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

Abstract

Uptake of transforming DNA by competent Bacillus subtilis cells in the presence of phage phi W-14 DNA (in which half the thymine residues are replaced by alpha-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 degrees C after DNA uptake at 30 degrees C in the presence of low concentrations of phi W-14 DNA (0.1 microgram/ml) demonstrated the presence of single-stranded transforming DNA molecules, typical for DNA taken up by B. subtilis. The intracellular effect of phi W-14 DNA was enhanced by an increase in its concentration (to 0.5-1 microgram/ml), or by increasing the temperature of uptake (to 37 degrees 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 microgram/ml) or untreated with phi W-14 DNA, and incubated for 20 min at 37 degrees 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 phi W-14 DNA. The influence of a high concentration of phi W-14 DNA on retention of transforming DNA label was correlated with its effect on transformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Three-dimensional Structure of MecI

“…The DNA was extracted as described (35) and used as a template for PCR amplification of the MecI coding sequence. Taq polymerase (Fermentas) was used with oligonucleotides 5Ј-GTTAAT-ACCATGGATAATAAAACGTATG-3Ј as forward primer and 5Ј-AAA-CACAACTAGTTTATTTTTTATTCAAT-3Ј as reverse primer (purchased from Roche Applied Science).…”