1887

Abstract

MexXY, a drug efflux pump in , confers resistance to aminoglycoside antibiotics. We recently reported that MexZ binds to the promoter region of the operon. Electrophoretic mobility shift assay (EMSA) using recombinant MexZ and oligonucleotide probes prepared from the intergenic region between and revealed that MexZ binds to a 20 bp palindromic sequence. Culture of in the presence of tetracycline induced higher levels of MexX and MexZ, as measured by immunoblotting and EMSA, than in the absence of antibiotics. When MexZ was expressed by a expression plasmid, the plasmid-borne MexZ repressed drug-induced MexX production, further confirming that MexZ acts as a repressor of the operon. PA5471 protein has been reported to be essential for drug-induced MexXY production. Similarly to that report, we observed that plasmid-borne PA5471 induced both MexX and MexZ production in PAO1 cells. Interestingly, interaction between MexZ and PA5471 was observed in a yeast two-hybrid assay. Furthermore, EMSA and transcription assays revealed that interaction between PA5471 and MexZ reduced MexZ DNA-binding ability, leading to transcription. These findings contribute to the understanding of the molecular mechanisms underlying the transcriptional regulation of and by drug-induced PA5471 expression.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.028993-0
2009-10-01
2019-09-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/10/3312.html?itemId=/content/journal/micro/10.1099/mic.0.028993-0&mimeType=html&fmt=ahah

References

  1. Aires, J. R., Kohler, T., Nikaido, H. & Plesiat, P. ( 1999; ). Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides. Antimicrob Agents Chemother 43, 2624–2628.
    [Google Scholar]
  2. Aramaki, H., Sagara, Y., Kabata, H., Shimamoto, N. & Horiuchi, T. ( 1995; ). Purification and characterization of a cam repressor (CamR) for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid. J Bacteriol 177, 3120–3127.
    [Google Scholar]
  3. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. ( 1995; ). Current Protocols in Molecular Biology. Chichester: John Wiley.
  4. Barcak, G. J., Chandler, M. S., Redfield, R. J. & Tomb, J. F. ( 1991; ). Genetic systems in Haemophilus influenzae. Methods Enzymol 204, 321–342.
    [Google Scholar]
  5. Bertrand, K. P., Postle, K., Wray, L. V., Jr & Reznikoff, W. S. ( 1983; ). Overlapping divergent promoters control expression of Tn10 tetracycline resistance. Gene 23, 149–156.[CrossRef]
    [Google Scholar]
  6. Daigle, D. M., Cao, L., Fraud, S., Wilke, M. S., Pacey, A., Klinoski, R., Strynadka, N. C., Dean, C. R. & Poole, K. ( 2007; ). A protein modulator of multidrug efflux gene expression in Pseudomonas aeruginosa. J Bacteriol 189, 5441–5451.[CrossRef]
    [Google Scholar]
  7. Dean, C. R., Visalli, M. A., Projan, S. J., Sum, P. E. & Bradford, P. A. ( 2003; ). Efflux-mediated resistance to tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1. Antimicrob Agents Chemother 47, 972–978.[CrossRef]
    [Google Scholar]
  8. Eckert, B. & Beck, C. F. ( 1989; ). Overproduction of transposon Tn10-encoded tetracycline resistance protein results in cell death and loss of membrane potential. J Bacteriol 171, 3557–3559.
    [Google Scholar]
  9. Forster, K., Helbl, V., Lederer, T., Urlinger, S., Wittenburg, N. & Hillen, W. ( 1999; ). Tetracycline-inducible expression systems with reduced basal activity in mammalian cells. Nucleic Acids Res 27, 708–710.[CrossRef]
    [Google Scholar]
  10. Freundlieb, S., Baron, U., Bonin, A. L., Gossen, M. & Bujard, H. ( 1997; ). Use of tetracycline-controlled gene expression systems to study mammalian cell cycle. Methods Enzymol 283, 159–173.
