Identification of new enzymes potentially involved in anaerobic naphthalene degradation by the sulfate-reducing enrichment culture N47

The sulfate-reducing highly enriched culture N47 is capable to anaerobically degrade naphthalene, 2-methylnaphthalene, and 2-naphthoic acid. A proteogenomic investigation was performed to elucidate the initial activation reaction of anaerobic naphthalene degradation. This lead to the identification of an alpha-subunit of a carboxylase protein that was two-fold up-regulated in naphthalene-grown cells compared to 2-methylnaphthalene-grown cells. The putative naphthalene carboxylase subunit showed 48% similarity to the anaerobic benzene carboxylase from an iron-reducing, benzene-degrading culture and 45% to alpha-subunit of phenylphosphate carboxylase of Aromatoleum aromaticum EbN1. A gene for the beta-subunit of putative naphthalene carboxylase was located nearby on the genome and was expressed with naphthalene. Similar to anaerobic benzene carboxylase, there were no genes for gamma- and delta-subunits of a putative carboxylase protein located on the genome which excludes participation in degradation of phenolic compounds. The genes identified for putative naphthalene carboxylase subunits showed only weak similarity to 4-hydroxybenzoate decarboxylase excluding ATP-independent carboxylation. Several ORFs were identified that possibly encode a 2-naphthoate-CoA ligase, which is obligate for activation before the subsequent ring reduction by naphthoyl-CoA reductase. One of these ligases was exclusively expressed on naphthalene and 2-naphthoic acid and might be the responsible naphthoate-CoA-ligase.     

Co-metabolic conversion of toluene in anaerobic n-alkane-degrading bacteria

Diverse microorganisms have been described to degrade petroleum hydrocarbons anaerobically. Strains able to utilize n-alkanes do not grow with aromatic hydrocarbons, whereas strains able to utilize aromatic hydrocarbons do not grow with n-alkanes. To investigate this specificity in more detail, three anaerobic n-alkane degraders (two denitrifying, one sulfate-reducing) and eight anaerobic alkylbenzene degraders (five denitrifying, three sulfate-reducing) were incubated with mixtures of n-alkanes and toluene. Whereas the toluene degradationers formed only the characteristic toluene-derived benzylsuccinate and benzoate, but no n-alkane-derived metabolites, the n-alkane degraders formed toluene-derived benzylsuccinate, 4-phenylbutanoate, phenylacetate and benzoate besides the regular n-alkane-derived (1-methylalkyl)succinates and methyl-branched alkanoates. The co-metabolic conversion of toluene by anaerobic n-alkane degraders to the level of benzoate obviously follows the anaerobic n-alkane degradation pathway with C-skeleton rearrangement and decarboxylation rather than the β-oxidation pathway of anaerobic toluene metabolism. Hence, petroleum-derived aromatic metabolites detectable in anoxic environments may not be exclusively formed by genuine alkylbenzene degraders. In addition, the hitherto largely unexplored fate of fumarate hydrogen during the activation reactions was examined with (2,3-(2) H(2) )fumarate as co-substrate. Deuterium was completely exchanged with hydrogen at the substituted carbon atom (C-2) of the succinate adducts of n-alkanes, whereas it is retained in toluene-derived benzylsuccinate, regardless of the type of enzyme catalysing the fumarate addition reaction.     

Anaerobic degradation of benzene by a marine sulfate-reducing enrichment culture, and cell hybridization of the dominant phylotype

The anaerobic biodegradation of benzene, a common constituent of petroleum and one of the least reactive aromatic hydrocarbons, is insufficiently understood with respect to the involved microorganisms and their metabolism. To study these aspects, sulfate-reducing bacteria were enriched with benzene as sole organic substrate using marine sediment as inoculum. Repeated subcultivation yielded a sediment-free enrichment culture constituted of mostly oval-shaped cells and showing benzene-dependent sulfate reduction and growth under strictly anoxic conditions. Amplification and sequencing of 16S rRNA genes from progressively diluted culture samples revealed an abundant phylotype; this was closely related to a clade of Deltaproteobacteria that includes sulfate-reducing bacteria able to degrade naphthalene or other aromatic hydrocarbons. Cell hybridization with two specifically designed 16S rRNA-targeted fluorescent oligonucleotide probes showed that the retrieved phylotype accounted for more than 85% of the cells detectable via DAPI staining (general cell staining) in the enrichment culture. The result suggests that the detected dominant phylotype is the 'candidate species' responsible for the anaerobic degradation of benzene. Quantitative growth experiments revealed complete oxidation of benzene with stoichiometric coupling to the reduction of sulfate to sulfide. Suspensions of benzene-grown cells did not show metabolic activity towards phenol or toluene. This observation suggests that benzene degradation by the enriched sulfate-reducing bacteria does not proceed via anaerobic hydroxylation (mediated through dehydrogenation) to free phenol or methylation to toluene, respectively, which are formerly proposed alternative mechanisms for benzene activation.     

