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The xylT gene product, a component of the xylene catabolic pathway of Pseudomonas putida mt2, has been recently characterized as a novel [2Fe-2S] ferredoxin which specifically reactivates oxygen-inactivated catechol 2,3-dioxygenase (XylE). In this study, three XylT-like proteins potentially involved in the catabolism of naphthalene (NahT) or cresols (PhhQ and DmpQ) have been overexpressed in Escherichia coli, purified, and compared with respect to their biochemical properties and interaction with XylE. The three XylT analogues show general spectroscopic characteristics common to plant-type [2Fe-2S] ferredoxins as well as distinctive features that appear to be typical for the XylT subgroup of these proteins. The midpoint redox potentials of the PhhQ and DmpQ proteins were -286 mV and -323 mV, respectively. Interestingly, all purified XylT-like proteins promoted in vitro reactivation of XylE almost as efficiently as XylT. The interaction of XylE with XylT and its analogues was studied by cross-linking experiments using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. A polypeptide band with an M(r) of 46,000, which corresponded to the cross-linked product between one XylE subunit and one molecule of ferredoxin, was obtained in all cases. The formation of the complex was affected by ionic strength, indicating that electrostatic forces are involved in the dioxygenase-ferredoxin interaction. In complementation experiments, plasmids expressing xylT or its analogues were introduced into an XylT-null mutant of P. putida which is unable to grow on p-methylbenzoate. All transconjugants regained the wild-type phenotype, indicating that all analogues can substitute for XylT in the in vivo reactivation of XylE. Our results provide evidence for a subgroup of [2Fe-2S] ferredoxins with distinct biochemical properties whose specific function is to reactivate intrinsically labile extradiol ring cleavage dioxygenases involved in the catabolism of various aromatic hydrocarbons.

The first step in the degradation of dibenzofuran and dibenzo-p-dioxin by Sphingomonas sp. strain RW1 is carried out by dioxin dioxygenase (DxnA1A2), a ring-dihydroxylating enzyme. An open reading frame (fdx3) that could potentially specify a new ferredoxin has been identified downstream of dxnA1A2, a two-cistron gene (J. Armengaud, B. Happe, and K. N. Timmis, J. Bacteriol. 180:3954-3966, 1998). In the present study, we report a biochemical analysis of Fdx3 produced in Escherichia coli. This third ferredoxin thus far identified in Sphingomonas sp. strain RW1 contained a putidaredoxin-type [2Fe-2S] cluster which was characterized by UV-visible absorption spectrophotometry and electron paramagnetic resonance spectroscopy. The midpoint redox potential of this ferredoxin (E'(0) = -247 +/- 10 mV versus normal hydrogen electrode at pH 8.0) is similar to that exhibited by Fdx1 (-245 mV), a homologous ferredoxin previously characterized in Sphingomonas sp. strain RW1. In in vitro assays, Fdx3 can be reduced by RedA2 (a reductase similar to class I cytochrome P-450 reductases), previously isolated from Sphingomonas sp. strain RW1. RedA2 exhibits a K(m) value of 3.2 +/- 0.3 microM for Fdx3. In vivo coexpression of fdx3 and redA2 with dxnA1A2 confirmed that Fdx3 can serve as an electron donor for the dioxin dioxygenase.

The TecA broad-spectrum chlorobenzene dioxygenase of Burkholderia sp. strain PS12 catalyzes the first step in the mineralization of 1,2,4, 5-tetrachlorobenzene. The catabolic genes were localized on a small plasmid that belongs to the IncPbeta incompatibility group. PCR analysis of the genetic environment of the tec genes indicated high similarity to the transposon-organized catabolic tcb chlorobenzene degradation genes of Pseudomonas sp. strain P51. Sequence analysis of the regions flanking the tecA genes revealed an upstream open reading frame (ORF) with high similarity to the todF 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase gene of Pseudomonas putida F1 and a discontinuous downstream ORF showing high similarity to the todE catechol 2,3-dioxygenase gene of strain F1. Both homologues in strain P51 exist only as deletion remnants. We suggest that different genetic events thus led to inactivation of the perturbing meta-cleavage enzymes in strains P51 and PS12 during the evolution of efficient chlorobenzene degradation pathways. Biochemical characterization of TodF-like protein TlpF and a genetically refunctionalized TodE-like protein, TlpE, produced in Escherichia coli provided data consistent with the proposed relationships.

