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Maleylacetate reductase from Pseudomonas sp. strain B13 is one of the enzymes of the modified ortho pathway that is involved in chlorocatechol degradation. Purification and characterization of the enzyme by high-performance liquid chromatography detected tfdF and tcbF genes on plasmids pJP4 of Alcaligenes eutrophus and pP51 of Pseudomonas strain P51. These genes were shown to have significant similarities with those on plasmid pAC27 of Pseudomonas putida AC866.

In contrast to the degradation of penta- and hexachlorobiphenyls in chemostat cultures, the metabolism of PCBs by Alcaligenes sp. JB1 wasshown to be restricted to PCBs with up to four chlorine substituentsin resting-cell assay, Among these, the PCB congeners containing ortho chlorine substituents on both phenyl rings were found to be least degraded. Monochloro-benzoates and dichlorobenzoates were detected asmetabolites. Resting cell assays with chlorobenzoates showed that JB1 could metabolize all three monochlorobenzoates and dichlorobenzoates containing only meta and para chlorine substituents, but not dichlorobenzoates possessing an ortho chlorine substituent. In enzyme activity assays, meta cleaving 2,3-dihydroxybiphenyl 1,2-dioxygenase and catechol 2,3-dioxygenase activities were constitutive, whereas benzoate dioxygenase and ortho cleaving catechol 1,2-dioxygenase activities,were induced by their substrates. No activity was found for pyrocatechase II, the enzyme that is specific for chlorocatechols. The data suggest that complete mineralization of PCBs with three or more chlorine substituents by Alcaligenes sp. JB1 is unlikely. [ABSTRACT FROM AUTHOR]

To improve the efficiency and to investigate the molecular determinants that direct substrate specificity of chlorocatechol 2,3-dioxygenase CbzE(GJ31) , several mutant enzymes were constructed. Loci for substitutions of amino acids were selected by sequence comparisons as well as by homology modelling of known chlorocatechol 2,3-dioxygenases (CbzE(BASF) , CbzE(SK1) and CbzE(16-6A)). Activity measurements with various catechols showed that most of the modifications influenced activity only to a minor degree. The amino acid at position 154 seems to be located at a non-important position in the enzyme with minor extension into the substrate tunnel. Similarly, the change of related amino acids such as D95E and Y223F did not influence the catalysis since both residues are far away from the catalytic centre and the substrate tunnel. Even the modification of isoleucine to threonine in position 310, located at the outer substrate tunnel, showed a significant alteration of activities. Position 196 seems to be of higher relevance since the modification of valine to alanine, i.e. the reduction of the side-chain, produced much alteration. The amino acid is located at the interface of inner to outer substrate tunnel. CbzE(V196A) showed high relative k(cat) for 3-chlorocatechol. A pronounced increase in activity for 3-chlorocatechol resulted by the change from alanine to valine and from aspartic acid to glycine laying in the outer substrate tunnel at position 211 and 212 respectively., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Pseudomonas putida GJ31 has been reported to grow on chlorobenzene using a meta-cleavage pathway with chlorocatechol 2,3-dioxygenase (CbzE) as a key enzyme. The CbzE-encoding gene was found to be localized on the 180 kb plasmid pKW1 in a cbzTEXGS cluster, which is flanked by transposases and encodes only a partial (chloro)catechol meta-cleavage pathway comprising ferredoxin reductase, chlorocatechol 2,3-dioxygenase, an unknown protein, 2-hydroxymuconic semialdehyde dehydrogenase and glutathione S-transferase. Downstream of cbzTEXGS are located cbzJ, encoding a novel type of 2-hydroxypent-2,4-dienoate hydratase, and a transposon region highly similar to Tn5501. Upstream of cbzTEXGS, traNEOFG transfer genes were found. The search for gene clusters possibly completing the (chloro)catechol metabolic pathway of GJ31 revealed the presence of two additional catabolic gene clusters on pKW1. The mhpRBCDFETP cluster encodes enzymes for the dissimilation of 2,3-dihydroxyphenylpropionate in a novel arrangement characterized by the absence of a gene encoding 3-(3-hydroxyphenyl)propionate monooxygenase and the presence of a GntR-type regulator, whereas the nahINLOMKJ cluster encodes part of the naphthalene metabolic pathway. Transcription studies supported their possible involvement in chlorobenzene degradation. The upper pathway cluster, comprising genes encoding a chlorobenzene dioxygenase and a chlorobenzene dihydrodiol dehydrogenase, was localized on the chromosome. A high level of transcription in response to chlorobenzene revealed it to be crucial for chlorobenzene degradation. The chlorobenzene degradation pathway in strain GJ31 is thus a mosaic encoded by four gene clusters.

An Arthrobacter strain, able to utilize 4-chlorobenzoic acid as the sole carbon and energy source, was isolated and characterized. The first step of the catabolic pathway was found to proceed via a hydrolytic dehalogenation that leads to the formation of 4-hydroxybenzoic acid. The dehalogenase encoding genes (fcb) were sequenced and found highly homologous to and organized as those of other 4-chlorobenzoic acid degrading Arthrobacter strains. The fcb genes were cloned and successfully expressed in the heterologous host Pseudomonas putida PaW340 and P. putida KT2442 upper TOL, which acquired the ability to grow on 4-chlorobenzoic acid and 4-chlorotoluene, respectively. The cloned dehalogenase displayed a high specificity for para-substituted haloaromatics with affinity Cl > Br > I >> F, in the order.

The reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di-, and trichlorinated (triCl) 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia sp. strain LB400 (DHBDLB400), DHBDP6-I and DHBDP6-III from Rhodococcus globerulus P6, and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. strain RW1 (THBDRW1). The specificity of each isozyme for particular DHBs differed by up to 3 orders of magnitude. Interestingly, the Kmapp values of each isozyme for the tested polychlorinated DHBs were invariably lower than those of monochlorinated DHBs. Moreover, each enzyme cleaved at least one of the tested chlorinated (Cl) DHBs better than it cleaved DHB (e.g., apparent specificity constants for 3',5'-dichlorinated [diCl] DHB were 2 to 13.4 times higher than for DHB). These results are consistent with structural data and modeling studies which indicate that the substrate-binding pocket of the DHBDs is hydrophobic and can accommodate the Cl DHBs, particularly in the distal portion of the pocket. Although the activity of DHBDP6-III was generally lower than that of the other three enzymes, six of eight tested Cl DHBs were better substrates for DHBDP6-III than was DHB. Indeed, DHBDP6-III had the highest apparent specificity for 4,3',5'-triCl DHB and cleaved this compound better than two of the other enzymes. Of the four enzymes, THBDRW1 had the highest specificity for 2'-Cl DHB and was at least five times more resistant to inactivation by 2'-Cl DHB, consistent with the similarity between the latter and 2,2',3-trihydroxybiphenyl. Nonetheless, THBDRW1 had the lowest specificity for 2',6'-diCl DHB and, like the other enzymes, was unable to cleave this critical PCB metabolite (kcatapp < 0.001 s(-1)).

Pseudomonas putida GJ31 harbors a degradative pathway for chlorobenzene via meta-cleavage of 3-chlorocatechol. Pseudomonads using this route for chlorobenzene degradation, which was previously thought to be generally unproductive, were isolated from various contaminated environments of distant locations. The new isolates, Pseudomonas fluorescens SK1 (DSM16274), Pseudomonas veronii 16-6A (DSM16273), Pseudomonas sp. strain MG61 (DSM16272), harbor a chlorocatechol 2,3-dioxygenase (CbzE). The cbzE-like genes were cloned, sequenced, and expressed from the isolates and a mixed culture. The chlorocatechol 2,3-dioxygenases shared 97% identical amino acids with CbzE from strain GJ31, forming a distinct family of catechol 2,3-dioxygenases. The chlorocatechol 2,3-dioxygenase, purified from chlorobenzene-grown cells of strain SK1, showed an identical N-terminal sequence with the amino acid sequence deduced from cloned cbzE. In all investigated chlorobenzene-degrading strains, cbzT-like genes encoding ferredoxins are located upstream of cbzE. The sequence data indicate that the ferredoxins are identical (one amino acid difference in CbzT of strain 16-6A compared to the others). In addition, the structure of the operon downstream of cbzE is identical in strains GJ31, 16-6A, and SK1 with genes cbzX (unknown function) and the known part of cbzG (2-hydroxymuconic semialdehyde dehydrogenase) and share 100% nucleotide sequence identity with the entire downstream region. The current study suggests that meta-cleavage of 3-chlorocatechol is not an atypical pathway for the degradation of chlorobenzene.

Rhodococcus globerulus strain P6 contains at least three genes, bphC1, bphC2, and bphC3, coding for 2,3-dihydroxybiphenyl 1,2-dioxygenases; the latter two specify enzymes of the family of one-domain extradiol dioxygenases. In order to assess the importance of these different isoenzymes for the broad catabolic activity of this organism towards the degradation of polychlorinated biphenyls (PCBs), the capacities of recombinant enzymes expressed in Escherichia coli to transform different chlorosubstituted dihydroxybiphenyls formed by the action of R. globerulus P6 biphenyl dioxygenase and biphenyl 2,3-dihydrodiol dehydrogenase were determined. Whereas both BphC2 and BphC3 showed similar activities for 2,3-dihydroxybiphenyl and all monochlorinated 2,3-dihydroxybiphenyls, BphC1 exhibited only weak activity for 2'-chloro-2,3-dihydroxybiphenyl. More highly chlorinated 2'-chlorosubstituted 2,3-dihydroxybiphenyls were also transformed at high rates by BphC2 and BphC3 but not BphC1. In R. globerulus P6, BphC2 was constitutively expressed, BphC1 expression was induced during growth on biphenyl, and BphC3 was not expressed at significant levels under the experimental conditions. Although we cannot rule out the expression of BphC3 under certain environmental conditions, it seems that the contrasting substrate specificities of BphC1 and BphC2 contribute significantly to the versatile PCB-degrading phenotype of R. globerulus P6.

