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-dichlorophenoxyacetic acid (2,4-D). When JMP134 was inoculated into a nonsterile soil microcosm amended with 1,000 g 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.

Summary 1.A ®eld study using transgenic plants with associated recombinant micro-organisms was conducted to assess the potential eects of genetically engineered organisms on soil ecosystems. Three genotypes of alfalfa plants (parental, transgenic aamylase-producing and transgenic lignin peroxidase-producing) were planted in an agricultural ®eld plot. Immediately prior to planting, the roots of the alfalfa plants were left uninoculated or were inoculated with a wild-type strain (PC), a recombinant strain with antibiotic resistances (RMB7201), or a recombinant strain with antibiotic resistances and enhanced nitrogen-®xation capability (RMBPC-2), of Sinorhizobium meliloti. 2. Analyses of the alfalfa plants and ®eld plot soil were made over two growing seasons and included: metabolic ®ngerprints and DNA ®ngerprints of soil bacterial communities; soil microbial respiration; population counts of indigenous soil bacteria, fungi, nematodes, protozoa and micro-arthropods; identi®cation of nematodes and micro-arthropods; plant shoot weight and chemistries; and soil chemistries and enzyme activities. 3. The lignin peroxidase transgenic plants had signi®cantly lower shoot weight, and higher nitrogen and phosphorus content, than the parental or transgenic amylase plants. Distinct metabolic ®ngerprints, based on patterns of substrate utilization in Biolog plates, were exhibited by the soil bacterial communities associated with the three alfalfa genotypes, and those for the lignin peroxidase plants were the most unique. Signi®cantly higher population levels of culturable, aerobic sporeforming and cellulose-utilizing bacteria, lower activity of the soil enzymes dehydrogenase and alkaline phosphatase, and higher soil pH levels, were also associated with the lignin peroxidase transgenic plants. Signi®cantly higher population levels of culturable, aerobic spore-forming bacteria were also measured in the treatments containing the recombinant RMBPC-2 S. meliloti. 4. Population levels of protozoa, nematodes and micro-arthropods, DNA ®nger-prints of indigenous soil bacteria, and rates of microbial substrate-induced respiration were not signi®cantly aected by the transgenic alfalfa and recombinant S. meliloti treatments. 5. These results suggest that the genetically engineered organisms caused detectable

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