Detection and characterization of plasmid pJP4 transfer to indigenous soil bacteria

Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism, Escherichia coli ATCC 15224, by plate mating with Ralstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2, 4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 10(7) and 10(8) transconjugants g of dry soil(-1) for samples supplemented with 500 and 1,000 microg of 2,4-D g of dry soil(-1), respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genus Burkholderia or the genus Pseudomonas. No mercury-resistant, 2, 4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.     

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.     

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.     

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.     

Frequency of horizontal gene transfer of a large catabolic plasmid (pJP4) in soil.

Limited work has been done to assess the bioremediation potential of transfer of plasmid-borne degradative genes from introduced to indigenous organisms in the environment. Here we demonstrate the transfer by conjugation of the catabolic plasmid pJP4, using a model system with donor and recipient organisms. The donor organism was Alcaligenes eutrophus JMP134 and the recipient organism was Variovorax paradoxus isolated from a toxic waste site. Plasmid pJP4 contains genes for mercury resistance and 2,4-dichlorophenoxyacetic (2,4-D) acid degradation. A transfer frequency of approximately 1/10(3) donor and recipient cells (parent cells) was observed on solid agar media, decreasing to 1/10(5) parent cells in sterile soil and finally 1/10(6) parent cells in 2,4-D-amended, nonsterile soil. Presumptive transconjugants were confirmed to be resistant to Hg, to be capable of degrading 2,4-D, and to contain a plasmid of size comparable to that of pJP4. In addition, we confirmed the transfer through PCR amplifications of the tfdB gene. Although transfer of pJP4 did occur at a high frequency in pure culture, the rate was significantly decreased by the introduction of abiotic (sterile soil) and biotic (nonsterile soil) stresses. An evaluation of the data from this model system implies that the reliance on plasmid transfer from a donor organism as a remediative strategy has limited potential.     

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.     

Plasmid-mediated bioaugmentation of sequencing batch reactors for enhancement of 2,4-dichlorophenoxyacetic acid removal in wastewater using plasmid pJP4

Plasmid-mediated bioaugmentation was demonstrated using sequencing batch reactors (SBRs) for enhancing 2,4-dichlorophenoxyacetic acid (2,4-D) removal by introducing Cupriavidus necator JMP134 and Escherichia coli HB101 harboring 2,4-D-degrading plasmid pJP4. C. necator JMP134(pJP4) can mineralize and grow on 2,4-D, while E. coli HB101(pJP4) cannot assimilate 2,4-D because it lacks the chromosomal genes to degrade the intermediates. The SBR with C. necator JMP134(pJP4) showed 100 % removal against 200 mg/l of 2,4-D just after its introduction, after which 2,4-D removal dropped to 0 % on day 7 with the decline in viability of the introduced strain. The SBR with E. coli HB101(pJP4) showed low 2,4-D removal, i.e., below 10 %, until day 7. Transconjugant strains of Pseudomonas and Achromobacter isolated on day 7 could not grow on 2,4-D. Both SBRs started removing 2,4-D at 100 % after day 16 with the appearance of 2,4-D-degrading transconjugants belonging to Achromobacter, Burkholderia, Cupriavidus, and Pandoraea. After the influent 2,4-D concentration was increased to 500 mg/l on day 65, the SBR with E. coli HB101(pJP4) maintained stable 2,4-D removal of more than 95 %. Although the SBR with C. necator JMP134(pJP4) showed a temporal depression of 2,4-D removal of 65 % on day 76, almost 100 % removal was achieved thereafter. During this period, transconjugants isolated from both SBRs were mainly Achromobacter with high 2,4-D-degrading capability. In conclusion, plasmid-mediated bioaugmentation can enhance the degradation capability of activated sludge regardless of the survival of introduced strains and their 2,4-D degradation capacity.     

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 (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.     

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)     

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.     

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.     