Ralstonia sp. strain PS12 is able to use 2,4-, 2,5-, and 3,4-dichlorotoluene as growth substrates. Dichloromethylcatechols are central intermediates that are formed by TecA tetrachlorobenzene dioxygenase-mediated activation at two adjacent unsubstituted carbon atoms followed by TecB chlorobenzene dihydrodiol dehydrogenase-catalyzed rearomatization and then are channeled into a chlorocatechol ortho cleavage pathway involving a chlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and dienelactone hydrolase. However, completely different metabolic routes were observed for the three dichloromethylcatechols analyzed. Whereas 3,4-dichloro-6-methylcatechol is quantitatively transformed into one dienelactone (5-chloro-2-methyldienelactone) and thus is degraded via a linear pathway, 3,5-dichloro-2-methylmuconate formed from 4,6-dichloro-3-methylcatechol is subject to both 1,4- and 3,6-cycloisomerization and thus is degraded via a branched metabolic route. 3,6-Dichloro-4-methylcatechol, on the first view, is transformed predominantly into one (2-chloro-3-methyl-trans-) dienelactone. In situ (1)H nuclear magnetic resonance analysis revealed the intermediate formation of 2,5-dichloro-4-methylmuconolactone, showing that both 1,4- and 3,6-cycloisomerization occur with this muconate and indicating a degradation of the muconolactone via a reversible cycloisomerization reaction and the dienelactone-forming branch of the pathway. Diastereomeric mixtures of two dichloromethylmuconolactones were prepared chemically to proof such a hypothesis. Chloromuconate cycloisomerase transformed 3,5-dichloro-2-methylmuconolactone into a mixture of 2-chloro-5-methyl-cis- and 3-chloro-2-methyldienelactone, affording evidence for a metabolic route of 3,5-dichloro-2-methylmuconolactone via 3,5-dichloro-2-methylmuconate into 2-chloro-5-methyl-cis-dienelactone. 2,5-Dichloro-3-methylmuconolactone was transformed nearly exclusively into 2-chloro-3-methyl-trans-dienelactone.

3-oxoadipate:succinyl-coenzyme A (CoA) transferase and 3-oxoadipyl-CoA thiolase carry out the ultimate steps in the conversion of benzoate and 3-chlorobenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the 3-oxoadipate pathway. This report describes the characterization of DNA fragments with the overall length of 5.9 kb from Pseudomonas sp. strain B13 that encode these enzymes. DNA sequence analysis revealed five open reading frames (ORFs) plus an incomplete one. ORF1, of unknown function, has a length of 414 bp. ORF2 (catI) encodes a polypeptide of 282 amino acids and starts at nucleotide 813. ORF3 (catJ) encodes a polypeptide of 260 amino acids and begins at nucleotide 1661. CatI and CatJ are the subunits of the 3-oxoadipate:succinyl-CoA transferase, whose activity was demonstrated when both genes were ligated into expression vector pET11a. ORF4, termed catF, codes for a protein of 401 amino acid residues with a predicted mass of 41,678 Da with 3-oxoadipyl-CoA thiolase activity. The last three ORFs seem to form an operon since they are oriented in the same direction and showed an overlapping of 1 bp between catI and catJ and of 4 bp between catJ and catF. Conserved functional groups important for the catalytic activity of CoA transferases and thiolases were identified in CatI, CatJ, and CatF. ORF5 (catD) encodes the 3-oxoadipate enol-lactone hydrolase. An incomplete ORF6 of 1,183 bp downstream of ORF5 and oriented in the opposite direction was found. The protein sequence deduced from ORF6 showed a putative AMP-binding domain signature.

The degradation of 3-oxoadipate in Pseudomonas sp. strain B13 was investigated and was shown to proceed through 3-oxoadipyl-coenzyme A (CoA) to give acetyl-CoA and succinyl-CoA. 3-Oxoadipate:succinyl-CoA transferase of strain B13 was purified by heat treatment and chromatography on phenyl-Sepharose, Mono-Q, and Superose 6 gels. Estimation of the native molecular mass gave a value of 115,000 +/- 5,000 Da with a Superose 12 column. Polyacrylamide gel electrophoresis under denaturing conditions resulted in two distinct bands of equal intensities. The subunit A and B values were 32,900 and 27,000 Da. Therefore it can be assumed that the enzyme is a heterotetramer of the type A2B2 with a molecular mass of 120,000 Da. The N-terminal amino acid sequences of both subunits are as follows: subunit A, AELLTLREAVERFVNDGTVALEGFTHLIPT; subunit B, SAYSTNEMMTVAAARRLKNGAVVFV. The pH optimum was 8.4. Km values were 0.4 and 0.2 mM for 3-oxoadipate and succinyl-CoA, respectively. Reversibility of the reaction with succinate was shown. The transferase of strain B13 failed to convert 2-chloro- and 2-methyl-3-oxoadipate. Some activity was observed with 4-methyl-3-oxoadipate. Even 2-oxoadipate and 3-oxoglutarate were shown to function as poor substrates of the transferase. 3-oxoadipyl-CoA thiolase was purified by chromatography on DEAE-Sepharose, blue 3GA, and reactive brown-agarose. Estimation of the native molecular mass gave 162,000 +/- 5,000 Da with a Superose 6 column. The molecular mass of the subunit of the denatured protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42 kDa. On the basis of these results, 3-oxoadipyl-CoA thiolase should be a tetramer of the type A4. The N-terminal amino acid sequence of 3-oxoadipyl-CoA thiolase was determined to be SREVYI-DAVRTPIGRFG. The pH optimum was 7.8. Km values were 0.15 and 0.01 mM for 3-oxoadipyl-CoA and CoA, respectively. Sequence analysis of the thiolase terminus revealed high percentages of identity (70 to 85%) with thiolases of different functions. The N termini of the transferase subunits showed about 30 to 35% identical amino acids with the glutaconate-CoA transferase of an anaerobic bacterium but only an identity of 25% with the respective transferases of aromatic compound-degrading organisms was found.

