Adaptation of soil microbial communities following exposure to 2,4-dichlorophenoxyacetic acid and introduction of tfd genes.

Abstract

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), has been used as a model compound for biodegradation studies. This compound is rapidly biodegraded in the environment by a diversity of microorganisms, many of which contain plasmids encoding its degradation. One such bacterium, Alcaligenes eutrophus JMP134, contains the well characterized 2,4-D plasmid pJP4. In this study, plasmid diversity in a population of 2,4-D-degrading soil bacteria isolated from a contaminated site was evaluated. In addition, the transfer of the well characterized 2,4-D plasmid pJP4 from A. eutrophus JMP134 to several indigenous soil microorganisms was studied as a model system for evaluating the potential of gene transfer as a remediative strategy. A population of 2,4-D-degrading (Tfd⁺) Variovorax paradoxus were isolated from a single sample of contaminated soil by enrichment culture. The isolates were found to contain plasmids of diverse number and size. Plasmid profile and restriction endonuclease analyses of parental, Tfd⁺ and Tfd ⁻ clones suggested that plasmids of varying size were linked to the ability to degrade 2,4-D and that variations in plasmids may have arisen from gene transfer or recombination. Transfer of plasmid pJP4 from A. eutrophus JMP134 to several indigenous soil microorganisms capable of expressing the tfd genes was demonstrated. Transfer of pJP4 to indigenous recipients was detected in soil inoculated with JMP134 and enriched with 1000 μg 2,4-D per gram of dry soil. The donor rapidly died off after introduction and the 2,4-D was completely degraded by transconjugants. Transconjugants were identified as Pseudomonas glathei GDD1, Burkholderia caryophyllii GDD2 and Burkholderia cepacia GDD3. These results suggest that catabolic gene transfer from an introduced donor to indigenous organisms may be a viable ecorestoration strategy.