Functionally Study On The Residues Of Multidrug Transporter QacA From Staphylococcus Aureus

Objective: To determine the importance of amino acid residues inα-helix1,2and TMS1of multidrug exporter QacA from Staphylococcusaureus.Methods:1. Cysteine site-directed mutagenesis was used to mutateresidues including I2，S3，F4，F5，T6，K7，D10，M11，R17，W18，T19，A20，V21，V22，V23，F29，D34，P43，E48，E50，P51and Q55.After transformation to E.coli DH5α, the mutants were screened by BamHIand EcoRI and verified by sequencing to confirm the desired mutation.2.The agar disk dilution method was used for Minimum inhibitoryconcentration (MIC) and Minimum bactericidal concentration (MBC) assay;transport assay was used to detect transport activity of QacA mutants.3.Western blot was performed to detect the mutant expression level of QacAprotein.Results:1.Twenty-two mutants were acquired by cysteine site-directedmutagenesis.2. MIC and MBC analysis suggested that the drug sensitivityof mutants S3C, T6C, R17C, T19C, A20C, F29C and E50C to the detectedsubstrates restored completely; the resistance of mutants F4C, F5C and P51C to the detected substrates reduced significantly. And the results of thefluorimetric transport assay were in agreement with those of MIC and MBCanalysis, which indicated that the mutants of S3C, F4C，F5C, T6C, R17C,T19C, A20C, F29C, E50C and P51C did not confer transport to the testedsubstrates.3. I2C, K7C, D10C, M11C, W18C, V21C, V22C, V23C, D34C,P43C, E48C, Q55C and QacA WT showed comparable protein expressionlevels.Conclusion: Our study identified that residues including S3，F4，F5，T6，R17，T19，A20，F29，E50and P51probably involved in the substratebinding and translocation in α-helix1,2and TMS1of multidrug exporterQacA.