Suppression of Escherichia coli dnaA46 mutations by integration of plasmid R100.1. derivatives: constraints imposed by the replication terminus

Abstract

We have studies the phenotypic suppression of a dnaA46 mutation by plasmid integration at preselected chromosomal sites after introducing homologous sequences (Mu prophages) onto both the chromosomes and the suppressive plasmid. The plasmids used were all derived from plasmid R100.1. We found that the conditions required to get viable suppressive integration varied as the plasmid integration site moved from the origin to the terminus of chromosome replication. Two constraints were observed. Both appeared to be linked to the new characteristics acquired by chromosome replication from the integrated plasmid. One constraint was that strains with integrative suppression near the terminus terC were viable only in minimal medium. The rich medium sensitivity of these strains was correlated with a loss of regulation of initiation. The other constraint was a requirement for a specific orientation in certain regions of the chromosome. The two branches defined by normally initiated replication, between oriC and terC, were also symmetrical with respect to these plasmid orientation constraints. In studying the possible reasons for a plasmid orientation constraint, we found that, of the two forks initiated in bidirectional replication from the integrated plasmid, one was capable of moving across the terC region with a higher movability than the other.