Characterization of catechol catabolic genes from Rhodococcus erythropolis 1CP.

The biochemical characterization of the muconate and the chloromuconate cycloisomerases of the chlorophenol-utilizing Rhodococcus erythropolis strain 1CP previously indicated that efficient chloromuconate conversion among the gram-positive bacteria might have evolved independently of that among gram-negative bacteria. Based on sequences of the N terminus and of tryptic peptides of the muconate cycloisomerase, a fragment of the corresponding gene has now been amplified and used as a probe for the cloning of catechol catabolic genes from R. erythropolis. The clone thus obtained expressed catechol 1,2-dioxygenase, muconate cycloisomerase, and muconolactone isomerase activities. Sequencing of the insert on the recombinant plasmid pRER1 revealed that the genes are transcribed in the order catA catB catC. Open reading frames downstream of catC may have a function in carbohydrate metabolism. The predicted protein sequence of the catechol 1,2-dioxygenase was identical to the one from Arthrobacter sp. strain mA3 in 59% of the positions. The chlorocatechol 1,2-dioxygenases and the chloromuconate cycloisomerases of gram-negative bacteria appear to be more closely related to the catechol 1,2-dioxygenases and muconate cycloisomerases of the gram-positive strains than to the corresponding enzymes of gram-negative bacteria.     

A new modified ortho cleavage pathway of 3-chlorocatechol degradation by Rhodococcus opacus 1CP: genetic and biochemical evidence

The 4-chloro- and 2,4-dichlorophenol-degrading strain Rhodococcus opacus 1CP has previously been shown to acquire, during prolonged adaptation, the ability to mineralize 2-chlorophenol. In addition, homogeneous chlorocatechol 1,2-dioxygenase from 2-chlorophenol-grown biomass has shown relatively high activity towards 3-chlorocatechol. Based on sequences of the N terminus and tryptic peptides of this enzyme, degenerate PCR primers were now designed and used for cloning of the respective gene from genomic DNA of strain 1CP. A 9.5-kb fragment containing nine open reading frames was obtained on pROP1. Besides other genes, a gene cluster consisting of four chlorocatechol catabolic genes was identified. As judged by sequence similarity and correspondence of predicted N termini with those of purified enzymes, the open reading frames correspond to genes for a second chlorocatechol 1,2-dioxygenase (ClcA2), a second chloromuconate cycloisomerase (ClcB2), a second dienelactone hydrolase (ClcD2), and a muconolactone isomerase-related enzyme (ClcF). All enzymes of this new cluster are only distantly related to the known chlorocatechol enzymes and appear to represent new evolutionary lines of these activities. UV overlay spectra as well as high-pressure liquid chromatography analyses confirmed that 2-chloro-cis,cis-muconate is transformed by ClcB2 to 5-chloromuconolactone, which during turnover by ClcF gives cis-dienelactone as the sole product. cis-Dienelactone was further hydrolyzed by ClcD2 to maleylacetate. ClcF, despite its sequence similarity to muconolactone isomerases, no longer showed muconolactone-isomerizing activity and thus represents an enzyme dedicated to its new function as a 5-chloromuconolactone dehalogenase. Thus, during 3-chlorocatechol degradation by R. opacus 1CP, dechlorination is catalyzed by a muconolactone isomerase-related enzyme rather than by a specialized chloromuconate cycloisomerase.     

Conversion of 2-Fluoromuconate to cis-Dienelactone by Purified Enzymes of Rhodococcus opacus 1cp

The present study describes the ¹⁹F nuclear magnetic resonance analysis of the conversion of 3-halocatechols to lactones by purified chlorocatechol 1,2-dioxygenase (ClcA2), chloromuconate cycloisomerase (ClcB2), and chloromuconolactone dehalogenase (ClcF) from Rhodococcus opacus 1cp grown on 2-chlorophenol. The 3-halocatechol substrates were produced from the corresponding 2-halophenols by either phenol hydroxylase from Trichosporon cutaneum or 2-hydroxybiphenyl 3-mono-oxygenase from Pseudomonas azelaica. Several fluoromuconates resulting from intradiol ring cleavage by ClcA2 were identified. ClcB2 converted 2-fluoromuconate to 5-fluoromuconolactone and 2-chloro-4-fluoromuconate to 2-chloro-4-fluoromuconolactone. Especially the cycloisomerization of 2-fluoromuconate is a new observation. ClcF catalyzed the dehalogenation of 5-fluoromuconolactone to cis-dienelactone. The ClcB2 and ClcF-mediated reactions are in line with the recent finding of a second cluster of chlorocatechol catabolic genes in R. opacus 1cp which provides a new route for the microbial dehalogenation of 3-chlorocatechol.     

