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The Journal of Microbiology

, Volume 47, Issue 3, pp 277–286 | Cite as

Molecular analysis of the copper-responsive CopRSCD of a pathogenic Pseudomonas fluorescens strain

  • Yong-hua Hu
  • Hua-lei Wang
  • Min Zhang
  • Li SunEmail author
Article

Abstract

CopRS/CopABCD is one of the known systems that control copper homeostasis in bacteria. Although CopRS/CopABCD homologues are found to exist in Pseudomonas fluorescens, the potential role of this system in P. fluorescens has not been investigated. In this study a genetic cluster, consisting of copR, S, C, and D but lacking copAB, was identified in a pathogenic P. fluorescens strain (TSS) isolated from diseased fish. The copRSCD cluster was demonstrated to be required for full copper resistance and regulated at the transcription level by Cu. Expression of copCD is regulated directly by the two-component response regulator CopR, which also regulates its own expression. Interruption of the regulated expression of copR affected bacterial growth, biofilm formation, and tissue dissemination and survival. A mutant CopR, which lacks the N-terminal signal receiver domain and is constitutively active, was found to have an attenuating effect on bacterial virulence when expressed in TSS. To our knowledge, this is the first report that suggests a link between CopR and bacterial pathogenicity in P. fluorescens.

Keywords

CopABCD copper resistance CopR Pseudomonas fluorescens virulence 

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References

  1. Adaikkalam, V. and S. Swarup. 2005. Characterization of copABCD operon from a copper-sensitive Pseudomonas putida strain. Can. J. Microbiol. 51, 209–216.PubMedCrossRefGoogle Scholar
  2. Barre, O., F. Mourlane, and M. Solioz. 2007. Copper-induction of lactate oxidase (LctO) of Lactococcus lactis: A novel metal stress-response. J. Bacteriol. 189, 5947–5954.PubMedCrossRefGoogle Scholar
  3. Brown, N.L., S.R. Barrett, J. Camakaris, B.T. Lee, and D.A. Rouch. 1995. Molecular genetics and transport analysis of the copper-resistance determinant (pco) from Escherichia coli plasmid pRJ1004. Mol. Microbiol. 17, 1153–1166.PubMedCrossRefGoogle Scholar
  4. Caille, O., C. Rossier, and K. Perron. 2007. A copper-activated two-component system interacts with zinc and imipenem resistance in Pseudomonas aeruginosa. J. Bacteriol. 189, 4561–4568.PubMedCrossRefGoogle Scholar
  5. Canovas, D., I. Cases, and V. de Lorenzo. 2003. Heavy metal tolerance and metal homeostasis in Pseudomonas putida as revealed by complete genome analysis. Environ. Microbiol. 5, 1242–1256.PubMedCrossRefGoogle Scholar
  6. Cha, J.S. and D.A. Cooksey. 1991. Copper resistance in Pseudomonas syringae mediated by periplasmic and outer membrane proteins. Proc. Natl. Acad. Sci. USA 88, 8915–8919.PubMedCrossRefGoogle Scholar
  7. Eagon, R.G. 1962. Pseudomonas natriegens, a marine bacterium with a generation time of less than 10 minutes. J. Bacteriol. 83, 736–737.PubMedGoogle Scholar
  8. Franke, S., G. Grass, C. Rensing, and D.H. Nies. 2003. Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli. J. Bacteriol. 185, 3804–3812.PubMedCrossRefGoogle Scholar
  9. Grass, G. and C. Rensing. 2001. Genes involved in copper homeostasis in Escherichia coli. J. Bacteriol. 183, 2145–2147.PubMedCrossRefGoogle Scholar
  10. Ha, U.H., J. Kim, H. Badrane, J. Jia, H.V. Baker, D. Wu, and S. Jin. 2004. An in vivo inducible gene of Pseudomonas aeruginosa encodes an anti-ExsA to suppress the type III secretion system. Mol. Microbiol. 54, 307–320.PubMedCrossRefGoogle Scholar
  11. Harrison, J.J., R.J. Turner, D.A. Joo, M.A. Stan, C.S. Chan, N.D. Allan, H.A. Vrionis, M.E. Olson, and H. Ceri. 2008. Copper and quaternary ammonium cations exert synergistic bactericidal and antibiofilm activity against Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 52, 2870–2881.PubMedCrossRefGoogle Scholar
  12. Lim, C.K. and D.A. Cooksey. 1993. Characterization of chromosomal homologs of the plasmid-borne copper resistance operon of Pseudomonas syringae. J. Bacteriol. 175, 4492–4498.PubMedGoogle Scholar
  13. Link, A.J., D. Phillips, and G.M. Church. 1997. Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. J. Bacteriol. 179, 6228–6237.PubMedGoogle Scholar
  14. Magnani, D., O. Barré, S.D. Gerber, and M. Solioz. 2008. Characterization of the CopR regulon of Lactococcus lactis IL1403. J. Bacteriol. 190, 536–545.PubMedCrossRefGoogle Scholar
  15. Marx, C.J. and M.E. Lidstrom. 2001. Development of improved versatile broad-host range vectors for use in methylotrophs and other Gram-negative bacteria. Microbiology 147, 2065–2075.PubMedGoogle Scholar
