Increased killing of Bacillus subtilis on the hair roots of transgenic T4 lysozyme-producing potatoes.

Transgenic potato plants expressing the phage T4 lysozyme gene which are resistant to the plant-pathogenic enterobacterium Erwinia carotovora subsp. carotovora have been constructed. The agricultural growth of these potatoes might have harmful effects on soil microbiota as a result of T4 lysozyme release into the rhizosphere. To assess the bactericidal effect of roots, we have developed a novel method to associate the cells of Bacillus subtilis with hair roots of plants and to quantify the survival of cells directly on the root surface by appropriate staining and fluorescence microscopy. With this technique, we found that the roots of potato plants (Désirée and transgenic control lines) without T4 lysozyme gene display measurable killing activity on root-adsorbed B. subtilis cells. Killing was largely independent of the plant age and growth of plants in greenhouse or field plots. Roots from potato lines expressing the T4 lysozyme gene always showed significantly (1.5- to 3.5-fold) higher killing. It is concluded that T4 lysozyme is released from the root epidermis cells and is active in the fluid film on the root surface. We discuss why strong negative effects of T4 lysozyme-producing potatoes on soil bacteria in field trials may not be observed. We propose that the novel method presented here to study interactions of bacteria with roots can be applied not only to bacterial killing but also to interactions leading to growth-sustaining effects of plants on bacteria.

A conditional suicide system in Escherichia coli based on the intracellular degradation of DNA.

The potential risks associated with the intentional or unintentional release of genetically engineered microorganisms led to the construction of biological containment systems by which bacteria are killed in a controlled suicide process. In previously published suicide systems, cell killing was caused by proteins destroying the cell membrane or cell wall. Here a conditional cell killing system based on the intracellular degradation of cellular DNA is presented. The nuclease gene used was that of the extracellular nuclease of Serratia marcescens. The nuclease gene was deleted for the leader-coding sequence, and the truncated gene was put under the control of the lambda pL promoter. Following thermoinduction of the nuclease gene cassette in Escherichia coli, cell survival dropped to 2 x 10(-5), and more than 80% of the radioactively labeled DNA was converted to acid-soluble material within 2.5 h in the absence of cell lysis. The majority (84%) of clones which survived thermoinduced killing turned out to be as sensitive to a second thermoinduction as the original strain. The other clones showed somewhat slower killing kinetics or slightly higher final levels of survivors. The suicide system described combines the regulated killing of cells with the destruction of intracellular DNA otherwise potentially available for horizontal gene transfer processes.

The extracellular nuclease of Serratia marcescens: studies on the activity in vitro and effect on transforming DNA in a groundwater aquifer microcosm.

A quantitative endonuclease assay, which relies on the introduction of single and double strand breaks into supercoiled plasmid DNA, was used to study the activity of the extracellular nuclease of Serratia marcescens SM6 in buffer and in groundwater. The parallel enzyme concentration-dependent production of relaxed and linear plasmid molecules suggests that the nuclease produces single and double strand breaks in duplex DNA. Bovine serum albumin stimulated the nuclease activity towards DNA and RNA and increased the stability of the enzyme against thermal inactivation. The DNase activity at 4 degrees C and 50 degrees C was almost half of that at the optimum temperature (37 degrees C). The nuclease was active in groundwater, although the specific activity was lower than in buffer. In a groundwater aquifer microcosm, mineral-adsorbed transforming DNA was substantially less accessible to the nuclease than was dissolved DNA. The data suggest that the extracellular nuclease of Serratia marcescens may contribute to DNA turnover in the environment and that adsorption of DNA to minerals provides protection against the nuclease.