Chloromethylmuconolactones as critical metabolites in the degradation of chloromethylcatechols: recalcitrance of 2-chlorotoluene

To elucidate possible reasons for the recalcitrance of 2-chlorotoluene, the metabolism of chloromethylcatechols, formed after dioxygenation and dehydrogenation by Ralstonia sp. strain PS12 tetrachlorobenzene dioxygenase and chlorobenzene dihydrodiol dehydrogenase, was monitored using chlorocatechol dioxygenases and chloromuconate cycloisomerases partly purified from Ralstonia sp. strain PS12 and Wautersia eutropha JMP134. Two chloromethylcatechols, 3-chloro-4-methylcatechol and 4-chloro-3-methylcatechol, were formed from 2-chlorotoluene. 3-Chloro-4-methylcatechol was transformed into 5-chloro-4-methylmuconolactone and 2-chloro-3-methylmuconolactone. For mechanistic reasons neither of these cycloisomerization products can be dehalogenated by chloromuconate cycloisomerases, with the result that 3-chloro-4-methylcatechol cannot be mineralized by reaction sequences related to catechol ortho-cleavage pathways known thus far. 4-Chloro-3-methylcatechol is only poorly dehalogenated during enzymatic processing due to the kinetic properties of the chloromuconate cycloisomerases. Thus, degradation of 2-chlorotoluene via a dioxygenolytic pathway is evidently problematic. In contrast, 5-chloro-3-methylcatechol, the major dioxygenation product formed from 3-chlorotoluene, is subject to quantitative dehalogenation after successive transformation by chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase, resulting in the formation of 2-methyldienelactone. 3-Chloro-5-methylcatechol is transformed to 2-chloro-4-methylmuconolactone.     

Two unusual chlorocatechol catabolic gene clusters in <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. TFD44

The genes responsible for the degradation of 2,4-dichlorophenoxyacetate (2,4-D) by -Proteobacteria have previously been difficult to detect by using gene probes or polymerase chain reaction (PCR) primers. PCR products of the chlorocatechol 1,2-dioxygenase gene, tfdC, now allowed cloning of two chlorocatechol gene clusters from the Sphingomonas sp. strain TFD44. Sequence characterization showed that the first cluster, tfdD,RFCE, comprises all the genes necessary for the conversion of 3,5-dichlorocatechol to 3-oxoadipate, including a presumed regulatory gene, tfdR, of the LysR-type family. The second gene cluster, tfdC2E2F2, is incomplete and appears to lack a chloromuconate cycloisomerase gene and a regulatory gene. Purification and N-terminal sequencing of selected enzymes suggests that at least representatives of both gene clusters (TfdD of cluster 1 and TfdC2 of cluster 2) are induced during the growth of strain TFD44 with 2,4-D. A mutant constructed to contain an insertion in the chloromuconate cycloisomerase gene tfdD still was able to grow with 2,4-D, but more slowly and with a longer lag phase. This, and the detection of additional activity peaks during protein purification suggest that strain TFD44 harbors at least another chloromuconate cycloisomerase gene. The sequence of the tfdCE region was almost identical to that of a partially characterized chlorocatechol catabolic gene cluster of Sphingomonas herbicidovorans MH, whereas the sequence of the tfdC2E2F2 cluster was different. The similarity of the predicted proteins of the tfdD,RFCE and tfdC2E2F2 clusters to known sequences of other Proteobacteria in the database ranged from 42 to 61% identical positions for the first cluster and from 45.5 to 58% identical positions for the second cluster. Between both clusters, the similarities of their predicted proteins ranged from 44.5 to 64% identical positions. Thus, both clusters (together with those of S. herbicidovorans MH) represent deep-branching lines in the respective dendrograms, and the sequence information will help future primer design for the detection of corresponding genes in the environment.     

