Measurement of the degradation capacity of activated sludge for a xenobiotic organic

Studies were conducted to evaluate the capacities of an activated sludge in degradation process for a xenobiotic. The results showed that during its acclimation to 2,4-D, the sludge sharply accumulated degradation capacities before degradation could proceed at a noticeable rate; moreover, during de-acclimation the sludge gradually lost some capacities in a manner resembling an exponential decay.     

Gene probe analysis of soil microbial populations selected by amendment with 2,4-dichlorophenoxyacetic acid.

Soils with a history of 2,4-dichlorophenoxyacetic acid (2,4-D) treatment at field application rates and control soils with no prior exposure to 2,4-D were amended with 2,4-D in the laboratory. Before and during these treatments, the populations of 2,4-D-degrading bacteria were monitored by most-probable-number (MPN) enumeration and hybridization analyses, using probes for the tfd genes of plasmid pJP4, which encode enzymes for 2,4-D degradation. Data obtained by these alternate methods were compared. Several months after the most recent field application of 2,4-D (approximately 1 ppm), soils with a 42-year history of 2,4-D treatment did not have significantly higher numbers of 2,4-D-degrading organisms than did control soils with no prior history of treatment. In response to laboratory amendments with 2,4-D, both the previously treated soils and those with no prior history of exposure exhibited a dramatic increase in the number of 2,4-D-metabolizing organisms. The MPN data indicate a 4- to 5-log population increase after one amendment with 250 ppm of 2,4-D and ultimately a 6- to 7-log increase after four additional amendments, each with 400 ppm of 2,4-D. Similarly, when total bacterial DNA from the soil microbial community of these samples was analyzed by using a probe for the tfdA gene (2,4-D monoxygenase) or the tfdB gene (2,4-dichlorophenol hydroxylase) a dramatic increase in the level of hybridization was observed in both soils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene probe analysis of soil microbial populations selected by amendment with 2,4-dichlorophenoxyacetic acid.

Soils with a history of 2,4-dichlorophenoxyacetic acid (2,4-D) treatment at field application rates and control soils with no prior exposure to 2,4-D were amended with 2,4-D in the laboratory. Before and during these treatments, the populations of 2,4-D-degrading bacteria were monitored by most-probable-number (MPN) enumeration and hybridization analyses, using probes for the tfd genes of plasmid pJP4, which encode enzymes for 2,4-D degradation. Data obtained by these alternate methods were compared. Several months after the most recent field application of 2,4-D (approximately 1 ppm), soils with a 42-year history of 2,4-D treatment did not have significantly higher numbers of 2,4-D-degrading organisms than did control soils with no prior history of treatment. In response to laboratory amendments with 2,4-D, both the previously treated soils and those with no prior history of exposure exhibited a dramatic increase in the number of 2,4-D-metabolizing organisms. The MPN data indicate a 4- to 5-log population increase after one amendment with 250 ppm of 2,4-D and ultimately a 6- to 7-log increase after four additional amendments, each with 400 ppm of 2,4-D. Similarly, when total bacterial DNA from the soil microbial community of these samples was analyzed by using a probe for the tfdA gene (2,4-D monoxygenase) or the tfdB gene (2,4-dichlorophenol hydroxylase) a dramatic increase in the level of hybridization was observed in both soils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of 2,4-dichlorophenoxyacetic acid degradation and plasmid transfer in soil resulting from bioaugmentation with two different pJP4 donors

A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory-generated strain amenable to donor counterselection, Escherichia coli D11. The R. eutropha strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the E. coli strain did not. The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors. To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of the Burkholderia and Ralstonia genera, suggesting multiple, if not successive, plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D. Upon reexposure, degradation was most rapid for the E. coli D11-inoculated treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts. In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful.     

Effect of Dissemination of 2,4-Dichlorophenoxyacetic Acid (2,4-D) Degradation Plasmids on 2,4-D Degradation and on Bacterial Community Structure in Two Different Soil Horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10⁵ CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10⁵ CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.     

Effect of dissemination of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids on 2,4-D degradation and on bacterial community structure in two different soil horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10(5) CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10(5) CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.     

Comparison of 2,4-Dichlorophenoxyacetic Acid Degradation and Plasmid Transfer in Soil Resulting from Bioaugmentation with Two Different pJP4 Donors

A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory-generated strain amenable to donor counterselection, Escherichia coli D11. The R. eutropha strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the E. coli strain did not. The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors. To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of the Burkholderia and Ralstonia genera, suggesting multiple, if not successive, plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D. Upon reexposure, degradation was most rapid for the E. coli D11-inoculated treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts. In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful.     

