Analysis of mutations in the Staphylococcus aureus clfB promoter leading to increased expression

A clfB: : tetK reporter was constructed in Staphylococcus aureus strains Newman and 8325-4, whereby the level of tetracycline resistance reflected the activity of the clfB promoter. Wild-type strains carrying a single copy of this construct exhibited a low level of tetracycline resistance, suggesting that the clfB promoter is weak. Spontaneous mutants that grew at higher tetracline concentrations were isolated. Some were due to point mutations in the clfB promoter that 1,A to increased expression of the tetK gene. The clfB promoter was identified by primer extension analysis and -35 and -10 elements were assigned. The promoter regions from the tetracycline-resistant mutants were sequenced and several had base changes within or adjacent to the -35 box. Three created the consensus -35 sequence TTGACA. The mutant clfB promoters were fused to lacZ. beta-Galactosiclase activity was six- to ninefold higher in the mutant strains compared to the wildtype. The wild-type clfB gene was placed under the control of the mutant promoters. ClfB expression was higher than the corresponding wild-type strains and the protein was present on bacteria from the stationary phase instead of being confined to the exponential phase. Therefore, mutations in the clfB promoter that cause changes in the -35 region produce a stronger promoter that is capable of increased transcription and, as a result, increased expression of ClfB.