Anaerobic degradation of the aromatic hydrocarbon biphenyl by a sulfate-reducing enrichment culture

The aromatic hydrocarbon biphenyl is a widely distributed environmental pollutant. Whereas the aerobic degradation of biphenyl has been extensively studied, knowledge of the anaerobic biphenyl-oxidizing bacteria and their biochemical degradation pathway is scarce. Here, we report on an enrichment culture that oxidized biphenyl completely to carbon dioxide under sulfate-reducing conditions. The biphenyl-degrading culture was dominated by two distinct bacterial species distantly affiliated with the Gram-positive genus Desulfotomaculum . Moreover, the enrichment culture has the ability to grow with benzene and a mixture of anthracene and phenanthrene as the sole source of carbon, but here the microbial community composition differed substantially from the biphenyl-grown culture. Biphenyl-4-carboxylic acid was identified as an intermediate in the biphenyl-degrading culture. Moreover, 4-fluorobiphenyl was converted cometabolically with biphenyl because in addition to the biphenyl-4-carboxylic acid, a compound identified as its fluorinated analog was observed. These findings are consistent with the general pattern in the anaerobic catabolism of many aromatic hydrocarbons where carboxylic acids are found to be central metabolites.     

Identification of intermediates formed during anaerobic benzene degradation by an iron-reducing enrichment culture

Anaerobic benzene degradation is an important process in contaminated aquifers but is poorly understood due to the scarcity of microbial cultures for study. We have enriched a ferric iron-reducing culture that completely mineralizes benzene to CO₂. With ¹³C₆-labelled benzene as the growth substrate, ring-labelled benzoate was identified as a major intermediate by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of culture supernatants. With increasing incubation time, ¹³C₇-labelled benzoate appeared, indicating that the carboxyl group of benzoate derived from CO₂ that was produced from mineralization of labelled benzene. This was confirmed by growing the culture in ¹³C-bicarbonate-buffered medium with unlabelled benzene as the substrate, as the label appeared in the carboxyl group of benzoate produced. Phenol was also identified as an intermediate at high concentration. However, it was clearly shown that phenol was formed abiotically by autoxidation of benzene during the sampling and analysis procedure as a result of exposure to air. The results suggest that, in our culture, anaerobic benzene degradation proceeds via carboxylation and that caution should be exercised in interpreting hydroxylated benzene derivatives as metabolic intermediates of anaerobic benzene degradation.     

Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron‐reducing enrichment culture

Anaerobic benzene degradation was studied with a highly enriched iron‐reducing culture (BF) composed of mainly Peptococcaceae‐related Gram‐positive microorganisms. The proteomes of benzene‐, phenol‐ and benzoate‐grown cells of culture BF were compared by SDS‐PAGE. A specific benzene‐expressed protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was subjected to N‐terminal sequence analysis. The first 31 amino acids revealed that the protein was encoded by ORF 138 in the shotgun sequenced metagenome of culture BF. ORF 138 showed 43% sequence identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A LC/ESI‐MS/MS‐based shotgun proteomic analysis revealed other specifically benzene‐expressed proteins with encoding genes located adjacent to ORF 138 on the metagenome. The protein products of ORF 137, ORF 139 and ORF 140 showed sequence identities of 37% to phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate‐CoA ligase (BamY) of Geobacter metallireducens and 67% to 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase (UbiD/UbiX) of A. aromaticum strain EbN1 respectively. These genes are proposed as constituents of a putative benzene degradation gene cluster (～17 kb) composed of carboxylase‐related genes. The identified gene sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a direct carboxylation of benzene to benzoate catalysed by putative anaerobic benzene carboxylase (Abc). The putative Abc probably consists of several subunits, two of which are encoded by ORFs 137 and 138, and belongs to a family of carboxylases including phenylphosphate carboxylase (Ppc) and 3‐octaprenyl‐4‐hydroxybenzoate carboxy‐lyase (UbiD/UbiX).     

