Expression of the Staphylococcus aureus UDP-N-acetylmuramoyl- L-alanyl-D-glutamate:L-lysine ligase in Escherichia coli and effects on peptidoglycan biosynthesis and cell growth

Abstract

The monomer units in the Escherichia coli and Staphylococcus aureus cell wall peptidoglycans differ in the nature of the third amino acid in the L-alanyl-gamma-D-glutamyl-X-D-alanyl-D-alanine side chain, where X is meso-diaminopimelic acid or L-lysine, respectively. The murE gene from S. aureus encoding the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: L-lysine ligase was identified and cloned into plasmid vectors. Induction of its overexpression in E. coli rapidly results in abnormal morphological changes and subsequent cell lysis. A reduction of 28% in the peptidoglycan content was observed in induced cells, and analysis of the peptidoglycan composition and structure showed that ca. 50% of the meso-diaminopimelic acid residues were replaced by L-lysine. Lysine was detected in both monomer and dimer fragments, but the acceptor units from the latter contained exclusively meso-diaminopimelic acid, suggesting that no transpeptidation could occur between the epsilon-amino group of L-lysine and the alpha-carboxyl group of D-alanine. The overall cross-linking of the macromolecule was only slightly decreased. Detection and analysis of meso-diaminopimelic acid- and L-lysine-containing peptidoglycan precursors confirmed the presence of L-lysine in precursors containing amino acids added after the reaction catalyzed by the MurE ligase and provided additional information about the specificity of the enzymes involved in these latter processes.

FIG. 1

Lytic effect of the expression of the S. aureus murE gene in E. coli cells. Cells of BL21(DE3)/pLysS/pMuSA2 were grown exponentially at 37°C in 2YT-ampicillin medium. At the time indicated by the arrow (OD = 0.4), IPTG was added at a final concentration of 1 mM. Growth of cells induced (○) or not induced (●) with IPTG was monitored at 600 nm.

FIG. 2

Overproduction of S. aureus MurE enzyme in E. coli cells. Cells of BL21(DE3)/pLysS/pMuSa2 were grown and induced for 1 h with IPTG as described in the legend to Fig. 1. Cells were harvested at an OD of 0.8 and were disrupted by sonication. The protein contents from both soluble and membrane fractions obtained after high-speed centrifugation of the crude extracts were analyzed by SDS-PAGE. Molecular weight standards (in thousands) indicated on the left are as follows: phosphorylase b, 94; bovine serum albumin, 67; ovalbumin 43; carbonic anhydrase, 30; and soybean trypsin, 20. Lanes: A and B, analysis of the soluble fractions from noninduced and IPTG-induced cells, respectively; C and D, analysis of the membrane fractions from noninduced and IPTG-induced cells, respectively. The arrow points to the overproduced S. aureus MurE enzyme.

FIG. 3

Separation of muropeptides by reversed-phase HPLC. Cells of BL21(DE3)/pLysS/pMuSa2 were grown and induced with IPTG as described in the legend to Fig. 1. The peptidoglycan from noninduced cells (A) and induced cells (B) was extracted and digested with muramidases, and the resulting fragments were reduced with NaBH₄ and separated by reversed-phase HPLC on a LiChrosorb RP-18 column (4 by 250 mm). Elution was performed at 0.5 ml · min⁻¹ with 50 mM sodium phosphate buffer (pH 4.5) and a linear gradient of methanol (0 to 15% from 0 to 100 min). Eluted compounds were detected at 220 nm at a sensitivity of 0.04 absorbance unit (full scale). MA₃, monomer tri(A₂pm); MA₄, monomer tetra(A₂pm); DAA, dimer tetra(A₂pm)-tetra(A₂pm); ML₃, monomer tri(lysine); ML₄, monomer tetra(lysine); DLA, dimer tetra(lysine)-tetra(A₂pm).

FIG. 4

Structures of the main muropeptides as separated in Fig. 3, based on amino acid and hexosamine composition. Glucosamine, Ala, Glu, A₂pm, and Lys were detected after acid hydrolysis of HPLC-purified muropeptides in the following ratios (taking Glu as reference): MA3, 0.95/0.92/1/0.95/0; ML3, 1.07/1.02/1/0/1.03; MA4, 0.97/1.95/1/1.05/0; ML4, 0.97/2.1/1/0/0.97; DAA, 0.95/1.97/1/1/0; and DLA, 1.04/1.9/1/0.48/0.45 (abbreviations are as defined for Fig. 3). When the two latter muropeptides were dinitrophenylated before acid hydrolysis, half of the A₂pm from DAA and all of the lysine, but not the A₂pm, from DLA were lost.