The DnaA inhibitor SirA acts in the same pathway as Soj (ParA) to facilitate oriC segregation during Bacillus subtilis sporulation

Summary DNA replication and chromosome segregation must be carefully regulated to ensure reproductive success. During Bacillus subtilis sporulation, chromosome copy number is reduced to two, and cells divide asymmetrically to produce the future spore (forespore) compartment. For successful sporulation, oriC must be captured in the forespore. New rounds of DNA replication are prevented in part by SirA, a protein that utilizes residues in its N‐terminus to directly target Domain I of the bacterial initiator, DnaA. Using a quantitative forespore chromosome organization assay, we show that SirA also acts in the same pathway as another DnaA regulator, Soj, to promote oriC capture in the forespore. By analyzing loss‐of‐function variants of both SirA and DnaA, we observe that SirA's ability to inhibit DNA replication can be genetically separated from its role in oriC capture. In addition, we identify substitutions near the C‐terminus of SirA and in DnaA Domain III that enhance interaction between the two proteins. One such variant, SirAP141T, remained functional in regard to inhibiting replication, but was unable to support oriC capture. Collectively, our results support a model in which SirA targets DnaA Domain I to inhibit DNA replication, and DnaA Domain III to facilitate Soj‐dependent oriC capture in the forespore. SirA inhibits DNA replication through interactions with DnaA Domain I; this activity is not required for oriC capture. SirA targets DnaA Domain III to maintain oriC in a state favorable for repositioning. In Model I, Soj acts upstream of SirA, generating a conformation of DnaA favorable for SirA association. The association of SirA with DnaA Domain III then permits oriC repositioning. In Model II, Soj acts downstream of SirA to facilitate oriC capture. Model I does not exclude the possibility that Soj may also be required to facilitate oriC capture through an independent, downstream mechanism (open arrow). During Bacillus subtilis sporulation, SirA inhibits new rounds of DNA replication by interacting directly with DnaA Domain I. Through an independent function, SirA facilitates Soj(ParA)‐dependent oriC segregation, thus ensuring oriC is captured on the forespore side of the septum following polar division.