The TecA chlorobenzene dioxygenase and the TodCBA toluene dioxygenase exhibit substantial sequence similarity yet have different substrate specificities. Escherichia coli cells producing recombinant TecA enzyme dioxygenate and simultaneously eliminate a halogen substituent from 1,2,4,5-tetrachlorobenzene but show no activity toward benzene, whereas those producing TodCBA dioxygenate benzene but not tetrachlorobenzene. A hybrid TecA dioxygenase variant containing the large alpha-subunit of the TodCBA dioxygenase exhibited a TodCBA dioxygenase specificity. Acquisition of dehalogenase activity was achieved by replacement of specific todC1 alpha-subunit subsequences by equivalent sequences of the tecA1 alpha-subunit. Substrate transformation specificities and rates by E. coli resting cells expressing hybrid systems were analyzed by high-performance liquid chromatography. This allowed the identification of both a single amino acid and potentially interacting regions required for dechlorination of tetrachlorobenzene. Hybrids with extended substrate ranges were generated that exhibited activity toward both benzene and tetrachlorobenzene. The regions determining substrate specificity in (chloro)benzene dioxygenases appear to be different from those previously identified in biphenyl dioxygenases.

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4), gtdA, from Sphingomonas sp. strain RW5 was cloned and sequenced. The gtdA gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the gtdA gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardioides sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488-6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in E. coli. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (Km) and specificity constants (Kcat/Km) of the enzyme were determined to be 15 microM and 511 s-1 M-1 x 10(4) for gentisate and 754 microM and 20 s-1 M-1 x 10(4) for 3, 6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of gtdA. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.

The dioxin dioxygenase of Sphingomonas sp. strain RW1 activates dibenzo-p-dioxin and dibenzofuran for further metabolism by introducing two atoms of oxygen at a pair of vicinal carbon atoms, one of which is involved in one of the bridges between the two aromatic rings, i.e., an angular dioxygenation. The dxnA1 and dxnA2 cistrons encoding this dioxygenase have been cloned and shown to be located just upstream of a hydrolase gene which specifies an enzyme involved in the subsequent step of the dibenzofuran biodegradative pathway. Genes encoding the electron supply system of the dioxygenase are not clustered with the dioxygenase gene but rather are located on two other distinct and separate genome segments. Moreover, whereas expression of dxnA1A2 is modulated according to the available carbon source, expression of the dbfB gene encoding the ring cleavage enzyme of the dibenzofuran pathway, which is located in the neighborhood of dxnA1A2 but oriented in the opposite direction, is constitutive. The scattering of genes for the component proteins of dioxin dioxygenase system around the genome of Sphingomonas sp. strain RW1, and the differential expression of dioxin pathway genes, is unusual and contrasts with the typical genetic organization of catabolic pathways where component cistrons tend to be clustered in multicistronic transcriptional units. The sequences of the alpha and beta subunits of the dioxin dioxygenase exhibit only weak similarity to other three component dioxygenases, but some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands are conserved. Dioxin dioxygenase activity in Escherichia coli cells containing the cloned dxnA1A2 gene was achieved only through coexpression of the cognate electron supply system from RW1. Under these conditions, exclusively angular dioxygenation of dibenzofuran and dibenzo-p-dioxin was obtained. The dioxin dioxygenase was not active in E. coli cells coexpressing a class IIB electron supply system. In the course of the isolation of the dxnA1 and dxnA2 cistrons, a number of other catabolic genes dispersed over different genome segments were identified, which may indicate greater catabolic potential than was previously suspected. This finding is consistent with the catabolic versatility of members of the genus Sphingomonas, which is becoming increasingly evident, and may indicate a less well evolved and regulated but more dynamic genetic organization in this organism than is the case for better-studied pathways in organisms such as Pseudomonas species.

Catechol 2,3-dioxygenase (XylE) is a component of the TOL plasmid-encoded pathway for the degradation of toluene and xylenes and catalyzes the dioxygenolytic cleavage of the aromatic ring. Purified XylE is oxygen-sensitive and unstable in vitro, particularly in the presence of substituted catechol substrates, but it is stabilized in vivo by another protein, XylT, encoded by the xylT gene located just upstream of xylE. In this study, we have purified to homogeneity the XylT product from a recombinant Escherichia coli strain containing a hyperexpressible xylT gene and characterized it as a novel [2Fe-2S] ferredoxin. It is the first example of a soluble ferredoxin with a net positive charge at neutral pH. The EPR signal of the iron sulfur cluster has rhombic symmetry as is the case for plant-type ferredoxins, but the XylT absorbance spectrum resembles more closely that of adrenodoxin. The midpoint redox potential was determined to be -373 +/- 6 mV, at pH 8. 5. XylT was unusually unstable for a [2Fe-2S] ferredoxin, with half-lives of 69 min at 25 degrees C in air and 70 min at 37 degrees C in argon. With photochemically reduced 5-deazaflavin for the controlled generation of reductant, it was demonstrated that XylT mediates the rapid reactivation of purified inactive catechol 2,3-dioxygenase in vitro. Inactivation of XylE by 4-methylcatechol resulted in oxidation of the active site iron to a high spin ferric state that was detectable by EPR. Spectroscopic evidence presented here demonstrates that XylT reactivates XylE through reduction of the iron atom in the active site of the enzyme. It is the first instance of a ferredoxin-mediated reactivation of an enzyme. The level of expression of XylT in Pseudomonas putida mt2 cells is low and the calculated XylT/XylE molar ratio is consistent with the proposal that XylE reactivation involves catalytic nonstoichiometric amounts of XylT.

