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The tecB gene, located downstream of tecA and encoding tetrachlorobenzene dioxygenase, in Ralstonia sp. strain PS12 was cloned into Escherichia coli DH5alpha together with the tecA gene. The identity of the tecB gene product as a chlorobenzene dihydrodiol dehydrogenase was verified by transformation into the respective catechols of chlorobenzene, the three isomeric dichlorobenzenes, as well as 1,2,3- and 1,2,4-trichlorobenzenes, all of which are transformed by TecA into the respective dihydrodihydroxy derivatives. Di- and trichlorotoluenes were either subject to TecA-mediated dioxygenation (the major or sole reaction observed for the 1,2,4-substituted 2,4-, 2,5-, and 3,4-dichlorotoluenes), resulting in the formation of the dihydrodihydroxy derivatives, or to monooxygenation of the methyl substituent (the major or sole reaction observed for 2,3-, 2,6-, and 3,5-dichloro- and 2,4,5-trichlorotoluenes), resulting in formation of the respective benzyl alcohols. All of the chlorotoluenes subject to dioxygenation by TecA were transformed, without intermediate accumulation of dihydrodihydroxy derivatives, into the respective catechols by TecAB, indicating that dehydrogenation is no bottleneck for chlorobenzene or chlorotoluene degradation. However, only those chlorotoluenes subject to a predominant dioxygenation were growth substrates for PS12, confirming that monooxygenation is an unproductive pathway in PS12.

The tcbR-tcbCDEF gene cluster, coding for the chlorocatechol ortho-cleavage pathway in Pseudomonas sp. strain P51, has been cloned into a Tn5-based minitransposon. The minitransposon carrying the tcb gene cluster and a kanamycin resistance gene was transferred to Pseudomonas putida KT2442, and chromosomal integration was monitored by selection either for growth on 3-chlorobenzoate or for kanamycin resistance. Transconjugants able to utilize 3-chlorobenzoate as a sole carbon source were obtained, although at a >100-fold lower frequency than kanamycin-resistant transconjugants. The vast majority of kanamycin-resistant transconjugants were not capable of growth on 3-chlorobenzoate. Southern blot analysis revealed that many transconjugants selected directly on 3-chlorobenzoate contained multiple chromosomal copies of the tcb gene cluster, whereas those selected for kanamycin resistance possessed a single copy. Subsequent selection of kanamycin resistance-selected single-copy transconjugants for growth on 3-chlorobenzoate yielded colonies capable of utilizing this carbon source, but no amplification of the tcb gene cluster was apparent. Introduction of two copies of the tcb gene cluster without prior 3-chlorobenzoate selection resulted in transconjugants able to grow on this carbon source. Expression of the tcb chlorocatechol catabolic operon in P. putida thus represents a useful model system for analysis of the relationship among gene dosage, enzyme expression level, and growth on chloroaromatic substrates.

The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4, tfdC(I)D(I)E(I)F(I) (module I) and tfdD(II)C(II)E(II)F(II) (module II), putatively encoding these enzymes. To assess the role of both tfd modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene. These constructs were introduced into R. eutropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,2-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I. The R. eutropha JMP222 derivative containing tfdR-tfdC(I)D(I)E(I)F(I) grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR-tfdD(II)C(II)E(II)F(II). In the case of P. putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB. These results indicate that efficient degradation of 3-CB by R. eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II.

Two genes, nahG and nahW, encoding two independent salicylate 1-hydroxylases have been identified in the naphthalene-degrading strain Pseudomonas stutzeri AN10. While nahG resides in the same transcriptional unit as the meta-cleavage pathway genes, forming the naphthalene degradation lower pathway, nahW is situated outside but in close proximity to this transcriptional unit. The nahG and nahW genes of P. stutzeri AN10 are induced and expressed upon incubation with salicylate, and the enzymes that are encoded, NahG and NahW, are involved in naphthalene and salicylate metabolism. Both genes, nahG and nahW, have been cloned in Escherichia coli JM109. The overexpression of these genes yields peptides with apparent molecular masses of 46 kDa (NahG) and 43 kDa (NahW), respectively. Both enzymes exhibit broad substrate specificities and metabolize salicylate, methylsalicylates, and chlorosalicylates. However, the relative rates by which the substituted analogs are transformed differ considerably.

