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.     

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)     

Isolation and characterization of a stable 2,4-dichlorophenoxyacetic acid degrading bacterium, Variovorax paradoxus, using chemostat culture

A strain of Variovorax paradoxus degrading 2,4-dichlorophenoxyacetic acid (2,4-D) was isolated from the Dijon area (France) using continuous chemostat culture. This strain, designated TV1, grew on up to 5 mM 2,4-D and efficiently degraded the herbicide as sole carbon source as well as in presence of soil extracts. It also degraded phenol and 2-methyl, 4-chlorophenoxyacetic acid at 3 mM and 2,4-dichlorophenol at 1 mM. This organism contained a stable 200 kb plasmid, designated pTV1, which showed no similarity in its restriction pattern with the archetypal 2,4-D catabolic plasmid pJP4. However, pTV1 contained an 11 kb BamHI fragment which hybridized at low stringency with the 2,4-D degradative genes tfdA, tfdB and tfdR from pJP4. PTV1 partial tfdA sequence showed 77 % similarity with the archetypal tfdA gene sequence from Ralstonia eutropha JMP134. Tn5 mutagenesis confirmed the involvement of this gene in the 2,4-D catabolic pathway. © Rapid Science Ltd. 1998     

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.     

Genetic Diversity through the Looking Glass: Effect of Enrichment Bias

The effect of enrichment bias on the diversity of 2,4-dichlorophenoxyacetate (2,4-D)-degrading (2,4-D(sup+)) bacteria recovered from soil was evaluated by comparing the diversity of isolates obtained by direct plating to the diversity of isolates obtained from 85 liquid batch cultures. By the two methods, a total of 159 isolates were purified from 1 g of soil and divided into populations based on repeated extragenic palindromic sequence PCR (rep-PCR) genomic fingerprints. Approximately 42% of the direct-plating isolates hybridized with the tfdA and tfdB genes from Alcaligenes eutrophus JMP134(pJP4), 27% hybridized with the tfdA and tfdB genes from Burkholderia sp. strain RASC, and 30% hybridized with none of the probes. In contrast, the enrichment isolates not only represented fewer populations than the isolates obtained by direct plating but also exhibited, almost exclusively, a single hybridization pattern with 2,4-D catabolic gene probes. Approximately 98% of the enrichment isolates possessed pJP4-type tfdA and tfdB genes, whereas isolates containing RASC-type tfdA and tfdB genes were obtained from only 2 of the 85 enrichment cultures. The skewed occurrence of the pJP4-type genes among the isolates obtained by enrichment suggests that the competitive fitness of 2,4-D(sup+) populations during growth with 2,4-D may be influenced either by specific tfd alleles or by genetic factors linked to these alleles. Moreover, the results indicate that evaluation of the diversity and distribution of catabolic pathways in nature can be highly distorted by the use of enrichment culture techniques.     

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.     

Characterization of a chromosomally encoded 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase from Burkholderia sp. strain RASC.

The findings of previous studies indicate that the genes required for metabolism of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) are typically encoded on broad-host-range plasmids. However, characterization of plasmid-cured strains of Burkholderia sp. strain RASC, as well as mutants obtained by transposon mutagenesis, suggested that the 2,4-D catabolic genes were located on the chromosome of this strain. Mutants of Burkholderia strain RASC unable to degrade 2,4-D (2,4-D- strains) were obtained by insertional inactivation with Tn5. One such mutant (d1) was shown to have Tn5 inserted in tfdARASC, which encodes 2,4-D/alpha-ketoglutarate dioxygenase. This is the first reported example of a chromosomally encoded tfdA. The tfdARASC gene was cloned from a library of wild-type Burkholderia strain RASC DNA and shown to express 2,4-D/alpha-ketoglutarate dioxygenase activity in Escherichia coli. The DNA sequence of the gene was determined and shown to be similar, although not identical, to those of isofunctional genes from other bacteria. Moreover, the gene product (TfdARASC) was purified and shown to be similar in molecular weight, amino-terminal sequence, and reaction mechanism to the canonical TfdA of Alcaligenes eutrophus JMP134. The data presented here indicate that tfdA genes can be found on the chromosome of some bacterial species and suggest that these catabolic genes are rather mobile and may be transferred by means other than conjugation.     

Gene transfer of Alcaligenes eutrophus JMP134 plasmid pJP4 to indigenous soil recipients.