Characterization of a Gene Cluster Involved in 4-Chlorocatechol Degradation by Pseudomonas reinekei MT1

Pseudomonas reinekei MT1 has previously been reported to degrade 4- and 5-chlorosalicylate by a pathway with 4-chlorocatechol, 3-chloromuconate, 4-chloromuconolactone, and maleylacetate as intermediates, and a gene cluster channeling various salicylates into an intradiol cleavage route has been reported. We now report that during growth on 5-chlorosalicylate, besides a novel (chloro)catechol 1,2-dioxygenase, C12O_ccaA, a novel (chloro)muconate cycloisomerase, MCI_ccaB, which showed features not yet reported, was induced. This cycloisomerase, which was practically inactive with muconate, evolved for the turnover of 3-substituted muconates and transforms 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, suggesting that it is a functional intermediate between chloromuconate cycloisomerases and muconate cycloisomerases. The corresponding genes, ccaA (C12O_ccaA) and ccaB (MCI_ccaB), were located in a 5.1-kb genomic region clustered with genes encoding trans-dienelactone hydrolase (ccaC) and maleylacetate reductase (ccaD) and a putative regulatory gene, ccaR, homologous to regulators of the IclR-type family. Thus, this region includes genes sufficient to enable MT1 to transform 4-chlorocatechol to 3-oxoadipate. Phylogenetic analysis showed that C12O_ccaA and MCI_ccaB are only distantly related to previously described catechol 1,2-dioxygenases and muconate cycloisomerases. Kinetic analysis indicated that MCI_ccaB and the previously identified C12O_salD, rather than C12O_ccaA, are crucial for 5-chlorosalicylate degradation. Thus, MT1 uses enzymes encoded by a completely novel gene cluster for degradation of chlorosalicylates, which, together with a gene cluster encoding enzymes for channeling salicylates into the ortho-cleavage pathway, form an effective pathway for 4- and 5-chlorosalicylate mineralization.The aerobic degradation of chloroaromatic compounds usually proceeds via chlorocatechols as central intermediates (20, 47), which in most of the cases reported thus far, are further degraded by enzymes of the chlorocatechol pathway (44). This pathway involves ortho-cleavage by a chlorocatechol 1,2-dioxygenase with high activity for chlorocatechols (12), a chloromuconate cycloisomerase with high activity for chloromuconates (54), a dienelactone hydrolase active with both cis- and trans-dienelactone (4-carboxymethylenebut-2-en-4-olide) (54), and a maleylacetate reductase (MAR) (28).However, it has become evident in recent years that microorganisms have evolved various alternative strategies to mineralize chlorocatechols. Pseudomonas putida GJ31 was found to degrade chlorobenzene rapidly via 3-chlorocatechol using a catechol meta-cleavage pathway (33). Two alternative pathways for 3- and 4-chlorocatechol degradation that involve reactions known from the chlorocatechol, as well as the 3-oxoadipate, pathway have recently been observed in Rhodococcus opacus 1CP (35) and Pseudomonas reinekei MT1 (39). In R. opacus 1CP, 3-chloro- and 2,4-dichloro-cis,cis-muconate (the ring cleavage products of 4-chlorocatechol and 3,5-dichlorocatechol, respectively) are converted to the respective cis-dienelactones (35, 58), similar to the reaction described for proteobacterial chloromuconate cycloisomerases (54). However, proteobacterial chloromuconate cycloisomerase can dehalogenate 2-chloromuconate (the ring cleavage product of 3-chlorocatechol) and transform this compound via 5-chloromuconolactone into trans-dienelactone (54, 65), whereas none of the described chloromuconate cycloisomerases of R. opacus 1CP can catalyze such a dehalogenation, and 5-chloromuconolactone is the product of the cycloisomerization reaction (35, 58). Dehalogenation is achieved by an enzyme with high sequence similarity to muconolactone isomerases (35), which in proteobacteria have been shown to be capable of dehalogenating 5-chloromuconolactone to cis-dienelactone (46).In P. reinekei MT1, a trans-dienelactone hydrolase (trans-DLH) was identified as the key enzyme involved in the degradation of 4- and 5-chlorosalicylate via 4-chlorocatechol as an intermediate (39). In contrast to all previously described dienelactone hydrolases involved in chlorocatechol degradation, which belong to the α/β hydrolase fold enzymes with a catalytic triad consisting of Cys, His, and Asp (10), trans-DLH was shown to be a zinc-dependent hydrolase (8). The function of this enzyme in the 4-chlorocatechol metabolic pathway was to interact with the muconate cycloisomerase (MCI)-mediated transformation of 3-chloromuconate into protoanemonin. By acting on the reaction intermediate 4-chloromuconolactone, trans-DLH prevents the formation of protoanemonin by catalyzing its hydrolysis to maleylacetate (39). Maleylacetate, in turn, is reduced by MAR to 3-oxoadipate.A more detailed genetic and biochemical analysis of the degradation of differently substituted salicylates (7) had shown the presence of two catabolic gene clusters in MT1. An archetype catRBCA gene cluster was shown to be involved in salicylate degradation. The second gene cluster (sal) had a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, clustered with the salCD genes, encoding MCI and catechol 1,2-dioxygenase (C12O), respectively. As these genes were expressed during growth on differently substituted salicylates, it was proposed that the function of the sal gene cluster is to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho-cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways. However, previous analyses had indicated the presence of an additional and thus third (chloro)muconate cycloisomerase in MT1 during growth on chlorosalicylate, which is distinct from both previously described MCIs encoded by the cat cluster (MCI_catB) and the sal cluster (MCI_salC), as it transforms 3-chloromuconate into approximately equal amounts of cis-dienelactone and protoanemonin (39). In the present report, this cycloisomerase is biochemically and genetically described and shown to be located in a third gene cluster involved in the degradation of 5-chlorosalicylate by strain MT1. This cluster comprises genes encoding a third C12O, trans-DLH (8), and a MAR. Evidently, P. reinekei MT1 is the first microorganism in which such a complex net of genes involved in chlorocatechol degradation has been described.     