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) is a key determinant in the aerobic transformation of polychlorinated biphenyls (PCBs) by Burkholderia sp. strain LB400 (S. Y. K. Seah, G. Labbé, S. Nerdinger, M. Johnson, V. Snieckus, and L. D. Eltis, J. Biol. Chem. 275:15701-15708, 2000). To determine whether this is also true in divergent biphenyl degraders, the homologous hydrolase of Rhodococcus globerulus P6, BphD(P6), was hyperexpressed, purified to apparent homogeneity, and studied by steady-state kinetics. BphD(P6) hydrolyzed HOPDA with a k(cat)/K(m) of 1.62 (+/- 0.03) x 10(7) M(-1) s(-1) (100 mM phosphate [pH 7.5], 25 degrees C), which is within 70% of that of BphD(LB400). BphD(P6) was also similar to BphD(LB400) in that it catalyzed the hydrolysis of HOPDAs bearing chloro substituents on the phenyl moiety at least 25 times more specifically than those bearing chloro substituents on the dienoate moiety. However, the rhodococcal enzyme was significantly more specific for 9-Cl and 10-Cl HOPDAs, catalyzing the hydrolysis of 9-Cl, 10-Cl, and 9,10-diCl HOPDAs two- to threefold respectively, more specifically than HOPDA. Moreover, 4-Cl HOPDA competitively inhibited BphD(P6) more effectively than 3-Cl HOPDA, which is the inverse of what was observed in BphD(LB400). These results demonstrate that BphD is a key determinant in the aerobic transformation of PCBs by divergent biphenyl degraders, but that there exists significant diversity in the specificity of these biphenyl hydrolases.

A gene designated thnD, which is required for biodegradation of the organic solvent tetralin by Sphingomonas macrogoltabidus strain TFA, has been identified. Sequence comparison analysis indicated that thnD codes for a carbon-carbon bond serine hydrolase showing highest similarity to hydrolases involved in biodegradation of biphenyl. An insertion mutant defective in ThnD accumulates the ring fission product which results from the extradiol cleavage of the aromatic ring of dihydroxytetralin. The gene product has been purified and characterized. ThnD is an octameric thermostable enzyme with an optimum reaction temperature at 65 degrees C. ThnD efficiently hydrolyzes the ring fission intermediate of the tetralin pathway and also 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the ring fission product of the biphenyl meta-cleavage pathway. However, it is not active towards the equivalent intermediates of meta-cleavage pathways of monoaromatic compounds which have small substituents in C-6. When ThnD hydrolyzes the intermediate in the tetralin pathway, it cleaves a C-C bond comprised within the alicyclic ring of tetralin instead of cleaving a linear C-C bond, as all other known hydrolases of meta-cleavage pathways do. The significance of this activity of ThnD for the requirement of other activities to mineralize tetralin is discussed.

The treatment of environmental pollution by microorganisms is a promising technology. Various genetic approaches have been developed and used to optimize the enzymes, metabolic pathways and organisms relevant for biodegradation. New information on the metabolic routes and bottlenecks of degradation is still accumulating, enlarging the available toolbox. With molecular methods allowing the characterization of microbial community structure and activities, the performance of microorganisms under in situ conditions and in concert with the indigenous microflora will become predictable.

A genomic region involved in tetralin biodegradation was recently identified in Sphingomonas strain TFA. We have cloned and sequenced from this region a gene designated thnC, which codes for an extradiol dioxygenase required for tetralin utilization. Comparison to similar sequences allowed us to define a subfamily of 1, 2-dihydroxynaphthalene extradiol dioxygenases, which comprises two clearly different groups, and to show that ThnC clusters within group 2 of this subfamily. 1,2-Dihydroxy-5,6,7, 8-tetrahydronaphthalene was found to be the metabolite accumulated by a thnC insertion mutant. The ring cleavage product of this metabolite exhibited behavior typical of a hydroxymuconic semialdehyde toward pH-dependent changes and derivatization with ammonium to give a quinoline derivative. The gene product has been purified, and its biochemical properties have been studied. The enzyme is a decamer which requires Fe(II) for activity and shows high activity toward its substrate (V(max), 40.5 U mg(-1); K(m), 18. 6 microM). The enzyme shows even higher activity with 1, 2-dihydroxynaphthalene and also significant activity toward 1, 2-dihydroxybiphenyl or methylated catechols. The broad substrate specificity of ThnC is consistent with that exhibited by other extradiol dioxygenases of the same group within the subfamily of 1, 2-dihydroxynaphthalene dioxygenases.

A strain designated TFA which very efficiently utilizes tetralin has been isolated from the Rhine river. The strain has been identified as Sphingomonas macrogoltabidus, based on 16S rDNA sequence similarity. Genetic analysis of tetralin biodegradation has been performed by insertion mutagenesis and by physical analysis and analysis of complementation between the mutants. The genes involved in tetralin utilization are clustered in a region of 9 kb, comprising at least five genes grouped in two divergently transcribed operons.