This study evaluated the potential for gene transfer of a large catabolic plasmid from an introduced organism to indigenous soil recipients. The donor organism Alcaligenes eutrophus JMP134 contained the 80-kb plasmid pJP4, which contains genes that code for mercury resistance. Genes on this plasmid plus chromosomal genes also allow degradation of 2,4-dichloruphenoxyacetic acid (2,4-D). When JMP134 was inoculated into a nonsterile soil microcosm amended with 1,000 micrograms of 2,4-D g-1, significant (10(6) g of soil-1) populations of indigenous recipients or transconjugants arose. These transconjugants all contained an 80-kb plasmid similar in size to pJP4, and all degraded 2,4-D. In addition, all transconjugants were resistant to mercury and contained the tfdB gene of pJP4 as detected by PCR. No mercury-resistant, 2,4-D-degrading organisms with large plasmids or the tfdB gene were found in the 2,4-D-amended but uninoculated control microcosm. These data clearly show that the plasmid pJP4 was transferred to indigenous soil recipients. Even more striking is the fact that not only did the indigenous transconjugant population survive and proliferate but also enhanced rates of 2,4-D degradation occurred relative to microcosms in which no such gene transfer occurred. Overall, these data indicate that gene transfer from introduced organisms is an effective means of bioaugmentation and that survival of the introduced organism is not a prerequisite for biodegradation that utilizes introduced biodegradative genes.     

Monitoring gene expression in mixed microbial communities by using DNA microarrays

A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested. Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Ralstonia eutropha JMP134, and the inducing agent 2,4-D. Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D. This strain was diluted into a constructed mixed microbial community derived from a laboratory scale sequencing batch reactor. Induction of two of five 2,4-D catabolic genes (tfdA and tfdC) from populations of JMP134 as low as 10(5) cells/ml was clearly detected against a background of 10(8) cells/ml. Induction of two others (tfdB and tfdE) was detected from populations of 10(6) cells/ml in the same background; however, the last gene, tfdF, showed no significant induction due to high variability. In another experiment, the induction of resin acid degradative genes was statistically detectable in sludge-fed pulp mill effluent exposed to dehydroabietic acid in batch experiments. We conclude that microarrays will be useful tools for the detection of bacterial gene expression in wastewaters and other complex systems.     

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.     

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.     

Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4.

Growth of Alcaligenes eutrophus JMP134 on 2,4-dichlorophenoxyacetate requires a 2,4-dichlorphenol hydroxylase encoded by gene tfdB. Catabolism of either 2,4-dichlorophenoxyacetate or 3-chlorobenzoate involves enzymes encoded by the chlorocatechol oxidative operon consisting of tfdCDEF, which converts 3-chloro- and 3,5-dichlorocatechol to maleylacetate and chloromaleylacetate, respectively. Transposon mutagenesis has localized tfdB and tfdCDEF to EcoRI fragment B of plasmid pJP4 (R. H. Don, A. J. Wieghtman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). We present the complete nucleotide sequence of tfdB and tfdCDEF contained within a 7,954-base-pair HindIII-SstI fragment from EcoRI fragment B. Sequence and expression analysis of tfdB in Escherichia coli suggested that 2,4-dichlorophenol hydroxylase consists of a single subunit of 65 kilodaltons. The amino acid sequences of proteins encoded by tfdD and tfdE were found to be 63 and 53% identical to those of functionally similar enzymes encoded by clcB and clcD, respectively, from plasmid pAC27 of Pseudomonas putida. P. putida(pAC27) can utilize 3-chlorocatechol but not dichlorinated catechols. A region of DNA adjacent to clcD in pAC27 was found to be 47% identical in amino acid sequence to tfdF, a gene important in catabolizing dichlorocatechols. The region in pAC27 does not appear to encode a protein, suggesting that the absence of a functional trans-chlorodienelactone isomerase may prevent P. putida(pAC27) from utilizing 3,5-dichlorocatechol.     

Analysis of duplicated gene sequences associated with tfdR and tfdS in Alcaligenes eutrophus JMP134.

Plasmid pJP4 of Alcaligenes eutrophus JMP134 encodes the degradation of 2,4-dichlorophenoxyacetic acid. A 1.2-kb BamHI-XhoI region of the restriction fragment BamHI-E has been proposed to contain the regulatory gene tfdR (A. R. Harker, R. H. Olsen, and R. J. Seidler, J. Bacteriol. 171:314-320, 1989; B. Kaphammer, J. J. Kukor, and R. H. Olsen, J. Bacteriol. 172:2280-2286, 1990). When sequenced and analyzed, the region is shown to contain two incomplete open reading frames (ORFs) positioned divergently. The complete DNA sequence for one of the two ORFs was obtained by sequencing the adjacent restriction fragment BamHI-F. The DNA sequence reveals 100% identify with the regulatory gene tfdS of pJP4. An XbaI-PstI fragment, containing the complete ORF, encodes a 32,000-Da protein which binds to the promoter regions upstream from tfdA and tfdDII. The deduced amino acid sequence of the complete ORF shows similarity with sequences of activator proteins TcbR, CatM, and CatR of the LysR family. The complete ORF represents the regulatory gene tfdR. The deduced amino acid sequence of the incomplete ORF, situated divergently from tfdR, indicates similarity to chloromuconate cycloisomerases produced by genes tfdD and tcbD of plasmids pJP4 and pP51, respectively. This ORF is identified as part of a putative isofunctional gene, tfdDII.     

