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

The monomer units in the Escherichia coli and Staphylococcus aureus cell wa ll peptidoglycans differ in the nature of the third amino acid in the L-ala nyl-gamma-D-glutamyl-X-D-alanyl-D-alanine side chain, where X is meso-diami nopimelic acid or L-lysine, respectively. The murE gene from S. aureus enco ding the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: L-lysine ligase was ide ntified and cloned into plasmid vectors. Induction of its overexpression in E. coli rapidly results in abnormal morphological changes and subsequent c ell 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 t he acceptor units from the latter contained exclusively meso-diaminopimelic acid, suggesting that no transpeptidation could occur between the E-amino group of L-lysine and the a-carboxyl group of D-alanine. The overall cross- linking of the macromolecule was only slightly decreased. Detection and ana lysis of meso-diaminopimelic acid- and L-lysine-containing peptidoglycan pr ecursors confirmed the presence of L-lysine in precursors containing amino acids added after the reaction catalyzed by the MurE ligase and provided ad ditional information about the specificity of the enzymes involved in these latter processes.