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. 1996 Jul;62(7):2470–2476. doi: 10.1128/aem.62.7.2470-2476.1996

Capture of a catabolic plasmid that encodes only 2,4-dichlorophenoxyacetic acid:alpha-ketoglutaric acid dioxygenase (TfdA) by genetic complementation.

E M Top 1, O V Maltseva 1, L J Forney 1
PMCID: PMC168029  PMID: 8779586

Abstract

The modular pathway for the metabolism of 2,4-dichlorophenoxyacetic acid (2,4-D) encoded on plasmid pJP4 of Alcaligenes eutrophus JMP134 appears to be an example in which two genes, tfdA and tfdB, have been recruited during the evolution of a catabolic pathway. The products of these genes act to convert 2,4-D to a chloro-substituted catechol that can be further metabolized by enzymes of a modified ortho-cleavage pathway encoded by tfdCDEF. Given that modified ortho-cleavage pathways are comparatively common and widely distributed among bacteria, we sought to determine if microbial populations in soil carry tfdA on plasmid vectors that lack tfdCDEF or tfdB. To capture such plasmids from soil populations, we used a recipient strain of A. eutrophus that was rifampin resistant and carried a derivative of plasmid pJP4 (called pBH501aE) in which the tfdA had been deleted. Upon mating with mixed bacterial populations from soil treated with 2,4-D, transconjugants that were resistant to rifampin yet able to grow on 2,4-D were obtained. Among the transconjugants obtained were clones that contained a ca. 75-kb plasmid, pEMT8. Bacterial hosts that carried this plasmid in addition to pBH501aE metabolized 2,4-D, whereas strains with only pEMT8 did not. Southern hybridization showed that pEMT8 encoded a gene with a low level of similarity to the tfdA gene from plasmid pJP4. Using oligonucleotide primers based on known tfdA sequences, we amplified a 330-bp fragment of the gene and determined that it was 77% similar to the tfdA gene of plasmid pJP4 and 94% similar to tfdA from Burkholderia sp. strain RASC. Plasmid pEMT8 lacked genes that exhibited significant levels of homology to tfdB and tfdCDEF. Moreover, cell extracts from A. eutrophus(pEMT8) cultures did not exhibit TfdB, TfdC, TfdD, and TfdE activities, whereas cell extracts from A. eutrophus(pEMT8)(pBH501aE) cultures did. These data suggest that pEMT8 encodes only tfdA and that this gene can effectively complement the tfdA deletion mutation of pBH501aE.

