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. 1995 Nov;177(22):6536–6544. doi: 10.1128/jb.177.22.6536-6544.1995

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.

S M Brown 1, M L Howell 1, M L Vasil 1, A J Anderson 1, D J Hassett 1
PMCID: PMC177506  PMID: 7592431

Abstract

Pseudomonas aeruginosa is an obligate aerobe that is virtually ubiquitous in the environment. During aerobic respiration, the metabolism of dioxygen can lead to the production of reactive oxygen intermediates, one of which includes hydrogen peroxide. To counteract the potentially toxic effects of this compound, P. aeruginosa possesses two heme-containing catalases which detoxify hydrogen peroxide. In this study, we have cloned katB, encoding one catalase gene of P. aeruginosa. The gene was cloned on a 5.4-kb EcoRI fragment and is composed of 1,539 bp, encoding 513 amino acids. The amino acid sequence of the P. aeruginosa katB was approximately 65% identical to that of a catalase from a related species, Pseudomonas syringae. The katB gene was mapped to the 71- to 75-min region of the P. aeruginosa chromosome, the identical region which harbors both sodA and sodB genes encoding both manganese and iron superoxide dismutases. When cloned into a catalase-deficient mutant of Escherichia coli (UM255), the recombinant P. aeruginosa KatB was expressed (229 U/mg) and afforded this strain resistance to hydrogen peroxide nearly equivalent to that of the wild-type E. coli strain (HB101). The KatB protein was purified to homogeneity and determined to be a tetramer of approximately 228 kDa, which was in good agreement with the predicted protein size derived from the translated katB gene. Interestingly, KatB was not produced during the normal P. aeruginosa growth cycle, and catalase activity was greater in nonmucoid than in mucoid, alginate-producing organisms. When exposed to hydrogen peroxide and, to a greater extent, paraquat, total catalase activity was elevated 7- to 16-fold, respectively. In addition, an increase in KatB activity caused a marked increase in resistance to hydrogen peroxide. KatB was localized to the cytoplasm, while KatA, the "housekeeping" enzyme, was detected in both cytoplasmic and periplasmic extracts. A P. aeruginosa katB mutant demonstrated 50% greater sensitivity to hydrogen peroxide than wild-type bacteria, suggesting that KatB is essential for optimal resistance of P. aeroginosa to exogenous hydrogen peroxide.

