Applied Microbiology and Biotechnology

, Volume 43, Issue 4, pp 656–666 | Cite as

Cloning, characterization and phenotypic expression in Escherichia coli of catF, which encodes the catalytic subunit of catalase isozyme CatF of Pseudomonas syringae

  • M. G. Klotz
  • Y. C. Kim
  • J. Katsuwon
  • A. J. Anderson
Original Paper


The phytopathogenic, gram-negative bacterium Pseudomonas syringae pv. syringae 61 contains three isozymes of catalase (EC, which have been proposed to play a role in the bacterium's responses to various environmental stresses. To study the role of individual isozymes, the gene coding for the catalytic subunit of one catalase isozyme was cloned from a cosmid library hosted in Escherichia coli DH5α by using a designed catalase-specific DNA probe for the screening. One out of four clones with a catalase-positive genotype was subcloned and a pUC19-based 2.7 × 103-base (2.7-kb) insert subclone, pMK3E5, was used to transform catalase-deficient E. coli strain UM255 (HPI, HPII). The transformants contained a single isozyme of catalase that had electrophoretic and enzymic properties similar to catalase isozyme CatF from P. syringae pv. syringae 61. Analysis of the sequenced 2.7-kb insert DNA revealed six putative open-reading frames (ORF). The 1542-base-pair DNA sequence of ORF2, called catF, encodes a peptide of 513 amino acid residues with a calculated molecular mass of 66.6 kDa. The amino acid sequence deduced from catF had homology to the primary structure of true catalases from mammals, plants, yeasts and bacteria. The activity of the recombinant catalase was inhibited by 3-amino-1,2,4-triazole and azide and stimulated by chloramphenicol. The N terminus contained a signal sequence of 26 amino acids necessary for secretion into the periplasm, a so-far unique property of Pseudomonas catalases.


