Anaerobic degradation of catechol by Desulfobacterium sp. strain Cat2 proceeds via carboxylation to protocatechuate.

Under anoxic conditions, most methoxylated mononuclear aromatic compounds are degraded by bacteria, with catechol being formed as an important intermediate. On the basis of our experiments with the sulfate-reducing bacterium Desulfobacterium sp. strain Cat2, we describe for the first time the enzymatic activities involved in the complete anaerobic oxidation of catechol and protocatechuate. Results obtained from experiments with dense cell suspensions of strain Cat2 demonstrated that all enzymes necessary for protocatechuate and benzoate degradation were induced during growth with catechol. In addition, anaerobic oxidation of catechol was found to be a CO2-dependent process. Phenol was not degraded in suspensions of cells grown with catechol. In cell extracts of Desulfobacterium sp. strain Cat2, protocatechuyl-coenzyme A (CoA) was formed from catechol, bicarbonate, and uncombined CoA. This oxygen-sensitive reaction requires high concentrations of both bicarbonate and protein, and only very low levels of enzyme were detected. In a second oxygen-sensitive step, protocatechuyl-CoA was reduced to 3-hydroxybenzoyl-CoA by reductive elimination of the p-hydroxyl group. Further dehydroxylation to benzoyl-CoA was not detectable. Key reactions described for anaerobic degradation of benzoate were catalyzed by cell extracts of strain Cat2, too.     

Two distinct pathways for anaerobic degradation of aromatic compounds in the denitrifying bacterium Thauera aromatica strain AR-1

Denitrifying bacteria degrade many different aromatic compounds anaerobically via the well-described benzoyl-CoA pathway. We have shown recently that the denitrifiers Azoarcus anaerobius and Thauera aromatica strain AR-1 use a different pathway for anaerobic degradation of resorcinol (1,3-dihydroxybenzene) and 3,5-dihydroxybenzoate, respectively. Both substrates are converted to hydroxyhydroquinone (1,2,4-trihydroxybenzene). In the membrane fraction of T. aromatica strain AR-1 cells grown with 3,5-dihydroxybenzoate, a hydroxyhydroquinone-dehydrogenating activity of 74 nmol min(-1)(mg protein)-1 was found. This activity was significantly lower in benzoate-grown cells. Benzoate-grown cells were not induced for degradation of 3,5-dihydroxybenzoate, and cells grown with 3,5-dihydroxybenzoate degraded benzoate only at a very low rate. With a substrate mixture of benzoate plus 3,5-dihydroxybenzoate, the cells showed diauxic growth. Benzoate was degraded first, while complete degradation of 3,5-dihydroxybenzoate occurred only after a long lag phase. The 3,5-dihydroxybenzoate-oxidizing and the hydroxyhydroquinone-dehydrogenating activities were fully induced only during 3,5-dihydroxybenzoate degradation. Synthesis of benzoyl-CoA reductase appeared to be significantly lower in 3,5-dihydroxybenzoate-grown cells as shown by immunoblotting. These results confirm that T. aromatica strain AR-1 harbors, in addition to the benzoyl-CoA pathway, a second, mechanistically distinct pathway for anaerobic degradation of aromatic compounds. This pathway is inducible and subject to catabolite repression by benzoate.     

New aerobic benzoate oxidation pathway via benzoyl-coenzyme A and 3-hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp.

A denitrifying Pseudomonas sp. is able to oxidize aromatic compounds compounds completely to CO2, both aerobically and anaerobically. It is shown that benzoate is aerobically oxidized by a new degradation pathway via benzoyl-coenzyme A (CoA) and 3-hydroxybenzoyl-CoA. The organism grew aerobically with benzoate, 3-hydroxybenzoate, and gentisate; catechol, 2-hydroxybenzoate, and protocatechuate were not used, and 4-hydroxybenzoate was a poor substrate. Mutants were obtained which were not able to utilize benzoate as the sole carbon source aerobically but still used 3-hydroxybenzoate or gentisate. Simultaneous adaptation experiments with whole cells seemingly suggested a sequential induction of enzymes of a benzoate oxidation pathway via 3-hydroxybenzoate and gentisate. Cells grown aerobically with benzoate contained a benzoate-CoA ligase (AMP forming) (0.1 mumol min-1 mg-1) which converted benzoate but not 3-hydroxybenzoate into its CoA thioester. The enzyme of 130 kDa composed of two identical subunits of 56 kDa was purified and characterized. Cells grown aerobically with 3-hydroxybenzoate contained a similarly active CoA ligase for 3-hydroxybenzoate, 3-hydroxybenzoate-CoA ligase (AMP forming). Extracts from cells grown aerobically with benzoate catalyzed a benzoyl-CoA- and flavin adenine dinucleotide-dependent oxidation of NADPH with a specific activity of at least 25 nmol NADPH oxidized min-1 mg of protein-1; NADH and benzoate were not used. This new enzyme, benzoyl-CoA 3-monooxygenase, was specifically induced during aerobic growth with benzoate and converted [U-14C]benzoyl-CoA stoichiometrically to [14C]3-hydroxybenzoyl-CoA.     

