Analysis of the FliM/FliG motor protein interaction by two-hybrid mutation suppression analysis

• Published in: Microbiology (Society for General Microbiology) 2008-03, Vol.154 (3), p.714-724
• Main Authors: Passmore, Steven E, Meas, Rithy, Marykwas, Donna L
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; Escherichia coli; Escherichia coli - genetics; Escherichia coli - physiology; Fundamental and applied biological sciences. Psychology; Microbiology; Models, Biological; Models, Molecular; Motility, taxis; Mutagenesis; Protein Interaction Mapping; Suppression, Genetic; Two-Hybrid System Techniques
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.2007/014597-0

Department of Biological Sciences, California State University, Long Beach, Long Beach, CA 90840, USA Correspondence Donna L. Marykwas dmarykwa{at}csulb.edu The Escherichia coli motor proteins FliM and FliG physically interact, presumably to control one or more of the functions of the bacterial flagellum clockwise/counterclockwise (CW/CCW) switch. We have previously demonstrated this interaction using the yeast two-hybrid system and have identified mutations in fliG that disrupt the interaction. Starting with the most interaction-defective of these fliG mutants, we mutagenized fliM to identify suppressor mutations that restore the FliM/FliG two-hybrid interaction. Certain fliM suppressor mutations exhibit allele specificity. These mutations help define a FliG-interaction surface on FliM. Moreover, the pattern of suppression suggests that two distinct sites on FliG interact with FliM, perhaps with two FliM molecules in a dimer per molecule of FliG. Abbreviations: CCW, counterclockwise; CW, clockwise Three supplementary figures, showing fliM mutagenesis to isolate suppressors of fliG mutations disrupting FliM/FliG interaction, mapping of fliM suppressor mutations by multifragment cloning in vivo , and an E. coli FliG model, a supplementary table listing primers used in this study, and the coordinates for the modelled structures of E. coli FliG and FliM, are available with the online version of this paper.