(A) Schematic of DnaA demonstrating three different possibilities in bacteria: species that have both the DnaD adaptor and SirA regulator, species that have DnaD but lack SirA, and species that lack DnaD and SirA. (B) B2H of T18-tagged SaDnaDWHD co-expressed with T25-tagged variants of SaDnaADI. (C) Sequence alignment of the DnaA hot spot from B. subtilis, S. aureus, and E. coli. The three hot spot residues mutated in the B2H assays are highlighted in yellow, with residues critical for the DnaD interaction in B. subtilis indicated by blue arrow heads. Residues critical for the direct interaction between E. coli DnaA and the replicative helicase are indicated by red arrow heads. The numbering at the top of the alignment refers to the B. subtilis sequence. (D) B2H of T18-tagged BsDnaDWHD variants co-expressed with T25-tagged BsDnaADI. (E) B2H of T25-tagged DnaADI from B. subtilis (Bs), S. aureus (Sa) or E. coli (Ec) co-expressed with T18-tagged BsDnaDWHD or SaDnaDWHD.