Initiator titration complex formed at datA with the aid of IHF regulates replication timing in Escherichia coli

Abstract

The initiation of replication in Escherichia coli is negatively controlled by a mechanism referred to as 'initiator titration', a process by which the initiator protein, DnaA, is titrated to newly replicated binding sequences on the chromosome to reduce the initiation potential for replication. Initiator titration occurs predominantly at the datA locus that binds exceptionally large amounts of DnaA molecules to prevent aberrant initiations. We found that this was enabled by integration host factor (IHF). Within datA, there is a consensus IHF recognition sequence between the two DnaA recognition sequences (DnaA boxes) essential for its function. Binding of IHF to this site was demonstrated both in vitro and in vivo. Disruption of the core sequence in the consensus of the IHF-binding resulted in increased origin concentration as observed in Delta datA cells. Furthermore, the number of DnaA molecules bound to datA was reduced in cells carrying a disruption in the IHF-binding core sequence. The IHF-binding site and the essential DnaA boxes had to be located at a proper distance and orientation to maintain the accurate initiation timing. Therefore, IHF is a unique element in the control of replication initiation that acts negatively at datA, while known to act as a positive regulator at oriC.