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     

Degradation of some phenols and hydroxybenzoates by the imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans: evidence for an operative gentisate pathway

The imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans degraded 3-hydroxybenzoic acid via gentisate which was the cleavage substrate. 4-Hydroxybenzoic acid was metabolized via protocatechuate. No cleavage enzyme for the latter was detected. In stead of this NADH- and NADPH-dependent monooxygenases were present. In cells grown at the expense of hydroquinone and 4-hydroxygenzoic acid, enzymes of the hydroxyhydroquinone variant of the 3-oxoadipate pathway were demonstrated, which also took part in the degradation of 2,4-dihydroxybenzoic acid byC. parapsilosis.Abbreviations HHQ Hydroxyhydroquinone (1,2,4-trihydroxybenzene) - GSH reduced Glutathione     

Proteomics analysis of aromatic catabolic pathways in thermophilic Geobacillus thermodenitrificans NG80-2

Geobacillus thermodenitrificans NG80-2 is a crude oil-degrading thermophilic bacterium isolated from an oil reservoir in China. In this study, the gene clusters and pathways for the degradation of benzoate (via benzoyl-CoA), phenylacetate (via phenylacetyl-CoA), 4-hydroxyphenylacetate (via 3,4-dihydroxyphenylacetate) and anthranilate (via 3-hydroxyanthranilate) were confirmed using combined in silico analysis and proteomics approaches. It was observed that synthesis of the enzymes catalyzing initial activation, ring oxidation and ring cleavage reactions were generally induced specifically by their respective substrates, while many of the enzymes catalyzing downstream reactions exhibited broader substrate specificities. Novel genes encoding benzoyl-CoA epoxidase and 3,4-dihydroxyphenylacetate 2,3-dioxygenase, and a paaX homologue that serves as a positive regulator of benzoate degradation were proposed. Downregulation of the glycolysis pathway, along with upregulation of the gluconeogenesis pathway and the glyoxylate bypass (phenylacetate) were detected in association with the utilization of the aromatics. This novel proteomics approach confirmed the presence of multiple metabolic pathways for aromatic compounds in NG80-2, which is highly advantageous to the survival of this thermophilic bacterium under reservoir conditions.     

Anaerobic metabolism of 2-hydroxybenzoic acid (salicylic acid) by a denitrifying bacterium

The anaerobic metabolism of 2-hydroxybenzoic acid (salicylic acid) was studied in a denitrifying bacterium. Cells grown with 2-hydroxybenzoate were simultaneously adapted to degrade benzoate. Extract of these cells formed benzoate or benzoyl-CoA when incubated under reducing conditions with salicylate, MgATP, and coenzyme A, suggesting a degradation of 2-hydroxybenzoate via benzoate or benzoyl-CoA. This suggestion was supported by enzyme activity measurements. In extracts of 2-hydroxybenzoate-grown cells, the following enzyme activities were detected: two CoA ligases, one specific for 2-hydroxybenzoate, the other for benzoate, and two different enzyme activities catalyzing the reductive transformation of 2-hydroxybenzoyl-CoA. These findings suggest a degradation of salicylic acid by two new enzymes, 2-hydroxybenzoate-CoA ligase (AMP-forming) and 2-hydroxybenzoyl-CoA reductase (dehydroxylating), catalyzing (1) 2-hydroxybenzoate + MgATP + CoASH → 2-hydroxybenzoyl-CoA + MgAMP + PP_i (2) 2-hydroxybenzoyl-CoA + 2[H] → benzoyl-CoA + H₂O Benzoyl-CoA was dearomatized by reduction of the ring. This represents another case in which benzoyl-CoA is a central intermediate in anaerobic aromatic metabolism. Received: 1 February 1996 / Accepted: 24 February 1996     

Degradation of phenol through ortho-cleavage pathway by Pseudomonas stutzeri strain SPC2

