Initial reactions involved in the dissimilation of mandelate by Rhodotorula graminis.

Rhodotorula graminis utilized DL-mandelate, L(+)-mandelate, and D(-)-mandelate as sole sources of carbon and energy. Growth on these aromatic substrates resulted in the induction of an NAD-dependent D(-)-mandelate dehydrogenase and a dye-linked L(+)-mandelate dehydrogenase, each catalyzing the stereospecific conversion of its respective enantiomer of mandelate to benzoylformate. Benzoylformate was oxidized to benzaldehyde, which was dehydrogenated to benzoate by an NAD-dependent benzaldehyde dehydrogenase. Benzoate was further metabolized through p-hydroxybenzoate and the protocatechuate branch of the 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.     

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.     

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.     

Peptide mass fingerprinting- and 2-DE/MS-based analysis of the biodegradation potential for monocyclic aromatic hydrocarbons in <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

The combined analysis of peptide mass fingerprinting and 2-DE/MS using the induced and selected protein spots following growth of Pseudomonas sp. DU102 on benzoate or p-hydroxybenzoate revealed not only alpha- and beta-subunits of protocatechuate 3,4-dioxygenase but also catechol 1,2-dioxygenase responsible for ortho-pathway through ring-cleavage of aromatic compounds. Toluate 1,2-dioxygenase and p-hydroxybenzoate hydroxylase were also identified. Purification of intradiol dioxygenases such as catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase from the benzoate or p-hydroxybenzoate culture makes it possible to trace the biodegradation pathway of strain DU102 for monocyclic aromatic hydrocarbons. Interestingly, vanillin-induced protocatechuate 3,4-dioxygenase was identical in amino acid sequences with protocatechuate 3,4-dioxygenase from p-hydroxybenzoate.     

Characterization of beta-ketoadipate pathway from multi-drug resistance bacterium, Acinetobacter baumannii DU202 by proteomic approach

In this study, the biodegradative activities of monocyclic aromatic compounds were determined from the multi-drug resistant (MDR) Acinetobacter baumannii, which were studied in the form of clinical isolates from a hospital in Korea. These bacteria were capable of biodegrading monocyclic aromatic compounds, such as benzoate and p-hydroxybenzoate. In order to determine which pathways are available for biodegradation in these stains, we conducted proteome analyses of benzoate and p-hydroxybenzoate-cultured A. baumannii DU202, using 2-DE/MS analysis. As genome DB of A. baumannii was not yet available, MS/MS analysis or de novo sequencing methods were employed in the identification of induced proteins. Benzoate branch enzymes [catechol 1,2-dioxygenase (CatA) and benzoate dioxygenase alpha subunit (BenA)] of the beta-ketoadipate pathway were identified under benzoate culture condition and p-hydroxybenzoate branch enzymes [protocatechuate 3,4-dioxygenase alpha subunit (PcaG) and 3-carboxy-cis,cis-muconate cycloisomerase (PcaB)] of the beta-ketoadipate pathway were identified under p-hydroxybenzoate culture condition, respectively, thereby suggesting that strain DU202 utilized the beta-ketoadipate pathway for the biodegradation of monocyclic aromatic compounds. The sequence analysis of two purified dioxygenases (CatA and PcaGH) indicated that CatA is closely associated with the CatA of Acinetobacter radiresistance, but PcaGH is only moderately associated with the PcaGH of Acinetobacter sp. ADP1. Interestingly, the fused form of PcaD and PcaC, carboxymuconolactone decarboxylase (PcaCD), was detected on benzoate-cultured A. baumannii DU202. These results indicate that A. baumannii DU202 exploits a different beta-ketoadipate pathway from other Acinetobacter species.     

Proteome analysis of Pseudomonas sp. K82 biodegradation pathways

Pseudomonas sp. K82 is a soil bacterium that can degrade and use monocyclic aromatic compounds including aniline, 3-methylaniline, 4-methylaniline, benzoate and p-hydroxybenzoate as its sole carbon and energy sources. In order to understand the impact of these aromatic compounds on metabolic pathways in Pseudomonas sp. K82, proteomes obtained from cultures exposed to different substrates were displayed by two-dimensional gel electrophoresis and were compared to search for differentially induced metabolic enzymes. Column separations of active fractions were performed to identify major biodegradation enzymes. More than thirty proteins involved in biodegradation and other types of metabolism were identified by electrospray ionization-quadrupole time of flight mass spectrometry. The proteome analysis suggested that Pseudomonas sp. K82 has three main metabolic pathways to degrade these aromatic compounds and induces specific metabolic pathways for each compound. The catechol 2,3-dioxygenase (CD2,3) pathway was the major pathway and the catechol 1,2-dioxygenase (beta-ketoadipate) pathway was the secondary pathway induced by aniline (aniline analogues) exposure. On the other hand, the catechol 1,2-dioxygenase pathway was the major pathway induced by benzoate exposure. For the degradation of p-hydroxybenzoate, the protocatechuate 4,5-dioxygenase pathway was the major degradation pathway induced. The nuclear magnetic resonance analysis of substrates demonstrated that Pseudomonas sp. K82 metabolizes some aromatic compounds more rapidly than others (benzoate > p-hydroxybenzoate > aniline) and that when combined, p-hydroxybenzoate metabolism is repressed by the presence of benzoate or aniline. These results suggest that proteome analysis can be useful in the high throughput study of bacterial metabolic pathways, including that of biodegradation, and that inter-relationships exist with respect to the metabolic pathways of aromatic compounds in Pseudomonas sp. K82.     