Pseudomonas putida GJ31 contains an unusual catechol 2,3-dioxygenase that converts 3-chlorocatechol and 3-methylcatechol, which enables the organism to use both chloroaromatics and methylaromatics for growth. A 3.1-kb region of genomic DNA of strain GJ31 containing the gene for this chlorocatechol 2,3-dioxygenase (cbzE) was cloned and sequenced. The cbzE gene appeared to be plasmid localized and was found in a region that also harbors genes encoding a transposase, a ferredoxin that was homologous to XylT, an open reading frame with similarity to a protein of a meta-cleavage pathway with unknown function, and a 2-hydroxymuconic semialdehyde dehydrogenase. CbzE was most similar to catechol 2,3-dioxygenases of the 2.C subfamily of type 1 extradiol dioxygenases (L. D. Eltis and J. T. Bolin, J. Bacteriol. 178:5930-5937, 1996). The substrate range and turnover capacity with 3-chlorocatechol were determined for CbzE and four related catechol 2,3-dioxygenases. The results showed that CbzE was the only enzyme that could productively convert 3-chlorocatechol. Besides, CbzE was less susceptible to inactivation by methylated catechols. Hybrid enzymes that were made of CzbE and the catechol 2, 3-dioxygenase of P. putida UCC2 (TdnC) showed that the resistance of CbzE to suicide inactivation and its substrate specificity were mainly determined by the C-terminal region of the protein.

The persistence of chloroaromatic compounds can be caused by various bottlenecks, such as incomplete degradative pathways or inappropriate regulation of these pathways. Patchwork assembly of existing pathways in novel combinations provides a general route for the development of strains degrading chloroaromatics. The recruitment of known complementary enzyme sequences in a suitable host organism by conjugative transfer of genes might generate a functioning hybrid pathway for the mineralization of some chloroaromatics not degraded by the parent organisms. The rational combination uses (a) peripheral, funneling degradation sequences originating from aromatics-degrading strains to fulfill the conversion of the respective analogous chloroaromatic compound to chlorocatechols as the central intermediates; (b) a central chlorocatechol degradation sequence, the so-called modified ortho pathway, which brings about elimination of chlorine substituents; and (c) steps of the 3-oxoadipate pathway to reach the tricarboxylic acid cycle. The genetic organization of these pathway segments has been well characterized. The specificity of enzymes of the xylene, benzene, biphenyl, and chlorocatechol pathways and the specificity of the induction systems for the chlorinated substrates are analyzed in various organisms to illustrate eventual bottlenecks and to provide alternatives that are effective in the conversion of the "new" substrate. Hybrid pathways are investigated in "new" strains degrading chlorinated benzoates, toluenes, benzenes, and biphenyls. Problems occurring after the conjugative DNA transfer and the "natural" solution of these are examined, such as the prevention of misrouting into the meta pathway, to give a functioning hybrid pathway. Some examples clearly indicate that patchwork assembly also happens in nature.

A purification procedure for a new kind of extradiol dioxygenase, termed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol productively was developed. Structural and kinetic properties of the enzyme, which is part of the degradative pathway used for growth of Pseudomonas putida GJ31 with chlorobenzene, were investigated. The enzyme has a subunit molecular mass of 33.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the native Mr value under nondenaturating conditions by gel filtration gave a molecular mass of 135 +/- 10 kDa, indicating a homotetrameric enzyme structure (4 x 33.4 kDa). The pI of the enzyme was estimated to be 7.1 +/- 0.1. The N-terminal amino acid sequence (43 residues) of the enzyme was determined and exhibits 70 to 42% identity with other extradiol dioxygenases. Fe(II) seems to be a cofactor of the enzyme, as it is for other catechol 2,3-dioxygenases. In contrast to other extradiol dioxygenases, the enzyme exhibited great sensitivity to temperatures above 40 degrees C. The reactivity of this enzyme toward various substituted catechols, especially 3-chlorocatechol, was different from that observed for other catechol 2,3-dioxygenases. Stoichiometric displacement of chloride occurred from 3-chlorocatechol, leading to the production of 2-hydroxymuconate.

Pseudomonas putida GJ31 is able to simultaneously grow on toluene and chlorobenzene. When cultures of this strain were inhibited with 3-fluorocatechol while growing on toluene or chlorobenzene, 3-methylcatechol or 3-chlorocatechol, respectively, accumulated in the medium. To establish the catabolic routes for these catechols, activities of enzymes of the (modified) ortho- and meta-cleavage pathways were measured in crude extracts of cells of P. putida GJ31 grown on various aromatic substrates, including chlorobenzene. The enzymes of the modified ortho-cleavage pathway were never present, while the enzymes of the meta-cleavage pathway were detected in all cultures. This indicated that chloroaromatics and methylaromatics are both converted via the meta-cleavage pathway. Meta cleavage of 3-chlorocatechol usually leads to the formation of a reactive acylchloride, which inactivates the catechol 2,3-dioxygenase and blocks further degradation of catechols. However, partially purified catechol 2,3-dioxygenase of P. putida GJ31 converted 3-chlorocatechol to 2-hydroxy-cis,cis-muconic acid. Apparently, P. putida GJ31 has a meta-cleavage enzyme which is resistant to inactivation by the acylchloride, providing this strain with the exceptional ability to degrade both toluene and chlorobenzene via the meta-cleavage pathway.

