Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1996 Mar;62(3):964–973. doi: 10.1128/aem.62.3.964-973.1996

Evidence for acetyl coenzyme A and cinnamoyl coenzyme A in the anaerobic toluene mineralization pathway in Azoarcus tolulyticus Tol-4.

J C Chee-Sanford 1, J W Frost 1, M R Fries 1, J Zhou 1, J M Tiedje 1
PMCID: PMC167860  PMID: 8975623

Abstract

A toluene-degrading denitrifier, Azoarcus tolulyticus Tol-4, was one of eight similar strains isolated from three petroleum-contaminated aquifer sediments. When the strain was grown anaerobically on toluene, 68% of the carbon from toluene was found as CO2 and 30% was found as biomass. Strain Tol-4 had a doubling time of 4.3 h, a Vmax of 50 micromol x min-1 x g of protein-1, and a cellular yield of 49.6 g x mol of toluene-1. Benzoate appeared to be an intermediate, since F-benzoates accumulated from F-toluenes and [14C]benzoate was produced from [14C]toluene in the presence of excess benzoate. Two metabolites, E-phenylitaconic acid (1 to 2%) and benzylsuccinic acid (<1%), accumulated from anaerobic toluene metabolism. These same products were also produced when cells were grown on hydrocinnamic acid and trans-cinnamic acid but were not produced from benzylalcohol, benzaldehyde, benzoate, p-cresol, or their hydroxylated analogs. The evidence supports an anaerobic toluene degradation pathway involving an initial acetyl coenzyme A (acetyl-CoA) attack in strain Tol-4, as proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for another toluene-degrading denitrifier, strain T1. Our findings support a modification of the proposed pathway in which cinnamoyl-CoA follows the oxidation of hydrocinnamoyl-CoA, analogous to the presumed oxidation of benzylsuccinic acid to form E-phenylitaconic acid. Cinnamic acid was detected in Tol-4 cultures growing in the presence of toluene and [14C]acetate. We further propose a second acetyl-CoA addition to cinnamoyl-CoA as the source of benzylsuccinic acid and E-phenylitaconic acid. This pathway is supported by the finding that monofluoroacetate added to toluene-growing cultures resulted in a significant increase in production of benzylsuccinic acid and E-phenylitaconic acid and by the finding that [14C]benzylsuccinic acid was detected after incubation of cells with toluene, [14C]acetate, and cinnamic acid. Evidence for anaerobic toluene metabolism by methyl group oxidation was not found, since benzylsuccinic acid and E-phenylitaconic acid were not detected after incubation with benzylalcohol and benzaldehyde, nor were benzylalcohol and benzaldehyde detected even in 14C trapping experiments.

Full Text

The Full Text of this article is available as a PDF (282.6 KB).