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Selected References

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  1. Amy P. S., Schulke J. W., Frazier L. M., Seidler R. J. Characterization of aquatic bacteria and cloning of genes specifying partial degradation of 2,4-dichlorophenoxyacetic acid. Appl Environ Microbiol. 1985 May;49(5):1237–1245. doi: 10.1128/aem.49.5.1237-1245.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bachmann B. J. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev. 1972 Dec;36(4):525–557. doi: 10.1128/br.36.4.525-557.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beadle C. A., Smith A. R. The purification and properties of 2,4-dichlorophenol hydroxylase from a strain of Acinetobacter species. Eur J Biochem. 1982 Apr 1;123(2):323–332. doi: 10.1111/j.1432-1033.1982.tb19771.x. [DOI] [PubMed] [Google Scholar]
  4. Burlage R. S., Bemis L. A., Layton A. C., Sayler G. S., Larimer F. Comparative genetic organization of incompatibility group P degradative plasmids. J Bacteriol. 1990 Dec;172(12):6818–6825. doi: 10.1128/jb.172.12.6818-6825.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Coco W. M., Rothmel R. K., Henikoff S., Chakrabarty A. M. Nucleotide sequence and initial functional characterization of the clcR gene encoding a LysR family activator of the clcABD chlorocatechol operon in Pseudomonas putida. J Bacteriol. 1993 Jan;175(2):417–427. doi: 10.1128/jb.175.2.417-427.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Datta N., Hedges R. W., Shaw E. J., Sykes R. B., Richmond M. H. Properties of an R factor from Pseudomonas aeruginosa. J Bacteriol. 1971 Dec;108(3):1244–1249. doi: 10.1128/jb.108.3.1244-1249.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Don R. H., Pemberton J. M. Properties of six pesticide degradation plasmids isolated from Alcaligenes paradoxus and Alcaligenes eutrophus. J Bacteriol. 1981 Feb;145(2):681–686. doi: 10.1128/jb.145.2.681-686.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Don R. H., Weightman A. J., Knackmuss H. J., Timmis K. N. Transposon mutagenesis and cloning analysis of the pathways for degradation of 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate in Alcaligenes eutrophus JMP134(pJP4). J Bacteriol. 1985 Jan;161(1):85–90. doi: 10.1128/jb.161.1.85-90.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dorn E., Knackmuss H. J. Chemical structure and biodegradability of halogenated aromatic compounds. Two catechol 1,2-dioxygenases from a 3-chlorobenzoate-grown pseudomonad. Biochem J. 1978 Jul 15;174(1):73–84. doi: 10.1042/bj1740073. [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. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Friedrich B., Meyer M., Schlegel H. G. Transfer and expression of the herbicide-degrading plasmid pJP4 in aerobic autotrophic bacteria. Arch Microbiol. 1983 Feb;134(2):92–97. doi: 10.1007/BF00407938. [DOI] [PubMed] [Google Scholar]
  13. Fukumori F., Hausinger R. P. Alcaligenes eutrophus JMP134 "2,4-dichlorophenoxyacetate monooxygenase" is an alpha-ketoglutarate-dependent dioxygenase. J Bacteriol. 1993 Apr;175(7):2083–2086. doi: 10.1128/jb.175.7.2083-2086.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fukumori F., Hausinger R. P. Purification and characterization of 2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenase. J Biol Chem. 1993 Nov 15;268(32):24311–24317. [PubMed] [Google Scholar]
  15. Fulthorpe R. R., McGowan C., Maltseva O. V., Holben W. E., Tiedje J. M. 2,4-Dichlorophenoxyacetic acid-degrading bacteria contain mosaics of catabolic genes. Appl Environ Microbiol. 1995 Sep;61(9):3274–3281. doi: 10.1128/aem.61.9.3274-3281.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fulthorpe R. R., Rhodes A. N., Tiedje J. M. Pristine soils mineralize 3-chlorobenzoate and 2,4-dichlorophenoxyacetate via different microbial populations. Appl Environ Microbiol. 1996 Apr;62(4):1159–1166. doi: 10.1128/aem.62.4.1159-1166.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Holben W. E., Schroeter B. M., Calabrese V. G., Olsen R. H., Kukor J. K., Biederbeck V. O., Smith A. E., Tiedje J. M. Gene probe analysis of soil microbial populations selected by amendment with 2,4-dichlorophenoxyacetic acid. Appl Environ Microbiol. 1992 Dec;58(12):3941–3948. doi: 10.1128/aem.58.12.3941-3948.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kado C. I., Liu S. T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol. 1981 Mar;145(3):1365–1373. doi: 10.1128/jb.145.3.1365-1373.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kaphammer B., Kukor J. J., Olsen R. H. Regulation of tfdCDEF by tfdR of the 2,4-dichlorophenoxyacetic acid degradation plasmid pJP4. J Bacteriol. 1990 May;172(5):2280–2286. doi: 10.1128/jb.172.5.2280-2286.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kaphammer B., Olsen R. H. Cloning and characterization of tfdS, the repressor-activator gene of tfdB, from the 2,4-dichlorophenoxyacetic acid catabolic plasmid pJP4. J Bacteriol. 1990 Oct;172(10):5856–5862. doi: 10.1128/jb.172.10.5856-5862.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kasberg T., Daubaras D. L., Chakrabarty A. M., Kinzelt D., Reineke W. Evidence that operons tcb, tfd, and clc encode maleylacetate reductase, the fourth enzyme of the modified ortho pathway. J Bacteriol. 1995 Jul;177(13):3885–3889. doi: 10.1128/jb.177.13.3885-3889.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kuhm A. E., Schlömann M., Knackmuss H. J., Pieper D. H. Purification and characterization of dichloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134. Biochem J. 1990 Mar 15;266(3):877–883. [PMC free article] [PubMed] [Google Scholar]
