A stable organic free radical in anaerobic benzylsuccinate synthase of Azoarcus sp. strain T

The novel enzyme benzylsuccinate synthase initiates anaerobic toluene metabolism by catalyzing the addition of toluene to fumarate, forming benzylsuccinate. Based primarily on its sequence similarity to the glycyl radical enzymes, pyruvate formate-lyase and anaerobic ribonucleotide reductase, benzylsuccinate synthase was speculated to be a glycyl radical enzyme. In this report we use EPR spectroscopy to demonstrate for the first time that active benzylsuccinate synthase from the denitrifying bacterium Azoarcus sp. strain T harbors an oxygen-sensitive stable organic free radical. The EPR signal of the radical was centered at g = 2.0021 and was characterized by a major 2-fold splitting of about 1.5 millitesla. The strong similarities between the EPR signal of the benzylsuccinate synthase radical and that of the glycyl radicals of pyruvate formate-lyase and anaerobic ribonucleotide reductase provide evidence that the benzylsuccinate synthase radical is located on a glycine residue, presumably glycine 828 in Azoarcus sp. strain T benzylsuccinate synthase.     

Operon structure and expression of the genes for benzylsuccinate synthase in<Emphasis Type="Italic"> Thauera aromatica</Emphasis> strain K172

The first step in anaerobic toluene degradation is the addition of a fumarate cosubstrate to the methyl group of toluene, as catalyzed by the glycyl radical enzyme benzylsuccinate synthase. The bssDCAB genes code for the subunits of benzylsuccinate synthase (BssA, B and C) and an additional enzyme implicated in activating the enzyme by introducing the glycyl radical (BssD). Quantitation of the amounts of benzylsuccinate synthase and activating enzyme showed that both proteins are only synthesized in toluene-grown cells, and that the activating enzyme is present in about 14-fold lower amounts. Two mRNA species of the bss gene cluster were identified, one beginning in front of bssD, and a second in front of bssC. Only the first mRNA 5'-end correlates with a toluene-induced promoter, which is similar to that preceding the bbs operon coding for the further enzymes of toluene catabolism of the same strain. The second mapped 5'-end appears to be generated by endonucleolytic processing. The mRNA segment containing the bssD gene is very short-lived, while that containing the bssCAB genes is more stable. The RNA stability data are consistent with the observed amounts of encoded gene products. Furthermore, the previously known bssDCAB genes are apparently cotranscribed with a fifth gene ( bssE) whose product may function as a putative ATP-dependent chaperone for assembly and/or activation of benzylsuccinate synthase.     

Degradation of <Emphasis Type="Italic">o</Emphasis>-xylene and <Emphasis Type="Italic">m</Emphasis>-xylene by a novel sulfate-reducer belonging to the genus <Emphasis Type="Italic">Desulfotomaculum</Emphasis>

A strictly anaerobic bacterium, strain OX39, was isolated with o-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. Apart from o-xylene, strain OX39 grew on m-xylene and toluene and all three substrates were oxidized completely to CO₂. Induction experiments indicated that o-xylene, m-xylene, and toluene degradation were initiated by different specific enzymes. Methylbenzylsuccinate was identified in supernatants of cultures grown on o-xylene and m-xylene, and benzylsuccinate was detected in supernatants of toluene-grown cells, thus indicating that degradation was initiated in all three cases by fumarate addition to the methyl group. Strain OX39 was sensitive towards sulfide and depended on Fe(II) in the medium as a scavenger of the produced sulfide. Analysis of the PCR-amplified 16S rRNA gene revealed that strain OX39 affiliates with the gram-positive endospore-forming sulfate reducers of the genus Desulfotomaculum and is the first hydrocarbon-oxidizing bacterium in this genus.     

