Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Feb;179(4):1112–1116. doi: 10.1128/jb.179.4.1112-1116.1997

Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH:flavin mononucleotide oxidoreductase.

Y Xu 1, M W Mortimer 1, T S Fisher 1, M L Kahn 1, F J Brockman 1, L Xun 1
PMCID: PMC178806  PMID: 9023192

Abstract

Nitrilotriacetate (NTA) is an important chelating agent in detergents and has also been used extensively in processing radionuclides. In Chelatobacter heintzii ATCC 29600, biodegradation of NTA is initiated by NTA monooxygenase that oxidizes NTA to iminodiacetate and glyoxylate. The NTA monooxygenase activity requires two component proteins, component A and component B, but the function of each component is unclear. We have cloned and sequenced a gene cluster encoding components A and B (nmoA and nmoB) and two additional open reading frames, nmoR and nmoT, downstream of nmoA. Based on sequence similarities, nmoR and nmoT probably encode a regulatory protein and a transposase, respectively. The NmoA sequence was similar to a monooxygenase that uses reduced flavin mononucleotide (FMNH2) as reductant; NmoB was similar to an NADH:flavin mononucleotide (FMN) oxidoreductase. On the basis of this information, we tested the function of each component. Purified component B was shown to be an NADH:FMN oxidoreductase, and its activity could be separated from that of component A. When the Photobacterium fischeri NADH:FMN oxidoreductase was substituted for component B in the complete reaction, NTA was oxidized, showing that the substrate specificity of the reaction resides in component A. Component A is therefore an NTA monooxygenase that uses FMNH2 and O2 to oxidize NTA, and component B is an NADH:FMN oxidoreductase that provides FMNH2 for NTA oxidation.

Full Text

The Full Text of this article is available as a PDF (483.4 KB).