Bacterial strain LW1, which belongs to the family Comamonadaceae, utilizes 1-chloro-4-nitrobenzene (1C4NB) as a sole source of carbon, nitrogen, and energy. Suspensions of 1C4NB-grown cells removed 1C4NB from culture fluids, and there was a concomitant release of ammonia and chloride. Under anaerobic conditions LW1 transformed 1C4NB into a product which was identified as 2-amino-5-chlorophenol by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. This transformation indicated that there was partial reduction of the nitro group to the hydroxylamino substituent, followed by Bamberger rearrangement. In the presence of oxygen but in the absence of NAD, fast transformation of 2-amino-5-chlorophenol into a transiently stable yellow product was observed with resting cells and cell extracts. This compound exhibited an absorption maximum at 395 nm and was further converted to a dead-end product with maxima at 226 and 272 nm. The compound formed was subsequently identified by 1H and 13C NMR spectroscopy and mass spectrometry as 5-chloropicolinic acid. In contrast, when NAD was added in the presence of oxygen, only minor amounts of 5-chloropicolinic acid were formed, and a new product, which exhibited an absorption maximum at 306 nm, accumulated.

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 bacterial strain RB1 has been isolated by enrichment cultivation with 2,4-dinitrophenol as the sole nitrogen, carbon, and energy source and characterized, on the basis of 16S rRNA gene sequence comparison, as a Rhodococcus species closely related to Rhodococcus opacus. Rhodococcus sp. strain RB1 degrades 2,4-dinitrophenol, releasing the two nitro groups from the compound as nitrite. The release of nitro groups from 2,4-dinitrophenol occurs in two steps. First, the 2-nitro group is removed as nitrite, with the production of an aliphatic nitro compound identified by 1H nuclear magnetic resonance and mass spectrometry as 3-nitroadipate. Then, this metabolic derivative is further metabolized, releasing its nitro group as nitrite. Full nitrite assimilation upon reduction to ammonia requires that an additional carbon source be supplied to the medium.

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.

Phanerochaete chrysosporium ATCC 34541 has been reported to be unable to mineralize 3,4-dichloroaniline (DCA). However, high mineralization is now shown to occur when a fermentation temperature of 37 degrees and gassing with oxygen are used. Mineralization did not correlate with lignin peroxidase activity. The latter was high under C limitation and low under N limitation, whereas the reverse was true for mineralization. The kinetics of DCA metabolism was studied in low-N and low-C and C- and N-rich culture media by metabolite analysis and 14CO2 determination. In all cases, DCA disappeared within 2 days, and a novel highly polar conjugate termed DCAX accumulated in the growth medium. This metabolite was a dead-end product under C and N enrichment. In oxygenated low-C medium and in much higher yield in oxygenated low-N medium, DCAX was converted to DCA-succinimide and then mineralized. DCAX was purified by high-performance liquid chromatography and identified as N-(3,4-dichlorophenyl)-alpha-ketoglutaryl-delta-amide by high-performance liquid chromatography and mass spectroscopy, gas chromatography and mass spectroscopy, and nuclear magnetic resonance spectroscopy. The formation of conjugate intermediates is proposed to facilitate mineralization because the sensitive amino group of DCA needs protection so that ring cleavage rather than oligomerization can occur.

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.

Protoanemonin is a toxic metabolite which may be formed during the degradation of some chloroaromatic compounds, such as polychlorinated biphenyls, by natural microbial consortia. We show here that protoanemonin can be transformed by dienelactone hydrolase of Pseudomonas sp. strain B13 to cis-acetylacrylate. Although similar Km values were observed for cis-dienelactone and protoanemonin, the turnover rate of protoanemonin was only 1% that of cis-dienelactone. This indicates that at least this percentage of the enzyme is in the active state, even in the absence of activation. The trans-dienelactone hydrolase of Pseudomonas sp. strain RW10 did not detectably transform protoanemonin. Obviously, Pseudomonas sp. strain B13 possesses at least two mechanisms to avoid protoanemonin toxicity, namely a highly active chloromuconate cycloisomerase, which routes most of the 3-chloro-cis,cis-muconate to the cis-dienelactone, thereby largely preventing protoanemonin formation, and dienelactone hydrolase, which detoxifies any small amount of protoanemonin that might nevertheless be formed.