P.Y. ANEEZ AHAMAD AND A.A.M. KUNHI. 1996. Generally pseudomonads degrade phenol through the meta -pathway, but Pseudomonas stutzeri strain SPC2 isolated by flask enrichment of municipal sewage degraded phenol through the ortho -pathway. The strain utilized up to 1200 ppm of phenol as a sole source of carbon and energy. The strain also degraded benzoate and 4-hydroxy and 3,4-dihydroxybenzoates via the ortho -pathway. Cell-free extracts of the strain grown on these substrates showed fairly good catechol 1,2-dioxygenase (C1,2-D) and protocatechuate 3,4-dioxyenase (PCA 3,4-D) activities, the induction of both activities being increased by benzoate. No meta -cleavage activities were detected.     

Anaerobic Degradation of the Benzene Nucleus by a Facultatively Anaerobic Microorganism

A bacterium was isolated by elective culture with p-hydroxybenzoate as substrate and nitrate as electron acceptor. It grew either aerobically or anaerobically, by nitrate respiration, on a range of aromatic compounds. The organism was identified as a pseudomonad and was given the trivial name Pseudomonas PN-1. Benzoate and p-hydroxybenzoate were metabolized aerobically via protocatechuate, followed by meta cleavage catalyzed by protocatechuic acid-4,5-oxygenase, to yield alpha-hydroxy-gamma-carboxymuconic semialdehyde. Pseudomonas PN-1 grew rapidly on p-hydroxybenzoate under strictly anaerobic conditions, provided nitrate was present, even though protocatechuic acid-4,5-oxygenase was repressed. Suspensions of cells grown anaerobically on p-hydroxybenzoate oxidized benzoate with nitrate and produced 4 to 5 mumoles of CO(2) per mumole of benzoate added; these cells did not oxidize benzoate aerobically. The patterns of the oxidation of aromatic substrates with oxygen or nitrate by cells grown aerobically or anaerobically on different aromatic compounds indicated that benzoate rather than protocatechuate was a key intermediate in the early stages of anaerobic metabolism. It was concluded that the pathway for the anaerobic breakdown of the aromatic ring is different and quite distinct from the aerobic pathway. Mechanisms for the anaerobic degradation of the benzene nucleus by Pseudomonas PN-1 are discussed.     

Catabolism of benzoate and monohydroxylated benzoates by Amycolatopsis and Streptomyces spp

Eight actinomycetes of the genera Amycolatopsis and Streptomyces were tested for the degradation of aromatic compounds by growth in a liquid medium containing benzoate, monohydroxylated benzoates, or quinate as the principal carbon source. Benzoate was converted to catechol. The key intermediate in the degradation of salicylate was either catechol or gentisate, while m-hydroxybenzoate was metabolized via gentisate or protocatechuate. p-Hydroxybenzoate and quinate were converted to protocatechuate. Catechol, gentisate, and protocatechuate were cleaved by catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, and protocatechuate 3,4-dioxygenase, respectively. The requirement for glutathione in the gentisate pathway was dependent on the substrate and the particular strain. The conversion of p-hydroxybenzoate to protocatechuate by p-hydroxybenzoate hydroxylase was gratuitously induced by all substrates that were metabolized via protocatechuate as an intermediate, while protocatechuate 3,4-dioxygenase was gratuitously induced by benzoate and salicylate in two Amycolatopsis strains.     

A novel pathway of aerobic benzoate catabolism in the bacteria Azoarcus evansii and Bacillus stearothermophilus

The aerobic catabolism of benzoate was studied in the Gram-negative proteobacterium Azoarcus evansii and in the Gram-positive bacterium Bacillus stearothermophilus. In contrast to earlier proposals, benzoate was not converted into hydroxybenzoate or gentisate. Rather, benzoyl-CoA was a product of benzoate catabolism in both microbial species under aerobic conditions in vivo. Benzoyl-CoA was converted into various CoA thioesters by cell extracts of both species in oxygen- and NADPH-dependent reactions. Using [ring-(13)C(6)]benzoyl-CoA as substrate, cis-3,4-[2,3,4,5,6-(13)C(5)]dehydroadipyl-CoA, trans-2,3-[2,3,4,5,6-(13)C(5)]dehydroadipyl-CoA, the 3,6-lactone of 3-[2,3,4,5,6-(13)C(5)]hydroxyadipyl-CoA, and 3-[2,3,4,5,6-(13)C(5)]hydroxyadipyl-CoA were identified as products by NMR spectroscopy. A protein mixture of A. evansii transformed [ring-(13)C(6)]benzoyl-CoA in an NADPH- and oxygen-dependent reaction into 6-[2,3,4,5,6-(13)C(5)]hydroxy-3-hexenoyl-CoA. The data suggest a novel aerobic pathway of benzoate catabolism via CoA intermediates leading to beta-ketoadipyl-CoA, an intermediate of the known beta-ketoadipate pathway.     

