Fate of transforming DNA following uptake by competent Bacillus subtilis: III. Formation and properties of products isolated from transformed cells which are derived entirely from donor DNA

Abstract

High molecular weight DNA labeled with [ 3H]thymine and in some cases deuterium oxide as well, was used to transform competent Bacillus subtilis cultures. Lysates were prepared from these transformed cultures and the properties of products derived entirely from the donor DNA were investigated. Three kinds of radioactive products were identified: 5′-TMP and metabolic derivatives, SSF ‡ ‡ Abbreviations used: SSF, DNA species consisting of single-stranded fragments; DSF, DNA species consisting of double-stranded fragments. and DSF. SSF consisted of single-stranded fragments with an approximate molecular weight of 1 to 2 × 10 5 daltons. DSF consisted of duplex fragments with an initial molecular weight of 9 × 10 6 daltons. The 5′-TMP was released quantitatively into the medium and could be detected starting about one minute after the addition of transforming DNA to the cells. It is tentatively concluded that the release of 5′-TMP is due to an exonuclease which functions outside the cell permeability barrier. Our evidence suggests that DSF is produced by endonucleolytic cleavage across both strands of the donor molecule. The endonuclease also presumably functions outside the permeability barrier since DSF is susceptible to extracellular nuclease. SSF does not appear to be an artifactual degradation product of higher molecular weight single-stranded DNA which has been generated during isolation. SSF has no transforming activity for a donor marker, and the activity of DSF is anomalously low compared to DNA with the same single strand molecular weight generated by the action of pancreatic deoxyribonuclease. Conversion of donor DNA to acid-soluble products and to SSF and DSF therefore explains the eclipse phenomenon in B. subtilis.