Graduate School of Frontier Biosciences, Osaka University

Japanese

Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex

Journal PLoS Biol (2017)
Authors Takuma Fukumura (1), Fumiaki Makino (1), Tobias Dietsche (2), Miki Kinoshita (1), Takayuki Kato (1), Samuel Wagner (2, 3), Keiichi Namba (1, 4), Katsumi Imada (5), Tohru Minamino (1)
  1. Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
  2. Interfactulty Institute of Microbiology and Infection Medicine, Section of Cellular and Molecular Microbiology, Eberhard Karls University Tübingen, Tübingen, Germany
  3. German Center for Infection Research (DZIF), Partner-site Tübingen, Tübingen, Germany
  4. Quantitative Biology Center, Riken, Suita, Osaka, Japan
  5. Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan
Title Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex
Laboratory Protonic NanoMachine Group 〈Prof. Namba〉
Abstract The bacterial flagellar type III export apparatus, which is required for flagellar assembly beyond the cell membranes, consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. FlhA, FlhB, FliP, FliQ and FliR form the gate complex inside the basal body MS ring although FliO is required for efficient export gate formation in Salmonella enterica. However, it remains unknown how they form the gate complex. Here we report that FliP forms a homo-hexameric ring with a diameter of 10 nm. Alanine substitutions of conserved Phe-137, Phe-150 and Glu-178 residues in the periplasmic domain of FliP (FliPP) inhibited FliP6 ring formation, suppressing flagellar protein export. FliO formed a 5 nm ring structure with three clamp-like structures that bind to the FliP6 ring. The crystal structure of FliPP derived from Thermotoga maritia and structure-based photo-crosslinking experiments revealed that Phe-150 and Ser-156 of FliPP are involved in the FliP-FliP interactions and that Phe-150, Arg-152, Ser-156 and Pro-158 are responsible for the FliP-FliO interactions. Over-expression of FliP restored motility of a ∆fliO mutant to the wild-type level, suggesting that the FliP6 ring is a functional unit in the export gate complex and that FliO is not part of the final gate structure. Co-purification assays revealed that FlhA, FlhB, FliQ and FliR are associated with the FliO/FliP complex. We propose that the assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR and FlhB and finally FlhA during MS ring formation.