Catabolism of benzoate and monohydroxylated benzoates by Amycolatopsis and Streptomyces spp.

Eight actinomycetes of the genera Amycolatopsis and Streptomyces were tested for the degradation of aromatic compounds by growth in a liquid medium containing benzoate, monohydroxylated benzoates, or quinate as the principal carbon source. Benzoate was converted to catechol. The key intermediate in the degradation of salicylate was either catechol or gentisate, while m-hydroxybenzoate was metabolized via gentisate or protocatechuate. p-Hydroxybenzoate and quinate were converted to protocatechuate. Catechol, gentisate, and protocatechuate were cleaved by catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, and protocatechuate 3,4-dioxygenase, respectively. The requirement for glutathione in the gentisate pathway was dependent on the substrate and the particular strain. The conversion of p-hydroxybenzoate to protocatechuate by p-hydroxybenzoate hydroxylase was gratuitously induced by all substrates that were metabolized via protocatechuate as an intermediate, while protocatechuate 3,4-dioxygenase was gratuitously induced by benzoate and salicylate in two Amycolatopsis strains.     

Anaerobic aniline degradation via reductive deamination of 4-aminobenzoyl-CoA in Desulfobacterium anilini

The initial reactions involved in anaerobic aniline degradation by the sulfate-reducing Desulfobacterium anilini were studied. Experiments for substrate induction indicated the presence of a common pathway for aniline and 4-aminobenzoate, different from that for degradation of 2-aminobenzoate, 2-hydroxybenzoate, 4-hydroxybenzoate, or phenol. Degradation of aniline by dense cell suspensions depended on CO₂ whereas 4-aminobenzoate degradation did not. If acetyl-CoA oxidation was inhibited by cyanide, benzoate accumulated during degradation of aniline or 4-aminobenzoate, indicating an initial carboxylation of aniline to 4-aminobenzoate, and further degradation via benzoate of both substrates. Extracts of alinine or 4-aminobenzoategrown cells activated 4-aminobenzoate to 4-aminobenzoyl-CoA in the presence of CoA, ATP and Mg²⁺. 4-Aminobenzoyl-CoA-synthetase showed a K _m for 4-aminobenzoate lower than 10 M and an activity of 15.8 nmol · min^-1 · mg^-1. 4-Aminobenzoyl-CoA was reductively deaminated to benzoyl-CoA by cell extracts in the presence of low-potential electron donors such as titanium citrate or cobalt sepulchrate (2.1 nmol · min^-1 · mg^-1). Lower activities for the reductive deamination were measured with NADH or NADPH. Reductive deamination was also indicated by benzoate accumulation during 4-aminobenzoate degradation in cell suspensions under sulfate limitation. The results provide evidence that aniline is degraded via carboxylation to 4-aminobenzoate, which is activated to 4-aminobenzoyl-CoA and further metabolized by reductive deamination to benzoyl-CoA.     

Cyclohexa-1,5-diene-1-carbonyl-coenzyme A (CoA) hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: Evidence for a common benzoyl-CoA degradation pathway in facultative and strict anaerobes

In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of beta-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km, 80 and 35 microM; Vmax, 350 and 550 micromol min(-1) mg(-1), respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.     

Novel nuclear magnetic resonance spectroscopy methods demonstrate preferential carbon source utilization by Acinetobacter calcoaceticus.

