title: A Periplasmic Complex of the Nitrite Reductase NirS, the Chaperone DnaK, and the Flagellum Protein FliC Is Essential for Flagellum Assembly and Motility in Pseudomonas aeruginosa.
abstract: Pseudomonas aeruginosa is a ubiquitously occurring environmental bacterium and opportunistic pathogen responsible for various acute and chronic infections. Obviously, anaerobic energy generation via denitrification contributes to its ecological success. To investigate the structural basis for the interconnection of the denitrification machinery to other essential cellular processes, we have sought to identify the protein interaction partners of the denitrification enzyme nitrite reductase NirS in the periplasm. We employed NirS as an affinity-purifiable bait to identify interacting proteins in vivo. Results obtained revealed that both the flagellar structural protein FliC and the protein chaperone DnaK form a complex with NirS in the periplasm. The interacting domains of NirS and FliC were tentatively identified. The NirS-interacting stretch of amino acids lies within its cytochrome c domain. Motility assays and ultrastructure analyses revealed that a nirS mutant was defective in the formation of flagella and correspondingly in swimming motility. In contrast, the fliC mutant revealed an intact denitrification pathway. However, deletion of the nirF gene, coding for a heme d1 biosynthetic enzyme, which leads to catalytically inactive NirS, did not abolish swimming ability. This pointed to a structural function for the NirS protein. FliC and NirS were found colocalized with DnaK at the cell surface of P. aeruginosa. A function of the detected periplasmic NirS-DnaK-FliC complex in flagellum formation and motility was concluded and discussed.
  
  
  OBJECTIVE
  Physiological functions in Gram-negative bacteria are connected with the cellular compartment of the periplasm and its membranes. Central enzymatic steps of anaerobic energy generation and the motility mediated by flagellar activity use these cellular structures in addition to multiple other processes. Almost nothing is known about the protein network functionally connecting these processes in the periplasm. Here, we demonstrate the existence of a ternary complex consisting of the denitrifying enzyme NirS, the chaperone DnaK, and the flagellar protein FliC in the periplasm of the pathogenic bacterium P. aeruginosa. The dependence of flagellum formation and motility on the presence of an intact NirS was shown, structurally connecting both cellular processes, which are important for biofilm formation and pathogenicity of the bacterium.
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Niedersachsen, Germany Environmental Microbiology Laboratory, Helmholtz Center for Infection Research, Braunschweig, Niedersachsen, Germany.
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Borrero-de Acuña
authors.affiliation: Environmental Microbiology Laboratory, Helmholtz Center for Infection Research, Braunschweig, Niedersachsen, Germany Central Facility for Microscopy, Helmholtz Center for Infection Research, Braunschweig, Niedersachsen, Germany.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Molinari
authors.affiliation: Central Facility for Microscopy, Helmholtz Center for Infection Research, Braunschweig, Niedersachsen, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Rohde
authors.affiliation: Institute for Physical and Theoretical Chemistry-NanoBioSciences, Technische Universität Braunschweig, Braunschweig, Niedersachsen, Germany.
authors.forename: Thorben
authors.initials: T
authors.lastname: Dammeyer
authors.affiliation: Cellular Proteome Research, Helmholtz Center for Infection Research, Braunschweig, Niedersachsen, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Wissing
authors.affiliation: Cellular Proteome Research, Helmholtz Center for Infection Research, Braunschweig, Niedersachsen, Germany.
authors.forename: Lothar
authors.initials: L
authors.lastname: Jänsch
authors.affiliation: Environmental Microbiology Laboratory, Helmholtz Center for Infection Research, Braunschweig, Niedersachsen, Germany.
authors.forename: Sagrario
authors.initials: S
authors.lastname: Arias
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Niedersachsen, Germany.
authors.forename: Martina
authors.identifier: http://orcid.org/0000-0002-8345-6550
authors.initials: M
authors.lastname: Jahn
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Niedersachsen, Germany.
authors.forename: Max
authors.initials: M
authors.lastname: Schobert
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Niedersachsen, Germany Environmental Microbiology Laboratory, Helmholtz Center for Infection Research, Braunschweig, Niedersachsen, Germany.
authors.forename: Kenneth N
authors.initials: KN
authors.lastname: Timmis
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Niedersachsen, Germany d.jahn@tu-bs.de.
authors.forename: Dieter
authors.initials: D
authors.lastname: Jahn
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009068:Movement; D009154:Mutation; D009572:Nitrite Reductases / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism*; D011487:Protein Conformation; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D009572:Nitrite Reductases