  16. Mellano, M.A. and D.A. Cooksey. 1988. Nucleotide sequence and organization of copper resistance genes from Pseudomonas syringae pv. tomato. J. Bacteriol. 170, 2879–2883.PubMedGoogle Scholar
  17. Miller, V.L. and J.J. Mekalanos. 1988. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170, 2575–2583.PubMedGoogle Scholar
  18. Mills, S.D., C.A. Jasalavich, and D.A. Cooksey. 1993. A two-component regulatory system required for copper-inducible expression of the copper resistance operon of Pseudomonas syringae. J. Bacteriol. 175, 1656–1664.PubMedGoogle Scholar
  19. Mills, S.D., C.K. Lim, and D.A. Cooksey. 1994. Purification and characterization of CopR, a transcriptional activator protein that binds to a conserved domain (cop box) in copper-inducible promoters of Pseudomonas syringae. Mol. Gen. Genet. 244, 341–351.PubMedCrossRefGoogle Scholar
  20. Munson, G.P., D.L. Lam, F.W. Outten, and T.V. O’Halloran. 2000. Identification of a copper-responsive two component system on the chromosome of Escherichia coli K-12. J. Bacteriol. 182, 5864–5871.PubMedCrossRefGoogle Scholar
  21. Nelson, K.E., C. Weinel, I.T. Paulsen, R.J. Dodson, H. Hilbert, V.A. Martins dos Santos, D.E. Fouts, S.R. Gill, M. Pop, M. Holmes, L. Brinkac, M. Beanan, R.T. DeBoy, S. Daugherty, J. Kolonay, R. Madupu, W. Nelson, O. White, J. Peterson, H. Khouri, et al. 2002. Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ. Microbiol. 4, 799–808.PubMedCrossRefGoogle Scholar
  22. Outten, F.W., D.L. Huffman, J.A. Hale, and T.V. O’Halloran. 2001. The independent cue and cus systems confer copper tolerance during aerobic growth in Escherichia coli. J. Biol. Chem. 27, 30670–30677.CrossRefGoogle Scholar
  23. Perron, K., O. Caille, C. Rossier, C. Van Delden, J.L. Dumas, and T. Köhler. 2004. CzcR-CzcS, a two-component system involved in heavy metal and carbapenem resistance in Pseudomonas aeruginosa. J. Biol. Chem. 279, 8761–8768.PubMedCrossRefGoogle Scholar
  24. Petersen, C. and L.B. Moller. 2000. Control of copper homeostasis in Escherichia coli by a P-type ATPase, CopA, and a MerR-like transcriptional activator, CopR. Gene 261, 289–298.PubMedCrossRefGoogle Scholar
  25. Rensing, C., B. Fan, R. Sharma, B. Mitra, and B.R. Rosen. 2000. CopA: an Escherichia coli Cu (I)-translocating Ptype ATPase. Proc. Natl. Acad. Sci. USA 97, 652–656.PubMedCrossRefGoogle Scholar
  26. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, N.Y., USA.Google Scholar
  27. Teitzel, G.M., A. Geddie, S.K. De Long, M.J. Kirisits, M. Whiteley, and M.R. Parsek. 2006. Survival and growth in the presence of elevated copper: transcriptional profiling of copper-stressed Pseudomonas aeruginosa. J. Bacteriol. 188, 7242–7256.PubMedCrossRefGoogle Scholar
  28. Wang, F., S. Cheng, K. Sun, and L. Sun. 2008. Molecular analysis of the fur (ferric uptake regulator) gene of a pathogenic Edwardsiella tarda strain. J. Microbiol. 46, 350–355.PubMedCrossRefGoogle Scholar
  29. West, A.H. and A.M. Stock. 2001. Histidine kinases and response regulator proteins in two-component signaling systems. Trends Biochem. Sci. 26, 369–376.PubMedCrossRefGoogle Scholar
  30. Winsor, G.L., R. Lo, S.J. Sui, K.S. Ung, S. Huang, D. Cheng, W.K. Ching, R.E. Hancock, and F.S. Brinkman. 2005. Pseudomonas aeruginosa genome database and PseudoCAP: facilitating community-based, continually updated, genome annotation. Nucleic Acids Res. 33(Database issue): D338–343.PubMedGoogle Scholar
  31. Yamamoto, K. and A. Ishihama. 2005. Transcriptional response of Escherichia coli to external copper. Mol. Microbiol. 56, 215–227.PubMedCrossRefGoogle Scholar
  32. Zhang, X.X. and P.B. Rainey. 2007. The role of a P1-type ATPase from Pseudomonas fluorescens SBW25 in copper homeostasis and plant colonization. Mol. Plant Microbe Interact 20, 581–588.PubMedCrossRefGoogle Scholar
  33. Zhang, W. and L. Sun. 2007. Cloning, characterization and molecular application of a beta-agarase gene from Vibrio sp. strain V134. Appl. Environ. Microbiol. 73, 2825–2831.PubMedCrossRefGoogle Scholar
  34. Zhang, M., K. Sun, and L. Sun. 2008. Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain. Microbiology 154, 2060–2069.PubMedCrossRefGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer-Verlag Berlin Heidelber GmbH 2009

Authors and Affiliations

  • Yong-hua Hu
    • 1
    • 2
  • Hua-lei Wang
    • 3
  • Min Zhang
    • 1
    • 2
  • Li Sun
    • 1
    Email author
  1. 1.Institute of OceanologyChinese Academy of SciencesQingdaoP. R. China
  2. 2.Graduate University of the Chinese Academy of SciencesBeijingP. R. China
  3. 3.Hang Zhou EnzySource Biotechnology Co., Ltd.HangzhouP. R. China

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