Skip to main content
SpringerLink
Log in

Induction of enzymes of 2,4-dichlorophenoxyacetate degradation in Burkholderia cepacia 2a and toxicity of metabolic intermediates

  • Original Paper
  • Published:
Biodegradation Aims and scope Submit manuscript

Abstract

Burkholderia cepacia 2a inducibly degraded 2,4-dichlorophenoxyacetate (2,4-D) sequentially via 2,4-dichlorophenol, 3,5-dichlorocatechol, 2,4-dichloromuconate, 2-chloromuconolactone and 2-chloromaleylacetate. Cells grown on nutrient agar or broth grew on 2,4-D-salts only if first passaged on 4-hydroxybenzoate- or succinate-salts agar. Buffered suspensions of 4-hydroxybenzoate-grown cells did not adapt to 2,4-D or 3,5-dichlorocatechol, but responded to 2,4-dichlorophenol at concentrations <0.4 mM. Uptake of 2,4-dichlorophenol by non-induced cells displayed a type S (cooperative uptake) uptake isotherm in which the accelerated uptake of the phenol began before the equivalent of a surface monolayer had been adsorbed, and growth inhibition corresponded with the acquisition of 2.2-fold excess of phenol required for the establishment of the monolayer. No evidence of saturation was seen even at 2 mM 2,4-dichlorophenol, possibly due to absorption by intracellular poly-β-hydroxybutyrate inclusions. With increasing concentration, 2,4-dichlorophenol caused progressive cell membrane damage and, sequentially, leakage of intracellular K+, Pi, ribose and material absorbing light at 260 nm (presumed nucleotide cofactors), until at 0.4 mM, protein synthesis and enzyme induction were forestalled. Growth of non-adapted cells was inhibited by 0.35 mM 2,4-dichlorophenol and 0.25 mM 3,5-dichlorocatechol; the corresponding minimum bacteriocidal concentrations were 0.45 and 0.35 mM. Strain 2a grew in chemostat culture on carbon-limited media containing 2,4-D, with an apparent growth yield coefficient of 0.23, and on 2,4-dichlorophenol. Growth on 3,5-dichlorocatechol did not occur without a supplement of succinate, probably due to accumulation of toxic quantities of quinonoid and polymerisation products. Cells grown on these compounds were active towards all three, but not when grown on other substrates. The enzymes of the pathway therefore appeared to be induced by 3,5-dichlorocatechol or some later metabolite. A possible reason is offered for the environmental persistence of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

2-CL:

2-Chlorodienelactone

2-CM:

2-Chloromaleylacetate

3,5-DCC:

3,5-Dichlorocatechol

2,4-DCM:

2,4-Dichloromuconate

2,4-DCP:

2,4-Dichlorophenol

2,4-D:

2,4-Dichlorophenoxyacetate

2,4,5-T:

2,4,5-Trichlorophenoxyacetate

DMG:

3,3′-Dimethylglutarate

4-HB:

4-Hydroxybenzoate

α-KG:

α-Ketoglutarate

MBC:

Minimum bacteriocidal concentration

MCPA:

4-Chloro-2-methylphenoxyacetate

MIC:

Minimum inhibitory concentration

Pi :

Inorganic phosphate

References

  • Alexander M (1965) Biodegradation: problems of molecular recalcitrance and microbial fallibility. Adv Appl Microbiol 7:35–80

    Article  PubMed  CAS  Google Scholar 

  • Audus LJ (1964) Herbicide behaviour in the soil. In: Audus LJ (ed) The physiology and biochemistry of herbicides. Academic Press, Oxford, pp 163–206

    Google Scholar 

  • Beadle CA, Smith ARW (1982) The purification and properties of 2,4-dichlorophenol hydroxylase from a strain of Acinetobacter species. Eur J Biochem 123:323–332

    Article  PubMed  CAS  Google Scholar 

  • Beadle CA, Kyprianou P, Smith ARW, Weight ML, Yon RJ (1984) Rapid purification of 2,4-dichlorophenol hydroxylase by biospecific desorption from 10-carboxydecylamino-Sepharose. Biochem Int 9:587–593

