Microbial interactions affecting the natural transformation of Bacillus subtilis in a model aquatic ecosystem.

The involvement of microbial interactions in natural transformation of bacteria was evaluated using an aquatic model system. For this purpose, the naturally transformable Bacillus subtilis was used as the model bacterium which was co-cultivated with the protist Tetrahymena thermophila (a consumer) and/or the hotosynthetic alga Euglena gracilis (a producer). Co-cultivation with as few as 10E+2 individuals ml-1 of T. thermophila lowered the number of transformants to less than the detectable level (<1x100ml-1), while co-cultivaton with E. gracilis did not.Metabolites from co-cultures of T. thermophila and B. subtilis also decreased the number of transformants to less than the detectable level, while metabolites from co-culture of T. thermophila and B. subtilis with E. gracilis did not. Thus, the introduction of transformation inhibitory factor(s) by the grazing of T. thermophila and the attenuation of this inhibitory factor(s) by E. gracilis is indicated. These observations suggest that biological components do affect the natural transformation of B. subtilis . The study described is the first to suggest that ecological interactions are responsible not only for the carbon and energy cycles, but also for the processes governing horizontal transfer of genes, in microbial ecosystems

Survival of genetically modified Escherichia coli carrying extraneous antibiotic resistance gene through microbial interactions.

遺伝子組み換え細菌の野外における生残性について研究を行った。遺伝子組み換え細菌の宿主は、栄養塩の競合あるいは培溶液中に老廃物を蓄積することにより遺伝子組み換え細菌を駆逐する効果を持つが、他の微生物、例えば植物プランクトンや動物プランクトンが存在することにより、宿主細菌が生存している環境において生残出来ることが分かった

Release of Extracellular Transformable Plasmid DNA from Escherichia coli Cocultivated with Algae

We studied the effects of cocultivation with either Euglena Gracilis (Euglenophyta), Microcystis aeruginosa (Cyanophyta), Chlamydonomas neglecta (Chlorophyta), or Carteria inversa (Chlorophyta) on the production of extracellular plasmid DNA by Excherichia coli LE392(pKZ105), Dot blot hybridization analysis showed a significant release of plasmid DNA by cocultivation with all the algae tested. Further analysis by electrotransformation confirmed the release of transformable plasmid DNA by cocultivation with either E.Gracilis, M. Aeruginosa, or C. inversa. These results suggest algal involvement in bacterial horizontal gene tranfer by stimulating the release of transformable DNA into aquatic environments

Use of the SYBR Green I fluorescent dye and a centrifugal filter device for rapid determination of dissolved DNA concentration in fresh water

A rapid fluorometric assay using the fluorescent dye SYBR Green I was established todetermine the concentration of dissolved nucleic acids in fresh water. The ensitivity of SYBR Green I to double stranded DNA (dsDNA) (lambda Hind lll digest) was as low as 50 pg, 200 times more sensitive than Hoechst 33258. SYBR Green I bound to dsDNA emitted 10 times stronger fluorescence than when bound to single stranded DNA or RNA,indicating its selectivity for dsDNA measurement. The dissolved DNA (dDNA) concentration in fresh water determined using SYBR Green I was almost the same as that obtained using Hoechst 33258. This suggests that the dDNA measured by SYBR GreenI is comparable to that determined by Hoechst 33258 in previous studies. To reduce preparation time,the dDNA in lake water filtrate was precipitated by ethanol and purified using a centrifugal filter device. The overall preparation process takes only a few hours and requires only 10 ml of water. The process described here may, therefore, facilitate \u27same day\u27measurement of dDNA dynamics in freshwater environments

Release of transforming plasmid DNA from actively growing genetically engineered Escherichia coli.

We studied the transforming ability of the extracellular plasmid DNA released from a genetically engineered Escherichia coli pEGFP and the culturing conditions for the release of transforming DNA. The transforming ability was evaluated by transformation of competent cells with filtrates of E. coli pEGFP cultures. The number of transformants increased with time when E. coli pEGFP cells grew exponentially in rich medium, but not in stationary phase or when inoculated in freshwater. These results suggested that crude extracellular plasmid DNA had transforming ability and this transforming DNA was mainly released by activelygrowing bacteria