Operon structure and nucleotide homology of the chlorocatechol oxidation genes of plasmids pJP4 and pAC27

Alcaligenes eutrophus harboring plasmid pJP4 (strain JMP134) is capable of growing on both 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-Cba), while Pseudomonas putida carrying plasmid pAC27 (strain AC867) can utilize only 3-Cba as the sole carbon source. The tfdCDEF operon of the pJP4 plasmid and the clcABD operon of plasmid pAC27 each encode enzymes for the degradation of chlorocatechols (Clc), key intermediates in the catabolism of 2,4-D and 3-Cba. Similarities in the nucleotide (nt) sequences of genes tfdC and clcA, encoding pyrocatechases, were reported earlier [Ghosal and You, Mol. Gen. Genet. 211 (1988a) 113-120]. Genes tfdD and clcB, encoding Clc-specific cycloisomerases, have been completely sequenced. The tfdD gene (1107 bp) is slightly smaller than gene clcB (1113 bp). Comparison of the two cycloisomerase-encoding genes reveals that the nt sequences are 63% homologous with 62% homology in the deduced amino acid (aa) sequences of the polypeptides they encode. Genes tfdD and tfdE are contiguous in the tfdCDEF operon, whereas the corresponding genes, clcB and clcD, of the clcABD operon, are known to be separated by a long open reading frame of unknown function. The predicted N-terminal aa sequences of the two hydrolase-encoding genes, tfdE and clcD, also show homology. The structural and nt homologies between the two Clc operons, tfdCDEF and clcABD, suggest their relatedness.     

Nucleotide sequence and expression of clcD, a plasmid-borne dienelactone hydrolase gene from Pseudomonas sp. strain B13.

The clcD structural gene encodes dienelactone hydrolase (EC 3.1.1.45), an enzyme that catalyzes the conversion of dienelactones to maleylacetate. The gene is part of the clc gene cluster involved in the utilization of chlorocatechol and is carried on a 4.3-kilobase-pair BglII fragment subcloned from the Pseudomonas degradative plasmid pAC27. A 1.9-kilobase-pair PstI-EcoRI segment subcloned from the BglII fragment was shown to carry the clcD gene, which was expressed inducibly under the tac promoter at levels similar to those found in 3-chlorobenzoate-grown Pseudomonas cells carrying the plasmid pAC27. In this study, we present the complete nucleotide sequence of the clcD gene and the amino acid sequence of dienelactone hydrolase deduced from the DNA sequence. The NH2-terminal amino acid sequence encoded by the clcD gene from plasmid pAC27 corresponds to a 33-residue sequence established for dienelactone hydrolase encoded by the Pseudomonas sp. strain B13 plasmid pWR1. A possible relationship between the clcD gene and pcaD, a Pseudomonas putida chromosomal gene encoding enol-lactone hydrolase (EC 3.1.1.24) is suggested by the fact that the gene products contain an apparently conserved pentapeptide neighboring a cysteinyl side chain that presumably lies at or near the active sites; the cysteinyl residue occupies position 60 in the predicted amino acid sequence of dienelactone hydrolase.     

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

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

Cloning and characterization of tfdS, the repressor-activator gene of tfdB, from the 2,4-dichlorophenoxyacetic acid catabolic plasmid pJP4.

Plasmid pRO101, a derivative of plasmid pJP4 which contains Tn1721 inserted into a nonessential region, is inducible for 2,4-dichlorophenol hydroxylase (DCPH) encoded by tfdB. Plasmid pRO103, which has a deletion in the BamHI-F--BamHI-E region of plasmid pRO101, has elevated basal levels of DCPH but is uninducible. The regulatory gene for tfdB, designated tfdS, was cloned as an 8.3-kilobase-pair EcoRI-E fragment. When the cloned tfdS gene was in trans with plasmid pRO103, the baseline DCPH levels were repressed to normal uninduced levels and were fully induced when this strain was grown in the presence of 2,4-dichlorophenoxyacetic acid, 2,4-dichlorophenol, or 4-chlorocatechol. However, when tfdS was in trans with tfdB in the absence of tfdCDEF, tfdB was repressed but could not be induced. When tfdS and tfdC1, which encodes chlorocatechol 1,2-dioxygenase, are in trans with tfdB, tfdB remained uninduced, indicating that a downstream metabolite of chloro-cis,cis-muconate, either 2-cis-chlorodiene lactone or chloromaleylacetic acid, is the effector. Collectively, these data demonstrate that the gene product of tfdS acts as a repressor of tfdB in the absence of an effector and as an activator of tfdB when an effector is present.     

