A method for demonstrating gene essentiality in Staphylococcus aureus

A method for demonstrating whether a gene of Staphylococcus aureus is essen tial 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 trip eptide synthetase required for peptidoglycan synthesis, is essential for gr owth in S. aureus. In this study, strain CYL368 was constructed from S. aur eus 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 a nd the Bacillus subtilis SPO1 phage promoter). To regulate the murE gene in CYL368, the E. coil lac1 gene was expressed from the B. licheniformis peni cillinase gene (pcn) promoter in plasmid pMJ8426. Strain CYL368(pMJ8426) gr ew normally in the presence of isopropyl-beta-D-thiogalactopyranoside but c ould not grow in the absence of the inducer. These results indicate that th e murE gene expressed from the spac promoter in CYL368(pMJ8426) is needed f or bacterial growth. We concluded that murE is an essential gene of S. aure us. (C) 2000 Academic Press.