The first step in the oxidation of the diaryl ethers dibenzo-p-dioxin and dibenzofuran by the bacterium Sphingomonas sp. RW1 is carried out by an atypical multi-component ring hydroxylating dioxygenase. This heteromeric enzyme requires the participation of a flavoprotein, reductase A2, and an iron-sulfur protein, Fdx1, to mediate the transfer of electrons from NADH to the dioxygenase for oxygen activation [Bünz, P. V. & Cook, A. M. (1993) J. Bacteriol. 175, 6467-6475]. From the type of ferredoxin (Fd) and flavoprotein, this complex is presumed to belong to the class-IIA dioxygenase system which has not been genetically analysed so far. The gene encoding the flavoprotein was identified by screening a genomic library constructed in pLAFR3 with a probe generated by PCR amplification. The nucleotide sequence of a 2.0-kb DNA fragment encompassing the reductase gene, redA2, was determined. The specified protein shares 37-40% identity with class-I cytochrome P450 reductases and 27-35% identity with reductases acting with class-IIB dioxygenases. An FAD-binding amino acid consensus sequence, as well as an NADH-binding site were detected by analogy beginning at residues 10 and 153, respectively. The redA2 gene is not linked to the dioxin dioxygenase cistrons. The rare start codon, GTG, of the reductase was changed to ATG and the modified gene hyperexpressed in Escherichia coli using the strong T7 polymerase promoter. The recombinant reductase was purified to homogeneity with an approximate yield of 3.3 mg/g wet mass cells. Flavin analysis confirmed the presence of 1 FAD/mol protein. The Km values for NADH and Fdx1 are 22 (+/- 3) microM and 3.7 (+/- 0.4) microM, respectively.

Bacterium Sphingomonas sp. strain RW1 is, under aerobic conditions, able to degrade dibenzofuran and dibenzo-p-dioxin. The first step of the pathway is performed by a ring-dihydroxylating enzyme. Bunz and Cook have reported the purification and characterization of this dioxin dioxygenase and a ferredoxin able to transfer electrons to the dioxygenase [Bunz, P. V. & Cook, A. M. (1993) J. Bacteriol. 175, 6467-6475]. The gene encoding this [2Fe-2S] ferredoxin was identified by screening a genomic library constructed in pLAFR3 with a probe generated by a nested-PCR amplification. Primers for the amplification were designed based on the N-terminus sequence of the purified ferredoxin and on sequence comparisons with related proteins. Several cosmids were obtained and the ferredoxin gene, fdx1, was subcloned from one of them. The nucleotide sequence of a 4.6-kb DNA fragment encompassing the ferredoxin gene was determined. While in the case of all known multi-component dioxygenases, genes encoding the alpha and beta subunits are found to be contiguous with the gene of the specific electron carrier, the fdx1 gene in Sphingomonas sp. RW1 does not appear to be directly linked with the dioxin dioxygenase genes. Rather, it is clustered with genes apparently encoding two atypical decarboxylases/isomerases and a glutathione S-transferase. The ferredoxin gene was hyperexpressed and the recombinant ferredoxin was purified. Spectroscopic characterization of Fdx1 demonstrated the presence of a putidaredoxin-type [2Fe-2S] cluster in this protein. Its redox potential was determined to be -245 (+/- 5) mV versus the normal hydrogen electrode at 25 degrees C, pH 8.0. Therefore, the protein is closely related to [2Fe-2S] ferredoxins known to be electron donors to monooxygenases involved in hydroxylation of aromatic compounds. Thus, this report provides clear evidence that a putidaredoxin-type [2Fe-2S] ferredoxin, namely Fdx1, is able to transfer electrons to the dioxin dioxygenase of Sphingomonas sp. RW1.