Isolation and characterization of a new plasmid from a Flavobacterium sp. which carries the genes for degradation of 2,4-dichlorophenoxyacetate.

A Flavobacterium sp. (strain 50001), capable of degrading 2,4-dichlorophenoxyacetate (2,4-D), 2-methyl-4-chlorophenoxyacetate, and 2-chlorobenzoate and imparting resistance to mercury, harbored a degradative plasmid, pRC10. Cured strains of the Flavobacterium sp. lost the plasmid as well as the ability to degrade these chlorinated compounds. Comparison of this plasmid with the well-characterized 2,4-D-degradative plasmid pJP4 from Alcaligenes eutrophus showed regions of homology between the two plasmids. Restriction fragments of plasmid pRC10 which shared homology with the regions conferring 2,4-D-degradative genes (tfd) of plasmid pJP4 were cloned into a broad-host-range plasmid and studied in Pseudomonas putida. From the results obtained, the cloned DNA fragment expressed the genes for 2,4-D monooxygenase (tfdA) and 2,4-dichlorophenol hydroxylase (tfdB). In spite of the similarity in function, the size (45 kilobases) and restriction pattern of plasmid pRC10 were considerably different from those of pJP4 (80 kilobases). This may be due to the difference in the microbial background during evolution of the two plasmids.     

Capture of a catabolic plasmid that encodes only 2,4-dichlorophenoxyacetic acid:alpha-ketoglutaric acid dioxygenase (TfdA) by genetic complementation.

The modular pathway for the metabolism of 2,4-dichlorophenoxyacetic acid (2,4-D) encoded on plasmid pJP4 of Alcaligenes eutrophus JMP134 appears to be an example in which two genes, tfdA and tfdB, have been recruited during the evolution of a catabolic pathway. The products of these genes act to convert 2,4-D to a chloro-substituted catechol that can be further metabolized by enzymes of a modified ortho-cleavage pathway encoded by tfdCDEF. Given that modified ortho-cleavage pathways are comparatively common and widely distributed among bacteria, we sought to determine if microbial populations in soil carry tfdA on plasmid vectors that lack tfdCDEF or tfdB. To capture such plasmids from soil populations, we used a recipient strain of A. eutrophus that was rifampin resistant and carried a derivative of plasmid pJP4 (called pBH501aE) in which the tfdA had been deleted. Upon mating with mixed bacterial populations from soil treated with 2,4-D, transconjugants that were resistant to rifampin yet able to grow on 2,4-D were obtained. Among the transconjugants obtained were clones that contained a ca. 75-kb plasmid, pEMT8. Bacterial hosts that carried this plasmid in addition to pBH501aE metabolized 2,4-D, whereas strains with only pEMT8 did not. Southern hybridization showed that pEMT8 encoded a gene with a low level of similarity to the tfdA gene from plasmid pJP4. Using oligonucleotide primers based on known tfdA sequences, we amplified a 330-bp fragment of the gene and determined that it was 77% similar to the tfdA gene of plasmid pJP4 and 94% similar to tfdA from Burkholderia sp. strain RASC. Plasmid pEMT8 lacked genes that exhibited significant levels of homology to tfdB and tfdCDEF. Moreover, cell extracts from A. eutrophus(pEMT8) cultures did not exhibit TfdB, TfdC, TfdD, and TfdE activities, whereas cell extracts from A. eutrophus(pEMT8)(pBH501aE) cultures did. These data suggest that pEMT8 encodes only tfdA and that this gene can effectively complement the tfdA deletion mutation of pBH501aE.     

