Regulation of chromosomal replication by DnaA protein availability in Escherichia coli: effects of the datA region

Initiation of chromosomal replication in Escherichia coli is dependent on a vailability of the initiator protein DnaA. We have introduced into E coli c ells plasmids carrying the chromosomal locus datA, which has a high affinit y for DnaA. To be able to monitor oriC initiation as a function of datA cop y number, we introduced a minichromosome which only replicates from oriC., using a host cell which replicates its chromosome independently of oriC. Ou r data show that a moderate increase in datA copy number is accompanied by increased DnaA protein synthesis that allows oriC initiation to occur norma lly, as measured by minichromosome copy number. As datA gene dosage is incr eased dnaA expression cannot be further derepressed, and the minichromosome copy number is dramatically reduced. Under these conditions the minichromo some was maintained by integration into the chromosome. These findings sugg est that the datA locus plays a significant role in regulating oriC initiat ion, by its capacity to bind DnaA. They also suggest that auto regulation o f the dnaA gene is of minor importance in regulation of chromosome initiati on. (C) 2001 Elsevier Science B.V. All rights reserved.