Role of tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II) gene modules in catabolism of 3-chlorobenzoate by Ralstonia eutropha JMP134(pJP4)

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.     

The filamentation pathway controlled by the Efg1 regulator protein is required for normal biofilm formation and development in Candida albicans

Ralstonia eutropha JMP134 (pJP4) grows on 3-chlorobenzoate (3-CB) or 2,4-dichlorophenoxyacetate (2,4-D). The copy number of chlorocatechol genes has been observed to be important for allowing growth of bacterial strains on chloroaromatic compounds. Despite the fact that two functional chlorocatechol degradation tfd gene clusters are harbored on plasmid pJP4, a single copy of the region comprising all tfd genes in strain JMP134-F was insufficient to allow growth on 3-CB, whereas growth on 2,4-D was only slightly retarded compared to the wild-type strain. Using competitive PCR, approximately five copies of pJP4 per genome were observed to be present in the wild-type strain, whereas only one copy of pJP4 was present per chromosome in strain JMP134-F. Therefore, several copies of pJP4 per chromosome are required for full expression of the tfd-encoded growth abilities in the wild-type R. eutropha strain.     

Role of tfdCIDIEIFI and tfdDIICIIEIIFII Gene Modules in Catabolism of 3-Chlorobenzoate by Ralstonia eutropha JMP134(pJP4)

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_ID_IE_IF_I (module I) and tfdD_IIC_IIE_IIF_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_ID_IE_IF_I grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR-tfdD_IIC_IIE_IIF_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.     

Importance of different tfd genes for degradation of chloroaromatics by Ralstonia eutropha JMP134

The tfdC(I)D(I)E(I)F(I,) and tfdD(II)C(II)E(II)F(II) gene modules of plasmid pJP4 of Ralstonia eutropha JMP134 encode complete sets of functional enzymes for the transformation of chlorocatechols into 3-oxoadipate, which are all expressed during growth on 2,4-dichlorophenoxyacetate (2,4-D). However, activity of tfd(I)-encoded enzymes was usually higher than that of tfd(II)-encoded enzymes, both in the wild-type strain grown on 2,4-D and in 3-chlorobenzoate-grown derivatives harboring only one tfd gene module. The tfdD(II)-encoded chloromuconate cycloisomerase exhibited special kinetic properties, with high activity against 3-chloromuconate and poor activity against 2-chloromuconate and unsubstituted muconate, thus explaining the different phenotypic behaviors of R. eutropha strains containing different tfd gene modules. The enzyme catalyzes the formation of an equilibrium between 2-chloromuconate and 5-chloro- and 2-chloromuconolactone and very inefficiently catalyzes dehalogenation to form trans-dienelactone as the major product, thus differing from all (chloro)muconate cycloisomerases described thus far.     

Molecular and population analyses of a recombination event in the catabolic plasmid pJP4

Cupriavidus necator JMP134(pJP4) harbors a catabolic plasmid, pJP4, which confers the ability to grow on chloroaromatic compounds. Repeated growth on 3-chlorobenzoate (3-CB) results in selection of a recombinant strain, which degrades 3-CB better but no longer grows on 2,4-dichlorophenoxyacetate (2,4-D). We have previously proposed that this phenotype is due to a double homologous recombination event between inverted repeats of the multicopies of this plasmid within the cell. One recombinant form of this plasmid (pJP4-F3) explains this phenotype, since it harbors two copies of the chlorocatechol degradation tfd gene clusters, which are essential to grow on 3-CB, but has lost the tfdA gene, encoding the first step in degradation of 2,4-D. The other recombinant plasmid (pJP4-FM) should harbor two copies of the tfdA gene but no copies of the tfd gene clusters. A molecular analysis using a multiplex PCR approach to distinguish the wild-type plasmid pJP4 from its two recombinant forms, was carried out. Expected PCR products confirming this recombination model were found and sequenced. Few recombinant plasmid forms in cultures grown in several carbon sources were detected. Kinetic studies indicated that cells containing the recombinant plasmid pJP4-FM were not selectable by sole carbon source growth pressure, whereas those cells harboring recombinant plasmid pJP4-F3 were selected upon growth on 3-CB. After 12 days of repeated growth on 3-CB, the complete plasmid population in C. necator JMP134 apparently corresponds to this form. However, wild-type plasmid forms could be recovered after growing this culture on 2,4-D, indicating that different plasmid forms can be found in C. necator JMP134 at the population level.     