Structural basis for the substrate specificity and the absence of dehalogenation activity in 2-chloromuconate cycloisomerase from Rhodococcus opacus 1CP

2-Chloromuconate cycloisomerase from the Gram-positive bacterium Rhodococcus opacus 1CP (Rho-2-CMCI) is an enzyme of a modified ortho-pathway, in which 2-chlorophenol is degraded using 3-chlorocatechol as the central intermediate. In general, the chloromuconate cycloisomerases catalyze not only the cycloisomerization, but also the process of dehalogenation of the chloromuconate to dienelactone. However Rho-2-CMCI, unlike the homologous enzymes from the Gram-negative bacteria, is very specific for only one position of the chloride on the substrate chloromuconate. Furthermore, Rho-2-CMCI is not able to dehalogenate the 5-chloromuconolactone and therefore it cannot generate the dienelactone.     

Characterization of muconate and chloromuconate cycloisomerase from Rhodococcus erythropolis 1CP: indications for functionally convergent evolution among bacterial cycloisomerases.

Muconate cycloisomerase (EC 5.5.1.1) and chloromuconate cycloisomerase (EC 5.5.1.7) were purified from extracts of Rhodococcus erythropolis 1CP cells grown with benzoate or 4-chlorophenol, respectively. Both enzymes discriminated between the two possible directions of 2-chloro-cis, cis-muconate cycloisomerization and converted this substrate to 5-chloromuconolactone as the only product. In contrast to chloromuconate cycloisomerases of gram-negative bacteria, the corresponding R. erythropolis enzyme is unable to catalyze elimination of chloride from (+)-5-chloromuconolactone. Moreover, in being unable to convert (+)-2-chloromuconolactone, the two cycloisomerases of R. erythropolis 1CP differ significantly from the known muconate and chloromuconate cycloisomerases of gram-negative strains. The catalytic properties indicate that efficient cycloisomerization of 3-chloro- and 2,4-dichloro-cis,cis-muconate might have evolved independently among gram-positive and gram-negative bacteria.     

Evolution of chlorocatechol catabolic pathways

The aerobic bacterial degradation of chloroaromatic compounds often involves chlorosubstituted catechols as central intermediates. They are converted to 3-oxoadipate in a series of reactions similar to that for catechol catabolism and therefore designated as modifiedortho-cleavage pathway. Among the enzymes of this catabolic route, the chlorocatechol 1,2-dioxygenases are known to have a relaxed substrate specificity. In contrast, several chloromuconate cycloisomerases are more specific, and the dienelactone hydrolases of chlorocatechol catabolic pathways do not even convert the corresponding intermediate of catechol degradation, 3-oxoadipate enol-lactone. While the sequences of chlorocatechol 1,2-dioxygenases and chloromuconate cycloisomerases are very similar to those of catechol 1,2-dioxygenases and muconate cycloisomerases, respectively, the relationship between dienelactone hydrolases and 3-oxoadipate enol-lactone hydrolases is more distant. They seem to share an / hydrolase fold, but the sequences comprising the fold are quite dissimilar. Therefore, for chlorocatechol catabolism, dienelactone hydrolases might have been recruited from some other, preexisting pathway. Their relationship to dienelactone (hydrolases identified in 4-fluorobenzoate utilizing strains ofAlcaligenes andBurkholderia (Pseudomonas) cepacia is investigated). Sequence evidence suggests that the chlorocatechol catabolic operons of the plasmids pJP4, pAC27, and pP51 have been derived from a common precursor. The latter seems to have evolved for the purpose of halocatechol catabolism, and may be considerably older than the chemical industry.     