Analysis of the 2,4-dichlorophenoxyacetic acid-degradative plasmid pEST4011 of Achromobacter xylosoxidans subsp. denitrificans strain EST4002

The 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative bacterium Achromobacter xylosoxidans subsp. denitrificans strain EST4002, isolated in Estonia more than 10years ago, was found to contain the 70kb plasmid pEST4011 that is responsible for the bacterium having had obtained a stable 2,4-D(+) phenotype. The tfd-like genes for 2, 4-D degradation of the strain EST4002 were located on a 10.5kb region of pEST4011, but without functional genes coding for chloromuconate cycloisomerase and chlorodienelactone hydrolase. The latter two genes are probably encoded by homologous, tcb-like genes, located elsewhere on pEST4011. We also present evidence of two copies of insertion element IS1071-like sequences on pEST4011. IS1071 is a class II (Tn3 family) insertion element, associated with different catabolic genes and operons and globally distributed in the recent past. We speculate that this insertion element might have had a role in the formation of plasmid pEST4011. The 28kb plasmid pEST4012 is generated by deletion from pEST4011 when cells of A. xylosoxidans EST4002 are grown in the absence of 2,4-D in growth medium. We propose that this is the result of homologous recombination between the two putative copies of IS1071-like sequences on pEST4011.     

New plasmids of herbicide 2,4-dichlorophenoxyacetic acid biodegradation

Three herbicide 2,4-D metabolizing bacterial strains were isolated from three independent soil samples of Estonia. The strains, although belonging to various species, contain 2,4-D degradative plasmids with identical restriction patterns. pEST4001 is a 78 kb conjugative plasmid. All Pseudomonas putida PaW340 2,4-D+ transconjugants obtained a 70 kb plasmid pEST4011 - a deletion derivative of the pEST4001. The restriction patterns of the plasmids mentioned above are considerably different from those of the other 2,4-D plasmids pJP4 and pRC10 reported previously.     

Physiological and genetic characteristics of two bacterial strains utilizing phenoxypropionate and phenoxyacetate herbicides

Two strains, Rhodoferax sp. P230 and Delftia (Comamonas) acidovorans MCI, have previously been shown to carry activities for the degradation of the two enantiomers of (RS)-2-(2,4-dichlorophenoxy-)propionate (dichlorprop) and (RS)-2-(4-chloro-2-methylphenoxy-)propionate (mecoprop) and, in addition, are capable of degrading phenoxyacetate derivatives 2.4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA). Metabolism of the herbicides is initiated by alpha-ketoglutarate-dependent dioxygenases for both enantiomers of the phenoxypropionate herbicides and for 2,4-D. These activities were constitutively expressed for both enantiomers of dichlorprop in strain MC1 and for the Renantiomer in strain P230. Enzyme activities for the complete degradation of phenoxyacetate and phenoxypropionate herbicides were induced during incubation on either of these herbicides. Strain MC1 has about threefold higher activities for the degradation of dichlorprop and for growth on this substrate (mumax = 0.15 h(-1)) than strain P230; the maximum growth rate on 2,4-D amounts to 0.045 h(-1) with strain MC1. Dichlorprop is utilized faster than mecoprop and the R-enantiomers are cleaved with higher rates than the S-enantiomers. The degradation of the chlorophenolic intermediates seems to proceed via the modified ortho cleavage pathway as indicated by activities of the respective enzymes. The enzymatic results were supported by genetic investigations by which the presence of the genes tfdB (encoding a dichlorophenol hydroxylase), tfdC (encoding a chlorocatechol 1,2-dioxygenase) and tfdD (encoding a chloromuconate cycloisomerase) could be demonstrated in both strains by PCR after application of respective primers. The presence of the tfdA gene (encoding a 2,4-D/alpha-ketoglutarate dioxygenase) was only shown for strain P230 but was lacking in strain MC1. Sequence analysis of the tfd gene fragments revealed high homology to the degradative genes of other proteobacterial strains degrading chloroaromatic compounds. Strain MC1 carries a plasmid of about 120 kb which apparently harbors herbicide degradative genes as concluded from deletion mutants which have lost 2,4-D[phenoxalkanoate]/alpha-ketoglutarate dioxygenase activities for cleavage of the R- and S-enantiomer, and of 2,4-D. For strain P230, no plasmid could be demonstrated; the activity was stably conserved in this strain during growth under nonselective conditions.     

