Transformation in Bacillus subtilis: biological and physical evidence for a novel DNA-intermediate in synchronously...

Competent B. subtilis cells exposed to transforming DNA in the presence of EDTA bind, but do not take up DNA. Rapid and almost synchronous uptake of the bound DNA is achieved by the addition of Mg2+ ions in excess of the EDTA. At 30 degrees and at 17 degrees comparable numbers of transformants are produced from cells pre-loaded with DNA at 30 degrees (after termination of uptake by the addition of DNA ase the samples were incubated at 37 degrees). However, almost no transformants are produced when cells are exposed to DNA at 17 degrees, although binding does take place then. Because DNA is taken up at 17 degrees after having loaded the cells at 30 degrees, whereas no uptake occurs after binding at 17 degrees, it is suggested that binding of DNA to the cellular surface involves at least two steps. In DNA re-extracted from cells at 17 degrees, pre-loaded with DNA at 30 degrees, little recombinant type activity is present, indicating that integration is blocked at 17 degrees. However, physico-chemical analysis of the re-extracted DNA indicates that a complex between single-stranded donor DNA and the recipient chromosome is formed at 17 degrees. This complex has a higher buoyant density than donor-recipient complexes formed at 30 degrees.