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Identification of a Staphylococcus aureus Efflux Pump Regulator Using a DNA–Protein Affinity Technique

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1700)

Abstract

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 words

Staphylococcus aureus Regulator Efflux pump Gel-shift assay Affinity binding assay In-frame gene deletion Protein purification qRT-PCRs 

Notes

Acknowledgments

This work was supported by the U.S. Public Health Service Grants R37-AI23988 from the National Institutes of Health to D.C.H.

References

  1. 1.
    Andersen JL, He GX, Kakarla P, Kc R, Kumar S, Lakra WS, Mukherjee MM, Ranaweera I, Shrestha U, Tran T, Varela MF (2015) Multidrug efflux pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus bacterial food pathogens. Int J Environ Res Public Health 12:1487–1547CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Floyd JL, Smith KP, Kumar SH, Floyd JT, Varela MF (2010) LmrS is a multidrug efflux pump of the major facilitator superfamily from Staphylococcus aureus. Antimicrob Agents Chemother 54:5406–5412CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Fournier B, Aras R, Hooper DC (2000) Expression of the multidrug resistance transporter NorA from Staphylococcus aureus is modified by a two-component regulatory system. J Bacteriol 182:664–671CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Villet RA, Truong-Bolduc QC, Wang Y, Estabrooks Z, Medeiros H, Hooper DC (2014) Regulation of expression of abcA and its response to environmental conditions. J Bacteriol 196:1532–1539CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Chien YT, Manna AC, Projan SJ, Cheung AL (1999) SarA, a global regulator of virulence determinants in Staphylococcus aureus, binds to a conserved motif essential for sar-dependent gene regulation. J Biol Chem 274:37169–37176CrossRefPubMedGoogle Scholar
  6. 6.
    Truong-Bolduc QC, Zhang X, Hooper DC (2003) Characterization of NorR protein, a multifunctional regulator of norA expression in Staphylococcus aureus. J Bacteriol 185:3127–3138CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Monk IR, Shah IM, Xu M, Tan MW, Foster TJ (2012) Transforming the untransformable: application of direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis. MBio 3:e00277-11CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ding Y, Onodera Y, Lee JC, Hooper DC (2008) NorB, an efflux pump in Staphylococcus aureus MW2, contributes to bacterial fitness in abscesses. J Bacteriol 190:7123–7129CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Ding Y, Fu Y, Lee JC, Hooper DC (2012) Staphylococcus aureus NorD, a putative efflux pump coregulated with the Opp1 oligopeptide permease, contributes selectively to fitness in vivo. J Bacteriol 194:6586–6593CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Truong-Bolduc QC, Ding Y, Hooper DC (2008) Posttranslational modification influences the effects of MgrA on norA expression in Staphylococcus aureus. J Bacteriol 190:7375–7381CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Zhang L, Fan F, Palmer LM, Lonetto MA, Petit C, Voelker LL, St John A, Bankosky B, Rosenberg M, McDevitt D (2000) Regulated gene expression in Staphylococcus aureus for identifying conditional lethal phenotypes and antibiotic mode of action. Gene 255:297–305CrossRefPubMedGoogle Scholar
  12. 12.
    Truong-Bolduc QC, Hooper DC (2010) Phosphorylation of MgrA and its efect on expression of the NorA and NorB efflux pumps of Staphylococcus aureus. J Bacteriol 192:2525–2534CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Chung CT, Niemela SL, Miller RH (1989) One-step prepration of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A 86:2172–2175CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Truong-Bolduc QC, Villet RA, Estabrooks ZA, Hooper DC (2014) Native efflux pumps contribute resistance to antimicrobials of skin and the ability of Staphylococcus aureus to colonize skin. J Infect Dis 209:1485–1493CrossRefPubMedGoogle Scholar
  15. 15.
    Truong-Bolduc QC, Hsing LC, Villet R, Bolduc GR, Estabrooks Z, Taguezem GF, Hooper DC (2012) Reduced aeration affects the expression of the NorB efflux pump of Staphylococcus aureus by posttranslational modification of MgrA. J Bacteriol 194:1823–1834CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Truong-Bolduc QC, Dunman PM, Strahilevitz J, Projan SJ, Hooper DC (2005) MgrA is a multiple regulator of two new efflux pumps in Staphylococcus aureus. J Bacteriol 187:2395–2405CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Division of Infectious Diseases and Medical Services, Massachusetts General HospitalHarvard Medical SchoolBostonUSA

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