A 3,167-bp PstI fragment of genomic DNA from Pseudomonas sp. strain B13 was cloned and sequenced. The gene clcE consists of 1,059 nucleotides encoding a protein of 352 amino acids with a calculated mass of 37,769 Da which showed maleylacetate reductase activity. The protein had significant sequence similarities with the polypeptides encoded by tcbF of pP51 (59.4% identical positions), tfdF of pJP4 (55.1%), and tftE of Burkholderia cepacia AC1100 (53.1%). The function of TcbF as maleylacetate reductase was established by an enzyme assay.

Chlorobenzoates (CBA) arise as intermediates during the degradation of polychlorinated biphenyls (PCBs) and some chlorinated herbicides. Since PCBs were produced as complex mixtures, a range of mono-, di-, and possibly trichloro-substituted benzoates would be formed. Chlorobenzoate degradation has been proposed to be one of the rate-limiting steps in the overall PCB-degradation process. Three hybrid bacteria constructed to have the ability to completely mineralise 2-, 3-, or 4-monochlorobiphenyl respectively, have been studied to establish the range of mono- and diCBAs that can be utilised. The three strains were able to mineralise one or more of the following CBAs: 2-, 3-, and 4-monochlorobenzoate and 3,5-dichlorobenzoate. No utilisation of 2,3-, 2,5-, 2,6-, or 3,4-diCBA was observed, and only a low concentration (0.11 mM) of 2,4-diCBA was mineralised. When the strain with the widest substrate range (Burkholderia cepacia JHR22) was simultaneously supplied with two CBAs, one that it could utilise plus one that it was unable to utilise, inhibitory effects were observed. The utilisation of 2-CBA (2.5 mM) by this strain was inhibited by 2,3-CBA (200 microM) and 3,4-CBA (50 microM). Although 2,5-cba and 2,6-cba were not utilised as carbon sources by strain jhr22, they did not inhibit 2-cba utilisation at the concentrations studied, whereas 2,4-cba was co-metabolised with 2-cba. The utilisation of 2-, 3-, and 4-chlorobiphenyl by strain JHR22 was also inhibited by the presence of 2,3- or 3,4-diCBA. We conclude that the effect of the formation of toxic intermediates is an important consideration when designing remediation strategies.

In contrast to the degradation of penta- and hexachlorobiphenyls in chemostat cultures, the metabolism of PCBs by Alcaligenes sp. JB1 was shown to be restricted to PCBs with up to four chlorine substituents in resting-cell assays. Among these, the PCB congeners containing ortho chlorine substituents on both phenyl rings were found to be least degraded. Monochloro-benzoates and dichlorobenzoates were detected as metabolites. Resting cell assays with chlorobenzoates showed that JB1 could metabolize all three monochlorobenzoates and dichlorobenzoates containing only meta and para chlorine substituents, but not dichlorobenzoates possessing an ortho chlorine substituent. In enzyme activity assays, meta cleaving 2,3-dihydroxybiphenyl 1,2-dioxygenase and catechol 2,3-dioxygenase activities were constitutive, whereas benzoate dioxygenase and ortho cleaving catechol 1,2-dioxygenase activities were induced by their substrates. No activity was found for pyrocatechase II, the enzyme that is specific for chlorocatechols. The data suggest that complete mineralization of PCBs with three or more chlorine substituents by Alcaligenes sp. JB1 is unlikely.

The maleylacetate reductases from Pseudomonas aeruginosa RHO1 and Alcaligenes eutrophus JMP134 were tested for activity and affinity to various maleylacetates as well as dechlorinating properties. The dechlorinating activity and the kcat/Km values revealed high-level similarity of these reductases to that of Pseudomonas sp. strain B13.

Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degradation of chloroaromatic compounds by channelling maleylacetate and some chlorinated derivatives into the 3-oxoadipate pathway. Several substituted maleylacetates were prepared in situ by alkaline or enzymatic hydrolysis of dienelactones as the precursor. The conversion of these methyl-, chloro-, fluoro-, and bromo-substituted maleylacetates by malelacetate reductase from 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. Two moles of NADH per mole of substrate was consumed for the conversion of maleylacetates which contain a halogen substituent in the 2 position. In contrast, only 1 mol of NADH was necessary to convert 1 mol of substrates without a halogen substituent in the 2 position. The conversion of 2-fluoro-, 2-chloro-, 2,3-dichloro-, 2,5-dichloro-, 2,3,5-trichloro-, 2-bromo-, 2,3-dibromo-, 2,5-dibromo-, 2-bromo-5-chloro-, 2-chloro-3-methyl-, and 2-chloro-5-methylmaleylacetate was accompanied by the elimination of halide from the 2 position and the temporary occurrence of the corresponding dehalogenated maleylacetate as an intermediate consuming the second mole equivalent of NADH. The properties of the halogen substituents influenced the affinity to the enzyme in the following manner. Km values increased with increasing van der Waals radii and with decreasing electronegativity of the halogen substituents (i.e., low steric hindrance and high electronegativity positively influenced the binding). The Km values obtained with 2-methyl-,3-methyl-, and 5-methylmaleylacetate showed that a methyl substituent negatively affected the affinity in the following order: 2 position >/ = 3 position >> 5 position. A reaction mechanism explaining the exclusive elimination of halogen substituents from the 2 position is proposed.