2,4-D impact on bacterial communities, and the activity and genetic potential of 2,4-D degrading communities in soil

The key role of telluric microorganisms in pesticide degradation is well recognized but the possible relationships between the biodiversity of soil microbial communities and their functions still remain poorly documented. If microorganisms influence the fate of pesticides, pesticide application may reciprocally affect soil microorganisms. The objective of our work was to estimate the impact of 2,4-D application on the genetic structure of bacterial communities and the 2,4-D-degrading genetic potential in relation to 2,4-D mineralization. Experiments combined isotope measurements with molecular analyses. The impact of 2,4-D on soil bacterial populations was followed with ribosomal intergenic spacer analysis. The 2,4-D degrading genetic potential was estimated by real-time PCR targeted on tfdA sequences coding an enzyme specifically involved in 2,4-D mineralization. The genetic structure of bacterial communities was significantly modified in response to 2,4-D application, but only during the intense phase of 2,4-D biodegradation. This effect disappeared 7 days after the treatment. The 2,4-D degrading genetic potential increased rapidly following 2,4-D application. There was a concomitant increase between the tfdA copy number and the 14C microbial biomass. The maximum of tfdA sequences corresponded to the maximum rate of 2,4-D mineralization. In this soil, 2,4-D degrading microbial communities seem preferentially to use the tfd pathway to degrade 2,4-D.     

The Pattern of Changes in Photosynthetic Apparatus in Response to Cold Acclimation and De-acclimation in Two Contrasting Cultivars of Oilseed Rape

In spring and winter cultivars of oilseed rape (Brassica napus var. oleifera), acclimation of photosynthetic apparatus to cold was connected with the increase in activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) and sucrose-phosphate synthase (SPS). Conversely, cold de-acclimation entailed the decline of RuBPCO and SPS activities. The rate of this photosynthetic de-acclimation might depend on day temperature. On the other hand, temperature rise during de-acclimation (identical during the day and night) resulted in the improvement of photosynthetic activity measured by means of chlorophyll fluorescence. An increase in SPS activity (and even transitory increase in RuBPCO activity) was observed when the elongation growth rate (EGR) accelerated during de-acclimation. Throughout re-acclimation, plants with high EGR were unable to maintain or recover higher photosynthetic capacity, despite the fact that SPS activity remained high or even increased during re-acclimation.     

Impacts of gene bioaugmentation with pJP4-harboring bacteria of 2,4-D-contaminated soil slurry on the indigenous microbial community

Gene bioaugmentation is a bioremediation strategy that enhances biodegradative potential via dissemination of degradative genes from introduced microorganisms to indigenous microorganisms. Bioremediation experiments using 2,4-dichlorophenoxyacetic acid (2,4-D)-contaminated soil slurry and strains of Pseudomonas putida or Escherichia coli harboring a self-transmissible 2,4-D degradative plasmid pJP4 were conducted in microcosms to assess possible effects of gene bioaugmentation on the overall microbial community structure and ecological functions (carbon source utilization and nitrogen transformation potentials). Although exogenous bacteria decreased rapidly, 2,4-D degradation was stimulated in bioaugmented microcosms, possibly because of the occurrence of transconjugants by the transfer of pJP4. Terminal restriction fragment length polymorphism analysis revealed that, although the bacterial community structure was disturbed immediately after introducing exogenous bacteria to the inoculated microcosms, it gradually approached that of the uninoculated microcosms. Biolog assay, nitrate reduction assay, and monitoring of the amoA gene of ammonia-oxidizing bacteria and nirK and nirS genes of denitrifying bacteria showed no irretrievable depressive effects of gene bioaugmentation on the carbon source utilization and nitrogen transformation potentials. These results may suggest that gene bioaugmentation with P. putida and E. coli strains harboring pJP4 is effective for the degradation of 2,4-D in soil without large impacts on the indigenous microbial community.     

Enhancement of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation in soil by dissemination of catabolic plasmids