The bacterium, Burkholderia (previously Pseudomonas) sp. strain PS12, reported earlier to degrade 1,2,4-trichlorobenzene is shown here to utilize also 1,2,4,5-tetrachlorobenzene (Cl4-benzene) as a growth substrate. To investigate the possibility that this organism attacks Cl4-benzene with a chlorobenzene dioxygenase which concomitantly causes dehalogenation, and to analyze the substrate range of the initial enzyme, a 5503-bp DNA fragment from PS12, exhibiting high similarity to genes coding for class IIB dioxygenases, was cloned and expressed in Escherichia coli. The sequence includes the tec genes coding for the alpha-subunit and beta-subunit of a terminal dioxygenase, a ferredoxin and a reductase. E. coli cells producing these proteins were able to dioxygenolytically attack a range of aromatic compounds including chlorinated benzenes and toluene, and also dinuclear aromatics such as biphenyl and dibenzo-p-dioxin. The enzyme was shown by (18)O2 incorporation experiments to dioxygenolytically attack a chlorosubstituted carbon atom of Cl4-benzene, thereby forming an unstable diol intermediate which spontaneously rearomatizes with concomitant chloride elimination to the corresponding 3,4,6-trichlorocatechol (Cl3-catechol).

The bphA1A2A3A4 gene cluster, encoding a biphenyl dioxygenase from Rhodococcus globerulus P6, a gram-positive microorganism able to degrade a wide spectrum of polychlorobiphenyls (PCBs), was expressed in Pseudomonas putida, thereby allowing characterization of chlorobiphenyl oxidation by this enzyme. While P6 biphenyl dioxygenase activity was observed in P. putida containing bphA1A2A3A4, no activity was detected in Escherichia coli cells containing the same gene cluster. In E. coli, transcription of genes bphB and bphCl, located downstream of bphA1A2A3A4, was shown to be driven solely by a vector promoter, which indicated that the lack of biphenyl dioxygenase activity was not due to a lack of mRNA synthesis. Radioactive labelling of bph gene products in E. coli implied inefficient translation of the bphA gene cluster or rapid degradation of the gene products. The biosynthesis of functional P6 biphenyl dioxygenase in P. putida cells containing the same plasmid construct that yielded no activity in E. coli emphasizes the importance of the host strain for heterologous expression and shows that synthesis, correct folding, and assembly of a Rhodococcus biphenyl dioxygenase can be achieved in a gram-negative organism. Dioxygenation of six mono- and dichlorinated PCB congeners by P. putida containing the P6 bphA gene cluster indicates the following ring substitution preference for this reaction (from most to least preferred): un-, meta-, para-, and ortho-substitution. No indications were found for dioxygenation of meta/para carbon pairs, or for hydroxylation of chlorinated carbons at any position of a monochlorinated ring, suggesting a strict specificity of this biphenyl dioxygenase for attack at nonhalogenated ortho/meta vicinal carbons. This contrasts the properties of an analogous enzyme from Pseudomonas sp. strain LB400, which can both dioxygenate at meta and para positions and dehalogenate substituted ortho carbons during ortho and meta dioxygenation.

In order to characterize the metabolites produced in vivo by biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase, the first two enzymes of the (polychloro)biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. LB400, recombinant E. coli strains expressing the respective genes were constructed. Biphenyl-2,3-dioxygenase attack on 2,2'- or 2,4'-dichlorobiphenyl was shown to give rise to virtually quantitative ortho-dechlorination of these congeners by hydroxylation at the chlorinated carbon 2 and its unsubstituted neighbour. Elimination of hydrochloric acid directly leads to 2,3-dihydroxy-chlorobiphenyls and obviates the need for biphenyl-2,3-dihydrodiol-2,3-dehydrogenase for the catabolism of such congeners.

Polychlorinated biphenyls (PCBs) typify a class of stable aromatic pollutants that are targeted by bioremediation strategies. In the aerobic degradation of biphenyl by bacteria, the key step of ring cleavage is catalyzed by an Fe(II)-dependent extradiol dioxygenase. The crystal structure of 2,3-dihydroxybiphenyl 1,2-dioxygenase from a PCB-degrading strain of Pseudomonas cepacia has been determined at 1.9 angstrom resolution. The monomer comprises amino- and carboxyl-terminal domains. Structural homology between and within the domains reveals evolutionary relationships within the extradiol dioxygenase family. The iron atom has five ligands in square pyramidal geometry: one glutamate and two histidine side chains, and two water molecules.

The 1H NMR spectra of an aromatic ring-cleaving extradiol dioxygenase, 2,2',3-trihydroxybiphenyl dioxygenase of the dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1, are reported. In the catalytically active reduced form of the monomeric enzyme (MW = 32 kDa), three broad strongly downfield shifted signals were observed, two of which disappeared in D2O solution. Their shifts and linewidths are consistent with ring NH and meta-like protons of coordinated histidines. These signals show strong sensitivity to the presence of the substrate. The oxidized form of the enzyme shows no hyperfine shifted signals. It is suggested that the high spin Fe(II) ion present in the active form of the enzyme is coordinated by at least two histidines. This is the first report of hyperfine shifted NMR signals being detected for an extradiol dioxygenase.