Novel nuclear magnetic resonance spectroscopy techniques, designated metabolic observation, were used to study aromatic compound degradation by the soil bacterium Acinetobacter calcoaceticus. Bacteria which had been rendered spectroscopically invisible by growth with deuterated (2H) medium were used to inoculate cultures in which natural-abundance 1H hydrogen isotopes were provided solely by aromatic carbon sources in an otherwise 2H medium. Samples taken during the incubation of these cultures were analyzed by proton nuclear magnetic resonance spectroscopy, and proton signals were correlated with the corresponding aromatic compounds or their metabolic descendants. This approach allowed the identification and quantitation of metabolites which accumulated during growth. This in vivo metabolic monitoring facilitated studies of catabolism in the presence of multiple carbon sources, a topic about which relatively little is known. A. calcoaceticus initiates aromatic compound dissimilation by forming catechol or protocatechuate from a variety of substrates. Degradation proceeds via the beta-ketoadipate pathway, comprising two discrete branches that convert catechol or protocatechuate to tricarboxylic acid cycle intermediates. As shown below, when provided with several carbon sources simultaneously, all degraded via the beta-ketoadipate pathway, A. calcoaceticus preferentially degraded specific compounds. For example, benzoate, degraded via the catechol branch, was consumed in preference to p-hydroxybenzoate, degraded via the protocatechuate branch, when both compounds were present. To determine if this preference were governed by metabolites unique to catechol degradation, pathway mutants were constructed. Studies of these mutants indicated that the product of catechol ring cleavage, cis,cis-muconate, inhibited the utilization of p-hydroxybenzoate in the presence of benzoate. The accumulation of high levels of cis,cis-muconate also appeared to be toxic to the cells.     

Evidence of Two Oxidative Reaction Steps Initiating Anaerobic Degradation of Resorcinol (1,3-Dihydroxybenzene) by the Denitrifying Bacterium Azoarcus anaerobius

The denitrifying bacterium Azoarcus anaerobius LuFRes1 grows anaerobically with resorcinol (1,3-dihydroxybenzene) as the sole source of carbon and energy. The anaerobic degradation of this compound was investigated in cell extracts. Resorcinol reductase, the key enzyme for resorcinol catabolism in fermenting bacteria, was not present in this organism. Instead, resorcinol was hydroxylated to hydroxyhydroquinone (HHQ; 1,2,4-trihydroxybenzene) with nitrate or K₃Fe(CN)₆ as the electron acceptor. HHQ was further oxidized with nitrate to 2-hydroxy-1,4-benzoquinone as identified by high-pressure liquid chromatography, UV/visible light spectroscopy, and mass spectroscopy. Average specific activities were 60 mU mg of protein⁻¹ for resorcinol hydroxylation and 150 mU mg of protein⁻¹ for HHQ dehydrogenation. Both activities were found nearly exclusively in the membrane fraction and were only barely detectable in extracts of cells grown with benzoate, indicating that both reactions were specific for resorcinol degradation. These findings suggest a new strategy of anaerobic degradation of aromatic compounds involving oxidative steps for destabilization of the aromatic ring, different from the reductive dearomatization mechanisms described so far.     

Alternative routes of aromatic catabolism in Pseudomonas acidovorans and Pseudomonas putida: gallic acid as a substrate and inhibitor of dioxygenases.

When 3,4-dihydroxyphenylacetic acid (homoprotocatechuic acid) was added to Pseudomonase acidovorans growing at the expense of succinate, enzymes required for degrading homoprotocatechuate to pyruvate and succinate semialdehyde were strongly induced. These enzymes were effectively absent from cell extracts of the organism grown with 4-hydroxyphenylacetic acid, and this substrate was metabolized by the catabolic enzymes of the homogentisate pathway. Two separate ring-fission dioxygenases for 3,4,5-trihydroxybenzoic acid (gallic acid) were present in cell extracts of Pseudomonas putida when grown with syringic acid, and gallate was degraded by reactions associated with meta fission. One of the two gallate dioxygenases also attacked 3-O-methylgallic acid; the other, which did not, was induced when cells were exposed to gallate. This organism possessed ortho fission enzymes, including protocatechuate 3,4-dioxygenase (EC 1.13.11.3) and cis,cis-carboxymuconate-lactonizing enzyme (EC 5.5.1.2), after induction with 3,4-dihydroxybenzoic acid (protocatechuic acid). Gallate was a substrate for protocatechuate 3,4-dioxygenase, with a Vmax about 3% of that of protocatechuate and with an apparent Km slightly lower. Gallate was a powerful competitive inhibitor of protocatechuate oxidation.     

Anaerobic degradation of 2-aminobenzoic acid (anthranilic acid) via benzoyl-coenzyme A (CoA) and cyclohex-1-enecarboxyl-CoA in a denitrifying bacterium.