Plasmids pJP4 and r68.45 Can Be Transferred between Populations of Bradyrhizobia in Nonsterile Soil

IncP plasmid r68.45, which carries several antibiotic resistance genes, and IncP plasmid pJP4, which contains genes for mercury resistance and 2,4-dichlorophenoxyacetic acid degradation, were evaluated for their ability to transfer to soil populations of rhizobia. Transfer of r68.45 was detected in nonsterile soil by using Bradyrhizobium japonicum USDA 123 as the plasmid donor and several Bradyrhizobium sp. strains as recipients. Plasmid transfer frequencies ranged up to 9.1 × 10^-5 in soil amended with 0.1% soybean meal and were highest after 7 days with strain 3G4b4-RS as the recipient. Transconjugants were detected in 7 of 500 soybean nodules tested, but the absence of both parental strains in these nodules suggests that plasmid transfer had occurred in the soil, in the rhizosphere, or on the root surface. Transfer of degradative plasmid pJP4 was also evaluated in nonsterile soil by using B. japonicum USDA 438 as the plasmid donor and several Bradyrhizobium sp. strains as recipients. Plasmid pJP4 was transferred only when strains USDA 110-ARS and 3G4b4-RS were the recipients. The plasmid transfer frequency was highest for strain 3G4b4-RS (up to 7.4 × 10^-6). Mercury additions to soil, ranging from 10 to 50 μg/g of soil, did not affect population levels of parental strains or the plasmid transfer frequency.     

Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage φR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10³ transconjugants per g of soil when 10⁷ donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx^r Rp^rP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

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.     

Transfer of the Pea Symbiotic Plasmid pJB5JI in Nonsterile Soil

Transfer of the pea (Pisum sativum L.) symbiotic plasmid pJB5JI between strains of rhizobia was examined in sterile and nonsterile silt loam soil. Sinorhizobium fredii USDA 201 and HH003 were used as plasmid donors, and symbiotic plasmid-cured Rhizobium leguminosarum 6015 was used as the recipient. The plasmid was carried but not expressed in S. fredii strains, whereas transfer of the plasmid to R. leguminosarum 6015 rendered the recipient capable of nodulating pea plants. Confirmation of plasmid transfer was obtained by acquisition of plasmid-encoded antibiotic resistance genes, nodulation of pea plants, and plasmid profiles. Plasmid transfer in nonsterile soil occurred at frequencies of up to 10⁻⁴ per recipient and appeared to be highest at soil temperatures and soil moisture levels optimal for rhizobial growth. Conjugation frequencies were usually higher in sterile soil than in nonsterile soil. In nonsterile soil, transconjugants were recovered only with strain USDA 201 as the plasmid donor. Increasing the inoculum levels of donor and recipient strains up to 10⁹ cells g of soil⁻¹ increased the number of transconjugants; peak plasmid transfer frequencies, however, were found at the lower inoculum level of 10⁷ cells g of soil⁻¹. Plasmid transfer frequencies were raised in the presence of the pea rhizosphere or by additions of plant material. Transconjugants formed by the USDA 201(pJB5JI) × 6015 mating in soil formed effective nodules on peas.     

Integration and excision of a 2,4-dichlorophenoxyacetic acid-degradative plasmid in Alcaligenes paradoxus and evidence of its natural intergeneric transfer.