Few studies have been done to evaluate the transfer of catabolic plasmids from an introduced donor strain to indigenous microbial populations as a means to remediate contaminated soils. In this work we determined the effect of the conjugative transfer of two 2,4-D degradative plasmids to indigenous soil bacterial populations on the rate of 2,4-D degradation in soil. We also assessed the influence of the presence of 2,4-D on the number of transconjugants formed. The two plasmids used, pEMT1k and pEMT3k, encode 2,4-D degradative genes (tfd) that differ in DNA sequence as well as gene organisation, and confer different growth rates to Ralstonia eutropha JMP228 when grown with 2,4-D as a sole carbon source. In an agricultural soil (Ardoyen) treated with 2,4-D (100 ppm) there were ca. 10⁷CFU of transconjugants per gram bearing pEMT1k as well as a high number of pEMT3k bearing transconjugants (ca. 10⁶ CFU/g). In this soil the formation of a high number of 2,4-D degrading transconjugants resulted in faster degradation of 2,4-D as compared to the uninoculated control soil. In contrast, only transconjugants with pEMT1k were detected (at a level of ca. 10³ CFU/g soil) in the untreated Ardoyen soil. High numbers of transconjugants that carried pEMT1k were also found in a second experiment done using forest soil (Lembeke) treated with 100 ppm 2,4-D. However, unlike in the Ardoyen soil, no transconjugants with pEMT3k were detected and the transfer of plasmid pEMT1k to indigenous bacteria did not result in a higher rate of decrease of 2,4-D. This may be because 2,4-D was readily metabolised by indigenous bacteria in this soil. The results indicate that bioaugmentation with catabolic plasmids may be a viable means to enhance the bioremediation of soils which lack an adequate intrinsic ability to degrade a given xenobiotic.     

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     

Stable transformation of tomato cell cultures after bombardment with plasmid and YAC DNA

Summary Stable transformants were obtained after microprojectile particle bombardment of tomato cell suspensions (Lycopersicon esculentum cv VFNT Cherry and L. pennellii). The suspensions were bombarded with tungsten particles coated with either plasmid (6.3 kb) or yeast artificial chromosome (YAC) (80 kb) DNA containing the ß-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. The YAC DNA contained an insert of approximately 50 kb of DNA from VFNT Cherry. L. pennellii suspensions were more amenable to transformation than VFNT Cherry; more kanamycin-resistant calli were recovered from L. pennelli after bombardment with plasmid DNA, and only L. pennellii cells produced transformants after bombardment with YAC DNA. DNA gel blot analysis confirmed the presence of the nptll and GUS genes. This analysis also confirmed the integration of YAC DNA into the genome of the kanamycin-resistant calli and suggested that the level of intactness of the integrated YAC DNA was fairly high in four of the five transformants examined. Microprojectile bombardment of regenerable cultures with YACs may ultimately aid in map-based cloning of agriculturally-important genes.Abbreviations YAC yeast artificial chromosome - MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indole-3-acetic acid - GUS ß-glucuronidase - nptII neomycin phosphotransferase II     

The influence of moisture on microbial transport, survival and 2,4-D biodegradation with a genetically marked Burkholderia cepacia in unsaturated soil columns

The influence of moisture on the survival, movement anddegradation activity of a 2,4-D degrading bacterium,Burkholderia cepacia strain BRI6001L, geneticallyengineered to contain bioluminescent and lactoseutilization genes, was studied in unsaturated soil columns.The distance traveled by BRI6001L was dependent on theclay content of the soil, higher clay contents beingresponsible for higher filtration coefficients. Long termsurvival, in excess of one year, was attributed to strainBRI6001L's ability to survive dry conditions. Changes inthe 2,4-D biodegradation rate showed a better correlationwith the BRI6001L population density than with the totalviable bacterial population. At moisture levels betweenfield capacity and 40% moisture (– 33 kPa to –100 kPa)2,4-D degradation was attributed mainly to BRI6001L. Atmoisture levels between 6 and 15%, 2,4-D disappearancewas attributed to the indigenous microbial population,with no degradation occurring at moisture levels below6%. Returning the moisture to above 40% led to anincrease of 4 orders of magnitude in the BRI6001Lpopulation density and to a 10-fold increase in the 2,4-Ddegradation rate. The ability to monitor a specificmicrobial population using reporter genes hasdemonstrated the importance of controlling moisturelevels for maximizing biodegradation rates in unsaturatedsoil environments.     

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.     

Chemotaxis of Ralstonia eutropha JMP134(pJP4) to the herbicide 2,4-dichlorophenoxyacetate

Ralstonia eutropha JMP134(pJP4) and several other species of motile bacteria can degrade the herbicide 2,4-dichlorophenoxyacetate (2,4-D), but it was not known if bacteria could sense and swim towards 2,4-D by the process of chemotaxis. Wild-type R. eutropha cells were chemotactically attracted to 2,4-D in swarm plate assays and qualitative capillary assays. The chemotactic response was induced by growth with 2,4-D and depended on the presence of the catabolic plasmid pJP4, which harbors the tfd genes for 2,4-D degradation. The tfd cluster also encodes a permease for 2,4-D named TfdK. A tfdK mutant was not chemotactic to 2,4-D, even though it grew at wild-type rates on 2,4-D.