Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Identification of Pseudomonas aeruginosa Genes Crucial for Hydrogen Peroxide Resistance

  • Published : 2007.08.30
Download PDF
⟨ Previous Next ⟩

Abstract

An opportunistic human pathogen, Pseudomonas aeruginosa, contains the major catalase KatA, which is required to cope with oxidative and osmotic stresses. As an attempt to uncover the $H_2O_2$-dependent regulatory mechanism delineating katA gene expression, four prototrophic $H_2O_2$-sensitive mutants were isolated from about 1,500 TnphoA mutant clones of P. aeruginosa strain PA14. Arbitrary PCR and direct cloning of the transposon insertion sites revealed that one insertion is located within the katA coding region and two are within the coding region of oxyR, which is responsible for transcriptional activation of several antioxidant enzyme genes in response to oxidative challenges. The fourth insertion was within PA3815 (IscR), which encodes a homolog of the Escherichia coli iron-sulfur assembly regulator, IscR. The levels of catalase and SOD activities were significantly reduced in the iscR mutant, but not in the oxyR mutant, during the normal planktonic culture conditions. These results suggest that both IscR and OxyR are required for the optimal resistance to $H_2O_2$, which involves the expression of multiple antioxidant enzymes including KatA.

Keywords

References

  1. Aslund, F., M. Zheng, J. Beckwith, and G. Storz. 1999. Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol-disulfide status. Proc. Natl. Acad. Sci. USA 96: 6161-6165
  2. Beauchamp, C. and I. Fridovich. 1971. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44: 276-287 https://doi.org/10.1016/0003-2697(71)90370-8
  3. Bodey, G. P., R. Bolivar, V. Fainstein, and L. Jadeja. 1983. Infections caused by Pseudomonas aeruginosa. Rev. Infect. Dis. 5: 279-313 https://doi.org/10.1093/clinids/5.2.279
  4. Brown, S. M., M. L. Howell, M. L. Vasil, A. J. Anderson, and D. J. Hassett. 1995. Cloning and characterization of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: Purification of KatB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide. J. Bacteriol. 177: 6536- 6544 https://doi.org/10.1128/jb.177.22.6536-6544.1995
  5. Choi, K. H., A. Kumar, and H. P. Schweizer. 2006. A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: Application for DNA fragment transfer between chromosomes and plasmid transformation. J. Microbiol. Methods 64: 391-397 https://doi.org/10.1016/j.mimet.2005.06.001
  6. Christman, M. F., G. Storz, and B. N. Ames. 1989. OxyR, a positive regulator of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium, is homologous to a family of bacterial regulatory proteins. Proc. Natl. Acad. Sci. USA 86: 3484-3488
  7. Farr, S. B. and T. Kogoma. 1991. Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbiol. Rev. 55: 561-585
  8. Gant, T. W., D. N. Rao, R. P. Mason, and G. M. Cohen. 1988. Redox cycling and sulphydryl arylation; their relative importance in the mechanism of quinone cytotoxicity to isolated hepatocytes. Chem. Biol. Interact. 65: 157-173 https://doi.org/10.1016/0009-2797(88)90052-X
  9. Ha, U. H., Y. Wang, and S. Jin. 2003. DsbA of Pseudomonas aeruginosa is essential for multiple virulence factors. Infect. Immun. 71: 1590-1595 https://doi.org/10.1128/IAI.71.3.1590-1595.2003
  10. Halliwell, B. and J. M. Gutteridge. 1990. The antioxidants of human extracellular fluids. Arch. Biochem. Biophys. 280: 1-8 https://doi.org/10.1016/0003-9861(90)90510-6
  11. Hassett, D. J. and M. S. Cohen. 1989. Bacterial adaptation to oxidative stress: Implications for pathogenesis and interaction with phagocytic cells. FASEB J. 3: 2574-2582 https://doi.org/10.1096/fasebj.3.14.2556311