Conversion of 2-fluoromuconate to cis-dienelactone by purified enzymes of Rhodococcus opacus 1cp

The present study describes the (19)F nuclear magnetic resonance analysis of the conversion of 3-halocatechols to lactones by purified chlorocatechol 1,2-dioxygenase (ClcA2), chloromuconate cycloisomerase (ClcB2), and chloromuconolactone dehalogenase (ClcF) from Rhodococcus opacus 1cp grown on 2-chlorophenol. The 3-halocatechol substrates were produced from the corresponding 2-halophenols by either phenol hydroxylase from Trichosporon cutaneum or 2-hydroxybiphenyl 3-mono-oxygenase from Pseudomonas azelaica. Several fluoromuconates resulting from intradiol ring cleavage by ClcA2 were identified. ClcB2 converted 2-fluoromuconate to 5-fluoromuconolactone and 2-chloro-4-fluoromuconate to 2-chloro-4-fluoromuconolactone. Especially the cycloisomerization of 2-fluoromuconate is a new observation. ClcF catalyzed the dehalogenation of 5-fluoromuconolactone to cis-dienelactone. The ClcB2 and ClcF-mediated reactions are in line with the recent finding of a second cluster of chlorocatechol catabolic genes in R. opacus 1cp which provides a new route for the microbial dehalogenation of 3-chlorocatechol.     

Sequence analysis of the Pseudomonas sp. strain P51 tcb gene cluster, which encodes metabolism of chlorinated catechols: evidence for specialization of catechol 1,2-dioxygenases for chlorinated substrates.

Pseudomonas sp. strain P51 contains two gene clusters located on catabolic plasmid pP51 that encode the degradation of chlorinated benzenes. The nucleotide sequence of a 5,499-bp region containing the chlorocatechol-oxidative gene cluster tcbCDEF was determined. The sequence contained five large open reading frames, which were all colinear. The functionality of these open reading frames was studied with various Escherichia coli expression systems and by analysis of enzyme activities. The first gene, tcbC, encodes a 27.5-kDa protein with chlorocatechol 1,2-dioxygenase activity. The tcbC gene is followed by tcbD, which encodes cycloisomerase II (39.5 kDa); a large open reading frame (ORF3) with an unknown function; tcbE, which encodes hydrolase II (25.8 kDa); and tcbF, which encodes a putative trans-dienelactone isomerase (37.5 kDa). The tcbCDEF gene cluster showed strong DNA homology (between 57.6 and 72.1% identity) and an organization similar to that of other known plasmid-encoded operons for chlorocatechol metabolism, e.g., clcABD of Pseudomonas putida and tfdCDEF of Alcaligenes eutrophus JMP134. The identity between amino acid sequences of functionally related enzymes of the three operons varied between 50.6 and 75.7%, with the tcbCDEF and tfdCDEF pair being the least similar of the three. Measurements of the specific activities of chlorocatechol 1,2-dioxygenases encoded by tcbC, clcA, and tfdC suggested that a specialization among type II enzymes has taken place. TcbC preferentially converts 3,4-dichlorocatechol relative to other chlorinated catechols, whereas TfdC has a higher activity toward 3,5-dichlorocatechol. ClcA takes an intermediate position, with the highest activity level for 3-chlorocatechol and the second-highest level for 3,5-dichlorocatechol.     

A new type of muconate cycloisomerase from Rhodococcus rhodochrous strain 89

Muconate cycloisomerase (MCI) was purified from Rhodococcus rhodochrous 89 grown on phenol. The enzyme appears to contain two different type subunits with molecular masses 35.5 and 37 kD. The N-terminal amino acid sequence of both subunits showed more similarity to corresponding enzymes from gram-negative bacteria than to one from Rhodococcus opacus 1CP. MCI from R. rhodochrous 89, like analogous enzymes from gram-negative bacteria, can convert 2-chloromuconate (2-CM) with the formation of both, 2- and 5-chloromuconolactones (CML) as intermediates. Nevertheless, its unique ability to convert 5-CML to cis- but not to trans-dienelactone sets it apart from all known chloromuconate cycloisomerases from gram-negative and gram-positive bacteria.     

