Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli

Abstract

The in vivo role of the Escherichia coli protein DnaA in the replication of plasmid pBR322 was investigated, using a plasmid derivative carrying an inducible dnaA+ gene. In LB medium without inducer, the replication of this plasmid, like that of pBR322, was inhibited by heat inactivation of chromosomal DnaA46 protein so that plasmid accumulation ceased 1 to 2 h after the temperature shift. This inhibition did not occur when the plasmid dnaA+ gene was expressed in the presence of the inducer isopropyl-1-thio-beta-D-galactopyranoside (IPTG). Inhibition was also not observed in glycerol minimal medium or in the presence of low concentrations of rifampicin or chloramphenicol. Deletion of the DnaA binding site and the primosome assembly sites (pas, rri) downstream of the replication origin did not affect the plasmid copy number during exponential growth at 30 degrees C, or after inactivation of DnaA by a shift to 42 degrees C in a dnaA46 host, or after oversupply of DnaA, indicating that these sites are not involved in a rate-limiting step for replication in vivo. The accumulation of the replication inhibitor, RNAI, was independent of DnaA activity, ruling out the possibility that DnaA acts as a repressor of RNAI synthesis, as has been suggested in the literature. Changes in the rate of plasmid replication in response to changes in DnaA activity (in LB medium) could be resolved into an early, rom-dependent, and a late, rom-independent component. Rom- plasmids show only the late effect. After heat inactivation of DnaC, plasmid replication ceased immediately.(ABSTRACT TRUNCATED AT 250 WORDS)