Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Dec;178(23):6802–6809. doi: 10.1128/jb.178.23.6802-6809.1996

Cloning and characterization of the katA gene of Rhizobium meliloti encoding a hydrogen peroxide-inducible catalase.

D Hérouart 1, S Sigaud 1, S Moreau 1, P Frendo 1, D Touati 1, A Puppo 1
PMCID: PMC178579  PMID: 8955300

Abstract

To investigate the involvement of bacterial catalases of the symbiotic gram-negative bacterium Rhizobium meliloti in the development of Medicago-Rhizobium functional nodules, we cloned a putative kat gene by screening a cosmid library with a catalase-specific DNA probe amplified by PCR from the R. meliloti genome. Nucleotide sequence analysis of a 1.8-kb DNA fragment revealed an open reading frame, called katA, encoding a peptide of 562 amino acid residues with a calculated molecular mass of 62.9 kDa. The predicted amino acid sequence showed a high homology with the primary structure of monofunctional catalases from eucaryotes and procaryotes. The katA gene was localized on the chromosome, and the katA gene product was essentially found in the periplasmic space. A katA::Tn5 mutant was obtained and showed a drastic sensitivity to hydrogen peroxide, indicating an essential protective role of KatA. However, neither Nod nor Fix phenotypes were impaired in the mutant, suggesting that KatA is not essential for nodulation and establishment of nitrogen fixation. Exposure to a sublethal concentration of H2O2 enhanced KatA activity (100-fold) and also increased survival to subsequent H2O2 exposure at higher concentrations. No protection is observed in katA::Tn5, indicating that KatA is the major component of an adaptive response.