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.     

Dissimilation of aromatic compounds by Alcaligenes eutrophus

The range of aromatic compounds that support the growth of Alcaligenes eutrophus has been determined, and the pathways used for the dissimilation of these substrates have been explored, largely by enzymatic analyses. The beta-ketoadipate pathway operates in the dissimilation of benzoate and p-hydroxybenzoate; the genetisate pathway, in the dissimilation of m-hydroxybenzoate; and the meta cleavage pathway, in the dissimilation of phenol and p-cresol. l-Tryptophan is oxidized via anthranilate; but the metabolic fate of anthranilate was not established. The metabolism of the three stereoisomers of muconic acid was also examined.     

Nitrobenzoates and aminobenzoates are chemoattractants for Pseudomonas strains

Three Pseudomonas strains were tested for the ability to sense and respond to nitrobenzoate and aminobenzoate isomers in chemotaxis assays. Pseudomonas putida PRS2000, a strain that grows on benzoate and 4-hydroxybenzoate by using the beta-ketoadipate pathway, has a well-characterized beta-ketoadipate-inducible chemotactic response to aromatic acids. PRS2000 was chemotactic to 3- and 4-nitrobenzoate and all three isomers of aminobenzoate when grown under conditions that induce the benzoate chemotactic response. P. putida TW3 and Pseudomonas sp. strain 4NT grow on 4-nitrotoluene and 4-nitrobenzoate by using the ortho (beta-ketoadipate) and meta pathways, respectively, to complete the degradation of protocatechuate derived from 4-nitrotoluene and 4-nitrobenzoate. However, based on results of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase assays, both strains were found to use the beta-ketoadipate pathway for the degradation of benzoate. Both strains were chemotactic to benzoate, 3- and 4-nitrobenzoate, and all three aminobenzoate isomers after growth with benzoate but not succinate. Strain TW3 was chemotactic to the same set of aromatic compounds after growth with 4-nitrotoluene or 4-nitrobenzoate. In contrast, strain 4NT did not respond to any aromatic acids when grown with 4-nitrotoluene or 4-nitrobenzoate, apparently because these substrates are not metabolized to the inducer (beta-ketoadipate) of the chemotaxis system. The results suggest that strains TW3 and 4NT have a beta-ketoadipate-inducible chemotaxis system that responds to a wide range of aromatic acids and is quite similar to that present in PRS2000. The broad specificity of this chemotaxis system works as an advantage in strains TW3 and 4NT because it functions to detect diverse carbon sources, including 4-nitrobenzoate.     

Aromatic acids are chemoattractants for Pseudomonas putida

A quantitative capillary assay was used to show that aromatic acids, compounds that are chemorepellents for Escherichia coli and Salmonella sp., are chemoattractants for Pseudomonas putida PRS2000. The most effective attractants were benzoate; p-hydroxybenzoate; the methylbenzoates; m-, p-, and o-toluate; salicylate; DL-mandelate; beta-phenylpyruvate; and benzoylformate. The chemotactic responses to these compounds were inducible. Taxis to benzoate and m-toluate was induced by beta-ketoadipate, a metabolic intermediate formed when benzoate is dissimilated via enzymes specified by chromosomal genes. Benzoylformate taxis was induced by benzoylformate and L(+)-mandelate. Taxis to mandelate, benzoylformate, and beta-phenylpyruvate was exhibited by cells grown on mandelate, but not by cells grown on benzoate. Cells grown on benzoate were chemotactic to benzoate, the toluates, p-hydroxybenzoate, and salicylate. These results show that P. putida synthesizes at least two distinct chemoreceptors for aromatic acids. Although DL-mandelate was an effective attractant in capillary assays, additional experiments indicated that the cells were actually responding to benzoylformate, a metabolite formed from mandelate. With the exception of mandelate taxis, chemotaxis to aromatic acids was not dependent on the expression of pathways for aromatic degradation. Therefore, the tactic responses exhibited by cells cannot be attributed to an effect of the oxidation of aromatic acids on the energy metabolism of cells.     

Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440

Analysis of the catabolic potential of Pseudomonas putida KT2440 against a wide range of natural aromatic compounds and sequence comparisons with the entire genome of this microorganism predicted the existence of at least four main pathways for the catabolism of central aromatic intermediates, that is, the protocatechuate (pca genes) and catechol (cat genes) branches of the beta-ketoadipate pathway, the homogentisate pathway (hmg/fah/mai genes) and the phenylacetate pathway (pha genes). Two additional gene clusters that might be involved in the catabolism of N-heterocyclic aromatic compounds (nic cluster) and in a central meta-cleavage pathway (pcm genes) were also identified. Furthermore, the genes encoding the peripheral pathways for the catabolism of p-hydroxybenzoate (pob), benzoate (ben), quinate (qui), phenylpropenoid compounds (fcs, ech, vdh, cal, van, acd and acs), phenylalanine and tyrosine (phh, hpd) and n-phenylalkanoic acids (fad) were mapped in the chromosome of P. putida KT2440. Although a repetitive extragenic palindromic (REP) element is usually associated with the gene clusters, a supraoperonic clustering of catabolic genes that channel different aromatic compounds into a common central pathway (catabolic island) was not observed in P. putida KT2440. The global view on the mineralization of aromatic compounds by P. putida KT2440 will facilitate the rational manipulation of this strain for improving biodegradation/biotransformation processes, and reveals this bacterium as a useful model system for studying biochemical, genetic, evolutionary and ecological aspects of the catabolism of aromatic compounds.     

Metabolism of aromatic compounds by Caulobacter crescentus.

Cultures of Caulobacter crescentus were found to grow on a variety of aromatic compounds. Degradation of benzoate, p-hydroxybenzoate, and phenol was found to occur via beta-ketoadipate. The induction of degradative enzymes such as benzoate 1,2-dioxygenase, the ring cleavage enzyme catechol 1,2-dioxygenase, and cis, cis-muconate lactonizing enzyme appeared similar to the control mechanism present in Pseudomonas spp. Both benzoate 1,2-dioxygenase and catechol 1,2-dioxygenase had stringent specificities, as revealed by their action toward substituted benzoates and substituted catechols, respectively.     

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.     

Constitutive synthesis of enzymes involved in 2-aminophenol metabolism and inducible synthesis of enzymes involved in benzoate, p-hydroxybenzoate, and protocatechuate metabolism in Pseudomonas sp. strain AP-3

Pseudomonas sp. strain AP-3 grows on benzoate, p-hydroxybenzoate, protocatechuate, and 2-aminophenol as sole carbon and energy source. This strain converted benzoate and p-hydroxybenzoate to catechol and protocatechuate respectively, which were metabolized via the ortho-cleavage pathway. The enzymes responsible for these reactions were shown to be inducible. In contrast, strain AP-3 constitutively expresses the enzymes involved in the metabolism of 2-aminophenol.     

Regulation of phenolic catabolism in Rhizobium leguminosarum biovar trifolii.

In members of the family Rhizobiaceae, many phenolic compounds are degraded by the protocatechuate branch of the beta-ketoadipate pathway. In this paper we describe a novel pattern of induction of protocatechuate (pca) genes in Rhizobium leguminosarum biovar trifolii. Isolation of pca mutant strains revealed that 4-hydroxybenzoate, quinate, and 4-coumarate are degraded via the protocatechuate pathway. At least three inducers govern catabolism of 4-hydroxybenzoate to succinyl coenzyme A and acetyl coenzyme A. The enzyme that catalyzes the initial step is induced by its substrate, whereas the catabolite beta-carboxy-cis,cis-muconate induces enzymes for the upper protocatechuate pathway, and beta-ketoadipate elicits expression of the enzyme for a subsequent step, beta-ketoadipate succinyl-coenzyme A transferase. Elucidation of the induction pattern relied in part on complementation of mutant Rhizobium strains by known subclones of Acinetobacter genes expressed off the lac promoter in a broad-host-range vector.     

Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 using a combined proteomic approach: 2-DE/MS and cleavable isotope-coded affinity tag analysis

Proteomic analysis of Pseudomonas putida KT2440 cultured in monocyclic aromatic compounds was performed using 2-DE/MS and cleavable isotope-coded affinity tag (ICAT) to determine whether proteins involved in aromatic compound degradation pathways were altered as predicted by genomic analysis (Jiménez et al., Environ Microbiol. 2002, 4, 824-841). Eighty unique proteins were identified by 2-DE/MS or MS/MS analysis from P. putida KT2440 cultured in the presence of six different organic compounds. Benzoate dioxygenase (BenA, BenD) and catechol 1,2-dioxygenase (CatA) were induced by benzoate. Protocatechuate 3,4-dixoygenase (PcaGH) was induced by p-hydroxybenzoate and vanilline. beta-Ketoadipyl CoA thiolase (PcaF) and 3-oxoadipate enol-lactone hydrolase (PcaD) were induced by benzoate, p-hydroxybenzoate and vanilline, suggesting that benzoate, p-hydroxybenzoate and vanilline were degraded by different dioxygenases and then converged in the same beta-ketoadipate degradation pathway. An additional 110 proteins, including 19 proteins from 2-DE analysis, were identified by cleavable ICAT analysis for benzoate-induced proteomes, which complemented the 2-DE results. Phenylethylamine exposure induced beta-ketoacyl CoA thiolase (PhaD) and ring-opening enzyme (PhaL), both enzymes of the phenylacetate (pha) biodegradation pathway. Phenylalanine induced 4-hydroxyphenyl-pyruvate dioxygenase (Hpd) and homogentisate 1,2-dioxygenase (HmgA), key enzymes in the homogentisate degradation pathway. Alkyl hydroperoxide reductase (AphC) was induced under all aromatic compounds conditions. These results suggest that proteome analysis complements and supports predictive information obtained by genomic sequence analysis.     

The metabolism of aromatic acids by micro-organisms. Metabolic pathways in the fungi

1. The metabolic pathways of aromatic-ring fission were examined in a range of fungal genera that utilize several compounds related to lignin. 2. Most of the genera, after growth on p-hydroxybenzoate, protocatechuate or compounds that are degraded to the latter (e.g. caffeate, ferulate or vanillate), rapidly oxidized these compounds, but not catechol. 3. Such genera possessed a protocatechuate 3,4-oxygenase and accumulated beta-carboxymuconate as the product of protocatechuate oxidation. This enzyme had a high pH optimum in most organisms; the Rhodotorula enzyme was competitively inhibited by catechol. 4. beta-Carboxymuconate was converted by all competent fungi into beta-carboxymuconolactone, which was isolated and characterized. None of the fungi produced or utilized at significant rates the corresponding bacterial intermediate gamma-carboxymuconolactone. 5. The lactonizing enzymes of Rhodotorula and Neurospora crassa had a pH optimum near 5.5 and approximate molecular weights of 19000 and 190000 respectively. 6. The fungi did not degrade the isomeric (+)-muconolactone, gamma-carboxymethylenebutanolide or beta-oxoadipate enol lactone at significant rates, and thus differ radically from bacteria, where beta-oxoadipate enol lactone is the precursor of beta-oxoadipate in all strains examined. 7. The end product of beta-carboxymuconolactone metabolism by extracts was beta-oxoadipate. 8. Evidence for a coenzyme A derivative of beta-oxoadipate was found during further metabolism of this keto acid. 9. A few anomalous fungi, after growth on p-hydroxybenzoate, had no protocatechuate 3,4-oxygenase, but possessed all the enzymes of the catechol pathway. Catechol was detected in the growth medium in one instance. 10. A strain of Penicillium sp. formed pyruvate but no beta-oxoadipate from protocatechuate, suggesting the existence also of a ;meta' type of ring cleavage among fungi.     

Characterization of the protocatechuic acid catabolic gene cluster from Streptomyces sp. strain 2065

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) catalyzes the ring cleavage step in the catabolism of aromatic compounds through the protocatechuate branch of the beta-ketoadipate pathway. A protocatechuate 3,4-dioxygenase was purified from Streptomyces sp. strain 2065 grown in p-hydroxybenzoate, and the N-terminal sequences of the beta- and alpha-subunits were obtained. PCR amplification was used for the cloning of the corresponding genes, and DNA sequencing of the flanking regions showed that the pcaGH genes belonged to a 6. 5-kb protocatechuate catabolic gene cluster; at least seven genes in the order pcaIJFHGBL appear to be transcribed unidirectionally. Analysis of the cluster revealed the presence of a pcaL homologue which encodes a fused gamma-carboxymuconolactone decarboxylase/beta-ketoadipate enol-lactone hydrolase previously identified in the pca gene cluster from Rhodococcus opacus 1CP. The pcaIJ genes encoded proteins with a striking similarity to succinyl-coenzyme A (CoA):3-oxoacid CoA transferases of eukaryotes and contained an indel which is strikingly similar between high-G+C gram-positive bacteria and eukaryotes.