EVIDENCE FOR ACETYL-COENZYME A AND CINNAMOYL COENZYME-A IN THE ANAEROBIC TOLUENE MINERALIZATION PATHWAY IN AZOARCUS-TOLULYTICUS TOL-4

A toluene-degrading denitrifier, Azoarcus tolulyticus Tol-4, was one o f eight similar strains isolated from three petroleum-contaminated aqu ifer sediments. When the strain was grown anaerobically on toluene, 68 % of the carbon from toluene was found as CO2 and 30% was found as bio mass, Strain Tol-4 had a doubling time of 4.3 h, a V'(max) of 50 mu mo l . min(-1). g of protein(-1), and a cellular yield of 49.6 g . mol of toluene(-1). Benzoate appeared to be an intermediate, since F-benzoat es accumulated from E-toluenes and [C-14]benzoate was produced from [C -14]toluene in the presence of excess benzoate. Two metabolites, E-phe nylitaconic acid (1 to 2%) and benzylsuccinic acid (<1%), accumulated from anaerobic toluene metabolism. These same products were also produ ced when cells were grown on hydrocinnamic acid and trans-cinnamic aci d but were not produced from benzylalcohol, benzaldehyde, benzoate, p- cresol, or their hydroxylated analogs. The evidence supports an anaero bic toluene degradation pathway involving an initial acetyl coenzyme A (acetyl-CoA) attack in strain Tol-4, as proposed by Evans and coworke rs (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Youn g, Appl. Environ. Microbiol. 58:496-501, 1992) for another toluene-deg rading denitrifier, strain T1. Our findings support a modification of the proposed pathway in which cinnamoyl-CoA follows the oxidation of h ydrocinnamoyl-CoA, analogous to the presumed oxidation of benzylsuccin ic acid to form E-phenylitaconic acid, Cinnamic acid was detected in T ol-4 cultures growing in the presence of toluene and [C-14]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-gr owing cultures resulted in a significant increase in production of ben zylsuccinic acid and E-phenylitaconic acid and by the finding that [C- 14]benzylsuccinic acid was detected after incubation of cells with tol uene, [C-14] acetate, and cinnamic acid. Evidence for anaerobic toluen e metabolism by methyl group oxidation was not found, since benzylsucc inic acid and E-phenylitaconic acid were not detected after incubation with benzylalcohol and benzaldehyde, nor were benzylalcohol and benza ldehyde detected even in C-14 trapping experiments.