The linker domain of the initiator DnaA contributes to its ATP binding and membrane association in E. coli chromosomal replication

DnaA, the initiator of Escherichia coli chromosomal replication, has in its adenosine triphosphatase (ATPase) domain residues required for adenosine 5 '-triphosphate (ATP) binding and membrane attachment. Here, we show that D118Q substitution in the DnaA linker domain, a domain known to be without major regulatory functions, influences ATP binding of DnaA and replication initiation in vivo. Although initiation defective by itself, overexpression of DnaA(D118Q) caused overinitiation of replication in dnaA46ts cells and prevented cell growth. The growth defect was rescued by overexpressing the initiation inhibitor, SeqA, indicating that the growth inhibition resulted from overinitiation. Small deletions within the linker showed another unexpected phenotype: cellular growth without requiring normal levels of anionic membrane lipids, a property found in DnaA mutated in its ATPase domain. The deleted proteins were defective in association with anionic membrane vesicles. These results show that changes in the linker domain can alter DnaA functions similarly to the previously shown changes in the ATPase domain.

Automated summary

This study found that the D118Q substitution in the linker domain of DnaA affects ATP binding and replication initiation. Overexpression of DnaA(D118Q) causes overinitiation of replication and prevents cell growth, which can be rescued by overexpression of SeqA. Additionally, small deletions in the linker domain allow cellular growth without requiring normal levels of anionic membrane lipids, similar to the properties of DnaA mutated in its ATPase domain.