The control of initiation of DNA replication in Escherichia coli.

Abstract

A new type of DNA-BNA hybridisation system was used to investigate two aspects of the initiation of replication in E.coli. A colony bank of recombinant plasmids carrying various fragments of the E.coli chromosome was constructed by manipulation in vitro. A number of plasmids carrying specific E.coli genes were selected by direct complementation analysis or by F'lac mediated mobilisation. These plasmids together with other similar ones were used as hybridisation probes. The conditions for hybridisation and the labelling of LNA.;vitrowere refined. DNA-DNA hybridisation and chemical DNA measurements were used to demonstrate that chloramphenicol induces a burst of extra initiation at the origin of a dnaA46 strain growing at a semi-permissive temperature. The temporal pattern of the initiation is consistent with the hypothesis that the stimulation of.Jiiitiation is the result of the stimulation of the. synthesis of an RNA species. It was shown that there is a disturbance in the pattern of replication of Hfr strain KL99 during exponential growth compared with that in a closely related F" strain. The pattern is consistent with initiation occuxring at the F origin. The LNA/mass ratio of Hfr AB313 is not changed relative to that of the isogenic F∼ strain, indicating that the F plasmid origin of replication is not active in this strain during exponential growth. As both Hfr strains are capable of giving rise to F' plasmids it is argued that the difference in the point of insertion of F in the chromosome is the cause of the difference in the initiation pattern between the two strains. These results, are interpreted as being a reflection of a copy number titration control mechanism for F replication. Possible mechanisms for the primary control and for the subsequent steps in the initiation process are discussed