Selected References

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

  1. Altschul S. F., Lipman D. J. Protein database searches for multiple alignments. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5509–5513. doi: 10.1073/pnas.87.14.5509. [DOI] [PMC free article] [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. Blanc V., Lagneaux D., Didier P., Gil P., Lacroix P., Crouzet J. Cloning and analysis of structural genes from Streptomyces pristinaespiralis encoding enzymes involved in the conversion of pristinamycin IIB to pristinamycin IIA (PIIA): PIIA synthase and NADH:riboflavin 5'-phosphate oxidoreductase. J Bacteriol. 1995 Sep;177(18):5206–5214. doi: 10.1128/jb.177.18.5206-5214.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bolton W. K. Goodpasture's syndrome. Kidney Int. 1996 Nov;50(5):1753–1766. doi: 10.1038/ki.1996.495. [DOI] [PubMed] [Google Scholar]
  5. Cripps R. E., Noble A. S. The metabolism of nitrilotriacetate by a pseudomonad. Biochem J. 1973 Dec;136(4):1059–1068. doi: 10.1042/bj1361059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Denome S. A., Oldfield C., Nash L. J., Young K. D. Characterization of the desulfurization genes from Rhodococcus sp. strain IGTS8. J Bacteriol. 1994 Nov;176(21):6707–6716. doi: 10.1128/jb.176.21.6707-6716.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Denome S. A., Olson E. S., Young K. D. Identification and Cloning of Genes Involved in Specific Desulfurization of Dibenzothiophene by Rhodococcus sp. Strain IGTS8. Appl Environ Microbiol. 1993 Sep;59(9):2837–2843. doi: 10.1128/aem.59.9.2837-2843.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ditta G., Schmidhauser T., Yakobson E., Lu P., Liang X. W., Finlay D. R., Guiney D., Helinski D. R. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid. 1985 Mar;13(2):149–153. doi: 10.1016/0147-619x(85)90068-x. [DOI] [PubMed] [Google Scholar]
  9. Dong J. M., Taylor J. S., Latour D. J., Iuchi S., Lin E. C. Three overlapping lct genes involved in L-lactate utilization by Escherichia coli. J Bacteriol. 1993 Oct;175(20):6671–6678. doi: 10.1128/jb.175.20.6671-6678.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Erickson B. D., Mondello F. J. Nucleotide sequencing and transcriptional mapping of the genes encoding biphenyl dioxygenase, a multicomponent polychlorinated-biphenyl-degrading enzyme in Pseudomonas strain LB400. J Bacteriol. 1992 May;174(9):2903–2912. doi: 10.1128/jb.174.9.2903-2912.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fernández-Moreno M. A., Martínez E., Boto L., Hopwood D. A., Malpartida F. Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase for the antibiotic actinorhodin. J Biol Chem. 1992 Sep 25;267(27):19278–19290. [PubMed] [Google Scholar]
  13. Firestone M. K., Tiedje J. M. Pathway of degradation of nitrilotriacetate by a Pseudomonas species. Appl Environ Microbiol. 1978 May;35(5):955–961. doi: 10.1128/aem.35.5.955-961.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Focht D. D., Joseph H. A. Bacterial degradation of nitrilotriacetic acid (NTA). Can J Microbiol. 1971 Dec;17(12):1553–1556. doi: 10.1139/m71-247. [DOI] [PubMed] [Google Scholar]
  15. Fujita Y., Fujita T. Identification and nucleotide sequence of the promoter region of the Bacillus subtilis gluconate operon. Nucleic Acids Res. 1986 Feb 11;14(3):1237–1252. doi: 10.1093/nar/14.3.1237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gergen J. P., Stern R. H., Wensink P. C. Filter replicas and permanent collections of recombinant DNA plasmids. Nucleic Acids Res. 1979 Dec 20;7(8):2115–2136. doi: 10.1093/nar/7.8.2115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Haydon D. J., Quail M. A., Guest J. R. A mutation causing constitutive synthesis of the pyruvate dehydrogenase complex in Escherichia coli is located within the pdhR gene. FEBS Lett. 1993 Dec 20;336(1):43–47. doi: 10.1016/0014-5793(93)81605-y. [DOI] [PubMed] [Google Scholar]
  18. Knobel H. R., Egli T., van der Meer J. R. Cloning and characterization of the genes encoding nitrilotriacetate monooxygenase of Chelatobacter heintzii ATCC 29600. J Bacteriol. 1996 Nov;178(21):6123–6132. doi: 10.1128/jb.178.21.6123-6132.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lei B., Tu S. C. Gene overexpression, purification, and identification of a desulfurization enzyme from Rhodococcus sp. strain IGTS8 as a sulfide/sulfoxide monooxygenase. J Bacteriol. 1996 Oct;178(19):5699–5705. doi: 10.1128/jb.178.19.5699-5705.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maas R. An improved colony hybridization method with significantly increased sensitivity for detection of single genes. Plasmid. 1983 Nov;10(3):296–298. doi: 10.1016/0147-619x(83)90045-8. [DOI] [PubMed] [Google Scholar]
  22. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  23. Matsutani S., Ohtsubo H., Maeda Y., Ohtsubo E. Isolation and characterization of IS elements repeated in the bacterial chromosome. J Mol Biol. 1987 Aug 5;196(3):445–455. doi: 10.1016/0022-2836(87)90023-4. [DOI] [PubMed] [Google Scholar]
  24. Menou G., Mahillon J., Lecadet M. M., Lereclus D. Structural and genetic organization of IS232, a new insertion sequence of Bacillus thuringiensis. J Bacteriol. 1990 Dec;172(12):6689–6696. doi: 10.1128/jb.172.12.6689-6696.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moos M., Jr, Nguyen N. Y., Liu T. Y. Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support. J Biol Chem. 1988 May 5;263(13):6005–6008. [PubMed] [Google Scholar]
  26. Piddington C. S., Kovacevich B. R., Rambosek J. Sequence and molecular characterization of a DNA region encoding the dibenzothiophene desulfurization operon of Rhodococcus sp. strain IGTS8. Appl Environ Microbiol. 1995 Feb;61(2):468–475. doi: 10.1128/aem.61.2.468-475.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Prieto M. A., Garcia J. L. Molecular characterization of 4-hydroxyphenylacetate 3-hydroxylase of Escherichia coli. A two-protein component enzyme. J Biol Chem. 1994 Sep 9;269(36):22823–22829. [PubMed] [Google Scholar]
  28. Reimmann C., Moore R., Little S., Savioz A., Willetts N. S., Haas D. Genetic structure, function and regulation of the transposable element IS21. Mol Gen Genet. 1989 Feb;215(3):416–424. doi: 10.1007/BF00427038. [DOI] [PubMed] [Google Scholar]
  29. Rogers M. B., Bennett T. K., Payne C. M., Smith C. J. Insertional activation of cepA leads to high-level beta-lactamase expression in Bacteroides fragilis clinical isolates. J Bacteriol. 1994 Jul;176(14):4376–4384. doi: 10.1128/jb.176.14.4376-4384.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Simonet J. M., Schneider D., Volff J. N., Dary A., Decaris B. Genetic instability in Streptomyces ambofaciens: inducibility and associated genome plasticity. Gene. 1992 Jun 15;115(1-2):49–54. doi: 10.1016/0378-1119(92)90539-2. [DOI] [PubMed] [Google Scholar]
  31. Spyrou G., Haggård-Ljungquist E., Krook M., Jörnvall H., Nilsson E., Reichard P. Characterization of the flavin reductase gene (fre) of Escherichia coli and construction of a plasmid for overproduction of the enzyme. J Bacteriol. 1991 Jun;173(12):3673–3679. doi: 10.1128/jb.173.12.3673-3679.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stephens P. E., Darlison M. G., Lewis H. M., Guest J. R. The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the pyruvate dehydrogenase component. Eur J Biochem. 1983 Jun 1;133(1):155–162. doi: 10.1111/j.1432-1033.1983.tb07441.x. [DOI] [PubMed] [Google Scholar]
  33. Thibaut D., Ratet N., Bisch D., Faucher D., Debussche L., Blanche F. Purification of the two-enzyme system catalyzing the oxidation of the D-proline residue of pristinamycin IIB during the last step of pristinamycin IIA biosynthesis. J Bacteriol. 1995 Sep;177(18):5199–5205. doi: 10.1128/jb.177.18.5199-5205.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tiedje J. M., Mason B. B., Warren C. B., Malec E. J. Metabolism of nitrilotriacetate by cells of Pseudomonas species. Appl Microbiol. 1973 May;25(5):811–818. doi: 10.1128/am.25.5.811-818.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tu S. C., Mager H. I. Biochemistry of bacterial bioluminescence. Photochem Photobiol. 1995 Oct;62(4):615–624. doi: 10.1111/j.1751-1097.1995.tb08708.x. [DOI] [PubMed] [Google Scholar]
  36. Uetz T., Schneider R., Snozzi M., Egli T. Purification and characterization of a two-component monooxygenase that hydroxylates nitrilotriacetate from "Chelatobacter" strain ATCC 29600. J Bacteriol. 1992 Feb;174(4):1179–1188. doi: 10.1128/jb.174.4.1179-1188.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zenno S., Saigo K. Identification of the genes encoding NAD(P)H-flavin oxidoreductases that are similar in sequence to Escherichia coli Fre in four species of luminous bacteria: Photorhabdus luminescens, Vibrio fischeri, Vibrio harveyi, and Vibrio orientalis. J Bacteriol. 1994 Jun;176(12):3544–3551. doi: 10.1128/jb.176.12.3544-3551.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES