Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1995 Dec;61(12):4284–4290. doi: 10.1128/aem.61.12.4284-4290.1995

Cloning and characterization of the genes for p-nitrobenzoate degradation from Pseudomonas pickettii YH105.

A V Yabannavar 1, G J Zylstra 1
PMCID: PMC167739  PMID: 8534095

Abstract

Pseudomonas pickettii YH105 was isolated for its ability to utilize p-nitrobenzoate as the sole source of carbon, nitrogen, and energy. Degradation of p-nitrobenzoate by this strain proceeds through a reductive route as evidenced by the accumulation of ammonia in the culture medium during growth on p-nitrobenzoate. Enzyme assays and high-performance liquid chromatography (HPLC) analysis of culture supernatants indicate that p-nitrobenzoate is degraded through p-hydroxylaminobenzoate and protocatechuate. In order to clone the genes responsible for the initial steps in the catabolic pathway, a cosmid library was constructed with P. pickettii YH105 genomic DNA. The library was screened for clones capable of transforming p-nitrobenzoate to protocatechuate, using a plate assay specific for diphenolic compounds. HPLC analysis of culture supernatants confirmed that the cosmid clones did indeed produce protocatechuate from p-nitrobenzoate. Five positive cosmid clones that possessed this activity were identified. Restriction digests of the cosmid clones indicated that all of the clones had two EcoRI fragments in common (3.9 and 1.0 kb). One of these cosmid clones, designated pGJZ1601, was chosen for further analysis. Subcloning and activity assay experiments localized the genes responsible for the conversion of p-nitrobenzoate to protocatechuate to a 1.4-kb SalI-SphI DNA fragment. Further subcloning experiments localized the gene coding for p-nitrobenzoate reductase, responsible for the first enzymatic step in the catabolic pathway, to a 0.8-kb SalI-ApaI DNA fragment. The gene for the second step in the catabolic pathway, coding for hydroxylaminolyase, was located adjacent to the gene for the p-nitrobenzoate reductase.

Full Text

The Full Text of this article is available as a PDF (219.4 KB).