Full Text

The Full Text of this article is available as a PDF (336.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baron C., Zambryski P. C. The plant response in pathogenesis, symbiosis, and wounding: variations on a common theme? Annu Rev Genet. 1995;29:107–129. doi: 10.1146/annurev.ge.29.120195.000543. [DOI] [PubMed] [Google Scholar]
  2. Becana M., Klucas R. V. Transition metals in legume root nodules: iron-dependent free radical production increases during nodule senescence. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8958–8962. doi: 10.1073/pnas.89.19.8958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bishai W. R., Smith H. O., Barcak G. J. A peroxide/ascorbate-inducible catalase from Haemophilus influenzae is homologous to the Escherichia coli katE gene product. J Bacteriol. 1994 May;176(10):2914–2921. doi: 10.1128/jb.176.10.2914-2921.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Brown S. M., Howell M. L., Vasil M. L., Anderson A. J., Hassett D. J. 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. 1995 Nov;177(22):6536–6544. doi: 10.1128/jb.177.22.6536-6544.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Christman M. F., Storz G., Ames B. N. 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 U S A. 1989 May;86(10):3484–3488. doi: 10.1073/pnas.86.10.3484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Dalton D. A., Post C. J., Langeberg L. Effects of ambient oxygen and of fixed nitrogen on concentrations of glutathione, ascrobate, and associated enzymes in soybean root nodules. Plant Physiol. 1991 Jul;96(3):812–818. doi: 10.1104/pp.96.3.812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davies M. J., Puppo A. Direct detection of a globin-derived radical in leghaemoglobin treated with peroxides. Biochem J. 1992 Jan 1;281(Pt 1):197–201. doi: 10.1042/bj2810197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Demple B., Halbrook J. Inducible repair of oxidative DNA damage in Escherichia coli. Nature. 1983 Aug 4;304(5925):466–468. doi: 10.1038/304466a0. [DOI] [PubMed] [Google Scholar]
  11. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Finan T. M., Hartweig E., LeMieux K., Bergman K., Walker G. C., Signer E. R. General transduction in Rhizobium meliloti. J Bacteriol. 1984 Jul;159(1):120–124. doi: 10.1128/jb.159.1.120-124.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Finan T. M., Kunkel B., De Vos G. F., Signer E. R. Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol. 1986 Jul;167(1):66–72. doi: 10.1128/jb.167.1.66-72.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fita I., Rossmann M. G. The NADPH binding site on beef liver catalase. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1604–1608. doi: 10.1073/pnas.82.6.1604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fita I., Rossmann M. G. The active center of catalase. J Mol Biol. 1985 Sep 5;185(1):21–37. doi: 10.1016/0022-2836(85)90180-9. [DOI] [PubMed] [Google Scholar]
  16. Francis A. J., Alexander M. Catalase activity and nitrogen fixation in legume root nodules. Can J Microbiol. 1972 Jun;18(6):861–864. doi: 10.1139/m72-132. [DOI] [PubMed] [Google Scholar]
  17. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  18. Glazebrook J., Walker G. C. Genetic techniques in Rhizobium meliloti. Methods Enzymol. 1991;204:398–418. doi: 10.1016/0076-6879(91)04021-f. [DOI] [PubMed] [Google Scholar]
  19. Halliwell B., Gutteridge J. M. Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys. 1986 May 1;246(2):501–514. doi: 10.1016/0003-9861(86)90305-x. [DOI] [PubMed] [Google Scholar]
  20. Heimberger A., Eisenstark A. Compartmentalization of catalases in Escherichia coli. Biochem Biophys Res Commun. 1988 Jul 15;154(1):392–397. doi: 10.1016/0006-291x(88)90698-5. [DOI] [PubMed] [Google Scholar]
  21. Honeycutt R. J., McClelland M., Sobral B. W. Physical map of the genome of Rhizobium meliloti 1021. J Bacteriol. 1993 Nov;175(21):6945–6952. doi: 10.1128/jb.175.21.6945-6952.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ivanova A., Miller C., Glinsky G., Eisenstark A. Role of rpoS (katF) in oxyR-independent regulation of hydroperoxidase I in Escherichia coli. Mol Microbiol. 1994 May;12(4):571–578. doi: 10.1111/j.1365-2958.1994.tb01043.x. [DOI] [PubMed] [Google Scholar]
  23. Jenkins D. E., Chaisson S. A., Matin A. Starvation-induced cross protection against osmotic challenge in Escherichia coli. J Bacteriol. 1990 May;172(5):2779–2781. doi: 10.1128/jb.172.5.2779-2781.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jones D. P. Intracellular catalase function: analysis of the catalatic activity by product formation in isolated liver cells. Arch Biochem Biophys. 1982 Apr 1;214(2):806–814. doi: 10.1016/0003-9861(82)90087-x. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Klotz M. G., Kim Y. C., Katsuwon J., Anderson A. J. Cloning, characterization and phenotypic expression in Escherichia coli of catF, which encodes the catalytic subunit of catalase isozyme CatF of Pseudomonas syringae. Appl Microbiol Biotechnol. 1995 Aug-Sep;43(4):656–666. doi: 10.1007/BF00164770. [DOI] [PubMed] [Google Scholar]
  27. Kuykendall L. D., Elkan G. H. Rhizobium japonicum derivatives differing in nitrogen-fixing efficiency and carbohydrate utilization. Appl Environ Microbiol. 1976 Oct;32(4):511–519. doi: 10.1128/aem.32.4.511-519.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Le Rudulier D., Gloux K., Riou N. Identification of an osmotically induced periplasmic glycine betaine-binding protein from Rhizobium meliloti. Biochim Biophys Acta. 1991 Jan 30;1061(2):197–205. doi: 10.1016/0005-2736(91)90285-g. [DOI] [PubMed] [Google Scholar]
  29. Loewen P. C., Hengge-Aronis R. The role of the sigma factor sigma S (KatF) in bacterial global regulation. Annu Rev Microbiol. 1994;48:53–80. doi: 10.1146/annurev.mi.48.100194.000413. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Long S. R. Rhizobium-legume nodulation: life together in the underground. Cell. 1989 Jan 27;56(2):203–214. doi: 10.1016/0092-8674(89)90893-3. [DOI] [PubMed] [Google Scholar]
  32. Mayfield J. E., Duvall M. R. Anomalous phylogenies based on bacterial catalase gene sequences. J Mol Evol. 1996 Apr;42(4):469–471. doi: 10.1007/BF02498641. [DOI] [PubMed] [Google Scholar]
  33. Mehdy M. C. Active Oxygen Species in Plant Defense against Pathogens. Plant Physiol. 1994 Jun;105(2):467–472. doi: 10.1104/pp.105.2.467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Murshudov G. N., Melik-Adamyan W. R., Grebenko A. I., Barynin V. V., Vagin A. A., Vainshtein B. K., Dauter Z., Wilson K. S. Three-dimensional structure of catalase from Micrococcus lysodeikticus at 1.5 A resolution. FEBS Lett. 1992 Nov 9;312(2-3):127–131. doi: 10.1016/0014-5793(92)80919-8. [DOI] [PubMed] [Google Scholar]
  35. Murthy M. R., Reid T. J., 3rd, Sicignano A., Tanaka N., Rossmann M. G. Structure of beef liver catalase. J Mol Biol. 1981 Oct 25;152(2):465–499. doi: 10.1016/0022-2836(81)90254-0. [DOI] [PubMed] [Google Scholar]
  36. Ruvkun G. B., Ausubel F. M. A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85–88. doi: 10.1038/289085a0. [DOI] [PubMed] [Google Scholar]
  37. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sha Z., Stabel T. J., Mayfield J. E. Brucella abortus catalase is a periplasmic protein lacking a standard signal sequence. J Bacteriol. 1994 Dec;176(23):7375–7377. doi: 10.1128/jb.176.23.7375-7377.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Triggs-Raine B. L., Doble B. W., Mulvey M. R., Sorby P. A., Loewen P. C. Nucleotide sequence of katG, encoding catalase HPI of Escherichia coli. J Bacteriol. 1988 Sep;170(9):4415–4419. doi: 10.1128/jb.170.9.4415-4419.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vainshtein B. K., Melik-Adamyan W. R., Barynin V. V., Vagin A. A., Grebenko A. I., Borisov V. V., Bartels K. S., Fita I., Rossmann M. G. Three-dimensional structure of catalase from Penicillium vitale at 2.0 A resolution. J Mol Biol. 1986 Mar 5;188(1):49–61. doi: 10.1016/0022-2836(86)90479-1. [DOI] [PubMed] [Google Scholar]
  41. Yanagi M., Yamasato K. Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer. FEMS Microbiol Lett. 1993 Feb 15;107(1):115–120. doi: 10.1111/j.1574-6968.1993.tb06014.x. [DOI] [PubMed] [Google Scholar]
  42. Yarosh O. K., Charles T. C., Finan T. M. Analysis of C4-dicarboxylate transport genes in Rhizobium meliloti. Mol Microbiol. 1989 Jun;3(6):813–823. doi: 10.1111/j.1365-2958.1989.tb00230.x. [DOI] [PubMed] [Google Scholar]
  43. van Slooten J. C., Cervantes E., Broughton W. J., Wong C. H., Stanley J. Sequence and analysis of the rpoN sigma factor gene of rhizobium sp. strain NGR234, a primary coregulator of symbiosis. J Bacteriol. 1990 Oct;172(10):5563–5574. doi: 10.1128/jb.172.10.5563-5574.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. von Ossowski I., Hausner G., Loewen P. C. Molecular evolutionary analysis based on the amino acid sequence of catalase. J Mol Evol. 1993 Jul;37(1):71–76. doi: 10.1007/BF00170464. [DOI] [PubMed] [Google Scholar]
  45. 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]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES