A novel nucleoid-associated protein coordinates asymmetric chromosome replication and chromosome partitioning

We searched for regulators of chromosome replication in the cell cycle model Caulobacter crescentus and found a novel DNA-binding protein that selectively aids both the initiation of chromosome replication and the initial steps of chromosome partitioning. We identified and purified a protein (OpaA) that binds the chromosome origin of replication (Cori) and its higher-affinity binding to mutated Cori-DNA that increases Cori-plasmid replication in vivo. opaA gene expression is essential for normal growth and sufficient OpaA levels are required for the correct timing of chromosome replication. Whole genome ChIP-seq identified dynamic DNA-binding distributions for OpaA, with the strongest associations at the partitioning (parABS) locus near Cori. Using molecular-genetic and fluorescence microscopy experiments, we showed that OpaA also promotes the first steps of chromosome partitioning, the initial separation of the duplicated parS loci following Cori replication. This separation occurs before the parABS-dependent partitioning and it coincides with the poorly defined mechanism(s) that establishes chromosome asymmetry: C. crescentus chromosomes are partitioned to distinct cell-poles which develop into replicating and non-replicating cell-types. We propose that OpaA coordinates chromosome replication with asymmetry-establishing chromosome separation, noting that both roles are consistent with the phylogenetic restriction of opaA to asymmetrically dividing bacteria that avoid multi-fork replication. Overall design: Examination of OpaA whole genome binding/occupency (ChIP-Seq) in WT (2 replicates) or ?opaA supp+ Caulobacter crescentus backgrounds. In addition, we addressed OpaA whole genome peaks redistribution from WT growing cultures treated with rifampicin, novobiocin or chloremphenicol (3 ChIP-Seq samples).