  12. Hassett, D. J., E. Alsabbagh, K. Parvatiyar, M. L. Howell, R. W. Wilmott, and U. A. Ochsner. 2000. A protease-resistant catalase, KatA, released upon cell lysis during stationary phase is essential for aerobic survival of a Pseudomonas aeruginosa oxyR mutant at low cell densities. J. Bacteriol. 182: 4557-4563 https://doi.org/10.1128/JB.182.16.4557-4563.2000
  13. Heo, Y.-J., S.-K. Kwan, J.-H. Song, and Y.-H. Cho. 2005. Profiling pyocins and competitive growth advantages of various Pseudomonas aeruginosa strains. J. Microbiol. Biotechnol. 15: 1369-1376
  14. Hoang, T. T., R. R. Karkhoff-Schweizer, A. J. Kutchma, and H. P. Schweizer. 1998. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: Application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212: 77-86 https://doi.org/10.1016/S0378-1119(98)00130-9
  15. Hogan, D. A. and R. Kolter. 2002. Pseudomonas-Candida interactions: An ecological role for virulence factors. Science 296: 2229-2232 https://doi.org/10.1126/science.1070784
  16. Jamet, A., E. Kiss, J. Batut, A. Puppo, and D. Herouart. 2005. The katA catalase gene is regulated by OxyR in both free-living and symbiotic Sinorhizobium meliloti. J. Bacteriol. 187: 376-378 https://doi.org/10.1128/JB.187.1.376-381.2005
  17. Kanazawa, Y. and T. Kuramata. 1966. A simple method for determination of ability of bacteria to inactivate chemotherapeutics using sensitivity disc. J. Antibiot. (Tokyo) 19: 272-277
  18. Kang, Y. S., Y. J. Kim, C. O. Jeon, and W. J. Park. 2006. Characterization of naphthalene-degrading Pseudomonas species isolated from pollutant-contaminated sites: Oxidative stress during their growth on naphthalene. J. Microbiol. Biotechnol. 16: 1819-1825
  19. Lau, G. W., B. E. Britigan, and D. J. Hassett. 2005. Pseudomonas aeruginosa OxyR is required for full virulence in rodent and insect models of infection and for resistance to human neutrophils. Infect. Immun. 73: 2550- 2553 https://doi.org/10.1128/IAI.73.4.2550-2553.2005
  20. Lee, J.-S., Y.-J. Heo, J. K. Lee, and Y.-H. Cho. 2005. KatA, the major catalase, is critical for osmoprotection and virulence in Pseudomonas aeruginosa PA14. Infect. Immun. 73: 4399-4403 https://doi.org/10.1128/IAI.73.7.4399-4403.2005
  21. Mahajan-Miklos, S., M. W. Tan, L. G. Rahme, and F. M. Ausubel. 1999. Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa-Caenorhabditis elegans pathogenesis model. Cell 96: 47-56 https://doi.org/10.1016/S0092-8674(00)80958-7
  22. Manoil, C. and J. Beckwith. 1985. TnphoA: A transposon probe for protein export signals. Proc. Natl. Acad. Sci. USA 82: 8129-8133
  23. Miller, J. H. 1992. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
  24. Mongkolsuk, S., W. Panmanee, S. Atichartpongkul, P. Vattanaviboon, W. Whangsuk, M. Fuangthong, W. Eiamphungporn, R. Sukchawalit, and S. Utamapongchai. 2002. The repressor for an organic peroxide-inducible operon is uniquely regulated at multiple levels. Mol. Microbiol. 44: 793-802 https://doi.org/10.1046/j.1365-2958.2002.02919.x
  25. Montgomery, K. T., G. Grills, L. Li, W. A. Brown, J. Decker, R. Elliot, et al. 2002. Pseudomonas aeruginosa strain UCBPP-PA14 whole genome shotgun sequencing project. Direct submission. Accession numbers AABQ06000000- AABQ06000008
  26. Newman, J. R. and C. Fuqua. 1999. Broad-host-range expression vectors that carry the L-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator. Gene 227: 197-203 https://doi.org/10.1016/S0378-1119(98)00601-5
  27. Ochsner, U. A., M. L. Vasil, E. Alsabbagh, K. Parvatiyar, and D. J. Hassett. 2000. Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF. J. Bacteriol. 182: 4533-4544 https://doi.org/10.1128/JB.182.16.4533-4544.2000
  28. Okura, M., R. Osawa, A. Iguchi, E. Arakawa, J. Terajima, and H. Watanabe. 2003. Genotypic analyses of Vibrio parahaemolyticus and development of a pandemic group specific multiplex PCR assay. J. Clin. Microbiol. 41: 4676- 4682 https://doi.org/10.1128/JCM.41.10.4676-4682.2003