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Selected References

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  1. BEERS R. F., Jr, SIZER I. W. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem. 1952 Mar;195(1):133–140. [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Buchmeier N. A., Libby S. J., Xu Y., Loewen P. C., Switala J., Guiney D. G., Fang F. C. DNA repair is more important than catalase for Salmonella virulence in mice. J Clin Invest. 1995 Mar;95(3):1047–1053. doi: 10.1172/JCI117750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Christman M. F., Morgan R. W., Jacobson F. S., Ames B. N. Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium. Cell. 1985 Jul;41(3):753–762. doi: 10.1016/s0092-8674(85)80056-8. [DOI] [PubMed] [Google Scholar]
  5. Clare D. A., Duong M. N., Darr D., Archibald F., Fridovich I. Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase. Anal Biochem. 1984 Aug 1;140(2):532–537. doi: 10.1016/0003-2697(84)90204-5. [DOI] [PubMed] [Google Scholar]
  6. Demple B., Halbrook J., Linn S. Escherichia coli xth mutants are hypersensitive to hydrogen peroxide. J Bacteriol. 1983 Feb;153(2):1079–1082. doi: 10.1128/jb.153.2.1079-1082.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Farr S. B., Touati D., Kogoma T. Effects of oxygen stress on membrane functions in Escherichia coli: role of HPI catalase. J Bacteriol. 1988 Apr;170(4):1837–1842. doi: 10.1128/jb.170.4.1837-1842.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fridovich I. The biology of oxygen radicals. Science. 1978 Sep 8;201(4359):875–880. doi: 10.1126/science.210504. [DOI] [PubMed] [Google Scholar]
  9. Hassan H. M., Fridovich I. Mechanism of the antibiotic action pyocyanine. J Bacteriol. 1980 Jan;141(1):156–163. doi: 10.1128/jb.141.1.156-163.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hassan H. M., Fridovich I. Paraquat and Escherichia coli. Mechanism of production of extracellular superoxide radical. J Biol Chem. 1979 Nov 10;254(21):10846–10852. [PubMed] [Google Scholar]
  11. Hassan H. M., Fridovich I. Regulation of the synthesis of catalase and peroxidase in Escherichia coli. J Biol Chem. 1978 Sep 25;253(18):6445–6420. [PubMed] [Google Scholar]
  12. Hassett D. J., Bean K., Biswas G., Cohen M. S. The role of hydroxyl radical in chromosomal and plasmid damage in Neisseria gonorrhoeae in vivo. Free Radic Res Commun. 1989;7(2):83–87. doi: 10.3109/10715768909087927. [DOI] [PubMed] [Google Scholar]
  13. Hassett D. J., Charniga L., Bean K., Ohman D. E., Cohen M. S. Response of Pseudomonas aeruginosa to pyocyanin: mechanisms of resistance, antioxidant defenses, and demonstration of a manganese-cofactored superoxide dismutase. Infect Immun. 1992 Feb;60(2):328–336. doi: 10.1128/iai.60.2.328-336.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hassett D. J., Charniga L., Cohen M. S. recA and catalase in H2O2-mediated toxicity in Neisseria gonorrhoeae. J Bacteriol. 1990 Dec;172(12):7293–7296. doi: 10.1128/jb.172.12.7293-7296.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hassett D. J., Cohen M. S. Bacterial adaptation to oxidative stress: implications for pathogenesis and interaction with phagocytic cells. FASEB J. 1989 Dec;3(14):2574–2582. doi: 10.1096/fasebj.3.14.2556311. [DOI] [PubMed] [Google Scholar]
  16. Hassett D. J., Woodruff W. A., Wozniak D. J., Vasil M. L., Cohen M. S., Ohman D. E. Cloning and characterization of the Pseudomonas aeruginosa sodA and sodB genes encoding manganese- and iron-cofactored superoxide dismutase: demonstration of increased manganese superoxide dismutase activity in alginate-producing bacteria. J Bacteriol. 1993 Dec;175(23):7658–7665. doi: 10.1128/jb.175.23.7658-7665.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hurst J. K., Barrette W. C., Jr Leukocytic oxygen activation and microbicidal oxidative toxins. Crit Rev Biochem Mol Biol. 1989;24(4):271–328. doi: 10.3109/10409238909082555. [DOI] [PubMed] [Google Scholar]
  18. Imlay J. A., Linn S. Bimodal pattern of killing of DNA-repair-defective or anoxically grown Escherichia coli by hydrogen peroxide. J Bacteriol. 1986 May;166(2):519–527. doi: 10.1128/jb.166.2.519-527.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Imlay J. A., Linn S. Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide. J Bacteriol. 1987 Jul;169(7):2967–2976. doi: 10.1128/jb.169.7.2967-2976.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Katsuwon J., Anderson A. J. Response of plant-colonizing pseudomonads to hydrogen peroxide. Appl Environ Microbiol. 1989 Nov;55(11):2985–2989. doi: 10.1128/aem.55.11.2985-2989.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Klotz M. G., Hutcheson S. W. Multiple periplasmic catalases in phytopathogenic strains of Pseudomonas syringae. Appl Environ Microbiol. 1992 Aug;58(8):2468–2473. doi: 10.1128/aem.58.8.2468-2473.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Loewen P. C. Isolation of catalase-deficient Escherichia coli mutants and genetic mapping of katE, a locus that affects catalase activity. J Bacteriol. 1984 Feb;157(2):622–626. doi: 10.1128/jb.157.2.622-626.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Loewen P. C., Switala J., Triggs-Raine B. L. Catalases HPI and HPII in Escherichia coli are induced independently. Arch Biochem Biophys. 1985 Nov 15;243(1):144–149. doi: 10.1016/0003-9861(85)90782-9. [DOI] [PubMed] [Google Scholar]