A self-transmissible 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative plasmid, pKA2, has been identified in a new 2,4-D-degrading strain, Alcaligenes paradoxus 2811P, isolated from agricultural soil. pKA2 occurred as a 42.9-kb plasmid in strain 2811P. A derivative strain, 2811C, was isolated from a stock culture in which the entire pKA2 plasmid was apparently integrated into the host chromosome without loss of the 2,4-D+ phenotype. This interpretation is based on the disappearance of a free plasmid DNA band, a shift in the tfdA-hybridizing band to the chromosome, loss of transmissibility of the 2,4-D+ trait, and appropriate shifts in Southern hybridization bands of plasmid DNA compared with whole-cell DNA. The integrated plasmid of strain 2811C was excised either precisely or imprecisely after continued transfer on 2,4-D-containing medium. This suggests that a chromosome-free plasmid cycle may occur to optimize fitness under conditions of specific resource fluctuation. Another new 2,4-D-degrading strain, Pseudomonas pickettii 712, which was isolated from the same field plot but at a different time, was found to carry a plasmid that is nearly identical to pKA2. The plasmid of this strain, pKA4, is 40.9 kb long and has features in common with pKA2, such as high self-transmissibility, hybridization only to the tfdA gene among the 2,4-D-metabolic genes of 2,4-D-degradative plasmid pJP4, and similar restriction endonuclease-generated fragments. Furthermore, the genetic homology between the two plasmids was high since all fragments of pKA2 hybridized to pKA4. These results suggest that these two plasmids are closely related and thus their occurrence in two genera in nature is the result of natural horizontal gene transfer.     

Earthworm Egg Capsules as Vectors for the Environmental Introduction of Biodegradative Bacteria

Earthworm egg capsules (cocoons) may acquire bacteria from the environment in which they are produced. We found that Ralstonia eutropha (pJP4) can be recovered from Eisenia fetida cocoons formed in soil inoculated with this bacterium. Plasmid pJP4 contains the genes necessary for 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (2,4-DCP) degradation. In this study we determined that the presence of R. eutropha (pJP4) within the developing earthworm cocoon can influence the degradation and toxicity of 2,4-D and 2,4-DCP, respectively. The addition of cocoons containing R. eutropha (pJP4) at either low or high densities (10² or 10⁵ CFU per cocoon, respectively) initiated degradation of 2,4-D in nonsterile soil microcosms. Loss of 2,4-D was observed within the first week of incubation, and respiking the soil with 2,4-D showed depletion within 24 h. Microbial analysis of the soil revealed the presence of approximately 10⁴ CFU R. eutropha (pJP4) g⁻¹ of soil. The toxicity of 2,4-DCP to developing earthworms was tested by using cocoons with or without R. eutropha (pJP4). Results showed that cocoons containing R. eutropha (pJP4) were able to tolerate higher levels of 2,4-DCP. Our results indicate that the biodegradation of 2,4-DCP by R. eutropha (pJP4) within the cocoons may be the mechanism contributing to toxicity reduction. These results suggest that the microbiota may influence the survival of developing earthworms exposed to toxic chemicals. In addition, cocoons can be used as inoculants for the introduction into the environment of beneficial bacteria, such as strains with biodegradative capabilities.     

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)     

Genetic and molecular analysis of a regulatory region of the herbicide 2,4-dichlorophenoxyacetate catabolic plasmid pJP4

In Alcaligenes eutrophus JMP134, pJP4 carries the genes coding for 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-Cba) degradation plus mercury resistance. The plasmid genes specifying 2,4-D and 3-Cba catabolism are organized in three operons: tfdA, tfdB, and tfdCDEF. Regulation of these operons by two unlinked genes, tfdR and tfdS, has been proposed. Physical and DNA sequence analyses revealed that the tfdR and tfdS genes were identical and were located within a longer inverted repeat of 1592bp. Similar stem-loop structures were observed among other 2,4-D plasmids. The tfdR gene is 888 bp long and capable of encoding a polypeptide of 32kDa. The deduced amino acid sequence of tfdR indicates that it is a member of the LysR-type activators. Investigation of the regulation of the catabolic gene clusters through the construction of a pJP4 defined deletion mutant, pYG1010, which lacks a 4.2 kilobase Xbal fragment containing the inverted repeat region carrying the tfdR and tfdS regulatory genes, showed that Pseudomonas cepacia strains containing pYG1010 became 2,4-D negative, but 3-Cba positive. In vivo recombinants of pYG1010 and a cloned tfdS gene rescued the 2,4-D phenotype, indicating that TfdS is a positive regulator of tfdA expression, but not for tfdCDEF expression.