    PubMed  CAS  Google Scholar 

  • Bean HS, Das A (1966) The absorption by Escherichia coli of phenols and their bactericidal activity. J Pharm Pharmacol 18:107S–113S

    Google Scholar 

  • Benndorf D, Babel W (2002) Assimilatory detoxification of herbicides by Delftia acidovorans MC1: induction of two chlorocatechol 1,2-dioxygenases as a response to chemostress. Microbiology 148:2883–2888

    PubMed  CAS  Google Scholar 

  • Benndorf D, Thiersch M, Loffhagen N, Kunath C, Harms H (2006) Pseudomonas putida KT2440 responds specifically to chlorophenoxy herbicides and their initial metabolites. Proteomics 6:3319–3329

    Article  PubMed  CAS  Google Scholar 

  • Bollag J-M, Briggs GG, Dawson JE, Alexander M (1968a) 2,4-D metabolism: enzymatic degradation of chlorocatechols. J Agric Food Chem 16:829–833

    Article  CAS  Google Scholar 

  • Bollag J-M, Helling CS, Alexander M (1968b) 2,4-D metabolism: enzymatic hydroxylation of chlorinated phenols. J Agric Food Chem 16:826–828

    Article  Google Scholar 

  • Carrington B (1994) The biochemistry of 2,4-D dissimilation by Pseudomonas cepacia. PhD thesis, University of Greenwich, London

  • Cases I, de Lorenzo V (2001) The black cat/white cat principle of signal integration in bacterial promoters. EMBO J 20:1–11

    Article  PubMed  CAS  Google Scholar 

  • Cooper CM, Fernstrom GA, Miller SA (1944) Performance of agitated gas-liquid contactors. Ind Eng Chem 36:504–509

    Article  CAS  Google Scholar 

  • Dawson RMC, Elliott EC, Elliott WH, Jones KM (1969) Data for Biochemical Research. 2nd edn. University Press, Oxford

    Google Scholar 

  • Don RH, Pemberton JM (1981) Properties of six pesticide degradation plasmids isolated from Alcaligenes paradoxus and Alcaligenes eutrophus. J Bacteriol 145:681–686

    PubMed  CAS  Google Scholar 

  • Don RH, Weightman AJ, Knackmuss H-J, Timmis KN (1985) 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 161:85–90

    PubMed  CAS  Google Scholar 

  • Dryer RL, Tammes AR, Routh JI (1957) Determination of phosphorus and phosphatase with N-phenyl-p-phenylenediamine. J Biol Chem 225:177–183

    PubMed  CAS  Google Scholar 

  • Evans WC, Smith BSW, Fernley HN, Davies JI (1971a) Bacterial metabolism of 2,4-dichlorophenoxyacetate. Biochem J 122:543–551

    PubMed  CAS  Google Scholar 

  • Evans WC, Smith BSW, Moss P, Fernley HN (1971b) Bacterial metabolism of 4-chlorophenoxyacetate. Biochem J 122:509–517

    PubMed  CAS  Google Scholar 

  • Filer K, Harker AR (1997) Identification of the inducing agent of the 2,4-dichlorophenoxyacetic acid pathway encoded by plasmid pJP4. Appl Environ Microbiol 63:317–320

    PubMed  CAS  Google Scholar 

  • Fisher PR, Appleton J, Pemberton JM (1978) Isolation and characterisation of the pesticide-degrading plasmid pJP1 from Alcaligenes paradoxus. J Bacteriol 135:798–804

    PubMed  CAS  Google Scholar 

  • Fukumori F, Hausinger RP (1993a) Alcaligenes eutrophus JMP134 2,4-dichlorophenoxyacetate monooxygenase is an α-ketoglutarate-dependent dioxygenase. J Bacteriol 175:2083–2086

    PubMed  CAS  Google Scholar 

  • Fukumori F, Hausinger RP (1993b) Purification and characterisation of 2,4-dichlorophenoxyacetate/α-ketoglutarate dioxygenase. J Biol Chem 268:24311–24317