Detection of chlorocatechol 1,2-dioxygenase genes in proteobacteria by PCR and gene probes

In various bacterial strains belonging to the β-subdivision of proteobacteria which are capable of degrading chlorinated monoaromatic compounds, chlorocatechol 1,2-dioxygenase genes were detected by PCR and Southern hybridization. Using PCR primers derived from the conserved sequence motifs of chlorocatechol 1,2-dioxygenase genes tfdC, clcA and tcbC, PCR products of the expected size were obtained with the test strains, but not with negative control strains. The specificity of the PCR products was verified by hybridization using an oligonucleotide probe for an internal sequence motif which is evolutionarily conserved among chlorocatechol 1,2-dioxygenases and some other dioxygenases that catalyze the intradiol aromatic-ring-cleavage. Hybridization with the tfdC PCR product from the 2,4-D degradative plasmid pJP4 under stringent conditions revealed different extents of homology of the chlorocatechol 1,2-dioxygenase genes to the canonical tfdC sequence in the various strains. These findings were confirmed by the nucleotide sequence analysis of the tfdC-specific PCR products. From our results, we conclude that the PCR primer set is more suitable than the hybridization with pJP4-derived gene probes for the detection of diverse chlorocatechol 1,2-dioxygenase genes in proteobacteria.     

Enzymology of the degradation of (di)chlorobenzenes by Xanthobacter flavus 14p1

Xanthobacter flavus 14p1 used 1,4-dichlorobenzene as the sole source of carbon and energy but did not grow on other (chloro)aromatic compounds. 1,4-Dichlorobenzene was attacked by a chlorobenzene dioxygenase, and the intermediate chlorocatechol was metabolized by the modified ortho pathway. All enzymes necessary to convert 1,4-dichlorobenzene to 3-oxoadipate showed a low substrate specificity and also accepted the respective intermediates of chlorobenzene or 1,3-dichlorobenzene degradation. Of the three compounds chlorobenzene, 1,4-dichlorobenzene, and 1,3-dichlorobenzene, the latter was the most toxic for X. flavus 14p1. Furthermore, 1,3-dichlorobenzene did not induce chlorocatechol 1,2-dioxygenase activity of the organism. Chlorobenzene, however, induced chlorocatechol 1,2-dioxygenase, dienelactone hydrolase, and maleylacetate reductase activities. As demonstrated by chloride release, also chlorobenzene dioxygenase, chlorobenzene cis-dihydrodiol dehydrogenase, and chloromuconate cycloisomerase activities were present in chlorobenzene-induced cells, but chlorobenzene failed to support growth. Presumably a toxic compound was formed from one of the intermediates. Received: 10 June 1996 / Revision received: 23 December 1996 / Accepted: 18 January 1997     

Characterization of the Maleylacetate Reductase MacA of Rhodococcus opacus 1CP and Evidence for the Presence of an Isofunctional Enzyme

