Site-Specific Photocrosslinking to Investigate Toxin Delivery Mediated by the Bacterial β-Barrel Assembly Machine.

Abstract

Contact-dependent inhibition (CDI) is a mechanism of interbacterial competition in Gram-negative organisms that relies on a specific interaction between a CdiA protein on the surface of one cell and a β-barrel protein on the surface of a neighboring cell. This interaction triggers the transport of a protein toxin into the neighboring cell where it exerts its lethal activity. Several classes of CdiA proteins that bind to different β-barrel receptors have been identified, but the molecular mechanism by which they deliver their toxins across the outer membranes of their target cells is poorly understood. Here we describe the use of site-specific photocrosslinking to characterize the interaction between a CdiA protein and its receptor. We describe the method for an E. coli CdiA that utilizes BamA as its receptor. BamA's central role in assembling β-barrel proteins in the outer membrane makes its role in CDI particularly intriguing; it suggests that these two different protein transport processes might share mechanistic features. Our in vitro photocrosslinking method is useful in elucidating early steps in the CDI mechanism, but it could be adapted to study later steps or to study other CdiA-receptor pairs.