Relationships between non-extractable DNA and the bacterial growth cycle

Abstract

Escherichia coli DNA has been fractionated into extractable and non-extractable DNA after deproteinization of detergent-lysed cell preparations with chloroform—isoamyl alcohol. The former was extracted with dilute buffered saline whereas the latter remained in the interphase layer associated with residual cellular debris from which almost 40% could be released by incubating with pronase. About 20–25% more amino acid residues were bound to the pronase-released DNA than to the extractable DNA, but the relative distribution of the residues in the two DNA samples was virtually identical. The specific activities and the relative amounts of denser (1.709 g · cm −3) and lighter (on the surface of CsCl gradients) DNA fractions from E. coli, grown in the presence of labeled thymidine, indicated that these two corresponded to extractable and non-extractable DNA, respectively. The relative amounts of the two fractions varied with the growth phase, primarily as a function of the growth rate. Age and metabolic state of cells in the culture or those used as inocula could modify this relative distribution. When growth rate was maximal, the ratio of the two remained at about 1. During lag phase when no appreciable net synthesis of DNA could be detected, there was a rapid and preferential incorporation of labeled thymidine into non-extractable DNA. A disproportionate increase in the fraction of the total DNA, which was extractable, was also observed but only when stationary phase cultures were used as inocula. Complete equilibration of the label in the two DNA fractions was attained only after cultures had reached mid-log phase of growth. Similar results were obtained when prelabeled cells were used. These data have been interpreted as suggesting that the rate of cell growth and DNA synthesis are related to the number or size of sites of attachment of DNA to some cellular structure. Newly synthesized DNA would be attached to different sites on this structure and initiation of DNA replication in lag phase would require reorientation of the two kinds of DNA. Small peptides which are firmly bound to the DNA and which vary quantitatively with the rate of DNA synthesis could perhaps be involved in the attachment to the sites.