    PubMed  CAS  Google Scholar 

  • Gaunt JK, Evans WC (1971a) Metabolism of 4-chloro-2-methylphenoxyacetate by a soil pseudomonad: preliminary evidence for the metabolic pathway. Biochem J 122:519–526

    PubMed  CAS  Google Scholar 

  • Gaunt JK, Evans WC (1971b) Metabolism of 4-chloro-2-methylphenoxyacetate by a soil pseudomonad: ring-fission, lactonizing and delactonizing enzymes. Biochem J 122:533–542

    PubMed  CAS  Google Scholar 

  • Gaur BK, Beevers H (1959) Respiratory and associated responses of carrot disks to substituted phenols. Plant Physiol 34:427–432

    PubMed  CAS  Google Scholar 

  • Giles CH, MacEwan TH, Nakhwa SN, Smith D (1960) Studies in adsorption. XI. A system of classification of solution adsorption isotherms, and its use in the diagnosis of adsorption mechanisms and in measurements of specific surface areas of solids. J Chem Soc 3973–3993

  • Hancock REW (1998) Resistance mechanisms in Pseudomonas aeruginosa and other nonfermentative Gram-negative bacteria. Clin Infect Dis 27(Suppl 1):S93-S99

    Article  PubMed  CAS  Google Scholar 

  • Herbert D, Phipps PJ, Strange RE (1971) Chemical analysis of microbial cells. In: Norris JR, Ribbons DW (eds) Methods in Microbiology, vol 5B. Academic Press, London & New York, pp 209–344

  • Hugo WB (1976) Survival of microbes exposed to chemical stress. In: Gray TRG, Postgate JR (eds) The survival of vegetative microbes. 26th Symp Soc Gen Microbiol. University Press, Cambridge, pp 383–413

    Google Scholar 

  • Kaphammer B, Kukor JJ, Olsen RH (1990) Regulation of tfdCDEF by tfdR of the 2,4-dichlorophenoxyacetic acid degradation plasmid pJP4. J Bacteriol 172:2280–2286

    PubMed  CAS  Google Scholar 

  • Kaphammer B, Olsen RH (1990) Cloning and characterisation of tfdS, the repressor-activator gene of tfdB, from the 2,4-dichlorophenoxyacetic acid catabolic plasmid pJP4. J Bacteriol 172:5856–5862

    PubMed  CAS  Google Scholar 

  • Lacoste RJ, Venable SH, Stone JC (1959) Modified 4-aminoantipyrine colorimetric method for phenols. Application to an acrylic monomer. Anal Chem 31:1246–1249

    Article  CAS  Google Scholar 

  • Lambert PA, Hammond SM (1973) Potassium fluxes, first indications of membrane damage in micro-organisms. Biochem Biophys Res Comm 54:796–799

    Article  PubMed  CAS  Google Scholar 

  • Lambert PA, Smith ARW (1976a) Antimicrobial action of dodecyldiethanolamine: induced membrane damage in Escherichia coli. Microbios 15:191–202

    CAS  Google Scholar 

  • Lambert PA, Smith ARW (1976b) Antimicrobial action of dodecyldiethanolamine: activation of ribonuclease I in Escherichia coli. Microbios 17:35–49

    PubMed  CAS  Google Scholar 

  • Liu T, Chapman PJ (1984) Purification and properties of a plasmid-encoded 2,4-dichlorophenol hydroxylase. FEBS Letters 173:314–318

    Article  PubMed  CAS  Google Scholar 

  • Mitchell AC (1924) Osmium tetroxide as a reagent for the estimation of tannins and their derivatives. Analyst 49:162–169

    Article  CAS  Google Scholar 

  • Pemberton JM, Corney B, Don RH (1979) Evolution and spread of pesticide-degrading ability among soil organisms. In: Kimmis KN, Puhler A (eds) Plasmids of Medical, Environmental and Commercial Importance. Elsevier/North Holland Biomedical Press, pp 287–299