Impacts of 2,4-D application on soil microbial community structure and on populations associated with 2,4-D degradation

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) application rate on microbial community structure and on the diversity of dominant 2,4-D degrading bacteria in an agricultural soil was examined using cultivation-independent molecular techniques coupled with traditional isolation and enumeration methods. Fingerprints of microbial communities established under increasing concentrations of 2,4-D (0-500 mg kg-1) in batch soil microcosms were obtained using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments. While a 2,4-D concentration of at least 100 mg kg-1 was required to obtain an apparent change in the community structure as visualized by DGGE, the greatest impact of 2,4-D concentration occurred in the 500 mg kg-1 treatment, resulting in significantly reduced diversity of the dominant populations and enrichment by Burkholderia-like populations. The greatest diversity of 2,4-D degrading isolates was cultivated from the 10 mg kg-1 treatment, indicating that under these conditions, cultivation was more sensitive than DGGE for detecting changes in community structure. Most of these isolates harbored homologs of Ralstonia eutrophus JMP134 and Burkholderia cepacia tfdA catabolic genes. Results from this study revealed that agriculturally relevant application rates of 2,4-D may provide a temporary selective advantage for organisms capable of utilizing 2,4-D as a carbon and energy source.     

Phenoxyacetic acid degradation by the 2,4-dichlorophenoxyacetic acid (TFD) pathway of plasmid pJP4: mapping and characterization of the TFD regulatory gene, tfdR.

Plasmid pJP4 enables Alcaligenes eutrophus JMP134 to degrade 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (TFD). Plasmid pRO101 is a derivative of pJP4 obtained by insertion of Tn1721 into a nonessential region of pJP4. Plasmid pRO101 was transferred by conjugation to several Pseudomonas strains and to A. eutrophus AEO106, a cured isolate of JMP134. AEO106(pRO101) and some Pseudomonas transconjugants grew on TFD. Transconjugants with a chromosomally encoded phenol hydroxylase also degraded phenoxyacetic acid (PAA) in the presence of an inducer of the TFD pathway, namely, TFD or 3-chlorobenzoate. A mutant of one such phenol-degrading strain, Pseudomonas putida PPO300(pRO101), grew on PAA as the sole carbon source in the absence of inducer. This isolate carried a mutant plasmid, designated pRO103, derived from pRO101 through the deletion of a 3.9-kilobase DNA fragment. Plasmid pRO103 constitutively expressed the TFD pathway, and this allowed the metabolism of PAA in the absence of the inducer, TFD. Complementation of pRO103 in trans by a DNA fragment corresponding to the fragment deleted in pRO101 indicates that a negative control-regulatory gene (tfdR) is located on the BamHI E fragment of pRO101. Other subcloning experiments resulted in the cloning of the tfdA monooxygenase gene on a 3.5-kilobase fragment derived from pRO101. This subclone, in the absence of other pRO101 DNA, constitutively expressed the tfdA gene and allowed PPO300 to grow on PAA. Preliminary evidence suggests that the monooxygenase activity encoded by this DNA fragment is feedback-inhibited by phenols.     

Evidence for Acquisition in Nature of a Chromosomal 2,4-Dichlorophenoxyacetic Acid/(alpha)-Ketoglutarate Dioxygenase Gene by Different Burkholderia spp

We characterized the gene required to initiate the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by the soil bacterium Burkholderia sp. strain TFD6, which hybridized to the tfdA gene of the canonical 2,4-D catabolic plasmid pJP4 under low-stringency conditions. Cleavage of the ether bond of 2,4-D by cell extracts of TFD6 proceeded by an (alpha)-ketoglutarate-dependent reaction, characteristic of TfdA (F. Fukumori and R. P. Hausinger, J. Bacteriol. 175:2083-2086, 1993). The TFD6 tfdA gene was identified in a recombinant plasmid which complemented a tfdA transposon mutant of TFD6 created by chromosomal insertion of Tn5. The plasmid also expressed TfdA activity in Escherichia coli DH5(alpha), as evidenced by enzyme assays with cell extracts. Sequence analysis of the tfdA gene and flanking regions from strain TFD6 showed 99.5% similarity to a tfdA gene cloned from the chromosome of a different Burkholderia species (strain RASC) isolated from a widely separated geographical area. This chromosomal gene has 77.2% sequence identity to tfdA from plasmid pJP4 (Y. Suwa, W. E. Holben, and L. J. Forney, abstr. Q-403, in Abstracts of the 94th General Meeting of the American Society for Microbiology 1994.). The tfdA homologs cloned from strains TFD6 and RASC are the first chromosomally encoded 2,4-D catabolic genes to be reported. The occurrence of highly similar tfdA genes in different bacterial species suggests that this chromosomal gene can be horizontally transferred.     

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.