PLASMID TRANSFER BETWEEN SPATIALLY SEPARATED DONOR AND RECIPIENT BACTERIA IN EARTHWORM-CONTAINING SOIL MICROCOSMS

Most gene transfer studies have been performed,vith relatively homogen eous soil systems in the absence of soil macrobiota, including inverte brates. In this study we examined the influence of earthworm activity (burrowing, casting, and feeding) on transfer of plasmid pJP4 between spatially separated donor (Alcaligenes eutrophus) and recipient (Pseud omonas fluorescens) bacteria in nonsterile soil columns. A model syste m was designed such that the activity of earthworms would act to media te cell contact and gene transfer. Three different earthworm species ( Aporrectodea trapezoides, Lumbricus rubellus, and Lumbricus terrestis) , representing each of the major ecological categories (endogeic, epig eic, and anecic), were evaluated. Inoculated soil microcosms, with and without added earthworms, were analyzed for donor, recipient, and tra nsconjugant bacteria at 5-cm-depth intervals by using selective platin g techniques. Transconjugants were confirmed by colony hybridization w ith a mel gene probe. The presence of earthworms significantly increas ed dispersal of the donor and recipient strains. In situ gene transfer of plasmid pJP4 from A. eutrophus to P. fluorescens was detected only in earthworm-containing microcosms, at a frequency of similar to 10(2 ) transconjugants per g of soil. The depth of recovery was dependent o n the burrowing behavior of each earthworm species; however, there was no significant difference in the total number of transconjugants amon g the earthworm species. Donor and recipient bacteria were recovered f rom earthworm feces (casts) of all three earthworm species, with numbe rs up to 10(6) and 10(4) bacteria per g of cast, respectively. A. trap ezoides egg capsules (cocoons) formed in the inoculated soil microcosm s contained up to 10(7) donor and 10(6) recipient bacteria per g of co coon. No transconjugant bacteria, however, were recovered from these m icrohabitats. To our knowledge, this is the first report of gene trans fer between physically isolated bacteria in nonsterile soil, using bur rowing earthworms as a biological factor to facilitate cell-to-cell co ntact.