  • Pieper DH, Reineke W, Engesser K-H, Knackmuss H-J (1988) Metabolism of 2,4-dichlorophenoxyacetic acid, 4-chloro-2-methylphenoxyacetic acid and 2-methylphenoxyacetic acid by Alcaligenes eutrophus JMP134. Arch Microbiol 150:95–102

    Article  CAS  Google Scholar 

  • Pirt SJ (1975) Principles of microbe and cell cultivation. Blackwell, Oxford, p 37

  • Poh RP, Smith ARW, Bruce IJ (2002) Complete characterisation of Tn5530 from Burkholderia cepacia strain 2a (pIJB1) and studies of 2,4-dichlorophenoxyacetate uptake by the organism. Plasmid 48:1–12

    Article  PubMed  CAS  Google Scholar 

  • Poh R, Xia X, Bruce IJ, Smith ARW (2001) 2,4-dichloro-phenoxyacetate/α-ketoglutarate dioxygenases from Burkholderia cepacia 2a and Ralstonia eutropha JMP134. Microbios 105:43–63

    PubMed  CAS  Google Scholar 

  • Schlömann M (1994) Evolution of chlorocatechol catabolic pathways: conclusions to be drawn from comparisons of lactone hydrolases. Biodegradation 5:301–321

    Article  PubMed  Google Scholar 

  • Schweigert N, Hunziker RW, Escher BI, Eggen RIK (2001) Acute toxicity of (chloro-)catechols and (chloro-)catechol-copper combinations in Escherichia coli corresponds to their membrane toxicity in vitro. Environ Toxicol Chem 20:239–247

    Article  PubMed  CAS  Google Scholar 

  • Sebastianelli A, Bruce IJ (2007) Tn5530 from Burkholderia cepacia strain 2a encodes a chloride channel protein essential for the catabolism of 2,4-dichlorophenoxyacetic acid. Environ Microbiol 9:256–265

    Article  PubMed  CAS  Google Scholar 

  • Sharpee KW, Duxbury JM, Alexander M (1973) 2,4-Dichlorophenoxyacetate metabolism by Arthrobacter sp.: accumulation of a chlorobutenolide. Appl Microbiol 26:445–447

    PubMed  CAS  Google Scholar 

  • Streber WG, Timmis KN, Zenk MH (1987) Analysis, cloning and high-level expression of 2,4-dichlorophenoxyacetate monooxygenase gene tfdA of Alcaligenes eutrophus JMP134. J Bacteriol 169:2950–2955

    PubMed  CAS  Google Scholar 

  • Tiedje JM, Alexander M (1969) Enzymatic cleavage of the ether bond of 2,4-dichlorophenoxyacetate. J Agric Food Chem 17:1080–1084

    Article  CAS  Google Scholar 

  • Tiedje JM, Duxbury JM, Alexander M, Dawson JE (1969) 2,4-D metabolism: pathway of degradation of chlorocatechols by Arthrobacter sp. J Agric Food Chem 17:1021–1026

    Article  CAS  Google Scholar 

  • Umbreit WW, Burris RH, Stauffer JF (1964) Manometric Techniques, 4th edn. Burgess, Minneapolis, p 157

Download references

Author information

Author notes

  1. Carol A. Beadle

    Present address: School of Arts & Humanities, Oxford Brookes University, Headington Campus, Gipsy Lane, Oxford, OX3 0BP, UK

Authors and Affiliations

  1. Department of Life Sciences, School of Science, University of Greenwich, Medway Campus, Pembroke, Central Avenue, Chatham Maritime, Kent, ME4 4TB, UK

    Anthony R. W. Smith & Carol A. Beadle

Authors
  1. Anthony R. W. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carol A. Beadle
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anthony R. W. Smith.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Smith, A.R.W., Beadle, C.A. Induction of enzymes of 2,4-dichlorophenoxyacetate degradation in Burkholderia cepacia 2a and toxicity of metabolic intermediates. Biodegradation 19, 669–681 (2008). https://doi.org/10.1007/s10532-007-9172-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10532-007-9172-0

Keywords

Search

Navigation