Metabolism of 21 chlorobiphenyls by the enzymes of the upper biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. strain LB400 was investigated by using recombinant strains harboring gene cassettes containing bphABC or bphABCD. The enzymes of the upper pathway were generally able to metabolize mono- and dichlorinated biphenyls but only partially transform most trichlorinated congeners investigated: 14 of 15 mono- and dichlorinated and 2 of 6 trichlorinated congeners were converted into benzoates. All mono- and at least 8 of 12 dichlorinated congeners were attacked by the bphA-encoded biphenyl dioxygenase virtually exclusively at ortho and meta carbons. This enzyme exhibited a high degree of selectivity for the aromatic ring to be attacked, with the order of ring preference being non- > ortho- > meta- > para-substituted for mono- and dichlorinated congeners. The influence of the chlorine substitution pattern of the metabolized ring on benzoate formation resembled its influence on the reactivity of initial dioxygenation, suggesting that the rate of benzoate formation may frequently be determined by the rate of initial attack. The absorption spectra of phenylhexadienoates formed correlated with the presence or absence of a chlorine substituent at an ortho position.

The Gram+ bacterium Rhodococcus globerulus P6 (RgP6) catabolizes a range of polychlorinated biphenyl (PCB) congeners, thus being of interest in bioelimination processes for PCB. The first step in the pathway, a dioxygenase attack of one of the biphenyl (BP) rings, is catalyzed by biphenyl dioxygenase (BDO). In this study, the nucleotide (nt) sequences of the four clustered cistrons, bphA1A2A3A4, encoding the subunits of BDO and forming part of the bph operon of RgP6 for BP degradation, were determined. A conserved motif proposed to bind a Rieske-type [2Fe-2S] cluster was identified in the deduced amino acid (aa) sequence of both the a subunit of the terminal oxygenase (BphA1) and ferredoxin (BphA3). The ferredoxin reductase subunit (BphA4) contains conserved sites for FAD and NADH binding. Deduced aa sequences of the BDO subunits shared homologies to multicomponent aromatic ring-hydroxylating dioxygenases from Gram- microorganisms. Stronger identity was found to toluene dioxygenase (TDO) of Pseudomonas putida F1 than to other BDO. Aa sequence comparisons suggest that BP degradation genes of RgP6 may have originated in Gram- microorganisms, probably Pseudomonas, and subsequently transferred to this Gram+ bacterium.

Catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida consists of four identical subunits, each containing one ferrous ion. The enzyme catalyzes ring cleavage of catechol, 3-methylcatechol, and 4-methylcatechol but shows only weak activity toward 4-ethylcatechol. Two mutants of catechol 2,3-dioxygenases (4ECR1 and 4ECR6) able to oxidize 4-ethylcatechol, one mutant (3MCS) which exhibits only weak activity toward 3-methylcatechol but retained the ability to cleave catechol and 4-methylcatechol, and one phenotypic revertant of 3MCS (3MCR) which had regained the ability to oxidize 3-methylcatechol were characterized by determining their Km and partition ratio (the ratio of productive catalysis to suicide catalysis). The amino acid substitutions in the four mutant enzymes were also identified by sequencing their structural genes. Wild-type catechol 2,3-dioxygenase was inactivated during the catalysis of 4-ethylcatechol and thus had a low partition ratio for this substrate, whereas the two mutant enzymes, 4ECR1 and 4ECR6, had higher partition ratios for it. Similarly, mutant enzyme 3MCS had a lower partition ratio for 3-methylcatechol than that of 3MCR. Molecular oxygen was required for the inactivation of the wild-type enzyme by 4-ethylcatechol and of 3MCS by 3-methylcatechol, and the inactivated enzymes could be reactivated by incubation with FeSO4 plus ascorbic acid. The enzyme inactivation is thus most likely mechanism based and occurred principally by oxidation and/or removal of the ferrous ion in the catalytic center. In general, partition ratios for catechols lower than 18,000 did not support bacterial growth. A possible meaning of the critical value of the partition ratio is discussed.