Genetic rearrangements in plasmids specifying total degradation of chlorinated benzoic acids

Summary Growth in a chemostat of the 3-chlorobenzoatepositive Pseudomonas putida cells harboring the plasmid pAC25, in presence of cells harboring the TOL plasmid, allows emergence of cells that can also utilize 4-chlorobenzoate (4Cba). Isolation of plasmid DNA from such cells demonstrate the deletion of a 11kb (Kilobase pair) EcoR1 fragment from the pAC25 plasmid; a portion of the TOL plasmid (41.5 kb TOL^*) is also found to be transposed onto the chromosome of such cells. Further enrichment of the 4-chlorobenzoate-positive cells with 3,5-dichlorobenzoate (3,5-Dcb) as a sole carbon source has produced cells that can also slowly utilize 3,5-dichlorobenzoate. Isolation of plasmid DNA from such cells demonstrates the appearance of a second plasmid (pAC29). Restriction hybridization of pAC29 EcoRI fragments with pAC25 and TOL demonstrates that pAC29 is derived primarily by duplication of a segment of the pAC27 plasmid and a fragment from TOL, with further mutational divergence. Southern hybridization of the EcoRI-digested chromosomal DNA with ³²P-labeled TOL, pTS11 and pTS71 plasmid DNAs demonstrates the presence of the TOL^* transposon containing xylD, G, E and F genes in both 4Cba⁺ (pAC27⁺) and 3,5-DCb⁺ (pAC27⁺, pAC29⁺) cells. Isolation of plasmid DNA from 3,5-Dcb⁺ faster growing variants, obtained from slow-growing pAC27⁺ pAC29⁺ cells, demonstrates the presence of a single type of plasmid, with identical size and EcoRI digestion profile as pAC27. The implications of gene duplications and subsequent homologous recombination with regard to the biochemical pathway of 3,5-dichlorobenzoate degradation have been discussed.     

Evidence that operons tcb, tfd, and clc encode maleylacetate reductase, the fourth enzyme of the modified ortho pathway.

The maleylacetate reductase from Pseudomonas sp. strain B13 functioning in the modified ortho pathway was purified and digested with trypsin. The polypeptides separated by high-performance liquid chromatography were sequenced. Alignments with the polypeptides predicted from the tfdF and tcbF genes located on plasmids pJP4 of the 2,4-dichlorophenoxyacetate-degrading Alcaligenes eutrophus JMP134 and pP51 of the 1,2,4-trichlorobenzene-degrading Pseudomonas sp. strain P51 as well as polypeptides predicted from the tftE gene located on the chromosome of the 2,4,5-trichlorophenoxyacetate-degrading Burkholderia cepacia AC1100 were obtained. In addition, the deduced protein sequence encoded by the nucleotide sequence downstream of clcD on plasmid pAC27 of the 3-chlorobenzoate-degrading Pseudomonas putida AC866 was tested for homology. Significant sequence similarities with the polypeptides encoded by the tfdF, tcbF, and tftE genes as well as the nucleotide sequence downstream of the clcD gene gave evidence that these genes might encode maleylacetate reductases. A NAD-binding motif in a beta alpha beta-element was detected.     

Sequence of the gene (pheA) encoding phenol monooxygenase from Pseudomonas sp. EST1001: expression in Escherichia coli and Pseudomonas putida.

The plasmid pEST1412 contains the genes, pheA and pheB, encoding phenol monooxygenase (PMO) and catechol 1,2-dioxygenase (C12]), respectively. Thse were originally cloned from the plasmid DNA of Pseudomonas sp. EST1001 [Kivisaar et al., Plasmid 24 (1990) 25-36]. Although pheA and pheB are cotranscribed using the promoter sequences derived from Tn4652 and the level of expression of C120 activities from pEST1412 was equal both in Escherichia coli and in Pseudomonas putida, the level of PMO activity measured in the cell-free extracts of E. coli was lower than that in P. putida. The nucleotide sequence of the 2.0-kb PstI-HindIII fragment of pEST1412 carrying pheA was determined. A 1821-bp ORF was found in this DNA. The structural gene (tfdB) encoding 2,4-dichlorophenol hydroxylase from pJP4 has been sequenced [Perkins et al., J. Bacteriol. 172 (1990) 2351-2359]. Comparison of the deduced amino acid sequences of tfdB and pheA revealed highly conserved regions in the protein products of these genes.     