    [Google Scholar]
  11. Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W. & Bujard, H. ( 1995; ). Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766–1769.[CrossRef]
    [Google Scholar]
  12. Grkovic, S., Brown, M. H., Schumacher, M. A., Brennan, R. G. & Skurray, R. A. ( 2001; ). The staphylococcal QacR multidrug regulator binds a correctly spaced operator as a pair of dimers. J Bacteriol 183, 7102–7109.[CrossRef]
    [Google Scholar]
  13. Hagman, K. E., Pan, W., Spratt, B. G., Balthazar, J. T., Judd, R. C. & Shafer, W. M. ( 1995; ). Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents is modulated by the mtrRCDE efflux system. Microbiology 141, 611–622.[CrossRef]
    [Google Scholar]
  14. Hillen, W. & Berens, C. ( 1994; ). Mechanisms underlying expression of Tn10 encoded tetracycline resistance. Annu Rev Microbiol 48, 345–369.[CrossRef]
    [Google Scholar]
  15. Hocquet, D., Nordmann, P., El Garch, F., Cabanne, L. & Plesiat, P. ( 2006; ). Involvement of the MexXY-OprM efflux system in emergence of cefepime resistance in clinical strains of Pseudomonas aeruginosa. Antimicrob Agents Chemother 50, 1347–1351.[CrossRef]
    [Google Scholar]
  16. Jeannot, K., Sobel, M. L., El Garch, F., Poole, K. & Plesiat, P. ( 2005; ). Induction of the MexXY efflux pump in Pseudomonas aeruginosa is dependent on drug–ribosome interaction. J Bacteriol 187, 5341–5346.[CrossRef]
    [Google Scholar]
  17. Klein, J. R., Henrich, B. & Plapp, R. ( 1991; ). Molecular analysis and nucleotide sequence of the envCD operon of Escherichia coli. Mol Gen Genet 230, 230–240.[CrossRef]
    [Google Scholar]
  18. Llanes, C., Hocquet, D., Vogne, C., Benali-Baitich, D., Neuwirth, C. & Plesiat, P. ( 2004; ). Clinical strains of Pseudomonas aeruginosa overproducing MexAB-OprM and MexXY efflux pumps simultaneously. Antimicrob Agents Chemother 48, 1797–1802.[CrossRef]
    [Google Scholar]
  19. Ma, D., Alberti, M., Lynch, C., Nikaido, H. & Hearst, J. E. ( 1996; ). The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stress signals. Mol Microbiol 19, 101–112.[CrossRef]
    [Google Scholar]
  20. Masuda, N., Sakagawa, E., Ohya, S., Gotoh, N., Tsujimoto, H. & Nishino, T. ( 2000a; ). Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux pumps in Pseudomonas aeruginosa. Antimicrob Agents Chemother 44, 3322–3327.[CrossRef]
    [Google Scholar]
  21. Masuda, N., Sakagawa, E., Ohya, S., Gotoh, N., Tsujimoto, H. & Nishino, T. ( 2000b; ). Contribution of the MexX-MexY-OprM efflux system to intrinsic resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 44, 2242–2246.[CrossRef]
    [Google Scholar]
  22. Matsuo, Y., Eda, S., Gotoh, N., Yoshihara, E. & Nakae, T. ( 2004; ). MexZ-mediated regulation of mexXY multidrug efflux pump expression in Pseudomonas aeruginosa by binding on the mexZ-mexX intergenic DNA. FEMS Microbiol Lett 238, 23–28.
    [Google Scholar]
  23. McDermott, P. F., Walker, R. D. & White, D. G. ( 2003; ). Antimicrobials: modes of action and mechanisms of resistance. Int J Toxicol 22, 135–143.[CrossRef]
    [Google Scholar]
  24. Mima, T., Sekiya, H., Mizushima, T., Kuroda, T. & Tsuchiya, T. ( 2005; ). Gene cloning and properties of the RND-type multidrug efflux pumps MexPQ-OpmE and MexMN-OprM from Pseudomonas aeruginosa. Microbiol Immunol 49, 999–1002.[CrossRef]
    [Google Scholar]
  25. Morita, Y., Sobel, M. L. & Poole, K. ( 2006; ). Antibiotic inducibility of the MexXY multidrug efflux system of Pseudomonas aeruginosa: involvement of the antibiotic-inducible PA5471 gene product. J Bacteriol 188, 1847–1855.[CrossRef]
    [Google Scholar]
  26. Okamoto, K., Gotoh, N. & Nishino, T. ( 2002; ). Alterations of susceptibility of Pseudomonas aeruginosa by overproduction of multidrug efflux systems, MexAB-OprM, MexCD-OprJ, and MexXY/OprM to carbapenems: substrate specificities of the efflux systems. J Infect Chemother 8, 371–373.[CrossRef]
    [Google Scholar]
  27. Pearson, J. P., Pesci, E. C. & Iglewski, B. H. ( 1997; ). Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J Bacteriol 179, 5756–5767.
