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. 1996 Nov;178(21):6123–6132. doi: 10.1128/jb.178.21.6123-6132.1996

Cloning and characterization of the genes encoding nitrilotriacetate monooxygenase of Chelatobacter heintzii ATCC 29600.

H R Knobel 1, T Egli 1, J R van der Meer 1
PMCID: PMC178480  PMID: 8892809

Abstract

A 6.2-kb DNA fragment containing the genes for the nitrilotriacetate (NTA) monooxygenase of Chelatobacter heintzii ATCC 29600 was cloned and characterized by DNA sequencing and expression studies. The nucleotide sequence contained three major open reading frames (ORFs). Two of the ORFs, which were oriented divergently with an intergenic region of 307 bp, could be assigned to the NTA monooxygenase components A and B. The predicted N-terminal amino acid sequences of these ORFs were identical with those determined for the purified components. We therefore named these genes ntaA (for component A of NTA monooxygenase) and ntaB (for component B). The ntaA and ntaB genes could be expressed in Escherichia coli DH5alpha, and the gene products were visualized after Western blotting (immunoblotting) and incubation with polyclonal antibodies against component A or B. By mixing overproduced NtaB from E. coli and purified component A from C. heintzii ATCC 29600, reconstitution of a functional NTA monooxygenase complex was possible. The deduced gene product of ntaA showed only significant homology to SoxA (involved in dibenzothiophene degradation) and to SnaA (involved in pristamycin synthesis); that of ntaB shared weak homologies in one domain with other NADH:flavine mononucleotide oxidoreductases. These homologies provide no conclusive answer as to the possible evolutionary origin of the NTA monooxygenase. The deduced gene product of the third ORF (ORF1) had homology in the N-terminal region with the GntR class of bacterial regulator proteins and therefore may encode a regulator protein, possibly involved in regulation of ntaA and ntaB expression.

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Selected References

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  1. Anderson R. L., Bishop W. E., Campbell R. L. A review of the environmental and mammalian toxicology of nitrilotriacetic acid. Crit Rev Toxicol. 1985;15(1):1–102. doi: 10.3109/10408448509023766. [DOI] [PubMed] [Google Scholar]
  2. Beck C. F., Warren R. A. Divergent promoters, a common form of gene organization. Microbiol Rev. 1988 Sep;52(3):318–326. doi: 10.1128/mr.52.3.318-326.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bedouelle H., Hofnung M. A DNA sequence containing the control regions of the malEFG and malK-lamB operons in Escherichia coli K12. Mol Gen Genet. 1982;185(1):82–87. doi: 10.1007/BF00333794. [DOI] [PubMed] [Google Scholar]
  4. 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]
  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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Crichton R. R., Charloteaux-Wauters M. Iron transport and storage. Eur J Biochem. 1987 May 4;164(3):485–506. doi: 10.1111/j.1432-1033.1987.tb11155.x. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Dairi T., Aisaka K., Katsumata R., Hasegawa M. A self-defense gene homologous to tetracycline effluxing gene essential for antibiotic production in Streptomyces aureofaciens. Biosci Biotechnol Biochem. 1995 Oct;59(10):1835–1841. doi: 10.1271/bbb.59.1835. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. DiMarco A. A., Averhoff B. A., Kim E. E., Ornston L. N. Evolutionary divergence of pobA, the structural gene encoding p-hydroxybenzoate hydroxylase in an Acinetobacter calcoaceticus strain well-suited for genetic analysis. Gene. 1993 Mar 15;125(1):25–33. doi: 10.1016/0378-1119(93)90741-k. [DOI] [PubMed] [Google Scholar]
  12. DiRusso C. C. Nucleotide sequence of the fadR gene, a multifunctional regulator of fatty acid metabolism in Escherichia coli. Nucleic Acids Res. 1988 Aug 25;16(16):7995–8009. doi: 10.1093/nar/16.16.7995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Firestone M. K., Aust S. D., Tiedje J. M. A nitrilotriacetic acid monooxygenase with conditional NADH-oxidase activity. Arch Biochem Biophys. 1978 Oct;190(2):617–623. doi: 10.1016/0003-9861(78)90318-1. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. Haydon D. J., Guest J. R. A new family of bacterial regulatory proteins. FEMS Microbiol Lett. 1991 Apr 15;63(2-3):291–295. doi: 10.1016/0378-1097(91)90101-f. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Kim I. C., Oriel P. J. Characterization of the Bacillus stearothermophilus BR219 phenol hydroxylase gene. Appl Environ Microbiol. 1995 Apr;61(4):1252–1256. doi: 10.1128/aem.61.4.1252-1256.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Neidhart D. J., Kenyon G. L., Gerlt J. A., Petsko G. A. Mandelate racemase and muconate lactonizing enzyme are mechanistically distinct and structurally homologous. Nature. 1990 Oct 18;347(6294):692–694. doi: 10.1038/347692a0. [DOI] [PubMed] [Google Scholar]
  23. Nishikiori T., Okuyama A., Naganawa H., Takita T., Hamada M., Takeuchi T., Aoyagi T., Umezawa H. Production by actinomycetes of (S,S)-N,N'-ethylenediamine-disuccinic acid, an inhibitor of phospholipase C. J Antibiot (Tokyo) 1984 Apr;37(4):426–427. doi: 10.7164/antibiotics.37.426. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Quail M. A., Dempsey C. E., Guest J. R. Identification of a fatty acyl responsive regulator (FarR) in Escherichia coli. FEBS Lett. 1994 Dec 19;356(2-3):183–187. doi: 10.1016/0014-5793(94)01264-4. [DOI] [PubMed] [Google Scholar]
  26. Raman N., DiRusso C. C. Analysis of acyl coenzyme A binding to the transcription factor FadR and identification of amino acid residues in the carboxyl terminus required for ligand binding. J Biol Chem. 1995 Jan 20;270(3):1092–1097. doi: 10.1074/jbc.270.3.1092. [DOI] [PubMed] [Google Scholar]
  27. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Schneider R. P., Zürcher F., Egli T., Hamer G. Determination of nitrilotriacetate in biological matrices using ion exclusion chromatography. Anal Biochem. 1988 Sep;173(2):278–284. doi: 10.1016/0003-2697(88)90190-x. [DOI] [PubMed] [Google Scholar]
  30. Schwacha A., Bender R. A. Nucleotide sequence of the gene encoding the repressor for the histidine utilization genes of Klebsiella aerogenes. J Bacteriol. 1990 Sep;172(9):5477–5481. doi: 10.1128/jb.172.9.5477-5481.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  32. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  33. Trijbels F., Vogels G. D. Degradation of allantoin by Pseudomonas acidovorans. Biochim Biophys Acta. 1966 Feb 14;113(2):292–301. doi: 10.1016/s0926-6593(66)80068-1. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Wanner U., Kemmler J., Weilenmann H. U., Egli T., el-Banna T., Auling G. Isolation and growth of a bacterium able to degrade nitrilotriacetic acid under denitrifying conditions. Biodegradation. 1990;1(1):31–41. doi: 10.1007/BF00117049. [DOI] [PubMed] [Google Scholar]
  36. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  37. van der Meer J. R., de Vos W. M., Harayama S., Zehnder A. J. Molecular mechanisms of genetic adaptation to xenobiotic compounds. Microbiol Rev. 1992 Dec;56(4):677–694. doi: 10.1128/mr.56.4.677-694.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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