Chlorophenol hydroxylases encoded by plasmid pJP4 differentially contribute to chlorophenoxyacetic acid degradation

Phenoxyalkanoic compounds are used worldwide as herbicides. Cupriavidus necator JMP134(pJP4) catabolizes 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), using tfd functions carried on plasmid pJP4. TfdA cleaves the ether bonds of these herbicides to produce 2,4-dichlorophenol (2,4-DCP) and 4-chloro-2-methylphenol (MCP), respectively. These intermediates can be degraded by two chlorophenol hydroxylases encoded by the tfdB(I) and tfdB(II) genes to produce the respective chlorocatechols. We studied the specific contribution of each of the TfdB enzymes to the 2,4-D/MCPA degradation pathway. To accomplish this, the tfdB(I) and tfdB(II) genes were independently inactivated, and growth on each chlorophenoxyacetate and total chlorophenol hydroxylase activity were measured for the mutant strains. The phenotype of these mutants shows that both TfdB enzymes are used for growth on 2,4-D or MCPA but that TfdB(I) contributes to a significantly higher extent than TfdB(II). Both enzymes showed similar specificity profiles, with 2,4-DCP, MCP, and 4-chlorophenol being the best substrates. An accumulation of chlorophenol was found to inhibit chlorophenoxyacetate degradation, and inactivation of the tfdB genes enhanced the toxic effect of 2,4-DCP on C. necator cells. Furthermore, increased chlorophenol production by overexpression of TfdA also had a negative effect on 2,4-D degradation by C. necator JMP134 and by a different host, Burkholderia xenovorans LB400, harboring plasmid pJP4. The results of this work indicate that codification and expression of the two tfdB genes in pJP4 are important to avoid toxic accumulations of chlorophenols during phenoxyacetic acid degradation and that a balance between chlorophenol-producing and chlorophenol-consuming reactions is necessary for growth on these compounds.     

Regulation of tfdCDEF by tfdR of the 2,4-dichlorophenoxyacetic acid degradation plasmid pJP4.

The closely linked structural genes tfdCDEF borne on the 2,4-dichlorophenoxyacetic acid (TFD) catabolic plasmid, pRO101, were cloned into vector pRO2321 as a 12.6-kilobase-pair BamHI C fragment and designated pRO2334. The first gene in this cluster, tfdC, encodes chlorocatechol 1,2-dioxygenase and was expressed constitutively. Chlorocatechol 1,2-dioxygenase expression by pRO2334 was repressed in trans by the negative regulatory element, tfdR, on plasmid pRO1949. Derepression of tfdC was achieved when Pseudomonas aeruginosa PAO4032 containing both plasmids pRO2334 and pRO1949 was grown in minimal glucose medium containing TFD, 2,4-dichlorophenol, or 4-chlorocatechol, suggesting that TFD and other pathway intermediates can act as inducing compounds. Genetic organization of the tfdCDEF cluster was established by deletion of the tfdC gene, which resulted in the loss of tfdD and tfdE activity, suggesting that genes tfdCDEF are organized in an operon transcribed from the negatively regulated promoter of tfdC. Deletion subcloning of pRO1949 was used to localize tfdR to a 1.2-kilobase-pair BamHI-XhoI region of the BamHI E fragment of plasmid pRO101. The tfdR gene product was shown not to regulate the expression of tfdB, which encodes 2,4-dichlorophenol hydroxylase.     

Bacterial metabolism of 2,4-dichlorophenoxyacetate

1. Two Pseudomonas strains isolated from soil metabolized 2,4-dichlorophenoxyacetate (2,4-D) as sole carbon source in mineral salts liquid medium. 2. 2,4-Dichlorophenoxyacetate cultures of Pseudomonas I (Smith, 1954) contained 2,4-dichlorophenol, 2-chlorophenol, 3,5-dichlorocatechol and alpha-chloromuconate, the last as a major metabolite. 3. Dechlorination at the 4(p)-position of the aromatic ring must therefore take place at some stages before ring fission. 4. Pseudomonas N.C.I.B. 9340 (Gaunt, 1962) cultures metabolizing 2,4-dichlorophenoxyacetate contained 2,4-dichloro-6-hydroxyphenoxyacetate, 2,4-dichlorophenol, 3,5-dichlorocatechol and an unstable compound, probably alphagamma-dichloromuconate. 5. Cell-free extracts of the latter organism grown in 2,4-dichlorophenoxyacetate cultures contained an oxygenase that converted 3,5-dichlorocatechol into alphagamma-dichloromuconate, a chlorolactonase that in the presence of Mn(2+) ions converted the dichloromuconate into gamma-carboxymethylene-alpha-chloro-Delta(alphabeta)-butenolide, and a delactonizing enzyme that gave alpha-chloromaleylacetate from this lactone. 6. Pathways of metabolism of 2,4-dichlorophenoxyacetate are discussed.     