Maleylacetate reductases (EC 1.3.1.32) have been shown to contribute not only to the bacterial catabolism of some usual aromatic compounds like quinol or resorcinol but also to the degradation of aromatic compounds carrying unusual substituents, such as halogen atoms or nitro groups. Genes coding for maleylacetate reductases so far have been analyzed mainly in chloroaromatic compound-utilizing proteobacteria, in which they were found to belong to specialized gene clusters for the turnover of chlorocatechols or 5-chlorohydroxyquinol. We have now cloned the gene macA, which codes for one of apparently (at least) two maleylacetate reductases in the gram-positive, chlorophenol-degrading strain Rhodococcus opacus 1CP. Sequencing of macA showed the gene product to be relatively distantly related to its proteobacterial counterparts (ca. 42 to 44% identical positions). Nevertheless, like the known enzymes from proteobacteria, the cloned Rhodococcus maleylacetate reductase was able to convert 2-chloromaleylacetate, an intermediate in the degradation of dichloroaromatic compounds, relatively fast and with reductive dehalogenation to maleylacetate. Among the genes ca. 3 kb up- and downstream of macA, none was found to code for an intradiol dioxygenase, a cycloisomerase, or a dienelactone hydrolase. Instead, the only gene which is likely to be cotranscribed with macA encodes a protein of the short-chain dehydrogenase/reductase family. Thus, the R. opacus maleylacetate reductase gene macA clearly is not part of a specialized chlorocatechol gene cluster.Maleylacetate reductases (EC 1.3.1.32) have long been known to be involved in the degradation of chloroaromatic compounds via chlorocatechols as intermediates (10, 31). By reduction of a carbon-carbon double bond they form 3-oxoadipate, a metabolite also of catechol catabolism, and thus compensate for the different oxidation states of chlorinated and nonchlorinated compounds. 2-Chloromaleylacetate, which is formed during turnover of several dichlorocatechols, is initially reductively dechlorinated and then reduced to 3-oxoadipate in a second reaction (22, 47).Corresponding to the biochemical function in chlorocatechol degradation, the following maleylacetate reductase genes have been shown to be associated with dioxygenase, cycloisomerase, and dienelactone hydrolase genes as components of specialized chlorocatechol catabolic operons: tfdF and tfdF_II on pJP4 from the 2,4-dichlorophenoxyacetate-utilizing strain Ralstonia eutropha (Alcaligenes eutrophus) JMP134 (29, 33, 37, 44), tcbF on pP51 from the 1,2,4-trichlorobenzene-degrading strain Pseudomonas sp. strain P51 (45), and clcE from the 3-chlorobenzoate catabolizing strains Pseudomonas sp. strain B13 and Pseudomonas putida AC866(pAC27) (15, 20, 21). Catechol degradation, in contrast, does not require a maleylacetate reductase activity, and corresponding genes do not belong to the known catechol operons. Thus, while at least two of the chlorocatechol catabolic enzymes, i.e., the dioxygenases and cycloisomerases, appear to have been recruited from catechol catabolism, maleylacetate reductase genes must have had a different origin and original function (34).The postulated original function of the maleylacetate reductases is still under discussion. In bacteria, these enzymes have been shown to play a role, for example, in quinol, resorcinol, and 2,4-dihydroxybenzoate degradation (6, 25, 41). Other aromatic growth substrates involving the action of maleylacetate reductase are more exotic, since they carry a fluorine substituent (35), a sulfo group (14), a nitro group (18, 40), or several chlorine substituents (8, 26, 48). Maleylacetate reductase genes have been shown to be part of a specialized gene cluster for 2,4,5-trichlorophenoxyacetate degradation (8, 9) and of a gene cluster for hydroxyquinol conversion which contributes to 4-nitrophenol turnover (4).The chlorocatechol pathway of the chlorophenol-utilizing strain Rhodococcus opacus (erythropolis) 1CP obviously evolved functionally convergent to the corresponding pathway in the proteobacteria mentioned above (13, 39). Thus, it is not surprising that the chlorocatechol gene cluster of strain 1CP is organized differently from the corresponding proteobacterial operons; in fact, its characterization showed that it does not comprise a maleylacetate reductase gene (13). Thus, the nature of the gene cluster(s) encoding a maleylacetate reductase in R. opacus remained to be elucidated. Such gene clusters could complement otherwise incomplete pathways, and they might also have provided the source from which the maleylacetate reductase gene was recruited during evolution of dedicated pathways, such as the proteobacterial chlorocatechol catabolic route.(Some of the results presented here have previously been reported in a preliminary communication [38].)     

Dioxygenases of chlorobiphenyl-degrading species <Emphasis Type="Italic">Rhodococcus wratislaviensis</Emphasis> G10 and chlorophenol-degrading species <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> 1CP induced in benzoate-grown cells and genes potentially involved in these processes

Dioxygenases induced during benzoate degradation by the actinobacterium Rhodococcus wratislaviensis G10 strain degrading haloaromatic compounds were studied. Rhodococcus wratislaviensis G10 completely degraded 2 g/liter benzoate during 30 h and 10 g/liter during 200 h. Washed cells grown on benzoate retained respiration activity for more than 90 days, and a high activity of benzoate dioxygenase was recorded for 10 days. Compared to the enzyme activities with benzoate, the activity of benzoate dioxygenases was 10-30% with 13 of 35 substituted benzoate analogs. Two dioxygenases capable of cleaving the aromatic ring were isolated and characterized: protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase. Catechol inhibited the activity of protocatechuate 3,4-dioxygenase. Protocatechuate did not affect the activity of catechol 1,2-dioxygenase. A high degree of identity was shown by MALDI-TOF mass spectrometry for protein peaks of the R. wratislaviensis G10 and Rhodococcus opacus 1CP cells grown on benzoate or LB. DNA from the R. wratislaviensis G10 strain was specifically amplified using specific primers to variable regions of genes coding αand β-subunits of protocatechuate 3,4-dioxygenase and to two genes of theR. opacus 1CP coding catechol 1,2-dioxygenase. The products were 99% identical with the corresponding regions of the R. opacus 1CP genes. This high identity (99%) between the genes coding degradation of aromatic compounds in the R. wratislaviensis G10 and R. opacus 1CP strains isolated from sites of remote location (1400 km) and at different time (20-year difference) indicates a common origin of biodegradation genes of these strains and a wide distribution of these genes among rhodococci.     