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.     

TfdD(II), one of the two chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4), cannot efficiently convert 2-chloro- cis,cis-muconate to trans-dienelactone to allow growth on 3-chlorobenzoate

Ralstonia eutropha JMP134 (pJP4) harbors two functional gene clusters for the degradation of chlorocatechols, i.e. tfdCDEF (in short: tfd (I)) and tfdD (II) C (II) E (II) F (II) (in short: tfd (II)), which are both present on the catabolic plasmid pJP4. In this study, we compared the function of both gene clusters for degradation of chlorocatechols by constructing isolated and hybrid tfd (I)- tfd (II) clusters on plasmids in R. eutropha, by activity assays of Tfd enzymes, and by HPLC/MS of individual enzymatic catalytic steps in chlorocatechol conversion. R. eutropha containing the tfd (II) cluster alone or hybrid tfd-clusters with tfdD (II) as sole gene for chloromuconate cycloisomerase were impaired in growth on 3-chlorobenzoate, in contrast to R. eutrophaharboring the complete tfd (I) cluster. Enzyme activities for TfdD(II) and for TfdE(II) were very low in R. eutropha when induced with 3-chlorobenzoate. By contrast, a relatively high enzyme activity was found for TfdF(II). Spectral conversion assays with extracts from R. eutropha strains expressing tfdD (II) all showed accumulation of a compound with a similar UV spectrum as 2-chloro- cis,cis-muconate from 3-chlorocatechol. HPLC analysis of in vitro assays in which each individual step in 3-chlorocatechol conversion was reproduced by sequentially adding cell extracts of an Escherichia coli expressing one Tfd enzyme only demonstrated that TfdD(II) was unable to cause conversion of 2-chloro- cis,cis-muconate. No accumulation of intermediates was observed with 4-chlorocatechol. From these results, we conclude that at least TfdD(II) is a bottleneck in conversion of 3-chlorocatechol and, therefore, in efficient metabolism of 3-chlorobenzoate. This study showed the subtle functional and expression differences between similar enzymes of the tfd-encoded pathway and demonstrated that extreme care has to be taken when inferring functionality from sequence data alone.     

Different types of dienelactone hydrolase in 4-fluorobenzoate-utilizing bacteria

Of various benzoate-utilizing bacteria tested, Alcaligenes eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, A. eutrophus JMP134, Alcaligenes strain A7, and Pseudomonas cepacia were able to grow with 4-fluorobenzoate as the sole source of carbon and energy. P. cepacia also utilizes 3-fluorobenzoate. Except for A. eutrophus JMP134, which is known to grow with 2,4-dichlorophenoxyacetate and 3-chlorobenzoate (R. H. Don and J. M. Pemberton, J. Bacteriol. 145:681-686, 1981), the strains were unable to grow at the expense of these compounds or 4-chlorobenzoate. Assays of cell extracts revealed that all strains express dienelactone hydrolase and maleylacetate reductase activities in addition to enzymes of the catechol branch of the 3-oxoadipate pathway when growing with 4-fluorobenzoate. Induction of dienelactone hydrolase and maleylacetate reductase apparently is not necessarily connected to synthesis of catechol 1,2-dioxygenase type II and chloromuconate cycloisomerase activities, which are indispensable for the degradation of chlorocatechols. Substrate specificities of the dienelactone hydrolases provisionally differentiate among three types of this activity. (i) Extracts of A. eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, and Alcaligenes strain A7 convert trans-4-carboxymethylenebut-2-en-4-olide (trans-dienelactone) much faster than the cis-isomer (type I). (ii) The enzyme present in P. cepacia shows the opposite preference for the isomeric substrates (type II). (iii) Cell extracts of A. eutrophus JMP134, as well as purified dienelactone hydrolase from Pseudomonas strain B13 (E. Schmidt and H.-J. Knackmuss, Biochem. J. 192:339-347, 1980), hydrolyze both dienelactones at rates that are of the same order of magnitude (type III). This classification implies that A. eutrophus JMP134 possesses at least two different dienelactone hydrolases, one of type III encoded by the plasmid pJP4 and one of type I, which is also present in the cured strain JMP222.     