  24. Ma M., Eaton J. W. Multicellular oxidant defense in unicellular organisms. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7924–7928. doi: 10.1073/pnas.89.17.7924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mandell G. L. Catalase, superoxide dismutase, and virulence of Staphylococcus aureus. In vitro and in vivo studies with emphasis on staphylococcal--leukocyte interaction. J Clin Invest. 1975 Mar;55(3):561–566. doi: 10.1172/JCI107963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McCord J. M., Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem. 1969 Nov 25;244(22):6049–6055. [PubMed] [Google Scholar]
  27. Meade H. M., Long S. R., Ruvkun G. B., Brown S. E., Ausubel F. M. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol. 1982 Jan;149(1):114–122. doi: 10.1128/jb.149.1.114-122.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moody C. S., Hassan H. M. Mutagenicity of oxygen free radicals. Proc Natl Acad Sci U S A. 1982 May;79(9):2855–2859. doi: 10.1073/pnas.79.9.2855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mulvey M. R., Sorby P. A., Triggs-Raine B. L., Loewen P. C. Cloning and physical characterization of katE and katF required for catalase HPII expression in Escherichia coli. Gene. 1988 Dec 20;73(2):337–345. doi: 10.1016/0378-1119(88)90498-2. [DOI] [PubMed] [Google Scholar]
  30. Schweizer H. D. Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis. Biotechniques. 1993 Nov;15(5):831–834. [PubMed] [Google Scholar]
  31. Schweizer H. P. Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol. 1992 May;6(9):1195–1204. doi: 10.1111/j.1365-2958.1992.tb01558.x. [DOI] [PubMed] [Google Scholar]
  32. Schweizer H. P. Two plasmids, X1918 and Z1918, for easy recovery of the xylE and lacZ reporter genes. Gene. 1993 Nov 30;134(1):89–91. doi: 10.1016/0378-1119(93)90178-6. [DOI] [PubMed] [Google Scholar]
  33. Shortridge V. D., Pato M. L., Vasil A. I., Vasil M. L. Physical mapping of virulence-associated genes in Pseudomonas aeruginosa by transverse alternating-field electrophoresis. Infect Immun. 1991 Oct;59(10):3596–3603. doi: 10.1128/iai.59.10.3596-3603.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  35. Speert D. P., Bond M., Woodman R. C., Curnutte J. T. Infection with Pseudomonas cepacia in chronic granulomatous disease: role of nonoxidative killing by neutrophils in host defense. J Infect Dis. 1994 Dec;170(6):1524–1531. doi: 10.1093/infdis/170.6.1524. [DOI] [PubMed] [Google Scholar]
  36. Stutts M. J., Knowles M. R., Gatzy J. T., Boucher R. C. Oxygen consumption and ouabain binding sites in cystic fibrosis nasal epithelium. Pediatr Res. 1986 Dec;20(12):1316–1320. doi: 10.1203/00006450-198612000-00026. [DOI] [PubMed] [Google Scholar]
  37. Terry J. M., Piña S. E., Mattingly S. J. Environmental conditions which influence mucoid conversion Pseudomonas aeruginosa PAO1. Infect Immun. 1991 Feb;59(2):471–477. doi: 10.1128/iai.59.2.471-477.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Terry J. M., Piña S. E., Mattingly S. J. Role of energy metabolism in conversion of nonmucoid Pseudomonas aeruginosa to the mucoid phenotype. Infect Immun. 1992 Apr;60(4):1329–1335. doi: 10.1128/iai.60.4.1329-1335.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Touati D., Jacques M., Tardat B., Bouchard L., Despied S. Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: protective role of superoxide dismutase. J Bacteriol. 1995 May;177(9):2305–2314. doi: 10.1128/jb.177.9.2305-2314.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  41. Wayne L. G., Diaz G. A. A double staining method for differentiating between two classes of mycobacterial catalase in polyacrylamide electrophoresis gels. Anal Biochem. 1986 Aug 15;157(1):89–92. doi: 10.1016/0003-2697(86)90200-9. [DOI] [PubMed] [Google Scholar]
  42. West S. E., Iglewski B. H. Codon usage in Pseudomonas aeruginosa. Nucleic Acids Res. 1988 Oct 11;16(19):9323–9335. doi: 10.1093/nar/16.19.9323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. von Ossowski I., Mulvey M. R., Leco P. A., Borys A., Loewen P. C. Nucleotide sequence of Escherichia coli katE, which encodes catalase HPII. J Bacteriol. 1991 Jan;173(2):514–520. doi: 10.1128/jb.173.2.514-520.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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