The polychlorobiphenyl-degrading bacterium Rhodococcus globerulus P6 contains three bphC genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenases. One of them, bphC1, is clustered with the bphB gene which encodes 2,3-dihydroxy-4-phenylhexa-4,6-diene dehydrogenase and constitutes part of the bph operon specifying the degradation of biphenyl. The nucleotide sequence of bphB and the three bphC genes has been determined. The protein products of the bphBC1 gene cluster were found to exhibit significant homology with the corresponding proteins of analogous degradative pathways in Gram-negative bacteria; the highest homology was in those of the toluene degradation pathway of Pseudomonas putida strain F1. No homology was found between bphC2 and bphC3 and any other sequence in the database. At least two of the three meta cleavage enzymes are inducible by biphenyl. 2,3-Dihydroxybiphenyl 1,2-dioxygenase II, encoded by the bphC2 gene, was purified to apparent homogeneity from a recombinant Escherichia coli strain. The enzyme differed from other extradiol dioxygenases in having a subunit molecular mass of 21 kDa and a hexameric structure. The enzyme contains one tightly bound iron per subunit. These characteristics demonstrate that the 2,3-dihydroxybiphenyl 1,2-dioxygenases encoded by bphC2 and bphC3 belong to a new class of extradiol dioxygenases.

A key enzyme in the degradation pathways of dibenzo-p-dioxin and dibenzofuran, namely, 2,2',3-trihydroxybiphenyl dioxygenase, which is responsible for meta cleavage of the first aromatic ring, has been genetically and biochemically analyzed. The dbfB gene of this enzyme has been cloned from a cosmid library of the dibenzo-p-dioxin- and dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 (R. M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) and sequenced. The amino acid sequence of this enzyme is typical of those of extradiol dioxygenases. This enzyme, which is extremely oxygen labile, was purified anaerobically to apparent homogeneity from an Escherichia coli strain that had been engineered to hyperexpress dbfB. Unlike most extradiol dioxygenases, which have an oligomeric quaternary structure, the 2,2',3-trihydroxybiphenyl dioxygenase is a monomeric protein. Kinetic measurements with the purified enzyme produced similar Km values for 2,2',3-trihydroxybiphenyl and 2,3-dihydroxybiphenyl, and both of these compounds exhibited strong substrate inhibition. 2,2',3-Trihydroxydiphenyl ether, catechol, 3-methylcatechol, and 4-methylcatechol were oxidized less efficiently and 3,4-dihydroxybiphenyl was oxidized considerably less efficiently.

The cistronic organization of the bph locus, encoding a biphenyl/polychlorinated biphenyl (PCB) degradation pathway in Pseudomonas sp. LB400, has been elucidated. Seven structural genes, encoding biphenyl dioxygenase (bphA1A2A3A4), biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (bphB), biphenyl-2,3-diol-1,2-dioxygenase (bphC) and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (bphD), have been located. The complete sequences of bphB, bphC and bphD are reported. Taken together with the data of Erickson and Mondello [J. Bacteriol. 174 (1992) 2903-2912], Pseudomonas sp. LB400 is now the first strain for which the sequences of all genes encoding the catabolism from biphenyls to benzoates have been determined. Comparisons of the deduced amino acid (aa) sequences of BphB, BphC and BphD with those of related proteins led to predictions about catalytically important aa residues. Six Bph have been detected and identified. Five of them could be obtained as the most abundant proteins when their genes were expressed in Escherichia coli.

Rhodococcus globerulus P6 (previously designated Acinetobacter sp. strain P6, Arthrobacter sp. strain M5, and Corynebacterium sp. strain MB1) is able to degrade a wide range of polychlorinated biphenyl (PCB) congeners. The genetic and biochemical analyses of the PCB catabolic pathway reported here have revealed the existence of a PCB gene cluster--bphBC1D--and two further bphC genes--bphC2 and bphC3--that encode three narrow-substrate-specificity enzymes (2,3-dihydroxybiphenyl dioxygenases) that meta cleave the first aromatic ring. None of the bphC genes show by hybridization homology to each other or to bphC genes in other bacteria, and the three bphC gene products have different kinetic parameters and sensitivities to inactivation by 3-chlorocatechol. This suggests that there exists a wide diversity in PCB meta cleavage enzymes.

2,3-Dihydroxybiphenyl 1,2-dioxygenase, an enzyme of the biphenyl biodegradation pathway that cleaves the first of the aromatic rings, was purified to apparent homogeneity from Pseudomonas sp. strain LB400 that had been engineered to hyperexpress the bphC gene. The enzyme had a subunit molecular mass of 33.2 kDa as determined by SDS-polyacrylamide electrophoresis. Kinetic studies indicate a KM of 7 +/- 1 microM for 2,3-dihydroxybiphenyl. The enzyme is strongly inhibited by substrate (Kss = 300 +/- 10 microM). Catechol, 3-methylcatechol, and 4-methylcatechol were cleaved less efficiently and showed weaker substrate inhibition. 3,4-Dihydroxybiphenyl was not a substrate for the enzyme. Ammonium sulfate and polyethylene glycol 6000 were used as precipitants to obtain two different crystal forms. Crystals grown from ammonium sulfate and polyethylene glycol 6000 had space groups of P4(2)2(1)2 and I222, respectively. Electron microscopy indicates that the enzyme is an octamer (265 kDa) consisting of subunits arranged in two planar tetramers in a staggered conformation.