The putative regulator of catechol catabolism in Rhodococcus opacus 1CP – an IclR-type, not a LysR-type transcriptional regulator

The catechol catabolic genes catABC from Rhodococcus opacus 1CP have previously been characterized by sequence analysis of the insert cloned on plasmid pRER1. Now, a 5.1-kb DNA fragment which overlaps with the insert of pRER1 was cloned, yielding pRER2, and subjected to sequencing. Besides three other open reading frames, a gene was detected ca 200 bp upstream of the catechol 1,2-dioxygenase gene catA, which is obviously transcribed divergently from catABC. The protein which can be deduced from this gene, CatR, resembles members of the PobR subfamily of IclR-type regulatory proteins. This finding was unexpected, as all catechol and chlorocatechol gene clusters known thus far from proteobacteria are under control of LysR-type regulators. It was not possible to inactivate catR by homologous recombination. However, heterologously expressed CatR in vitro bound specifically to the intergenic region between catR and catA thereby providing a first indication for a possible involvement of CatR in the regulation of catechol catabolism.     

Patchwork assembly of nag-like nitroarene dioxygenase genes and the 3-chlorocatechol degradation cluster for evolution of the 2-chloronitrobenzene catabolism pathway in Pseudomonas stutzeri ZWLR2-1

Pseudomonas stutzeri ZWLR2-1 utilizes 2-chloronitrobenzene (2CNB) as a sole source of carbon, nitrogen, and energy. To identify genes involved in this pathway, a 16.2-kb DNA fragment containing putative 2CNB dioxygenase genes was cloned and sequenced. Of the products from the 19 open reading frames that resulted from this fragment, CnbAc and CnbAd exhibited striking identities to the respective α and β subunits of the Nag-like ring-hydroxylating dioxygenases involved in the metabolism of nitrotoluene, nitrobenzene, and naphthalene. The encoding genes were also flanked by two copies of insertion sequence IS6100. CnbAa and CnbAb are similar to the ferredoxin reductase and ferredoxin for anthranilate 1,2-dioxygenase from Burkholderia cepacia DBO1. Escherichia coli cells expressing cnbAaAbAcAd converted 2CNB to 3-chlorocatechol with concomitant nitrite release. Cell extracts of E. coli/pCNBC exhibited chlorocatechol 1,2-dioxygenase activity. The cnbCDEF gene cluster, homologous to a 3-chlorocatechol degradation cluster in Sphingomonas sp. strain TFD44, probably contains all of the genes necessary for the conversion of 3-chlorocatechol to 3-oxoadipate. The patchwork-like structure of this catabolic cluster suggests that the cnb cluster for 2CNB degradation evolved by recruiting two catabolic clusters encoding a nitroarene dioxygenase and a chlorocatechol degradation pathway. This provides another example to help elucidate the bacterial evolution of catabolic pathways in response to xenobiotic chemicals.     

Biodegradation of 3-nitrotoluene by Rhodococcus sp. strain ZWL3NT

A pure bacterial culture was isolated by its ability to utilize 3-nitrotoluene (3NT) as the sole source of carbon, nitrogen, and energy for growth. Analysis of its 16S rRNA gene showed that the organism (strain ZWL3NT) belongs to the genus Rhodococcus. A rapid disappearance of 3NT with concomitant release of nitrite was observed when strain ZWL3NT was grown on 3NT. The isolate also grew on 2-nitrotoluene, 3-methylcatechol and catechol. Two metabolites, 3-methylcatechol and 2-methyl-cis,cis-muconate, in the reaction mixture were detected after incubation of cells of strain ZWL3NT with 3NT. Enzyme assays showed the presence of both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase in strain ZWL3NT. In addition, a catechol degradation gene cluster (catRABC cluster) for catechol ortho-cleavage pathway was cloned from this strain and cell extracts of Escherichia coli expressing CatA and CatB exhibited catechol 1,2-dioxygenase activity and cis,cis-muconate cycloisomerase activity, respectively. These experimental evidences suggest a novel pathway for 3NT degradation with 3-methylcatechol as a key metabolite by Rhodococcus sp. strain ZWL3NT.