A derivative of Pseudomonas putida strain PaW1 bearing the TOL plasmid pWW0 was isolated from a culture which has grown unlimited on toluene. In contrast to the parent strain PaW1, the derivative, strain CG220, is unable to grow with xylenes and toluates, while toluene and benzoate served as substrates. Strain CG220 had a remarkable growth advantage against the wild type when grown with toluene. Biochemical analysis showed that in strain CG220 toluene was metabolised through the TOL plasmid upper pathway to benzoate and the latter to amphibolic intermediates by the chromosomal encoded ortho-cleavage pathway. No activities of the TOL plasmid encoded toluate dioxygenase and catechol 2,3-dioxygenase were detectable in strain CG220. No reversion of strain CG220 to growth with xylenes or toluates was observed. Hybridisation experiments with TOL plasmid-derived gene probes and oligonucleotides revealed that genes xylY to xylG were absent, while xylX and xylK were still present.

Maleylacetate reductase of Pseudomonas sp. strain B13 was purified to homogeneity by chromatography on DEAE-cellulose, Butyl-Sepharose, Blue-Sepharose, and Sephacryl S100. The final preparation gave a single band by polyacrylamide gel electrophoresis under denaturing conditions and a single symmetrical peak by gel filtration under nondenaturing conditions. The subunit M(r) value was 37,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Estimation of the native M(r) value by gel filtration gave a value of 74,000 with a Superose 6 column, indicating that the enzyme is dimeric. The pH and temperature optima were 5.4 and 50 degrees C, respectively. The pI of the enzyme was estimated to be 7.0. The apparent Km values for maleylacetate and NADH were 58 and 30 microM, respectively, and the maximum velocity was 832 U/mg of protein for maleylacetate. Maleylacetate and various substituted maleylacetates, such as 2-chloro- and 2-methyl-maleylacetate, were reduced at significant rates. NADPH was also used as a cofactor instead of NADH with nearly the same Vmax value, but its Km value was estimated to be 77 microM. Reductase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group in the enzyme.

A defined mixed culture, consisting of an Arthrobacter sp. and a Micrococcus sp. and able to grow with 4-chloroacetophenone as a sole source of carbon and energy, was isolated. 4-Chlorophenyl acetate, 4-chlorophenol, and 4-chlorocatechol were identified as metabolites through comparison of retention times and UV spectra with those of standard substances. The proposed pathway was further confirmed by investigation of enzymes. The roles of the two collaborating strains were studied by growth experiments and on the level of enzymes. If transient accumulation of 4-chlorophenol was avoided either by the use of phenol-absorbing substances or by careful supplement of 4-chloroacetophenone, the Arthrobacter sp. was able to grow as a pure culture with 4-chloroacetophenone as a sole source of carbon and energy. Several mono-, di-, and trichlorinated acetophenones were mineralized by the Arthrobacter sp.

The hybrid Pseudomonas cepacia strain JHR22 was tested for its ability to degrade Aroclor 1221 in soil. The influence of supplements--mineral salts and trace elements--on the degradation was investigated. Disappearance of Aroclor 1221 congeners, occurrence of metabolites, and release of chloride were measured under different conditions. After 45 days the hybrid organism, strain JHR22, was still present at high numbers in soil, independently of whether the soil had been sterilized prior to inoculation or not. There was only a minor difference in degradation efficiency between sterilized and untreated soil with about 70% release of chloride when 10(7) cells/g soil were inoculated. The whole hybrid pathway, originating from three different strains, was found to be stable under the conditions tested. Mineral salts did not significantly affect the degradation rate or survival of the hybrid strain.

The activities of the TOL plasmid-coded xylene oxygenase, benzylalcohol dehydrogenase, benzaldehyde dehydrogenase of Pseudomonas putida strain PaW1 were tested with substituted toluenes, benzylalcohols and benzaldehydes, respectively, as substrates. Several chlorinated toluenes were shown to induce enzymes of the xylene degradation sequence. Conjugative transfer of the TOL plasmid from Pseudomonas putida strain PaW1 to Pseudomonas sp. strain B13 and Pseudomonas cepacia strain JH230 allowed the isolation of hybrid strains capable of growing in the presence of 3-chloro-, 4-chloro- and 3,5-dichlorotoluene. Hybrid strains revealed new ways to prevent the dead-end meta-pathway for cholorocatechols.

The maleylacetate reductase of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 has been purified 50-fold. The enzyme converted 2-chloromaleylacetate to 3-oxoadipate with temporary occurrence of maleylacetate; 1 mol of chloride was eliminated during the conversion of 1 mol of 2-chloro- and 2,3-dichloromaleylacetate; 2 mol of NADH were consumed per mol of 2-chloro- and 2,3-dichloromaleylacetate while only 1 mol was necessary to catalyze the conversion of maleylacetate or 2-methylmaleylacetate. The maleylacetate reductase failed to use fumarylacetate as a substrate. The role of the enzyme in the chloroaromatics degradation is discussed.

