title: A gateway-based system for fast evaluation of protein-protein interactions in bacteria.
abstract: Protein-protein interactions are important layers of regulation in all kingdoms of life. Identification 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 interactions 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 interactions via Fluorescence Resonance Energy Transfer (FRET), Bacterial two Hybrid (B2H) and split Gaussia 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 interaction 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 chemotaxis response regulator CheY and its phosphatase CheZ.
authors: Junior Research Group 3, Robert Koch-Institute, Wernigerode Branch, Wernigerode, Germany.
authors: Thorsten
authors: T
authors: Wille
authors: Division of Microbiology, School of Biology/Chemistry, University of Osnabrück, Osnabrück, Germany.
authors: Britta
authors: B
authors: Barlag
authors: Center for Molecular Biology at the University of Heidelberg (ZMBH), DKFZ (German Cancer Research Center) -ZMBH Alliance, Heidelberg, Germany.
authors: Vladimir
authors: V
authors: Jakovljevic
authors: Division of Microbiology, School of Biology/Chemistry, University of Osnabrück, Osnabrück, Germany.
authors: Michael
authors: M
authors: Hensel
authors: Center for Molecular Biology at the University of Heidelberg (ZMBH), DKFZ (German Cancer Research Center) -ZMBH Alliance, Heidelberg, Germany; Max Planck Institute for Terrestrial Microbiology & LOEWE (state offensive for the development of scientific and economic excellence) Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany.
authors: Victor
authors: V
authors: Sourjik
authors: Junior Research Group 3, Robert Koch-Institute, Wernigerode Branch, Wernigerode, Germany.
authors: Roman G
authors: RG
authors: Gerlach
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D031541:Fluorescence Resonance Energy Transfer; D005822:Genetic Vectors; D008156:Luciferases / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D060066:Protein Interaction Maps / Q000235:genetics*; D012475:Salmonella / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D008840:Microfilament Proteins; C118287:SipA protein, Salmonella; C000607606:cheY protein, E coli; D008156:Luciferases