Novel transposon and plasmid-based broad-host-range expression systems have been developed to facilitate the genetic analysis of gene products of Pseudomonas and related Gram- bacteria. The properties of lacIq/Ptrp-lac were used to construct mini-Tn5 expression vector transposons and RSF1010-derived plasmids for controlled expression and generation of conditional phenotypes. These plasmids were used to hyper-express the XylS regulator of the meta operon of the TOL plasmid of P. putida or the bphB and bphC genes of the polychlorobiphenyl-degrading pathway of Pseudomonas sp. LB400 in different strains of Pseudomonas instead of in Escherichia coli. Specific activity of 2.3 dihydroxybiphenyl dioxygenase (bphC gene product) was increased tenfold when hyperproduced in its native host as compared to E. coli, but under the same in vivo conditions, the XylS regulator formed protein aggregates. The other lacIq/Ptrp-lac-based expression vector presented here, transposon mini-Tn5 lacIq/Ptrc, facilitates the insertion of genetic cassettes containing heterologous genes under the control of lac inducers in the chromosome of target bacteria, as shown by monitoring expression of a lacZ reporter cloned in mini-Tn5 lacIq/Ptrc and inserted in the chromosome of P. putida.

The conversion of substituted benzoates into 1,2-cis-dihydroxycyclohexa-3,5-diene carboxylic acids (cis-diols) was effected by using Escherichia coli and Pseudomonas recombinants carrying the xylXYZ genes originating from the Pseudomonas putida mt-2 TOL plasmid, thus producing toluate-1,2-dioxygenase. Pseudomonas oleovorans GPo12 recombinants readily produced meta- and para-substituted cis-diols, but were limited in their oxidation of ortho-substituted substrates.

Stimulation of transcription from positively regulated promoters involves regulatory proteins that have been activated, generally as a consequence of binding low molecular weight effector molecules. To define essential structural features of effectors for one positively acting gene regulator, the xylS-encoded protein, which activates the TOL plasmid meta-cleavage pathway operon promoters, effector activities of a wide range of benzoate derivatives have been systematically analyzed, and mutant xylS-encoded proteins exhibiting altered effector specificities have been generated and characterized. Cloned mutant xylS genes were trans dominant in partial diploids containing the wild-type xylS allele and could therefore be used to effect expansion of the range of aromatic compounds completely or partially degraded by Pseudomonas bacteria. The method developed to isolate mutant xylS-encoded proteins has general applicability and could in principle be used to isolate gene regulator specificity mutants of any inducible regulatory system.

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.

DNA fragments containing the xylD and xylL genes of TOL plasmid pWW0 -161 of Pseudomonas putida, which code for the catabolic enzymes toluate 1,2-dioxygenase and dihydrodihydroxybenzoic acid dehydrogenase, respectively, and the nahG gene of the NAH plasmid NAH7 , which codes for salicylate hydroxylase, were cloned in pBR322 vector plasmid. Deletion and insertion mutagenesis were used to localize these genes with respect to crucial endonuclease cleavage sites. The pBR322-based plasmids were ligated to the broad host range cloning vector pKT231 , or derivatives of it, and the hybrid plasmids were introduced into Pseudomonas sp. B13( WR1 ), a bacterium able to degrade 3-chlorobenzoate but not 4-chlorobenzoate, 3,5- dichlorobenzoate , salicylate, or chlorosalicylates . The cloned xylD gene expanded the catabolic range of WR1 to include 4-chlorobenzoate, whereas the cloned xylD - xylL genes enabled the isolation of derivatives of WR1 that degraded 3-chlorobenzoate, 4-chlorobenzoate, and 3,5- dichlorobenzoate . The cloned nahG gene extended the catabolic range of WR1 to include salicylate and 3-, 4-, and 5- chlorosalicylate .

