RESEARCH ARTICLE A Gateway-Based System for Fast Evaluation of Protein-Protein Interactions in Bacteria

Protein-protein interactions are important layers of regulation in all kingdoms of life. Identifi-cation and characterization of these interactions is one challenging task of the post-genomic era and crucial for understanding of molecular processes within a cell. Several methods have been successfully employed during the past decades to identify protein-protein inter-actions in bacteria, but most of them include tedious and time-consuming manipulations of DNA. In contrast, the MultiSite Gateway system is a fast tool for transfer of multiple DNA fragments between plasmids enabling simultaneous and site directed cloning of up to four fragments into one construct. Here we developed a new set of Gateway vectors including custom made entry vectors and modular Destination vectors for studying protein-protein in-teractions via Fluorescence Resonance Energy Transfer (FRET), Bacterial two Hybrid (B2H) and splitGaussia luciferase (Gluc), as well as for fusions with SNAP-tag and HaloTag for dual-color super-resolution microscopy. As proof of principle, we characterized the inter-action between the Salmonella effector SipA and its chaperone InvB via split Gluc and B2H approach. The suitability for FRET analysis as well as functionality of fusions with SNAP-and HaloTag could be demonstrated by studying the transient interaction between chemo-taxis response regulator CheY and its phosphatase CheZ