Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. II. Identification of the essential regions for inhibitory functions shown by the DNA segment containing the replication origin.

A BamHI restriction endonuclease fragment B7, which contains the replication origin of the Bacillus subtilis chromosome, showed inhibitory effects on cell growth and plasmid replication in Escherichia coli and Bacillus subtilis, when B7 was inserted into a composite plasmid pMS102' and introduced into these cells. In order to localize these properties in more limited regions within the B7 fragment, we developed a new and widely applicable method for deletion of DNA segments of various lengths from one or other end of a given region of the plasmid molecule. Using a set of deletions in the B7 fragments of pMS102'-B7, we determined the loci responsible for the inhibitory effects of B7 as described below. (1) Stickiness appearing in E. coli cells was caused by a segment residing in a region of approximately 2.2 kilobase pairs (kbp) overlapping the E19 and E22 fragments. (2) instability of the plasmid in E. coli was due to a segment localized in the 440 bp region of the E19-side terminal portion of the 2.2 kbp region. (3) The same 440 bp were also responsible for inhibition of the replication of the plasmid in B. subtilis. Hybridization of the cloned DNA fragments containing the 2.2 kbp region with the whole B. subtilis chromosome revealed that several regions of the chromosome are homologous to this characteristic sequence.