DE3)/plysS cells carrying the pet21b+MurGhistag plasmid with IPTG induction. (B) E. coli BL21(DE3)/plysS cells carrying the pet21b+MurGhistag plasmid without IPTG induction. (C) E. coli BL21(DE3)/plysS cells carrying the empty pet21b plasmid with IPTG induction. (D) E. coli BL21(DE3)/plysS cells carrying the pet21b+MurG (without His tag) plasmid with IPTG induction.

FIG. 5.

Freeze fracture electron micrograph of lysate of E. coli BL21(DE3)/plysS cells carrying the pet21b+MurGhistag plasmid with IPTG induction.

FIG. 6.

MurG activity on lipid I incorporated in pure lipid vesicles. His-tagged MurG (100 ng) was incubated with 0.3 μmol of lipid vesicles containing 2 P_i% lipid I and a certain percentage of TOCL or DOPG normalized for the amount of phosphate. This was done for 0.5 to 16 min at room temperature in the presence of 0.33 mM UDP-GlcNAc (of which 1% was ¹⁴C labeled), 6.7 mM MgCl₂, 20 mM Tris-HCl, pH 8, and 50 mM NaCl. All measurements were performed in duplicate. The MurG activity was calculated as the amount of lipid II formed per min per mg of protein, based on the trend line through the data points. The error bars indicate standard deviations based on two or three measurements.