Initial reactions of anaerobic metabolism of alkylbenzenes in denitrifying and sulfate-reducing bacteria

The initial activation reactions of anaerobic oxidation of the aromatic hydrocarbons toluene and ethylbenzene were investigated in cell extracts of a toluene-degrading, sulfate-reducing bacterium, Desulfobacula toluolica, and in cell extracts of strain EbN1, a denitrifying bacterium capable of degrading toluene and ethylbenzene. Extracts of toluene-grown cells of both species catalysed the addition of fumarate to the methyl group of [phenyl-¹⁴C]-toluene and formed [¹⁴C]-labeled benzylsuccinate. Extracts of ethylbenzene-grown cells of strain EbN1 did not catalyse this reaction, but catalysed the formation of 1-phenylethanol and acetophenone from [methylene-¹⁴C]-ethylbenzene. Toluene-grown cells of D. toluolica and strain EbN1 synthesised highly induced polypeptides corresponding to the large subunits of benzylsuccinate synthase from Thauera aromatica. These polypeptides were absent in strain EbN1 after growth on ethylbenzene, although a number of different polypeptides were highly induced. Thus, formation of benzylsuccinate from toluene and fumarate appears to be the general initiating step in anaerobic toluene degradation by bacteria affiliated with the phylogenetically distinct β-subclass (strain EbN1 and T. aromatica) and δ-subclass (D. toluolica) of the Proteobacteria. Anaerobic ethylbenzene oxidation proceeds via a different pathway involving a two-step oxidation of the methylene group to an alcohol and an oxo group; these steps are most probably followed by a biotin-independent carboxylation reaction and thiolytic cleavage. Received: 16 March 1998 / Accepted: 27 June 1998     

Stable Isotope Fractionation Caused by Glycyl Radical Enzymes during Bacterial Degradation of Aromatic Compounds

Stable isotope fractionation was studied during the degradation of m-xylene, o-xylene, m-cresol, and p-cresol with two pure cultures of sulfate-reducing bacteria. Degradation of all four compounds is initiated by a fumarate addition reaction by a glycyl radical enzyme, analogous to the well-studied benzylsuccinate synthase reaction in toluene degradation. The extent of stable carbon isotope fractionation caused by these radical-type reactions was between enrichment factors () of −1.5 and −3.9‰, which is in the same order of magnitude as data provided before for anaerobic toluene degradation. Based on our results, an analysis of isotope fractionation should be applicable for the evaluation of in situ bioremediation of all contaminants degraded by glycyl radical enzyme mechanisms that are smaller than 14 carbon atoms. In order to compare carbon isotope fractionations upon the degradation of various substrates whose numbers of carbon atoms differ, intrinsic (_intrinsic) were calculated. A comparison of _intrinsic at the single carbon atoms of the molecule where the benzylsuccinate synthase reaction took place with compound-specific elucidated that both varied on average to the same extent. Despite variations during the degradation of different substrates, the range of found for glycyl radical reactions was reasonably narrow to propose that rough estimates of biodegradation in situ might be given by using an average if no fractionation factor is available for single compounds.     

Transformation ofo-xylene too-methyl benzoic acid by a denitrifying enrichment culture using toluene as the primary substrate

A highly enriched denitrifying mixed culture transformedo-xylene cometabolically along with toluene by methyl group oxidation.o-Methyl benzaldehyde ando-methyl benzoic acid accumulated transiently as metabolic products ofo-xylene transformation. Transformation ofo-methyl benzyl alcohol ando-methyl benzaldehyde occurred independently of toluene degradation and resulted in the formation of a compound coeluting witho-methyl benzoic acid on a gas chromatograph. The cometabolic relationship between toluene ando-xylene could be attributed to a mechanism linked to the initial oxidation of the methyl group.     

Modelling of cometabolic transformation ofortho-xylene in a denitrifying biofilm system

A model describing the cometabolic biotransformation ofo-xylene with toluene as primary carbon source in a continuously fed fixed biofilm reactor is presented. The model is based on the concept of competitive inhibition betweeno-xylene and toluene. The proposed model simulated successfully the transformation ofo-xylene and the associated by-products formation, as well as the toluene degradation. However, it appears that an accurate measurement of active biomass density and distribution in the biofilm is needed, since these factors dramatically affects the modelling. The modelling of various kinetic experiments indicates that the active biomass (or toluene degraders) is accumulated on the top of the biofilm, leading to the conclusion that only a minor part of the biofilm thickness was active. The calibrated model is able to predict the removal of toluene ando-xylene for concentrations ranging from 0 to 30 mg/L. For higher concentrations toxicity phenomena may decrease the accuracy of the model.