Nucleotide homology and organization of chlorocatechol oxidation genes of plasmids pJP4 and pAC27

Summary The 2,4-dichlorophenoxyacetate (2,4-D) catabolic plasmid pJP4 of Alcaligenes eutrophus JMP134 contains two sets of nonidentical chlorocatechol oxidation gene sequences physically separated by a 7 kb DNA region. We determined the nucleotide sequence of the 1.6 kb HindIII fragment containing the known genes tfdC and tfdD (Don et al. 1985) which encode pyrocatechase and cycloisomerase, respectively. The 1.3 kb BglII-HindIII segment of recombinant plasmid pDC25 containing at least three chlorocatechol (clc) oxidation genes of the pAC27 plasmid in Pseudomonas putida AC868 (Ghosal et al. 1985a; Frantz and Chakrabarty 1986), was also sequenced. When the tfdC gene of the pJP4 plasmid was compared with gene clcA of plasmid pAC27, which encodes the chlorocatechol specific pyrocatechase (pyrocatechase II), the two genes showed 63% nucleotide sequence homology with 60% homology in their amino acid sequences. In both plasmid pJP4 and pAC27, the two genes encoding the pyrocatechase and the cycloisomerase showed a 4 bp overlap spanning the initiation codon of the cycloisomerase gene and the termination codon of the pyrocatechase gene. The sizes of the polypeptides encoded by the isofunctional genes tfdC and clcA are very similar and thus reflect their functional homology.     

Identification and characterization of a new plasmid carrying genes for degradation of 2,4-dichlorophenoxyacetate from Pseudomonas cepacia CSV90.

Pseudomonas cepacia CSV90 is able to utilize 2,4-dichlorophenoxyacetate (2,4-D) and 2-methyl-4-chlorophenoxyacetate as sole sources of carbon and energy. Mutants of the strain CSV90 which had lost this ability appeared spontaneously on a nonselective medium. The wild-type strain harbored a 90-kb plasmid, pMAB1, whereas 2,4-D-negative mutants either lost the plasmid or had a 70-kb plasmid, pMAB2. The plasmid pMAB2 was found to have undergone a deletion of a 20-kb fragment of pMAB1. The plasmid-free mutants regained the ability to degrade 2,4-D after introduction of purified pMAB1 by electroporation. Cloning in Escherichia coli of a 10-kb BamHI fragment from pMAB1, the region absent in pMAB2, resulted in the expression of the gene tfdC encoding 3,5-dichlorocatechol 1,2-dioxygenase. After subcloning, the tfdC gene was located in a 1.6-kb HindIII fragment. The nucleotide sequence of the tfdC gene and the restriction map of its contiguous region are identical to those of the well-characterized 2,4-D-degradative plasmid pJP4 of Alcaligenes eutrophus, whereas the overall restriction maps of the two plasmids are different. The N-terminal 44-amino-acid sequence of the enzyme purified from the strain CSV90 confirmed the reading frame in the DNA sequence for tfdC and indicated that the initiation codon GUG is read as methionine instead of valine.     

Induction of enzymes of 2,4-dichlorophenoxyacetate degradation in Burkholderia cepacia 2a and toxicity of metabolic intermediates