Selected References

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

  1. Altenschmidt U., Fuchs G. Anaerobic degradation of toluene in denitrifying Pseudomonas sp.: indication for toluene methylhydroxylation and benzoyl-CoA as central aromatic intermediate. Arch Microbiol. 1991;156(2):152–158. doi: 10.1007/BF00290990. [DOI] [PubMed] [Google Scholar]
  2. Altenschmidt U., Fuchs G. Anaerobic toluene oxidation to benzyl alcohol and benzaldehyde in a denitrifying Pseudomonas strain. J Bacteriol. 1992 Jul;174(14):4860–4862. doi: 10.1128/jb.174.14.4860-4862.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beller H. R., Grbić-Galić D., Reinhard M. Microbial degradation of toluene under sulfate-reducing conditions and the influence of iron on the process. Appl Environ Microbiol. 1992 Mar;58(3):786–793. doi: 10.1128/aem.58.3.786-793.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brock T. D., Od'ea K. Amorphous ferrous sulfide as a reducing agent for culture of anaerobes. Appl Environ Microbiol. 1977 Feb;33(2):254–256. doi: 10.1128/aem.33.2.254-256.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dean B. J. Recent findings on the genetic toxicology of benzene, toluene, xylenes and phenols. Mutat Res. 1985 Nov;154(3):153–181. doi: 10.1016/0165-1110(85)90016-8. [DOI] [PubMed] [Google Scholar]
  6. Dolfing J., Zeyer J., Binder-Eicher P., Schwarzenbach R. P. Isolation and characterization of a bacterium that mineralizes toluene in the absence of molecular oxygen. Arch Microbiol. 1990;154(4):336–341. doi: 10.1007/BF00276528. [DOI] [PubMed] [Google Scholar]
  7. Edwards E. A., Edwards A. M., Grbić-Galić D. A method for detection of aromatic metabolites at very low concentrations: application to detection of metabolites of anaerobic toluene degradation. Appl Environ Microbiol. 1994 Jan;60(1):323–327. doi: 10.1128/aem.60.1.323-327.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Edwards E. A., Grbić-Galić D. Anaerobic degradation of toluene and o-xylene by a methanogenic consortium. Appl Environ Microbiol. 1994 Jan;60(1):313–322. doi: 10.1128/aem.60.1.313-322.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elder D. J., Kelly D. J. The bacterial degradation of benzoic acid and benzenoid compounds under anaerobic conditions: unifying trends and new perspectives. FEMS Microbiol Rev. 1994 Apr;13(4):441–468. doi: 10.1111/j.1574-6976.1994.tb00061.x. [DOI] [PubMed] [Google Scholar]
  10. Elder D. J., Morgan P., Kelly D. J. Anaerobic degradation of trans-cinnamate and omega-phenylalkane carboxylic acids by the photosynthetic bacterium Rhodopseudomonas palustris: evidence for a beta-oxidation mechanism. Arch Microbiol. 1992;157(2):148–154. doi: 10.1007/BF00245283. [DOI] [PubMed] [Google Scholar]
  11. Evans P. J., Ling W., Goldschmidt B., Ritter E. R., Young L. Y. Metabolites formed during anaerobic transformation of toluene and o-xylene and their proposed relationship to the initial steps of toluene mineralization. Appl Environ Microbiol. 1992 Feb;58(2):496–501. doi: 10.1128/aem.58.2.496-501.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Evans P. J., Mang D. T., Kim K. S., Young L. Y. Anaerobic degradation of toluene by a denitrifying bacterium. Appl Environ Microbiol. 1991 Apr;57(4):1139–1145. doi: 10.1128/aem.57.4.1139-1145.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Evans W. C., Fuchs G. Anaerobic degradation of aromatic compounds. Annu Rev Microbiol. 1988;42:289–317. doi: 10.1146/annurev.mi.42.100188.001445. [DOI] [PubMed] [Google Scholar]
  14. Frazer A. C., Ling W., Young L. Y. Substrate induction and metabolite accumulation during anaerobic toluene utilization by the denitrifying strain T1. Appl Environ Microbiol. 1993 Sep;59(9):3157–3160. doi: 10.1128/aem.59.9.3157-3160.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fries M. R., Zhou J., Chee-Sanford J., Tiedje J. M. Isolation, characterization, and distribution of denitrifying toluene degraders from a variety of habitats. Appl Environ Microbiol. 1994 Aug;60(8):2802–2810. doi: 10.1128/aem.60.8.2802-2810.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grbić-Galić D., Vogel T. M. Transformation of toluene and benzene by mixed methanogenic cultures. Appl Environ Microbiol. 1987 Feb;53(2):254–260. doi: 10.1128/aem.53.2.254-260.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lochmeyer C., Koch J., Fuchs G. Anaerobic degradation of 2-aminobenzoic acid (anthranilic acid) via benzoyl-coenzyme A (CoA) and cyclohex-1-enecarboxyl-CoA in a denitrifying bacterium. J Bacteriol. 1992 Jun;174(11):3621–3628. doi: 10.1128/jb.174.11.3621-3628.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lovley D. R., Lonergan D. J. Anaerobic Oxidation of Toluene, Phenol, and p-Cresol by the Dissimilatory Iron-Reducing Organism, GS-15. Appl Environ Microbiol. 1990 Jun;56(6):1858–1864. doi: 10.1128/aem.56.6.1858-1864.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Migaud M. E., Chee-Sanford J. C., Tiedje J. M., Frost J. W. Benzylfumaric, benzylmaleic, and Z- and E-phenylitaconic acids: synthesis, characterization, and correlation with a metabolite generated by Azoarcus tolulyticus Tol-4 during anaerobic toluene degradation. Appl Environ Microbiol. 1996 Mar;62(3):974–978. doi: 10.1128/aem.62.3.974-978.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schocher R. J., Seyfried B., Vazquez F., Zeyer J. Anaerobic degradation of toluene by pure cultures of denitrifying bacteria. Arch Microbiol. 1991;157(1):7–12. doi: 10.1007/BF00245327. [DOI] [PubMed] [Google Scholar]
  21. Seyfried B., Glod G., Schocher R., Tschech A., Zeyer J. Initial reactions in the anaerobic oxidation of toluene and m-xylene by denitrifying bacteria. Appl Environ Microbiol. 1994 Nov;60(11):4047–4052. doi: 10.1128/aem.60.11.4047-4052.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tschech A., Fuchs G. Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads. Arch Microbiol. 1987 Sep;148(3):213–217. doi: 10.1007/BF00414814. [DOI] [PubMed] [Google Scholar]
  23. Vogel T. M., Grbìc-Galìc D. Incorporation of Oxygen from Water into Toluene and Benzene during Anaerobic Fermentative Transformation. Appl Environ Microbiol. 1986 Jul;52(1):200–202. doi: 10.1128/aem.52.1.200-202.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Zhou J., Fries M. R., Chee-Sanford J. C., Tiedje J. M. Phylogenetic analyses of a new group of denitrifiers capable of anaerobic growth of toluene and description of Azoarcus tolulyticus sp. nov. Int J Syst Bacteriol. 1995 Jul;45(3):500–506. doi: 10.1099/00207713-45-3-500. [DOI] [PubMed] [Google Scholar]

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

RESOURCES