Intracellular effects of phage ϕW-14 DNA on transformation of Bacillus subtilis

Summary

Uptake of transforming DNA by competent Bacillus subtilis cells in the presence of phage ϕW-14 DNA (in which half the thymine residues are replaced by α-putrescinyl-thymine) is accompanied by a decrease in the amount of trichloracetic acid-precipitable label of the former retained by recipient cells during subsequent incubation. Fractionation of lysates of cells incubated for 0.5 min at 37°C after DNA uptake at 30°C in the presence of low concentrations of ϕW-14 DNA (0.1 μg/ml) demonstrated the presence of single-stranded transforming DNA molecules, typical for DNA taken up by B. subtilis. The intracellular effect of ϕW-14 DNA was enhanced by an increase in its concentration (to 0.5–1 μg/ml), or by increasing the temperature of uptake (to 37°C). With either of these treatments transforming DNA taken up was found in the form of a broad asymmetric band, indicative of degradation, and partially located at the density characteristic for single-stranded molecules. Fractionation of lysates of cells treated (0.1 μg/ml) or untreated with ϕW-14 DNA, and incubated for 20 min at 37°C after DNA uptake, showed disappearance of the single-stranded band. Donor DNA label was then found exclusively in the recipient DNA band, its amount being lower in samples treated with ϕW-14 DNA. The influence of a high concentration of ϕW-14 DNA on retention of transforming DNA label was correlated with its effect on transformation. On exposure to low concentrations of phage DNA, such a correlation was observed only after longer periods of incubation, due to slower intracellular degradation of homologous DNA taken up. The results are consistent with the proposal that ϕW-14 DNA-induced reduction in efficiency of transformation is due to intracellular stimulation of transforming DNA degradation, leading to a decrease in the number of donor molecules available for recombination with the recipient chromosome.