Burkholderia cepacia 2a inducibly degraded 2,4-dichlorophenoxyacetate (2,4-D) sequentially via 2,4-dichlorophenol, 3,5-dichlorocatechol, 2,4-dichloromuconate, 2-chloromuconolactone and 2-chloromaleylacetate. Cells grown on nutrient agar or broth grew on 2,4-D-salts only if first passaged on 4-hydroxybenzoate- or succinate-salts agar. Buffered suspensions of 4-hydroxybenzoate-grown cells did not adapt to 2,4-D or 3,5-dichlorocatechol, but responded to 2,4-dichlorophenol at concentrations <0.4 mM. Uptake of 2,4-dichlorophenol by non-induced cells displayed a type S (cooperative uptake) uptake isotherm in which the accelerated uptake of the phenol began before the equivalent of a surface monolayer had been adsorbed, and growth inhibition corresponded with the acquisition of 2.2-fold excess of phenol required for the establishment of the monolayer. No evidence of saturation was seen even at 2 mM 2,4-dichlorophenol, possibly due to absorption by intracellular poly-beta-hydroxybutyrate inclusions. With increasing concentration, 2,4-dichlorophenol caused progressive cell membrane damage and, sequentially, leakage of intracellular K(+), P(i), ribose and material absorbing light at 260 nm (presumed nucleotide cofactors), until at 0.4 mM, protein synthesis and enzyme induction were forestalled. Growth of non-adapted cells was inhibited by 0.35 mM 2,4-dichlorophenol and 0.25 mM 3,5-dichlorocatechol; the corresponding minimum bacteriocidal concentrations were 0.45 and 0.35 mM. Strain 2a grew in chemostat culture on carbon-limited media containing 2,4-D, with an apparent growth yield coefficient of 0.23, and on 2,4-dichlorophenol. Growth on 3,5-dichlorocatechol did not occur without a supplement of succinate, probably due to accumulation of toxic quantities of quinonoid and polymerisation products. Cells grown on these compounds were active towards all three, but not when grown on other substrates. The enzymes of the pathway therefore appeared to be induced by 3,5-dichlorocatechol or some later metabolite. A possible reason is offered for the environmental persistence of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).     

Degradation of chlorosubstituted aromatic compounds by Pseudomonas sp. strain B13: fate of 3,5-dichlorocatechol

The degradation of 3,5-dichlorocatechol by enzymes of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. The following compounds were formed from 3,5-dichlorocatechol: trans-2-chloro-4-carboxymethylenebut-2-en-4-olide, cis-2-chloro-4-carboxymethylenebut-2-en-4-olide, and chloroacetylacrylate as the decarboxylation product of 2-chloromaleylacetate. They were identified by chromatographic and spectroscopic methods (UV, MS, PMR). An enzyme activity converting trans-2-chloro-4-carboxymethylenebut-2-en-4-olide into the cis-isomer was observed.Abbreviations 3CB 3-chlorobenzoate - 4CB 4-chlorobenzoate - 3,5DCB 3,5-dichlorobenzoate - 2,4D 2,4-dichlorophenoxyacetate - NOE Nuclear-Overhauser-Effect     

Transposon mutagenesis and cloning analysis of the pathways for degradation of 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate in Alcaligenes eutrophus JMP134(pJP4).

Plasmid pJP4 permits its host bacterium, strain JMP134, to degrade and utilize as sole sources of carbon and energy 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (R. H. Don and J. M. Pemberton, J. Bacteriol. 145:681-686, 1981). Mutagenesis of pJP4 by transposons Tn5 and Tn1771 enabled localization of five genes for enzymes involved in these catabolic pathways. Four of the genes, tfdB, tfdC, tfdD, and tfdE, encoded 2,4-dichlorophenol hydroxylase, dichlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and chlorodienelactone hydrolase, respectively. No function has been assigned to the fifth gene, tfdF, although it may encode a trans-chlorodiene-lactone isomerase. Inactivation of genes tfdC, tfdD, and tfdE, which encode the transformation of dichlorocatechol to chloromaleylacetic acid, prevented host strain JMP134 from degrading both 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid, which indicates that the pathways for these two substrates utilize common enzymes for the dissimilation of chlorocatechols. Studies with cloned catabolic genes from pJP4 indicated that whereas all essential steps in the degradation of 2,4-dichlorophenoxyacetic acid are plasmid encoded, the conversion of 3-chlorobenzoate to chlorocatechol is specified by chromosomal genes.     

Purification and properties of a plasmid-encoded 2,4-dichlorophenol hydroxylase

2,4-Dichlorophenol hydroxylase, an enzyme involved in the bacterial degradation of the herbicide 2,4-dichlorophenoxyacetate (2,4-D) was purified from two bacterial strains that harbored the same 2,4-D plasmid, pJP4. The purified enzymes (Mr 224 000) from the two transconjugants were indistinguishable; they contained FAD and were composed of non-identical subunits, Mr 67 000 and 45 000, respectively. Various substituted phenols were hydroxylated, using either NADH or NADPH. The amino acid composition of the native enzyme was determined.