Characterization of the Replication Terminus of the Bacillus subtilis Chromosome

Abstract

DNA in the terminal region of the chromosome of B. subtilis was labeled by a procedure in which cells in sporulation-inducing conditions were pulse-labeled with [3H]thymidine and then treated with p-hydroxyphenylazouracil, an inhibitor of DNA synthesis. The labeled DNA in isolated spores yielded a small number of restriction fragments. About 14 EcoRI fragments with a total length of 80 kilobases (kb) were labeled in a 2.5 min pulse. A fragment of 4.0 kb had the highest specific radioactivity in terminally labeled DNA from several strains. One of these strains lacked the 120 kb prophage of SP.beta. that is normally integrated close to the terminus. Loss of the 120 kb prophage did not affect the point of termination which must therefore be regarded as a specific stop sequence. Labeled terminus DNA was used to identify .lambda. (Charon 4A) clones containing sequences derived from the terminal region. The total length of the restriction fragments present was 150 kb and adds another 90 kb to the 150 kb region mapped previously. Only 1 group of these sequences was present in a B. subtilis strain (CU 1695) carrying a deletion spanning from SP.beta. to the right of the terminus and gltA. Thus, the terminator sequence found in the wild-type can be deleted, but presumably this strain has an alternative mechanism of termination.