The enzymes catalyzing the initial reactions in the anaerobic degradation of 2-aminobenzoic acid (anthranilic acid) were studied with a denitrifying Pseudomonas sp. anaerobically grown with 2-aminobenzoate and nitrate as the sole carbon and energy sources. Cells grown on 2-aminobenzoate are simultaneously adapted to growth with benzoate, whereas cells grown on benzoate degrade 2-aminobenzoate several times less efficiently than benzoate. Evidence for a new reductive pathway of aromatic metabolism and for four enzymes catalyzing the initial steps is presented. The organism contains 2-aminobenzoate-coenzyme A ligase (2-aminobenzoate-CoA ligase), which forms 2-aminobenzoyl-CoA. 2-Aminobenzoyl-CoA is then reductively deaminated to benzoyl-CoA by an oxygen-sensitive enzyme, 2-aminobenzoyl-CoA reductase (deaminating), which requires a low potential reductant [Ti(III)]. The specific activity is 15 nmol of 2-aminobenzoyl-CoA reduced min-1 mg-1 of protein at an optimal pH of 7. The two enzymes are induced by the substrate under anaerobic conditions only. Benzoyl-CoA is further converted in vitro by reduction with Ti(III) to six products; the same products are formed when benzoyl-CoA or 2-aminobenzoyl-CoA is incubated under reducing conditions. Two of them were identified preliminarily. One product is cyclohex-1-enecarboxyl-CoA, the other is trans-2-hydroxycyclohexane-carboxyl-CoA. The complex transformation of benzoyl-CoA is ascribed to at least two enzymes, benzoyl-CoA reductase (aromatic ring reducing) and cyclohex-1-enecarboxyl-CoA hydratase. The reduction of benzoyl-CoA to alicyclic compounds is catalyzed by extracts from cells grown anaerobically on either 2-aminobenzoate or benzoate at almost the same rate (10 to 15 nmol min-1 mg-1 of protein). In contrast, extracts from cells grown anaerobically on acetate or grown aerobically on benzoate or 2-aminobenzoate are inactive. This suggests a sequential induction of the enzymes.     

Key aromatic-ring-cleaving enzyme, protocatechuate 3,4-dioxygenase, in the ecologically important marine Roseobacter lineage

Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the alpha-proteobacteria was investigated. Initial screens suggested that isolates in the Roseobacter lineage can degrade aromatic compounds via the beta-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Six Roseobacter isolates were screened for the presence of protocatechuate 3,4-dioxygenase, a key enzyme in the beta-ketoadipate pathway. All six isolates were capable of growth on at least three of the eight aromatic monomers presented (anthranilate, benzoate, p-hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuate, and coumarate). Four of the Roseobacter group isolates had inducible protocatechuate 3, 4-dioxygenase activity in cell extracts when grown on p-hydroxybenzoate. The pcaGH genes encoding this ring cleavage enzyme were cloned and sequenced from two isolates, Sagittula stellata E-37 and isolate Y3F, and in both cases the genes could be expressed in Escherichia coli to yield dioxygenase activity. Additional genes involved in the protocatechuate branch of the beta-ketoadipate pathway (pcaC, pcaQ, and pobA) were found to cluster with pcaGH in these two isolates. Pairwise sequence analysis of the pca genes revealed greater similarity between the two Roseobacter group isolates than between genes from either Roseobacter strain and soil bacteria. A degenerate PCR primer set targeting a conserved region within PcaH successfully amplified a fragment of pcaH from two additional Roseobacter group isolates, and Southern hybridization indicated the presence of pcaH in the remaining two isolates. This evidence of protocatechuate 3, 4-dioxygenase and the beta-ketoadipate pathway was found in all six Roseobacter isolates, suggesting widespread abilities to degrade aromatic compounds in this marine lineage.     

Energetics and biochemistry of fermentative benzoate degradation by Syntrophus gentianae

The pathway of fermentative benzoate degradation by the syntrophically fermenting bacterium Syntrophus gentianae was studied by measurement of enzyme activities in cell-free extracts. Benzoate was activated by a benzoate-CoA ligase reaction, forming AMP and pyrophosphate, which was subsequently cleaved by a membrane-bound proton-translocating pyrophosphatase. Glutaconyl-CoA (formed from hypothetical pimelyl-CoA and glutaryl-CoA intermediates) was decarboxylated to crotonyl-CoA by a sodium-ion-dependent membrane-bound glutaconyl-CoA decarboxylase, a biotin enzyme that could be inhibited by avidin. The overall energy budget of this fermentation could be balanced only if the dearomatizing reduction of benzoyl-CoA is assumed to produce cyclohexene carboxyl-CoA rather than cyclohexadiene carboxyl-CoA, although experimental evidence of this reaction is still insufficient. With this assumption, benzoate degradation by S. gentianae can be balanced to yield one-third to two-thirds of an ATP unit per benzoate degraded, in accordance with earlier measurements of whole-cell energetics. Received: 5 August 1998 / Accepted: 18 February 1999