Initiation of anaerobic degradation of p-cresol by formation of 4-hydroxybenzylsuccinate in desulfobacterium cetonicum

Abstract

The anaerobic bacterium Desulfobacterium cetonicum oxidized p-cresol completely to CO(2) with sulfate as the electron acceptor. During growth, 4-hydroxybenzylsuccinate accumulated in the medium. This finding indicated that the methyl group of p-cresol is activated by addition to fumarate, analogous to anaerobic toluene, m-xylene, and m-cresol degradation. In cell extracts, the formation of 4-hydroxybenzylsuccinate from p-cresol and fumarate was detected at an initial rate of 0.57 nmol min(-1) (mg of protein)(-1). This activity was specific for extracts of p-cresol-grown cells. 4-Hydroxybenzylsuccinate was degraded further to 4-hydroxybenzoyl-coenzyme A (CoA), most likely via beta-oxidation. 4-Hydroxybenzoyl-CoA was reductively dehydroxylated to benzoyl-CoA. There was no evidence of degradation of p-cresol via methyl group oxidation by p-cresol-methylhydroxylase in this bacterium.

FIG. 1

Identification of 4-HBS in supernatants of p-cresol-converting cultures of D. cetonicum. (A) On-line UV spectra of chemically synthesized 4-HBS (A) and of 4-HBS from culture supernatant (B) in acetonitrile (10%) and 10 mM potassium phosphate buffer (pH 2.2). The maxima are at 232 and 278 nm. (B and C) Mass analysis by GC/MS of chemically synthesized (B) 4-methoxybenzylsuccinate dimethyl ester and (C) 4-methoxybenzylsuccinate dimethyl ester obtained from culture supernatant; mass units are daltons.

FIG. 2

Conversion of p-cresol (■) to 4-HBS (□) by cell extracts of D. cetonicum.

FIG. 3

Degradation of p-cresol (■) in the presence of fumarate (●). Formation of sulfide (▿) and succinate (○) and increase in OD (▵) are also shown. d, days.

FIG. 4

Proposed initial reactions of p-cresol degradation by D. cetonicum. Compounds with a question mark (4-hydroxyphenylitaconyl-CoA and 4-hydroxybenzoylsuccinyl-CoA) were not identified.