Detection of Protein–Protein Interactions in <i>Escherichia coli</i> With Single Molecule Sensitivity

Protein-protein interactions are central in all biological processes. Methods capable of detecting interactions within living, intact cells have been particularly useful to identify and characterize protein interaction networks. We describe here an exquisitely sensitive regulatory circuit that can detect in bacteria, protein-protein interaction with single-molecule sensitivity. This approach involves the interaction-mediated reconstitution of a cyclic AMP signaling cascade in Escherichia coli taking advantage of the high catalytic activity of the adenylate cyclase (AC) from Bordetella pertussis upon activation by its natural activator, calmodulin (CaM). We show that less than one complex of interacting hybrid proteins per cell on average, is enough to confer a selectable trait to the host. This exquisitely sensitive adenylate cyclase hybrid (ESACH) system allows for direct selection, in living bacteria, of ligands exhibiting high affinity for given targets or for studying interactions involving toxic proteins. The extreme sensitivity of the AC/CaM/cAMP signaling cascade may thus be harnessed to interrogate biological processes with single-molecule resolution in live bacteria and could be exploited to design novel synthetic regulatory networks operating at, or even below, the theoretical threshold limit of one molecule per cell.