Anaerobic degradation of monoaromatic hydrocarbons

Over the last two decades significant advances have been made in our understanding of the anaerobic biodegradability of monoaromatic hydrocarbons. It is now known that compounds such as benzene, toluene, ethylbenzene, and all three xylene isomers can be biodegraded in the absence of oxygen by a broad diversity of organisms. These compounds have been shown to serve as carbon and energy sources for bacteria growing phototrophically, or respiratorily with nitrate, manganese, ferric iron, sulfate, or carbon dioxide as the sole electron acceptor. In addition, it has also been recently shown that complete degradation of monoaromatic hydrocarbons can also be coupled to the respiration of oxyanions of chlorine such as perchlorate or chlorate, or to the reduction of the quinone moieties of humic substances. Many pure cultures of hydrocarbon-degrading anaerobes now exist and some novel biochemical and genetic pathways have been identified. In general, a fumarate addition reaction is used as the initial activation step of the catabolic process of the corresponding monoaromatic hydrocarbon compounds. However, other reactions may alternatively be involved depending on the electron acceptor utilized or the compound being degraded. In the case of toluene, fumarate addition to the methyl group mediated by benzylsuccinate synthase appears to be the universal mechanism of activation and is now known to be utilized by anoxygenic phototrophs, nitrate-reducing, Fe(III)-reducing, sulfate-reducing, and methanogenic cultures. Many of these biochemical pathways produce unique extracellular intermediates that can be utilized as biomarkers for the monitoring of hydrocarbon degradation in anaerobic natural environments.     

Inhibition of benzene,toluene, phenol and benzoate in single and combination on Anammox activity: implication to the denitrification–Anammox synergy

A previous study demonstrated that denitrification synergized with Anammox could accelerate the anaerobic degradation of benzene. The inhibitory effects of benzene, toluene, phenol and benzoate in single and combination on Anammox activity were investigated by short-term batch tests. The results indicated that the inhibition of single compounds on Anammox could be well fitted with the extended non-competitive and Luong inhibition kinetic models. The inhibitions of the individual compound were in order as follows: benzene?>?toluene?>?phenol?>?benzoate. The joint inhibitions of bi-component mixtures of benzene with toluene, benzene with phenol and benzene with benzoate on Anammox activity were additive; the joint inhibition of a tri-component mixture (benzene, toluene and phenol) was partly additive; and the joint inhibition of a multicomponent mixture (benzene, toluene, phenol and benzoate) was synergistic. The effect of benzoate on the denitrification–Anammox synergy for benzene degradation was evaluated using a long-term test. Although the average rate of benzene degradation decreased by 13% with the addition of 10 mg L^?1 benzoate, the average rate of NO₃^? and NH₄⁺ increased by approximately 1- and 0.56-fold, respectively, suggesting that benzoate favors the stability of the denitrification–Anammox synergy. The carboxylation of benzene would be a more favorable pathway for the anaerobic degradation of benzene under denitrification synergized with Anammox.     

Anaerobic Benzene Oxidation via Phenol in Geobacter metallireducens

Anaerobic activation of benzene is expected to represent a novel biochemistry of environmental significance. Therefore, benzene metabolism was investigated in Geobacter metallireducens, the only genetically tractable organism known to anaerobically degrade benzene. Trace amounts (<0.5 μM) of phenol accumulated in cultures of Geobacter metallireducens anaerobically oxidizing benzene to carbon dioxide with the reduction of Fe(III). Phenol was not detected in cell-free controls or in Fe(II)- and benzene-containing cultures of Geobacter sulfurreducens, a Geobacter species that cannot metabolize benzene. The phenol produced in G. metallireducens cultures was labeled with ¹⁸O during growth in H₂¹⁸O, as expected for anaerobic conversion of benzene to phenol. Analysis of whole-genome gene expression patterns indicated that genes for phenol metabolism were upregulated during growth on benzene but that genes for benzoate or toluene metabolism were not, further suggesting that phenol was an intermediate in benzene metabolism. Deletion of the genes for PpsA or PpcB, subunits of two enzymes specifically required for the metabolism of phenol, removed the capacity for benzene metabolism. These results demonstrate that benzene hydroxylation to phenol is an alternative to carboxylation for anaerobic benzene activation and suggest that this may be an important metabolic route for benzene removal in petroleum-contaminated groundwaters, in which Geobacter species are considered to play an important role in anaerobic benzene degradation.     

Metabolic by-products of anaerobic toluene degradation by sulfate-reducing enrichment cultures.

Two dead-end metabolites of anaerobic toluene transformation, benzylsuccinic acid and benzylfumaric acid, accumulated in sulfate-reducing enrichment cultures that were fed toluene as the sole carbon source. Stable isotope-labeled toluene and gas chromatography-mass spectrometry were used to confirm that the compounds resulted from toluene metabolism. The two metabolites constituted less than 10% of the toluene carbon (over 80% was mineralized to carbon dioxide, according to a previous study). This study demonstrates that the novel nonproductive pathway 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 a denitrifying pure culture applies to disparate anaerobic bacteria.     

4-Hydroxybenzoate-coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation.

Anaerobic metabolism of most aromatic acids is initiated by coenzyme A thioester formation. Rhodopseudomonas palustris grows well under anaerobic, phototrophic conditions with many aromatic acids, including benzoate and 4-hydroxybenzoate, as a carbon source. A coenzyme A ligase that reacts with 4-hydroxybenzoate was purified from 4-hydroxybenzoate-grown cells of R. palustris. This enzyme required MgATP, reduced coenzyme A, and 4-hydroxybenzoate, benzoate, or cyclohex-1,4-dienecarboxylate for optimal activity but also used phosphopantetheine, cyclohex-2,5-dienecarboxylate, and 4-fluorobenzoate at lower rates. The 4-hydroxybenzoate-coenzyme A ligase differed in molecular characteristics from a previously described benzoate-coenzyme A ligase from R. palustris, and the two ligases did not cross-react immunologically. The gene encoding the 4-hydroxybenzoate enzyme was cloned and sequenced. The deduced gene product showed about 20% amino acid identity with bacterial coenzyme A ligases involved in aerobic degradation of aromatic acids. An R. palustris mutant carrying a disrupted 4-hydroxybenzoate-coenzyme A ligase gene was unable to grow with 4-hydroxybenzoate under anaerobic conditions, indicating that the enzyme is essential for anaerobic degradation of this compound.     

Metabolic by-products of anaerobic toluene degradation by sulfate-reducing enrichment cultures.

Two dead-end metabolites of anaerobic toluene transformation, benzylsuccinic acid and benzylfumaric acid, accumulated in sulfate-reducing enrichment cultures that were fed toluene as the sole carbon source. Stable isotope-labeled toluene and gas chromatography-mass spectrometry were used to confirm that the compounds resulted from toluene metabolism. The two metabolites constituted less than 10% of the toluene carbon (over 80% was mineralized to carbon dioxide, according to a previous study). This study demonstrates that the novel nonproductive pathway 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 a denitrifying pure culture applies to disparate anaerobic bacteria.     

Syntrophorhabdus aromaticivorans gen. nov., sp. nov., the first cultured anaerobe capable of degrading phenol to acetate in obligate syntrophic associations with a hydrogenotrophic methanogen

Phenol degradation under methanogenic conditions has long been studied, but the anaerobes responsible for the degradation reaction are still largely unknown. An anaerobe, designated strain UI(T), was isolated in a pure syntrophic culture. This isolate is the first tangible, obligately anaerobic, syntrophic substrate-degrading organism capable of oxidizing phenol in association with an H(2)-scavenging methanogen partner. Besides phenol, it could metabolize p-cresol, 4-hydroxybenzoate, isophthalate, and benzoate. During the degradation of phenol, a small amount of 4-hydroxybenzoate (a maximum of 4 microM) and benzoate (a maximum of 11 microM) were formed as transient intermediates. When 4-hydroxybenzoate was used as the substrate, phenol (maximum, 20 microM) and benzoate (maximum, 92 microM) were detected as intermediates, which were then further degraded to acetate and methane by the coculture. No substrates were found to support the fermentative growth of strain UI(T) in pure culture, although 88 different substrates were tested for growth. 16S rRNA gene sequence analysis indicated that strain UI(T) belongs to an uncultured clone cluster (group TA) at the family (or order) level in the class Deltaproteobacteria. Syntrophorhabdus aromaticivorans gen. nov., sp. nov., is proposed for strain UI(T), and the novel family Syntrophorhabdaceae fam. nov. is described. Peripheral 16S rRNA gene sequences in the databases indicated that the proposed new family Syntrophorhabdaceae is largely represented by abundant bacteria within anaerobic ecosystems mainly decomposing aromatic compounds.     

Anaerobic Benzene Biodegradation: A Microcosm Survey

Benzene-amended microcosms prepared with saturated soil or sediment from five hydrocarbon-contaminated sites and one pristine site were monitored for a year and a half to determine the rate of benzene biodegradation under a variety of electron-accepting conditions. Sustainable benzene degradation was observed under specific conditions in microcosms from four of the six sites. Significant differences were observed between sites with respect to lag times before the onset of degradation, rates of degradation, sustainability of the activity, and environmental conditions supporting degradation. Benzene degradation was observed under sulfate-reducing, nitrate-reducing, and iron(III)-reducing conditions, but not under methanogenic conditions. The presence of competing substrates such as toluene, xylenes, and ethylbenzene was found to inhibit anaerobic benzene degradation in microcosms where sulfate or possibly nitrate was the electron acceptor for benzene degradation, but not in microcosms from where iron(III) was the electron acceptor. The presence of organic matter, indicated by a high fraction organic carbon (f_oc), also appeared to inhibit the biodegradation of benzene; microcosms constructed with soils with the highest f_oc exhibited the longest lag times before the onset of benzene degradation. The initial extent of hydrocarbon contamination did not appear to correlate with anaerobic benzene-degrading activity.     

Influence of Alternate Electron Acceptors on the Metabolic Fate of Hydroxybenzoate Isomers in Anoxic Aquifer Slurries

The biodegradation of hydroxybenzoate isomers was investigated with samples obtained from two sites within a shallow anoxic aquifer. The metabolic fates of the substrates were compared in denitrifying, sulfate-reducing, and methanogenic incubations. Under the latter two conditions, phenol was detected as a major intermediate of p-hydroxybenzoate, but no metabolites were initially found with m- or o-hydroxybenzoate. However, benzoate accumulation was noted when metabolic inhibitors were used with these samples. About 9 to 17 days was required for >95% removal of the parent isomers under these conditions. When aquifer slurries were amended with nitrate, the equivalent removal of the hydroxybenzoates occurred within 4 days. In the denitrifying incubations, phenol was formed from all three hydroxybenzoates and accounted for about 30% of the initial substrate amendment. No benzoate was measured in these samples. All metabolites were identified by chromatographic mobility, mass spectral profiles, or both. Autoclaved controls were uniformly incapable of transforming the parent substrates. These results suggest that the anaerobic fate of hydroxybenzoate isomers depends on the relative substitution pattern and the prevailing ecological conditions. Furthermore, since these compounds are central metabolites formed during the breakdown of many aromatic chemicals, our findings may help provide guidelines for the reliable extrapolation of metabolic fate information from diverse anaerobic environments.     

Anaerobic benzene degradation

Although many studies have indicated that benzene persists under anaerobic conditions in petroleum-contaminated environments, it has recently been documented that benzene can be anaerobically oxidized with most commonlyconsidered electron acceptors for anaerobic respiration. These include: Fe(III),sulfate, nitrate, and possibly humic substances. Benzene can also be convertedto methane and carbon dioxide under methanogenic conditions. There is evidencethat benzene can be degraded under in situ conditions in petroleum-contaminatedaquifers in which either Fe(III) reduction or methane production is the predominant terminal electron-accepting process. Furthermore, evidence from laboratory studies suggests that benzene may be anaerobically degraded in petroleum-contaminated marine sediments under sulfate-reducing conditions. Laboratory studies have suggested that within the Fe(III) reduction zone of petroleum-contaminated aquifers, benzene degradation can be stimulated with the addition of synthetic chelators which make Fe(III) more available for microbial reduction. The addition of humic substances and other compounds that contain quinone moieties can also stimulate anaerobic benzene degradation in laboratory incubations of Fe(III)-reducing aquifer sediments by providing an electron shuttle between Fe(III)-reducing microorganisms and insoluble Fe(III) oxides. Anaerobic benzene degradation in aquifer sediments can be stimulated with the addition of sulfate, but in some instances an inoculum of benzene-oxidizing,sulfate-reducing microorganisms must also be added. In a field trial, sulfate addition to the methanogenic zone of a petroleum-contaminated aquifer stimulated the growth and activity of sulfate-reducing microorganisms and enhanced benzene removal. Molecular phylogenetic studies have provided indications of what microorganisms might be involved in anaerobic benzene degradation in aquifers. The major factor limiting further understanding of anaerobic benzene degradation is the lack of a pure culture of an organism capable of anaerobic benzene degradation.     

Biodegradation of Phenol: Mechanisms and Applications

Phenol, or hydroxybenzene, is both a synthetically and naturally produced aromatic compound. Microorganisms capable of degrading phenol are common and include both aerobes and anaerobes. Many aerobic phenol-degrading microorganisms have been isolated and the pathways for the aerobic degradation of phenol are now firmly established. The first steps include oxygenation of phenol by phenol hydroxylase enzymes to form catechol, followed by ring cleavage adjacent to or in between the two hydroxyl groups of catechol. Phenol hydroxylases ranging from simple flavoprotein monooxygenases to multicomponent hydroxylases, as well as the genes coding for these enzymes, have been described for a number of aerobic phenol-degrading microorganisms. Phenol can also be degraded in the absence of oxygen. Our knowledge of this process is less advanced than that of the aerobic process, and only a few anaerobic phenol-degrading bacteria have been isolated to date. Convincing evidence from both pure culture studies with the denitrifying organism Thauera aromatica K172 and with two Clostridium species, as well as from mixed culture studies, indicates that the first step in anaerobic phenol degradation is carboxylation in the para-position to form 4-hydroxybenzoate. Following para-carboxylation, thioesterification of 4-hydroxybenzoate to co-enzyme A allows subsequent ring reduction, hydration, and fission. Para-carboxylation appears to be involved in the anaerobic degradation of a number of aromatic compounds. Numerous practical applications exist for microbial phenol degradation. These include the exploitation of indigenous anaerobic phenol-degrading bacteria in the in situ bioremediation of creosote-contaminated subsurface environments, and the use of phenol as a co-substrate for indigenous aerobic phenol-degrading bacteria to enhance in situ biodegradation of chlorinated solvents.     

Anaerobic degradation ofm-cresol via methyl oxidation to 3-hydroxybenzoate by a denitrifying bacterium

The anaerobic degradation of m-cresol was studied in a denitrifying bacterium. In the initial studies, hypothetical intermediates of m-cresol degradation were tested in growth experiments and in adaptation studies with dense cell suspensions. Results suggested a degradation of m-cresol via 3-hydroxybenzoate. To verify this, the degradation of m-cresol was followed in concentrated cell suspensions in the presence of metabolic inhibitors. Fluoroacetate treatment resulted in the transient accumulation of substantial amounts of 3-hydroxybenzoate. In the presence of iodoacetamide, not only was 3-hydroxybenzoate transiently formed, but benzoate was also accumulated. These findings support a degradation of m-cresol via initial anaerobic methyl oxidation to 3-hydroxybenzoate, followed by reductive dehydroxylation to benzoate or benzoyl-CoA. Studies with extracts of m-cresol-grown cells showed the presence of several enzyme activities to be postulated for this pathway. No evidence was found for a carboxylation, hydroxylation of the aromatic ring, or direct ring reduction as the initial step in m-cresol metabolism. Received: 29 November 1994 / Accepted: 7 March 1995