Regulated gene expression in Staphylococcus aureus for identifying conditional lethal phenotypes and antibiotic mode of action

Selectively regulating gene expression in bacteria has provided an importan t tool for studying gene function. However, well-regulated gene control sys tems have been restricted primarily for use in laboratory non-pathogenic st rains of bacteria (e.g. Escherichia coli, Bacillus subtilis). The developme nt of analogous systems for use in bacterial pathogens such as Staphylococc us aureus would significantly enhance our ability to examine the contributi on of any given gene product to pathogen growth and viability. In this repo rt, we adapt, examine and compare three regulated gene expression systems i n S. aureus, which had previously been used in B. subtilis. We demonstrate that all three systems function and exhibit titratable induction, together covering a dynamic range of gene expression of similar to 3000-fold. This d ynamic range correlates well with the physiological expression levels of ce llular proteins. Importantly, we show that one of these systems, the Spac s ystem, is particularly useful for examining gene essentiality and creating specific conditional lethal phenotypes. Moreover, we find that titration of selective target gene products using this system allows direct demonstrati on of antibiotic mode of action. (C) 2000 Elsevier Science B.V. All rights reserved.