Relationship of R6K replicating forms to the folded chromosome of Escherichia coli

Abstract

An examination of the relationship of both nonreplicating and replicating forms of R6K plasmid DNA to the Escherichia coli folded chromosome showed that both forms cosediment with the chromosome in neutral sucrose gradients. Approximately 20% of the nonreplicating molecules was found as freely sedimenting forms when the folded configuration of the chromosomes was preserved. However, under the same conditions negligible amounts of the replicating forms were found as freely sedimenting molecules. Thus, it is concluded that the replicating forms, when compared with nonreplicating molecules, are preferentially associated with the folded chromosomal structure. Exposure of the folded chromosomal structure to RNase resulted in an unfolding of the chromosome and a concomitant increase in the amount of freely sedimenting replicating and nonreplicating forms of R6K DNA. Analyses of the single-stranded length of RNase-released nascent molecules suggest that they replicate in continuous association with the folded chromosome complex. Nonenzymatic unfolding of the chromosomes by progressively lowering the sodium ion concentration during lysis resulted in a progressive increase in the release of nonreplicating molecules. Replicating molecules were not released by unfolding the chromosome in this fashion. was used. Streptomycin (200,ug/ml) and ampicillin (40 ytg/ml) were used to select for cells harboring R6K.

Biochemical characterization of nonintegrated plasmid-folded chromosome complexes: sex factor F and the Escherichia coli nucleoid

“…Archibold et al (1) found that lowering the NaCl concentration to 0.25 M released R6K monomers from folded chromosome complexes and concomitantly decreased the sedimentation coefficient of the chromosome to 700S. R6K replicative intermediates, however, remain bound to the 700S entity but can be released subsequently by RNase or protease treatment.…”

“…Moreover, the biochemical nature of complexes may differ from plasmid species to plasmid species. However, knowledge that plasmid-chromosome complexes do exist and are mediated by RNA in a number of bacterial genera is proving quite useful in biochemical and physiological studies of plasmid maintenance (1,13).…”