Selected References

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

  1. Angermaier L., Simon H. On nitroaryl reductase activities in several Clostridia. Hoppe Seylers Z Physiol Chem. 1983 Dec;364(12):1653–1663. doi: 10.1515/bchm2.1983.364.2.1653. [DOI] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blasco R., Castillo F. Characterization of a nitrophenol reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1. Appl Environ Microbiol. 1993 Jun;59(6):1774–1778. doi: 10.1128/aem.59.6.1774-1778.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Bryant C., DeLuca M. Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae. J Biol Chem. 1991 Mar 5;266(7):4119–4125. [PubMed] [Google Scholar]
  7. Bryant C., Hubbard L., McElroy W. D. Cloning, nucleotide sequence, and expression of the nitroreductase gene from Enterobacter cloacae. J Biol Chem. 1991 Mar 5;266(7):4126–4130. [PubMed] [Google Scholar]
  8. CAIN R. B. The microbial metabolism of nitro-aromatic compounds. J Gen Microbiol. 1958 Aug;19(1):1–14. doi: 10.1099/00221287-19-1-1. [DOI] [PubMed] [Google Scholar]
  9. DURHAM N. N. Studies on the metabolism of p-nitrobenzoic acid. Can J Microbiol. 1958 Apr;4(2):141–148. doi: 10.1139/m58-015. [DOI] [PubMed] [Google Scholar]
  10. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gorontzy T., Drzyzga O., Kahl M. W., Bruns-Nagel D., Breitung J., von Loew E., Blotevogel K. H. Microbial degradation of explosives and related compounds. Crit Rev Microbiol. 1994;20(4):265–284. doi: 10.3109/10408419409113559. [DOI] [PubMed] [Google Scholar]
  12. Groenewegen P. E., Breeuwer P., van Helvoort J. M., Langenhoff A. A., de Vries F. P., de Bont J. A. Novel degradative pathway of 4-nitrobenzoate in Comamonas acidovorans NBA-10. J Gen Microbiol. 1992 Aug;138(Pt 8):1599–1605. doi: 10.1099/00221287-138-8-1599. [DOI] [PubMed] [Google Scholar]
  13. Haigler B. E., Spain J. C. Biodegradation of 4-nitrotoluene by Pseudomonas sp. strain 4NT. Appl Environ Microbiol. 1993 Jul;59(7):2239–2243. doi: 10.1128/aem.59.7.2239-2243.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Haigler B. E., Wallace W. H., Spain J. C. Biodegradation of 2-nitrotoluene by Pseudomonas sp. strain JS42. Appl Environ Microbiol. 1994 Sep;60(9):3466–3469. doi: 10.1128/aem.60.9.3466-3469.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  16. Higson F. K. Microbial degradation of nitroaromatic compounds. Adv Appl Microbiol. 1992;37:1–19. doi: 10.1016/s0065-2164(08)70250-8. [DOI] [PubMed] [Google Scholar]
  17. Hohn B., Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene. 1980 Nov;11(3-4):291–298. doi: 10.1016/0378-1119(80)90069-4. [DOI] [PubMed] [Google Scholar]
  18. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jain R. K., Dreisbach J. H., Spain J. C. Biodegradation of p-nitrophenol via 1,2,4-benzenetriol by an Arthrobacter sp. Appl Environ Microbiol. 1994 Aug;60(8):3030–3032. doi: 10.1128/aem.60.8.3030-3032.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. KE Y. H., GEE L. L., DURHAM N. N. Mechanism involved in the metabolism of nitrophenyl-carboxylic acid compounds by microorganisms. J Bacteriol. 1959 May;77(5):593–598. doi: 10.1128/jb.77.5.593-598.1959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Keen N. T., Tamaki S., Kobayashi D., Trollinger D. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene. 1988 Oct 15;70(1):191–197. doi: 10.1016/0378-1119(88)90117-5. [DOI] [PubMed] [Google Scholar]
  22. Kinouchi T., Ohnishi Y. Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis. Appl Environ Microbiol. 1983 Sep;46(3):596–604. doi: 10.1128/aem.46.3.596-604.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kitamura S., Narai N., Tatsumi K. Studies on bacterial nitroreductases. Enzymes involved in reduction of aromatic nitro compounds in Escherichia coli. J Pharmacobiodyn. 1983 Jan;6(1):18–24. doi: 10.1248/bpb1978.6.18. [DOI] [PubMed] [Google Scholar]
  24. LENNOX E. S. Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1955 Jul;1(2):190–206. doi: 10.1016/0042-6822(55)90016-7. [DOI] [PubMed] [Google Scholar]
  25. Lenke H., Knackmuss H. J. Initial hydrogenation during catabolism of picric acid by Rhodococcus erythropolis HL 24-2. Appl Environ Microbiol. 1992 Sep;58(9):2933–2937. doi: 10.1128/aem.58.9.2933-2937.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Marvin-Sikkema F. D., de Bont J. A. Degradation of nitroaromatic compounds by microorganisms. Appl Microbiol Biotechnol. 1994 Dec;42(4):499–507. doi: 10.1007/BF00173912. [DOI] [PubMed] [Google Scholar]
  27. McCormick N. G., Feeherry F. E., Levinson H. S. Microbial transformation of 2,4,6-trinitrotoluene and other nitroaromatic compounds. Appl Environ Microbiol. 1976 Jun;31(6):949–958. doi: 10.1128/aem.31.6.949-958.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nishino S. F., Spain J. C. Degradation of nitrobenzene by a Pseudomonas pseudoalcaligenes. Appl Environ Microbiol. 1993 Aug;59(8):2520–2525. doi: 10.1128/aem.59.8.2520-2525.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Olsen R. H., DeBusscher G., McCombie W. R. Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome. J Bacteriol. 1982 Apr;150(1):60–69. doi: 10.1128/jb.150.1.60-69.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Parke D. Application of p-Toluidine in Chromogenic Detection of Catechol and Protocatechuate, Diphenolic Intermediates in Catabolism of Aromatic Compounds. Appl Environ Microbiol. 1992 Aug;58(8):2694–2697. doi: 10.1128/aem.58.8.2694-2697.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rafil F., Franklin W., Heflich R. H., Cerniglia C. E. Reduction of nitroaromatic compounds by anaerobic bacteria isolated from the human gastrointestinal tract. Appl Environ Microbiol. 1991 Apr;57(4):962–968. doi: 10.1128/aem.57.4.962-968.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rhys-Williams W., Taylor S. C., Williams P. A. A novel pathway for the catabolism of 4-nitrotoluene by Pseudomonas. J Gen Microbiol. 1993 Sep;139(9):1967–1972. doi: 10.1099/00221287-139-9-1967. [DOI] [PubMed] [Google Scholar]
  33. Rieble S., Joshi D. K., Gold M. H. Aromatic nitroreductase from the basidiomycete Phanerochaete chrysosporium. Biochem Biophys Res Commun. 1994 Nov 30;205(1):298–304. doi: 10.1006/bbrc.1994.2664. [DOI] [PubMed] [Google Scholar]
  34. Spain J. C., Gibson D. T. Pathway for Biodegradation of p-Nitrophenol in a Moraxella sp. Appl Environ Microbiol. 1991 Mar;57(3):812–819. doi: 10.1128/aem.57.3.812-819.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Spanggord R. J., Spain J. C., Nishino S. F., Mortelmans K. E. Biodegradation of 2,4-dinitrotoluene by a Pseudomonas sp. Appl Environ Microbiol. 1991 Nov;57(11):3200–3205. doi: 10.1128/aem.57.11.3200-3205.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stanier R. Y., Palleroni N. J., Doudoroff M. The aerobic pseudomonads: a taxonomic study. J Gen Microbiol. 1966 May;43(2):159–271. doi: 10.1099/00221287-43-2-159. [DOI] [PubMed] [Google Scholar]
  37. Suen W. C., Spain J. C. Cloning and characterization of Pseudomonas sp. strain DNT genes for 2,4-dinitrotoluene degradation. J Bacteriol. 1993 Mar;175(6):1831–1837. doi: 10.1128/jb.175.6.1831-1837.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Vorbeck C., Lenke H., Fischer P., Knackmuss H. J. Identification of a hydride-Meisenheimer complex as a metabolite of 2,4,6-trinitrotoluene by a Mycobacterium strain. J Bacteriol. 1994 Feb;176(3):932–934. doi: 10.1128/jb.176.3.932-934.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Watanabe M., Ishidate M., Jr, Nohmi T. Nucleotide sequence of Salmonella typhimurium nitroreductase gene. Nucleic Acids Res. 1990 Feb 25;18(4):1059–1059. doi: 10.1093/nar/18.4.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zeyer J., Kocher H. P., Timmis K. N. Influence of para-substituents on the oxidative metabolism of o-nitrophenols by Pseudomonas putida B2. Appl Environ Microbiol. 1986 Aug;52(2):334–339. doi: 10.1128/aem.52.2.334-339.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES