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.     

Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads

From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO₂ with nitrate as the terminal electron acceptor. All strains grew in defined mineral salts medium; two of them were further characterized. The bacteria were facultatively anaerobic Gramnegative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor. The isolates were tentatively identified as pseudomonads. Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate. While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only. Reduced alicyclic compounds were not degraded. Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate.     

2-Ketocyclohexanecarboxyl Coenzyme A Hydrolase, the Ring Cleavage Enzyme Required for Anaerobic Benzoate Degradation by Rhodopseudomonas palustris

2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium Rhodopseudomonas palustris by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography. The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the badI gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of badI indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins. Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration. This indicates that the native form of the enzyme is a homotetramer. The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 μmol min⁻¹ mg of protein⁻¹. Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate. An R. palustris mutant, carrying a chromosomal disruption of badI, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate. These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by R. palustris.     

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 Metabolism of Catechol by the Denitrifying Bacterium Thauera aromatica—a Result of Promiscuous Enzymes and Regulators?

The anaerobic metabolism of catechol (1,2-dihydroxybenzene) was studied in the betaproteobacterium Thauera aromatica that was grown with CO₂ as a cosubstrate and nitrate as an electron acceptor. Based on different lines of evidence and on our knowledge of enzymes and genes involved in the anaerobic metabolism of other aromatic substrates, the following pathway is proposed. Catechol is converted to catechylphosphate by phenylphosphate synthase, which is followed by carboxylation by phenylphosphate carboxylase at the para position to the phosphorylated phenolic hydroxyl group. The product, protocatechuate (3,4-dihydroxybenzoate), is converted to its coenzyme A (CoA) thioester by 3-hydroxybenzoate-CoA ligase. Protocatechuyl-CoA is reductively dehydroxylated to 3-hydroxybenzoyl-CoA, possibly by 4-hydroxybenzoyl-CoA reductase. 3-Hydroxybenzoyl-CoA is further metabolized by reduction of the aromatic ring catalyzed by an ATP-driven benzoyl-CoA reductase. Hence, the promiscuity of several enzymes and regulatory proteins may be sufficient to create the catechol pathway that is made up of elements of phenol, 3-hydroxybenzoate, 4-hydroxybenzoate, and benzoate metabolism.     

Anaerobic degradation of phenylacetate and 4-hydroxyphenylacetate by denitrifying bacteria

From various oxic or anoxic habitats anaerobic enrichment cultures were set up which completely oxidized aromatic amino acids to CO₂ with nitrate as electron acceptor. Tyrosine and tryptophan at first were degraded to phenol and indole, respectively, prior to utilization of the aromatic ring; with phenylalanine no intermediates were detected. Attempts to isolate denitrifying bacteria able to completely degrade aromatic amino acids were unsuccessful. Starting with these enrichments several strains of denitrifying bacteria were anaerobically enriched and isolated with known fermentation products of amino acids (phenylacetate, 4-OH-phenylacetate, 2-OH-benzoate) plus nitrate as sole sources of carbon and energy.Three strains were characterized further. They grew well in defined mineral salts medium, were gram-negative and facultatively anaerobic with strictly oxidative metabolism; molecular oxygen, nitrate or nitrite served as electron acceptors. The isolates were tentatively identified as pseudomonads, but could not be aligned to known species. They oxidized a variety of aromatic compounds completely to CO₂ anaerobically and, with some exceptions, also aerobically. The substrates included among others: (4-OH)-phenylacetate, (4-OH)-phenylglyoxylate, benzoate, 2-aminobenzoate, phenol, OH-benzoates, indole and notably toluene. Reduced alicyclic compounds were not utilized. During anaerobic degradation of (4-OH)-phenylacetate transient accumulation of (4-OH)-phenylglyoxylate was observed.It is proposed that anaerobic -oxidation of the-CH₂–COOH side chain to -CO–COOH initiates anaerobic degradation of (4-OH)-phenylacetate. This implies a novel type of anaerobic -hydroxylation with water as the oxygen donor. Abbreviation. Hydroxyl groups were abbreviated as OH     

Reductive Dehalogenation and Conversion of 2-Chlorophenol to 3-Chlorobenzoate in a Methanogenic Sediment Community: Implications for Predicting the Environmental Fate of Chlorinated Pollutants

Biotransformation of 2-chlorophenol by a methanogenic sediment community resulted in the transient accumulation of phenol and benzoate. 3-Chlorobenzoate was a more persistent product of 2-chlorophenol metabolism. The anaerobic biotransformation of phenol to benzoate presumably occurred via para-carboxylation and dehydroxylation reactions, which may also explain the observed conversion of 2-chlorophenol to 3-chlorobenzoate.     

Acidocella aromatica sp. nov.: an acidophilic heterotrophic alphaproteobacterium with unusual phenotypic traits

Three obligately heterotrophic bacterial isolates were identified as strains of a proposed novel species of extremely acidophilic, mesophilic Alphaproteobacteria, Acidocella aromatica. They utilized a restricted range of organic substrates, which included fructose (but none of the other monosaccharides tested), acetate and several aromatic compounds (benzoate, benzyl alcohol and phenol). No growth was obtained on complex organic substrates, such as yeast extract and tryptone. Tolerance of the proposed type strain of the species (PFBC) to acetic acid was much greater than that typically reported for acidophiles. The bacteria grew aerobically, and catalyzed the dissimilatory reductive dissolution of the ferric iron mineral schwertmannite under both micro-aerobic and anaerobic conditions. Strain PFBC did not grow anaerobically via ferric iron respiration, though it has been reported to grow in co-culture with acid-tolerant sulfidogenic bacteria under strictly anoxic conditions. Tolerance of strains of Acidocella aromatica to nickel were about two orders of magnitude greater than those of other Acidocella spp., though similar levels of tolerance to other metals tested was observed. The use of this novel acidophile in solid media designed to promote the isolation and growth of other (aerobic and anaerobic) acidophilic heterotrophs is discussed.     

Development of a PCR method for the detection and quantification of benzoyl-CoA reductase genes and its application to monitored natural attenuation

Benzoyl coenzyme A reductase (BCR) catalyzes dearomatization of benzoyl coenzyme A (benzoyl-CoA), which is the central step in the anaerobic degradative pathways for a variety of aromatic compounds. This study developed a PCR method for the detection and quantification of BCR genes in bacterial strains and environmental samples. PCR primers were designed by aligning known BCR genes in Thauera, Azoarcus and Rhodopseudomonas species, and their utility was assessed by amplifying BCR fragments from aromatic-hydrocarbon degrading anaerobes and other bacteria. BCR fragments with the expected sizes were obtained from denitrifying and phototrophic aromatics degraders. The positive signals were also obtained from Geobacter metallireducens and xylene-degrading sulfate-reducing bacterium (strain mXyS1) but not from other aromatics-degrading sulfate-reducing bacteria and aerobic bacteria. When the PCR was used for analyzing a natural attenuation (NA) site, the positive signal was obtained only from gasoline-contaminated groundwater; sequence analysis of these amplicons revealed that most of them exhibited substantial similarities to the known BCRs. Quantitative competitive PCR analysis estimated BCR-gene copies to account for 10–40% of bacterial 16S rRNA gene copies in the contaminated groundwater, indicating that bacteria possessing BCR genes were highly enriched in the contaminated groundwater. In microcosm bioremediation tests using the contaminated groundwater, the copy number of BCR gene was approximately 10-fold increased in the course of aromatics degradation under denitrifying conditions but not under sulfidogenic conditions. These results suggest the utility of the PCR method for assessing the potential of denitrifying bacteria for aromatic-compound degradation in groundwater.     

Anaerobic degradation of aromatic compounds coupled to Fe(III) reduction by Ferroglobus placidus

Aromatic compounds are an important component of the organic matter in some of the anaerobic environments that hyperthermophilic microorganisms inhabit, but the potential for hyperthermophilic microorganisms to metabolize aromatic compounds has not been described previously. In this study, aromatic metabolism was investigated in the hyperthermophile Ferroglobus placidus . F. placidus grew at 85°C in anaerobic medium with a variety of aromatic compounds as the sole electron donor and poorly crystalline Fe(III) oxide as the electron acceptor. Growth coincided with Fe(III) reduction. Aromatic compounds supporting growth included benzoate, phenol, 4-hydroxybenzoate, benzaldehyde, p -hydroxybenzaldehyde and t -cinnamic acid (3-phenyl-2-propenoic acid). These aromatic compounds did not support growth when nitrate was provided as the electron acceptor, even though nitrate supports the growth of this organism with Fe(II) or H₂ as the electron donor. The stoichiometry of benzoate and phenol uptake and Fe(III) reduction indicated that F. placidus completely oxidized these aromatic compounds to carbon dioxide, with Fe(III) serving as the sole electron acceptor. This is the first example of an Archaea that can anaerobically oxidize an aromatic compound. These results also demonstrate for the first time that hyperthermophilic microorganisms can anaerobically oxidize aromatic compounds and suggest that hyperthermophiles may metabolize aromatic compounds in hot environments such as the deep hot subsurface and in marine and terrestrial hydrothermal zones in which Fe(III) is available as an electron acceptor.     

Acetate Oxidation Coupled to Fe(III) Reduction in Hyperthermophilic Microorganisms

No hyperthermophilic microorganisms have previously been shown to anaerobically oxidize acetate, the key extracellular intermediate in the anaerobic oxidation of organic matter. Here we report that two hyperthermophiles, Ferroglobus placidus and “Geoglobus ahangari,” grow at 85°C by oxidizing acetate to carbon dioxide, with Fe(III) serving as the electron acceptor. These results demonstrate that acetate could potentially be metabolized within the hot microbial ecosystems in which hyperthermophiles predominate, rather than diffusing to cooler environments prior to degradation as has been previously proposed.     

Photocatabolism of Aromatic Compounds by the Phototrophic Purple Bacterium Rhodomicrobium vannielii

The phototrophic purple non-sulfur bacterium Rhodomicrobium vannielii grew phototrophically (illuminated anaerobic conditions) on a variety of aromatic compounds (in the presence of CO₂). Benzoate was universally photocatabolized by all five strains of R. vannielii examined, and benzyl alcohol was photocatabolized by four of the five strains. Catabolism of benzyl alcohol by phototrophic bacteria has not been previously reported. Other aromatic substrates supporting reasonably good growth of R. vannielii strains were the methoxylated benzoate derivatives vanillate (4-hydroxy-3-methoxybenzoate) and syringate (4-hydroxy-3,5-dimethoxybenzoate). However, catabolism of vanillate and syringate led to significant inhibition of bacteriochlorophyll synthesis in R. vannielii cells, eventually causing cultures to cease growing. No such effect on photopigment synthesis in cells grown on benzoate or benzyl alcohol was observed. Along with a handful of other species of anoxygenic phototrophic bacteria, the ability of the species R. vannielii to photocatabolize aromatic compounds indicates that this organism may also be ecologically significant as a consumer of aromatic derivatives in illuminated anaerobic habitats in nature.