Using living bacteria and in vitro analysis with purified proteins; we have identified step-by step interactions between the initiator protein, DnaA, and the E. coli chromosomal replication origin, oriC, as well as several DNA bending proteins that modulate these DNA-protein complexes. Recently we have also identified instruction sets for DnaA oligomer assembly, in the form of symmetrically arranged recognition site motifs that are built into the oriC nucleotide sequence. Studies are ongoing to understand how these motifs direct the orderly growth of DnaA oligomers, and whether any of these recognition sites play a role in timing chromosome replication during the cell cycle. Studies are also underway to determine whether several novel DnaA sub-complexes are required to unwind oriC DNA during an early step of DNA synthesis. We hope to learn the specific functional role for each DnaA molecule and determine how each step of the DNA-protein complex is choreographed under different bacterial growth rates.
As a long-term goal, our efforts to dissect this fundamental bacterial growth regulatory machinery should lead to the identification of new targets for novel inhibitors of bacterial pathogens. Since the regulators we study are highly conserved and are part of larger complexes assembled step-wise during the cell cycle, it should be possible to identify the steps during bacterial growth that are most sensitive to inhibition.