Molecular Mass Pseudomonas Amino Acid Residue Catalase Azide 
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  1. Anderson AJ, Guerra D (1985) Responses of bean to root colonization with Pseudomonas putida in a hydroponic system. Phytopathology 75:992–995Google Scholar
  2. Bishai WR, Smith HO, Barcak GJ (1994) A peroxide/ascorbate-inducible catalase from Haemophilus influenzae is homologous to the Escherichia coli katE gene product. J Bacteriol 176:2914–2921Google Scholar
  3. Bol DK, Yasbin RE (1991) The isolation, cloning and identification of a vegetative catalase gene from Bacillus subtilis. Gene 109:31–37Google Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254Google Scholar
  5. Brown S, Anderson A, Ohman DE, Hassett DJ (1994) Cloning of the catalase gene (katE) and characterization of the purified enzyme in Pseudomonas aeruginosa: localization of katE near sodA and sodB on the genomic map. In: General meeting of the American Society for Microbiology Abstracts. American Society for Microbiology, Washington, DC, p 34Google Scholar
  6. Claiborne A, Fridovich I (1979) Purification of the o-diasidine peroxidase from Escherichia coli B. J Biol Chem 254:4245–4252Google Scholar
  7. Del Sal G, Manfioletti G, Schneider C (1989) The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagmids, phages, or plasmids suitable for sequencing. Biotechniques 7:514–519Google Scholar
  8. Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395Google Scholar
  9. Dye DW, Bradbury JF, Goto M, Hayward AC, Lelliott RA, Schroth MN (1980) International standards for naming pathovars of phytopathogenic bacteria and a list of pathovar names and pathotype strains. Rev Plant Pathol 59:153–168Google Scholar
  10. Goldberg I, Hochman A (1989) Purification and characterization of a novel type of catalase from the bacterium Klebsiella pneumoniae. Biochim Biophys Acta 991:330–336Google Scholar
  11. Haas A, Brehm K, Kreft J, Goebel W (1991) Cloning, characterization, and expression in Escherichia coli of a gene encoding Listeria seeligeri catalase, a bacterial enzyme highly homologous to mammalian catalases. J Bacteriol 173:5159–5167Google Scholar
  12. Hawley DK, McClure WR (1983) Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 11:2237–2255Google Scholar
  13. Hedrick JL, Smith AJ (1968) Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys 128:155–164Google Scholar
  14. Heym B, Zhang Y, Poulet S, Young D, Cole ST (1993) Characterization of the katG gene encoding a catalase-peroxidase required for the ioniazid susceptibility of Mycobacterium tuberculosis. J Bacteriol 175:4255–4259Google Scholar
  15. Katsuwon J (1993) Characterization of catalases from a root-colonizing bacterium Pseudomonas putida. Ph D Thesis Utah State University, Utah, USAGoogle Scholar
  16. Katsuwon J, Anderson AJ (1989) Responses of plant-colonizing pseudomonads to hydrogen peroxide. Appl Environ Microbiol 55:2985–2989Google Scholar
  17. Katsuwon J, Anderson AJ (1992) Characterization of catalase activities in a root-colonizing isolate of Pseudomonas putida. Can J Microbiol 38:1026–1032Google Scholar
  18. Keppler DL, Baker CJ, Atkinson MM (1989) Active oxygen production during bacteria-induced hypersensitive reaction in tobacco suspension cells. Phytopathology 79:974–978Google Scholar
  19. Klotz MG (1993) The importance of bacterial growth phase for in planta virulence and pathogenicity testing: coordinated stress response regulation in fluorescent pseudomonads? Can J Microbiol 39:948–957Google Scholar
  20. Klotz MG, Anderson AJ (1994) The role of catalase isozymes in the culturability of the root colonizer Pseudomonas putida after exposure to hydrogen peroxide and antibiotics. Can J Microbiol 40:382–387Google Scholar
  21. Klotz MG, Hutcheson SW (1992) Multiple periplasmic catalases in phytopathogenic strains of Pseudomonas syringae. Appl Environ Microbiol 58:2468–2473Google Scholar
  22. Knauf HJ, Vogel RF, Hammes WP (1992) Cloning, sequence, and phenotypic expression of katA, which encodes the catalase of Lactobacillus sake LTH677. Appl Environ Microbiol 58:832–839Google Scholar
  23. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  24. Loewen PC, Stauffer GV (1990) Nucleotide sequence of katG of Salmonella typhimurium LT2 and characterization of its product, hydroperoxidase I. Mol Gen Genet 224:147–151Google Scholar
  25. Loprasert S, Negoro S, Okada H (1988) Thermostable peroxidase from Bacillus stearothermophilus. J Gen Microbiol 134:1971–1976Google Scholar
  26. Margoliash E, Novogrodski A, Schejter A (1960) Irreversible reaction of 3-amino-1:2:4-triazole and related inhibitors with the protein of catalase. Biochem J 74:339–350Google Scholar
  27. Morris SL, Nair J, Rouse DA (1992) The catalase-peroxidase of Mycobacterium intracellulare: nucleotide sequence analysis and expression in Escherichia coli. J Gen Microbiol 138:2363–2370Google Scholar
  28. Murshudov GN, Melik-Adamyan WR, Grebenkow AI, Barynin VV, Vagin AA, Vainshtein BK, Dauter Z, Wilson KS (1992) Three-dimensional structure of catalase from Micrococcus lysodeikticus at 1.5 Å resolution. FEBS Lett 312:127–131Google Scholar
  29. Nadler V, Goldberg I, Hochman A (1986) Comparative study of bacterial catalases. Biochim Biophys Acta 882:234–241Google Scholar
  30. Platt T (1986) Transcription termination and the regulation of gene expression. Annu Rev Biochem 55:339–372Google Scholar
  31. Pugsley AP (1993) The complete secretory pathway in gram-negative bacteria. Microbiol Rev 57:50–108Google Scholar
  32. Ossowski I von, Mulvey MR, Leco PA, Borys A, Loewen PC (1991) Nucleotide sequence of Escherichia coli katE, which encodes catalase HPII. J Bacteriol 173:514–520Google Scholar
  33. Ossowski I von, Hausner G, Loewen PC (1993) Molecular evolutionary analysis based on the amino acid sequence of catalase. J Mol Evol 37:71–76Google Scholar
  34. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A la laboratory manual, 2nd Cold String Harbor Press, Cold Spring Harbor, NYGoogle Scholar
  35. Scandalios JG (1993) Oxygen stress and superoxide dismutases. Plant Physiol 101:7–12Google Scholar
  36. Staskawicz BJ, Dahlbeck D, Keen NT, Napoli C (1987) Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol 169:5789–5794Google Scholar
  37. Sutherland MW (1991) The generation of oxygen radicals during host plant responses to infection. Physiol Mol Plant Pathol 39:79–93Google Scholar
  38. Triggs-Raine BL, Doble BW, Mulvey MR, Sorby PA, Loewen PC (1988) Nucleotide sequence of katG, encoding catalase HPI of Escherichia coli. J Bacteriol 170:4415–4419Google Scholar
  39. Sha Z, Stabel TJ, Mayfield JE (1994) J Bacteriol 176:7375–7377Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • M. G. Klotz
    • 1
  • Y. C. Kim
    • 1
  • J. Katsuwon
    • 1
  • A. J. Anderson
    • 1
  1. 1.Biology DepartmentUniversity of Colorado at DenverDenverUSA

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