Identification of a Staphylococcus aureus Efflux Pump Regulator Using a DNA–Protein Affinity Technique
In this chapter, we describe the step-by-step identification of a putative regulator protein and demonstrate the function of this protein as a repressor of the expression of a specific efflux pump, causing resistance to quinolones in Staphylococcus aureus. We show that the knockout gene mutant has an increase in transcript levels of the target efflux pump when compared to that of the S. aureus parental strain RN6390. We provide a detailed protocol that includes the identification of the DNA-binding transcriptional regulatory protein from S. aureus cell extracts using DNA sequences linked to magnetic beads. In addition, we describe the real-time qRT-PCR assays and MIC testing to evaluate the effects of the regulator on S. aureus drug resistance phenotype.
Key wordsStaphylococcus aureus Regulator Efflux pump Gel-shift assay Affinity binding assay In-frame gene deletion Protein purification qRT-PCRs
This work was supported by the U.S. Public Health Service Grants R37-AI23988 from the National Institutes of Health to D.C.H.
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