Cocultures consisting of strains converting chlorobiphenyls to the respective benzoates or catechols and of chlorobenzoate degraders were investigated for the mineralization of chlorobiphenyls. Stable mixed cultures were obtained with 4-chlorobiphenyl, while those with 2-chloro- or 3-chlorobiphenyl were found to be unstable and released only low yields of chloride. When both sets of enzyme sequences were combined in one organism, Pseudomonas cepacia strain JH230, by conjugative transfer of genes of the biphenyl degradation sequence, the total degradation of 2-chloro-, 3-chloro-, 4-chloro-, 2,4-dichloro-, and 3,5-dichlorobiphenyl was achieved.

Pseudomonas sp. strain RHO1 able to use chloro- and 1,4-dichlorobenzene as growth substrates was tested towards sensitivity against chlorobenzene. Concentrations of chlorobenzene higher than 3.5 mM were found to be toxic to cells independent of pregrowth with chlorobenzene or nutrient broth. Below this concentration, sensitivity towards chlorobenzene depended on the precultivation of the cells, i.e. type of growth substrate (chlorobenzene or nutrient broth) and the concentration of chlorobenzene as the growth substrate. Cells grown in continuous culture were especially sensitive with a threshold concentration of 2.5 mM chlorobenzene. In addition to chlorobenzene, metabolites also seem to function as toxic compounds. 2-Chlorophenol and 3-chlorocatechol were isolated from cell extracts. Cleavage of 3-chlorocatechol by catechol 1,2-dioxygenase seems to be the critical step in the metabolism of chlorobenzene.

3-Chlorobiphenyl-degrading bacteria were obtained from the mating between Pseudomonas putida strain BN10 and Pseudomonas sp. strain B13. Strains such as BN210 resulted from the transfer of the genes coding the enzyme sequence for the degradation of chlorocatechols from B13 into BN10, whereas B13 derivatives such as B131 have acquired the biphenyl degradation sequence from BN10. During growth of the hybrid strains on 3-chlorobiphenyl 90% chloride was released. Activities of phenylcatechol 2,3-dioxygenase, benzoate dioxygenase, catechol 1,2-dioxygenase, chloromuconate cyloisomerase and 4-carboxymethylenebut-2-en-4-olide hydrolase were found in 3-chlorobiphenyl-grown cells. The hybrid strains were found to convert some congeners of the Aroclor 1221 mixture such as mono- and dichloro-substituted biphenyls.

Derivatives of Pseudomonas sp. B13 which had acquired the capability to utilize 4-chloro- and 3,5-dichlorobenzoate as a consequence of the introduction of genes of the TOL plasmid of Pseudomonas putida mt-2 were studied. The utilization of these substrates, a property not shared by the parent strains, was shown to depend upon the combined activities of enzymes from the donor and from the recipient. During growth on 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate, predominantly the toluate 1,2-deoxygenase and both dihydrodihydroxybenzoate dehydrogenases of the parent strains were induced. On the other hand, no catechol 2,3-dioxygenase from P. putida mt-2 was detectable, so that degradation of chlorocatechols by the nonproductive meta-cleavage pathway was avoided. Instead of that, chlorocatechols were subject to ortho cleavage and totally degraded by the preexisting enzymes of Pseudomonas sp. B13.

The hybrid pathway for chlorobenzoate metabolism was studied in WR211 and WR216, which were derived from Pseudomonas sp. B13 by acquisition of TOL plasmid pWW0 from Pseudomonas putida mt-2. Chlorobenzoates are utilized readily by these strains when meta cleavage of chlorocatechols is suppressed. When WR211 utilizes 3-chlorobenzoate (3CB), the expression of catechol 2,3-dioxygenase (C23O) and the catabolic activities for chloroaromatics via the ortho pathway coexist as a consequence of inactivation of the meta cleavage activity by 3-chlorocatechol. Utilization of 4-chlorobenzoate (4CB) by WR216 presupposes the suppression of C23O by a spontaneous mutation in the structural gene, so that 4-chlorocatechol is not misrouted into the meta pathway. Such C23O- mutants were also selected when WR211 was grown continuously on 3CB. Our data explain why the phenotypic characters 3CB+ and Mtol+ (m-toluate) are compatible, whereas 4CB+ and Mtol+ are incompatible.

WR211 and WR216 are derivatives of halobenzoate-degrading Pseudomonas sp. strain B13 into which the 117-kilobase TOL degradative plasmid pWW0 has been transferred from Pseudomonas putida mt-2. WR211 has lost the ability to grow on the TOL-specific substrate m-xylene but retains the ability to grow on its metabolite, m-toluate. An analysis of the induction of enzymes was consistent with WR211 carrying a nonfunctional regulatory gene, xy1R, WR216 is a spontaneous derivative of WR211 which grows on one of the TOL substrates and yet expresses the nonspecific toluate oxidase, which enables it to grow on the novel substrate 4-chlorobenzoate. In addition to the xy1R lesion inherited from WR211, WR216 appears to carry a mutation in the structural gene for catechol 2,3-oxygenase, xy1E. The plasmids in both strains were analyzed by restriction endonuclease digestion. pWW0-1211 in WR211 has a large deletion (39 kilobases) compared with pWW0 and appears to be identical to a previously described plasmid (pWW0-8) which encodes none of the TOL degradative functions. pWW0-1216 in WR216 has undergone a major structural reorganization relative to its parent, pWW0-1211. This plasmid has a smaller deletion (19 kilobases), which is staggered relative to the deletion in pWW0-1211, and in addition it has two 3-kilobase insertions of unknown origin, one of which appears to cause the xylE mutation.