  23. Lejeune P., Mergeay M., Van Gijsegem F., Faelen M., Gerits J., Toussaint A. Chromosome transfer and R-prime plasmid formation mediated by plasmid pULB113 (RP4::mini-Mu) in Alcaligenes eutrophus CH34 and Pseudomonas fluorescens 6.2. J Bacteriol. 1983 Sep;155(3):1015–1026. doi: 10.1128/jb.155.3.1015-1026.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leveau J. H., de Vos W. M., van der Meer J. R. Analysis of the binding site of the LysR-type transcriptional activator TcbR on the tcbR and tcbC divergent promoter sequences. J Bacteriol. 1994 Apr;176(7):1850–1856. doi: 10.1128/jb.176.7.1850-1856.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Matheson V. G., Forney L. J., Suwa Y., Nakatsu C. H., Sexstone A. J., Holben W. E. Evidence for Acquisition in Nature of a Chromosomal 2,4-Dichlorophenoxyacetic Acid/(alpha)-Ketoglutarate Dioxygenase Gene by Different Burkholderia spp. Appl Environ Microbiol. 1996 Jul;62(7):2457–2463. doi: 10.1128/aem.62.7.2457-2463.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matrubutham U., Harker A. R. Analysis of duplicated gene sequences associated with tfdR and tfdS in Alcaligenes eutrophus JMP134. J Bacteriol. 1994 Apr;176(8):2348–2353. doi: 10.1128/jb.176.8.2348-2353.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nurk A., Kasak L., Kivisaar M. Sequence of the gene (pheA) encoding phenol monooxygenase from Pseudomonas sp. EST1001: expression in Escherichia coli and Pseudomonas putida. Gene. 1991 Jun 15;102(1):13–18. doi: 10.1016/0378-1119(91)90531-f. [DOI] [PubMed] [Google Scholar]
  28. Perkins E. J., Gordon M. P., Caceres O., Lurquin P. F. Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4. J Bacteriol. 1990 May;172(5):2351–2359. doi: 10.1128/jb.172.5.2351-2359.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pieper D. H., Stadler-Fritzsche K., Engesser K. H., Knackmuss H. J. Metabolism of 2-chloro-4-methylphenoxyacetate by Alcaligenes eutrophus JMP 134. Arch Microbiol. 1993;160(3):169–178. doi: 10.1007/BF00249121. [DOI] [PubMed] [Google Scholar]
  30. Schlömann M. Evolution of chlorocatechol catabolic pathways. Conclusions to be drawn from comparisons of lactone hydrolases. Biodegradation. 1994 Dec;5(3-4):301–321. doi: 10.1007/BF00696467. [DOI] [PubMed] [Google Scholar]
  31. Schlömann M., Schmidt E., Knackmuss H. J. Different types of dienelactone hydrolase in 4-fluorobenzoate-utilizing bacteria. J Bacteriol. 1990 Sep;172(9):5112–5118. doi: 10.1128/jb.172.9.5112-5118.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schmidt E., Remberg G., Knackmuss H. J. Chemical structure and biodegradability of halogenated aromatic compounds. Halogenated muconic acids as intermediates. Biochem J. 1980 Oct 15;192(1):331–337. doi: 10.1042/bj1920331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Streber W. R., Timmis K. N., Zenk M. H. Analysis, cloning, and high-level expression of 2,4-dichlorophenoxyacetate monooxygenase gene tfdA of Alcaligenes eutrophus JMP134. J Bacteriol. 1987 Jul;169(7):2950–2955. doi: 10.1128/jb.169.7.2950-2955.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Suwa Y., Wright A. D., Fukimori F., Nummy K. A., Hausinger R. P., Holben W. E., Forney L. J. Characterization of a chromosomally encoded 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase from Burkholderia sp. strain RASC. Appl Environ Microbiol. 1996 Jul;62(7):2464–2469. doi: 10.1128/aem.62.7.2464-2469.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Top E. M., Holben W. E., Forney L. J. Characterization of diverse 2,4-dichlorophenoxyacetic acid-degradative plasmids isolated from soil by complementation. Appl Environ Microbiol. 1995 May;61(5):1691–1698. doi: 10.1128/aem.61.5.1691-1698.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Top E., Mergeay M., Springael D., Verstraete W. Gene escape model: transfer of heavy metal resistance genes from Escherichia coli to Alcaligenes eutrophus on agar plates and in soil samples. Appl Environ Microbiol. 1990 Aug;56(8):2471–2479. doi: 10.1128/aem.56.8.2471-2479.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Versalovic J., Koeuth T., Lupski J. R. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 1991 Dec 25;19(24):6823–6831. doi: 10.1093/nar/19.24.6823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. de Bruijn F. J. Use of repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus) sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Appl Environ Microbiol. 1992 Jul;58(7):2180–2187. doi: 10.1128/aem.58.7.2180-2187.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. de Lorenzo V., Herrero M., Jakubzik U., Timmis K. N. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990 Nov;172(11):6568–6572. doi: 10.1128/jb.172.11.6568-6572.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. van der Meer J. R., Frijters A. C., Leveau J. H., Eggen R. I., Zehnder A. J., de Vos W. M. Characterization of the Pseudomonas sp. strain P51 gene tcbR, a LysR-type transcriptional activator of the tcbCDEF chlorocatechol oxidative operon, and analysis of the regulatory region. J Bacteriol. 1991 Jun;173(12):3700–3708. doi: 10.1128/jb.173.12.3700-3708.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. van der Meer J. R., Zehnder A. J., de Vos W. M. Identification of a novel composite transposable element, Tn5280, carrying chlorobenzene dioxygenase genes of Pseudomonas sp. strain P51. J Bacteriol. 1991 Nov;173(22):7077–7083. doi: 10.1128/jb.173.22.7077-7083.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. van der Meer J. R., van Neerven A. R., de Vries E. J., de Vos W. M., Zehnder A. J. Cloning and characterization of plasmid-encoded genes for the degradation of 1,2-dichloro-, 1,4-dichloro-, and 1,2,4-trichlorobenzene of Pseudomonas sp. strain P51. J Bacteriol. 1991 Jan;173(1):6–15. doi: 10.1128/jb.173.1.6-15.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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