A method for demonstrating gene essentiality in Staphylococcus aureus

A method for demonstrating whether a gene of Staphylococcus aureus is essential for growth in a rich medium is described. We have used this method to determine whether the murE gene, which encodes the UDP-N-acetylmuramyl tripeptide synthetase required for peptidoglycan synthesis, is essential for growth in S. aureus. In this study, strain CYL368 was constructed from S. aureus RN4220 by placing the murE gene in the chromosome under the control of the spae promoter (a hybrid promoter of the Escherichia coli lac operator and the Bacillus subtilis SPO1 phage promoter). To regulate the murE gene in CYL368, the E. coli lac1 gene was expressed from the B. licheniformis penicillinase gene (pcn) promoter in plasmid pMJ8426. Strain CYL368(pMJ8426) grew normally in the presence of isopropyl-beta-D-thiogalactopyranoside but could not grow in the absence of the inducer. These results indicate that the murE gene expressed from the spac promoter in CYL368(pMJ8426) is needed for bacterial growth. We concluded that murE is an essential gene of S. aureus. (C) 2000 Academic Press.