    [Google Scholar]
  28. Poole, K. ( 2001; ). Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms. J Mol Microbiol Biotechnol 3, 255–264.
    [Google Scholar]
  29. Poole, K. ( 2002a; ). Outer membranes and efflux: the path to multidrug resistance in Gram-negative bacteria. Curr Pharm Biotechnol 3, 77–98.[CrossRef]
    [Google Scholar]
  30. Poole, K. ( 2002b; ). Mechanisms of bacterial biocide and antibiotic resistance. J Appl Microbiol 92 (Suppl), 55S–64S.[CrossRef]
    [Google Scholar]
  31. Poole, K. ( 2005a; ). Efflux-mediated antimicrobial resistance. J Antimicrob Chemother 56, 20–51.[CrossRef]
    [Google Scholar]
  32. Poole, K. ( 2005b; ). Aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 49, 479–487.[CrossRef]
    [Google Scholar]
  33. Sanchez, P., Alonso, A. & Martinez, J. L. ( 2002a; ). Cloning and characterization of SmeT, a repressor of the Stenotrophomonas maltophilia multidrug efflux pump SmeDEF. Antimicrob Agents Chemother 46, 3386–3393.[CrossRef]
    [Google Scholar]
  34. Sanchez, P., Rojo, F. & Martinez, J. L. ( 2002b; ). Transcriptional regulation of mexR, the repressor of Pseudomonas aeruginosa mexAB-oprM multidrug efflux pump. FEMS Microbiol Lett 207, 63–68.[CrossRef]
    [Google Scholar]
  35. Schnappinger, D. & Hillen, W. ( 1996; ). Tetracyclines: antibiotic action, uptake, and resistance mechanisms. Arch Microbiol 165, 359–369.[CrossRef]
    [Google Scholar]
  36. Smith, A. W. & Iglewski, B. H. ( 1989; ). Transformation of Pseudomonas aeruginosa by electroporation. Nucleic Acids Res 17, 10509 [CrossRef]
    [Google Scholar]
  37. Sobel, M. L., McKay, G. A. & Poole, K. ( 2003; ). Contribution of the MexXY multidrug transporter to aminoglycoside resistance in Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother 47, 3202–3207.[CrossRef]
    [Google Scholar]
  38. Teran, W., Krell, T., Ramos, J. L. & Gallegos, M. T. ( 2006; ). Effector-repressor interactions, binding of a single effector molecule to the operator-bound TtgR homodimer mediates derepression. J Biol Chem 281, 7102–7109.[CrossRef]
    [Google Scholar]
  39. Vogne, C., Aires, J. R., Bailly, C., Hocquet, D. & Plesiat, P. ( 2004; ). Role of the multidrug efflux system MexXY in the emergence of moderate resistance to aminoglycosides among Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Antimicrob Agents Chemother 48, 1676–1680.[CrossRef]
    [Google Scholar]
  40. Westbrock-Wadman, S., Sherman, D. R., Hickey, M. J., Coulter, S. N., Zhu, Y. Q., Warrener, P., Nguyen, L. Y., Shawar, R. M., Folger, K. R. & Stover, C. K. ( 1999; ). Characterization of a Pseudomonas aeruginosa efflux pump contributing to aminoglycoside impermeability. Antimicrob Agents Chemother 43, 2975–2983.
    [Google Scholar]
  41. Wright, G. D. ( 2003; ). Mechanisms of resistance to antibiotics. Curr Opin Chem Biol 7, 563–569.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.028993-0
Loading
/content/journal/micro/10.1099/mic.0.028993-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error