Efficient degradation of 2,4,6-Trichlorophenol requires a set of catabolic genes related to tcp genes from Ralstonia eutropha JMP134(pJP4)

2,4,6-Trichlorophenol (2,4,6-TCP) is a hazardous pollutant. Several aerobic bacteria are known to degrade this compound. One of these, Ralstonia eutropha JMP134(pJP4), a well-known, versatile chloroaromatic compound degrader, is able to grow in 2,4,6-TCP by converting it to 2,6-dichlorohydroquinone, 6-chlorohydroxyquinol, 2-chloromaleylacetate, maleylacetate, and beta-ketoadipate. Three enzyme activities encoded by tcp genes, 2,4,6-TCP monooxygenase (tcpA), 6-chlorohydroxyquinol 1,2-dioxygenase (tcpC), and maleylacetate reductase (tcpD), are involved in this catabolic pathway. Here we provide evidence that all these tcp genes are clustered in the R. eutropha JMP134(pJP4) chromosome, forming the putative catabolic operon tcpRXABCYD. We studied the presence of tcp-like gene sequences in several other 2,4,6-TCP-degrading bacterial strains and found two types of strains. One type includes strains belonging to the Ralstonia genus and possessing a set of tcp-like genes, which efficiently degrade 2,4,6-TCP and therefore grow in liquid cultures containing this chlorophenol as a sole carbon source. The other type includes strains belonging to the genera Pseudomonas, Sphingomonas, or Sphingopixis, which do not have tcp-like gene sequences and degrade this pollutant less efficiently and which therefore grow only as small colonies on plates with 2,4,6-TCP. Other than strain JMP134, none of the bacterial strains whose genomes have been sequenced possesses a full set of tcp-like gene sequences.     

Characterization of diverse 2,4-dichlorophenoxyacetic acid-degradative plasmids isolated from soil by complementation.

The diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative plasmids in the microbial community of an agricultural soil was examined by complementation. This technique involved mixing a suitable Alcaligenes eutrophus (Rifr) recipient strain with the indigenous microbial populations extracted from soil. After incubation of this mixture, Rifr recipient strains which grow with 2,4-D as the only C source were selected. Two A. eutrophus strains were used as recipients: JMP228 (2,4-D-), which was previously derived from A. eutrophus JMP134 by curing of the 2,4-D-degradative plasmid pJP4, and JMP228 carrying pBH501aE (a plasmid derived from pJP4 by deletion of a large part of the tfdA gene which encodes the first step in the mineralization of 2,4-D). By using agricultural soil that had been treated with 2,4-D for several years, transconjugants were obtained with both recipients. However, when untreated control soil was used, no transconjugants were isolated. The various transconjugants had plasmids with seven different EcoRI restriction patterns. The corresponding plasmids are designated pEMT1 to pEMT7. Unlike pJP4, pEMT1 appeared not to be an IncP1 plasmid, but all the others (pEMT2 to pEMT7) belong to the IncP1 group. Hybridization with individual probes for the tfdA to tfdF genes of pJP4 demonstrated that all plasmids showed high degrees of homology to the tfdA gene. Only pEMT1 showed a high degree of homology to tfdB, tfdC, tfdD, tfdE, and tfdF, while the others showed only moderate degrees of homology to tfdB and low degrees of homology to tfdC.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Analysis, cloning, and high-level expression of 2,4-dichlorophenoxyacetate monooxygenase gene tfdA of Alcaligenes eutrophus JMP134.

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.     

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.     

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

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

Deletions of mob and tra pJP4 transfer functions after mating of Ralstonia eutropha JMP134 (pJP4) with Escherichia coli harboring F'::Tn10

One-tenth of Escherichia coli transconjugants resulting from the transfer of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 to E. coli XL1Blue, contained pJP4 derivatives with deletions (approximately 15-30 kb). The occurrence of these deletions is probably associated with the presence of Tn10 in the recipient. DNA endonuclease restriction analysis of the pJP4 deletion derivatives showed the absence of SphI and EcoRI fragments previously reported to hybridize with IncP Tra DNA probes. Moreover, these pJP4 deletion derivatives are not able to self-transfer, nor are they able to be mobilized. Accordingly, these pJP4 deletion derivatives lack transfer functions.     

Role of eukaryotic microbiota in soil survival and catabolic performance of the 2,4-D herbicide degrading bacteria <Emphasis Type="Italic">Cupriavidus necator</Emphasis> JMP134

Cupriavidus necator (formerly Ralstonia eutropha) JMP134, harbouring the catabolic plasmid pJP4, is the best-studied 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide degrading bacterium. A study of the survival and catabolic performance of strain JMP134 in agricultural soil microcosms exposed to high levels of 2,4-D was carried out. When C. necator JMP134 was introduced into soil microcosms, the rate of 2,4-D removal increased only slightly. This correlated with the poor survival of the strain, as judged by 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) profiles, and the semi-quantitative detection of the pJP4-borne tfdA gene sequence, encoding the first step in 2,4-D degradation. After 3 days of incubation in irradiated soil microcosms, the survival of strain JMP134 dramatically improved and the herbicide was completely removed. The introduction of strain JMP134 into native soil microcosms did not produce detectable changes in the structure of the bacterial community, as judged by 16S rRNA gene T-RFLP profiles, but provoked a transient increase of signals putatively corresponding to protozoa, as indicated by 18S rRNA gene T-RFLP profiling. Accordingly, a ciliate able to feed on C.␣necator JMP134 could be isolated after soil enrichment. In␣native soil microcosms, C. necator JMP134 survived better than Escherichia coli DH5α (pJP4) and similarly to Pseudomonas putida KT2442 (pJP4), indicating that species specific factors control the survival of strains harbouring pJP4. The addition of cycloheximide to soil microcosms strongly improved survival of these three strains, indicating that the eukaryotic microbiota has a strong negative effect in bioaugmentation with catabolic bacteria.     

Duplication of a 2,4-dichlorophenoxyacetic acid monooxygenase gene in Alcaligenes eutrophus JMP134(pJP4).

The Alcaligenes eutrophus JMP134 plasmid pJP4 contains genes necessary for the complete degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoic acid. tfdA encodes 2,4-D monooxygenase, the initial enzyme in the 2,4-D catabolic pathway. The tfdA locus has recently been localized to a region on pJP4 13 kilobases away from a cluster of five genes, tfdB to tfdF, which encode the enzymes responsible for the further degradation of 2,4-D to chloromaleylacetic acid (W.R. Streber, K. N. Timmis, and M. H. Zenk, J. Bacteriol. 169:2950-2955, 1987). A second, dissimilar locus on pJP4, tfdAII, has been observed which encodes 2,4-D monooxygenase activity. Gas chromatographic analysis of the 2,4-D metabolites of A. eutrophus harboring pJP4 or subclones thereof localized tfdAII to within a 9-kilobase SstI fragment of pJP4 which also carries the genes tfdBCDEF. This fragment was further characterized in Escherichia coli by deletion and subcloning analysis. A region of 2.5 kilobases, adjacent to tfdC, enabled E. coli extracts to degrade 2,4-D to 2,4-dichlorophenol. Hybridization under low-stringency conditions was observed between tfdA and tfdAII, signifying that the 2,4-D monooxygenase gene was present as two related copies on pJP4.     

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

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