Toluate 1,2-dioxygenase is the first enzyme of a meta-cleavage pathway for the oxidative catabolism of benzoate and substituted benzoates to Krebs cycle intermediates that is specified by TOL plasmid pWW0 of Pseudomonas putida. A collection of derivatives harbouring Tn1000 insertions and defective in toluate dioxygenase have been isolated from pPL392, a pBR322-based hybrid plasmid carrying the TOL plasmid meta-cleavage pathway operon. In parallel, a series of N-methyl-N'-nitro-N-nitro-soguanidine-induced mutant plasmids defective in this enzyme activity were isolated from pNM72, a pKT231-based hybrid plasmid carrying the same operon. Pairs of mutant plasmids, consisting of one Tn1000 derivative and one nitrosoguanidine-induced derivative, were used for complementation analysis of toluate dioxygenase in Escherichia coli recA bacteria, in which the formation of 2-hydroxymuconic semialdehyde from benzoate was examined. Four cistrons for toluate 1,2-dioxygenase were thus identified. DNA fragments containing nitrosoguanidine-induced mutant cistrons plus the other meta-cleavage operon genes were cloned into pOT5, an R388-based vector, and complementation tests between different nitrosoguanidine-induced mutant cistrons were carried out in Pseudomonas putida cells, this time scoring for growth on p-toluate. This analysis also identified four cistrons. Examination of the products of these cistrons, by means of E. coli minicells containing pPL392 or its Tn1000 insertion derivatives, indicated that the first two cistrons of the operon comprise a single gene, xylX, which encodes a 57 kilodalton protein, and that the third cistron, xylY, encodes a 20 kilodalton protein.

TOL plasmid pWW0 specifies enzymes for the oxidative catabolism of toluene and xylenes. The upper pathway converts the aromatic hydrocarbons to aromatic carboxylic acids via corresponding alcohols and aldehydes and involves three enzymes: xylene oxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. The synthesis of these enzymes is positively regulated by the product of xylR. Determination of upper pathway enzyme levels in bacteria carrying Tn5 insertion mutant derivatives of plasmid pWW0-161 has shown that the genes for upper pathway enzymes are organized in an operon with the following order: promoter-xylC (benzaldehyde dehydrogenase gene[s])-xylA (xylene oxygenase gene[s])-xylB (benzyl alcohol dehydrogenase gene). Subcloning of the upper pathway genes in a lambda pL promoter-containing vector and analysis of their expression in Escherichia coli K-12 confirmed this order. Two distinct enzymes were found to attack benzyl alcohol, namely, xylene oxygenase and benzyl alcohol dehydrogenase; and their catalytic activities were additive in the conversion of benzyl alcohol to benzaldehyde. The fact that benzyl alcohol is both a product and a substrate of xylene oxygenase indicates that this enzyme has a relaxed substrate specificity.

Plasmid pJP4 of Alcaligenes eutrophus JMP134 contains all genes for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D). Five of these genes, tfdB, tfdC, tfdD, tfdE, and tfdF, have recently been localized and cloned (R. H. Don, A. J. Weightman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). Gene tfdA, which codes for the 2,4-D monooxygenase, has now been found by mutagenesis with transposon Tn5. A 3-kilobase fragment of pJP4 cloned in a broad-host-range vector could complement the 2,4-D-negative phenotype of two mutants which lacked 2,4-D monooxygenase activity. The cloned tfdA gene was also transferred to A. eutrophus JMP222, which is a cured derivative of JMP134. The recombinant strain could utilize phenoxyacetic acid as a sole source of carbon and energy. Pseudomonas sp. strain B13, containing the cloned tfdA, was able to degrade phenoxyacetic acid and 4-chlorophenoxyacetic acid. Gene tfdA was subcloned and analyzed by deletions. Expression of 2,4-D monooxygenase in Escherichia coli containing a 1.4-kilobase subfragment was demonstrated by radioisotopic enzyme assay, and a protein of 32,000-dalton molecular mass was detected by labeling experiments. A 2-kilobase subfragment containing tfdA has been sequenced. Sequence analysis revealed an open reading frame of 861 bases which was identified as the coding region of tfdA by insertion mutagenesis.

Pseudomonas putida B2 is able to grow on o-nitrophenol (ONP) as the sole source of carbon and nitrogen. ONP was converted by a nitrophenol oxygenase to nitrite and catechol. Catechol was then attacked by a catechol 1,2-dioxygenase and further degraded through an ortho-cleavage pathway. ONP derivatives which were para-substituted with a methyl-, chloro-, carboxy-, formyl- or nitro-group failed to support growth of strain B2. Relevant catabolic enzymes were characterized to analyze why these derivatives were not mineralized. Nitrophenol oxygenase of strain B2 is a soluble, NADPH-dependent enzyme that is stimulated by magnesium, manganese, and calcium ions. It is active toward ONP, 4-methyl-, 4-chloro-, and to a lesser extent, 4-formyl-ONP but not toward 4-carboxy- or 4-nitro-ONP. In addition, 4-formyl-, 4-carboxy-, and 4-nitro-ONP failed to induce the formation of nitrophenol oxygenase. Catechol 1,2-dioxygenase of strain B2 is active toward catechol and 4-methyl-catechol but only poorly active toward chlorinated catechols. 4-Methyl-catechol is likely to be degraded to methyl-lactones, which are often dead-end metabolites in bacteria. Thus, of the compounds tested, only unsubstituted ONP acts as an inducer and substrate for all of the enzymes of a productive catabolic pathway.