  29. O'Toole, G. A., L. A. Pratt, P. I. Watnick, D. K. Newman, V. B. Weaver, and R. Kolter. 1999. Genetic approaches to study of biofilms. Methods Enzymol. 310: 91-109 https://doi.org/10.1016/S0076-6879(99)10008-9
  30. Park, S.-Y., Y.-J. Heo, Y.-S. Choi, E. Déziel, and Y.-H. Cho. 2005. Conserved virulence factors of Pseudomonas aeruginosa are required for killing Bacillus subtilis. J. Microbiol. 43: 443-450
  31. Rahme, L. G., E. J. Stevens, S. F. Wolfort, J. Shao, R. G. Tompkins, and F. M. Ausubel. 1995. Common virulence factors for bacterial pathogenicity in plants and animals. Science 268: 1899-1902 https://doi.org/10.1126/science.7604262
  32. Rahme, L. G., M. W. Tan, L. Le, S. M. Wong, R. G. Tompkins, S. B. Calderwood, and F. M. Ausubel. 1997. Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors. Proc. Natl. Acad. Sci. USA 94: 13245-13250
  33. Sambrook, J., E. F. Fritsh, and T. Maniatis. 2001. Molecular Cloning; A Laboratory Manual, 3rd Ed. Laboratory Press, Cold Spring Harbor, N.Y
  34. Schell, M. A. 1993. Molecular biology of the LysR family of transcriptional regulators. Annu. Rev. Microbiol. 47: 597-626 https://doi.org/10.1146/annurev.mi.47.100193.003121
  35. Schwartz, C. J., J. L. Giel, T. Patschkowski, C. Luther, F. J. Ruzicka, H. Beinert, and P. J. Kiley. 2001. IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins. Proc. Natl. Acad. Sci. USA 98: 14895-14900
  36. Schweizer, H. P. 1991. Escherichia-Pseudomonas shuttle vectors derived from pUC18/19. Gene 97: 109-121 https://doi.org/10.1016/0378-1119(91)90016-5
  37. Seib, K. L., H. J. Wu, S. P. Kidd, M. A. Apicella, M. P. Jennings, and A. G. McEwan. 2006. Defenses against oxidative stress in Neisseria gonorrhoeae: A system tailored for a challenging environment. Microbiol. Mol. Biol. Rev. 70: 344-361 https://doi.org/10.1128/MMBR.00044-05
  38. Simon, R., U. Priefer, and A. Pühler. 1983. A broad host range mobilization for in vivo genetic engineering: Transposon mutagenesis in Gram-negative bacteria. Biotechnology 28: 37-45
  39. Storz, G. and L. A. Tartaglia. 1992. OxyR: A regulator of antioxidant genes. J. Nutr. 122: 627-630 https://doi.org/10.1093/jn/122.suppl_3.627
  40. Taylor, R. K., C. Manoil, and J. J. Mekalanos. 1989. Broadhost- range vectors for delivery of TnphoA: Use in genetic analysis of secreted virulence determinants of Vibrio cholerae. J. Bacteriol. 171: 1870-1878 https://doi.org/10.1128/jb.171.4.1870-1878.1989
  41. Wayne, L. G. and G. A. Diaz. 1986. A double staining method for differentiating between two classes of mycobacterial catalase in polyacrylamide electrophoresis gels. Anal. Biochem. 157: 89-92 https://doi.org/10.1016/0003-2697(86)90200-9
  42. Xu, B., Y. J. Yang, and Z. X. Huang. 2006. Cloning and overexpression of gene encoding the pullulanase from Bacillus naganoensis in Pichia pastoris. J. Microbiol. Biotechnol. 16: 1185-1191
  43. Yang, H. Y., H. S. Lee, J. H. Ko, S. W. Yeon, T. Y. Kim, B. Y. Hwang, S. S. Kang, J. Chun, and S. K. Hong. 2006. Identification of 3'-hydroxymelanetin and liquiritigenin as akt protein kinase inhibitors. J. Microbiol. Biotechnol. 16: 1384-1391
  44. Yorgey, P., L. G. Rahme, M. W. Tan, and F. M. Ausubel. 2001. The roles of mucD and alginate in the virulence of Pseudomonas aeruginosa in plants, nematodes and mice. Mol. Microbiol. 41: 1063-1076 https://doi.org/10.1046/j.1365-2958.2001.02580.x
  45. Zheng, M. and G. Storz. 2000. Redox sensing by prokaryotic transcription factors. Biochem. Pharmacol. 59: 1-6 https://doi.org/10.1016/S0006-2952(99)00289-0
  46. Zheng, M., F. Åslund, and G. Storz. 1998. Activation of the OxyR transcription factor by reversible disulfide bond formation. Science 279: 1655-1656 https://doi.org/10.1126/science.279.5357.1655