Chromosome-membrane association in Bacillus subtilis III. Isolation and characterization of a DNA-protein complex carrying replication origin markers*

A chromosomal fragment containing pur A, a genetic marker near the replication origin of the Bacillus subtilis chromosome, was found in two different forms. One was tightly associated with the membrane (M-complex) while the other was a complex (S-complex) containing proteins that was easily solubilized during cell lysis. The S-complex had a markedly higher sedimentation rate (70 to 120 S) than the bulk of the supernatant DNA (40 S). High-salt concentrations, Pronase, and ionic detergents reduced the rate to 40 S, indistinguishable from that of the bulk DNA. Its characteristic sedimentation rate allowed us to isolate a single DNA fragment carrying genetic markers near the origin (e.g. pur A) in a highly purified state. From the ratio of pur A to his A (a middle marker of the chromosome) in the purified complex, the purity of the pur A-DNA fragment was estimated to be 70 to 80%. No genetic markers other than pur A and those closely linked to it were found in the S-complex. The origin-labelled DNA was concentrated in it but the replicating point was not. Lysates obtained without using detergent and mechanical shearing yielded the same amount of S-complex as these treatments, suggesting that the S-complex is not a partially degraded product of the membrane-bound pur A-DNA. Biochemical evidence suggests that the complex is an intermolecular aggregate of pur A-DNA-protein complex. This was directly proved by electron microscopic observation of purified S-complex. Aggregates of several DNA molecules (average 3·4) form a structure containing loops, bushes which are sensitive to RNAase, and amorphous materials stained black. These were seen by electron microscopy when the complex was fixed by glutaraldehyde. The assumption that the DNA in the S-complex carries a particular region of the chromosome containing the pur A marker was confirmed by site-specific cleavage of the DNA with restriction endonucleases. Hind III and Hae II produced at least four and ten major fragments, respectively, in about equal molar ratios. In both cases the sum of the molecular weights of the fragments was approximately 2×10 7 .