title: Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus.
abstract: Oxidative phosphorylation using multiple-component, membrane-associated protein complexes is the most effective way for a cell to generate energy. Here, we systematically investigated the multiple protein-protein interactions of the denitrification apparatus of the pathogenic bacterium Pseudomonas aeruginosa During denitrification, nitrate (Nar), nitrite (Nir), nitric oxide (Nor), and nitrous oxide (Nos) reductases catalyze the reaction cascade of NO(3-)→ NO(2-)→ NO → N2O → N2 Genetic experiments suggested that the nitric oxide reductase NorBC and the regulatory protein NosR are the nucleus of the denitrification protein network. We utilized membrane interactomics in combination with electron microscopy colocalization studies to elucidate the corresponding protein-protein interactions. The integral membrane proteins NorC, NorB, and NosR form the core assembly platform that binds the nitrate reductase NarGHI and the periplasmic nitrite reductase NirS via its maturation factor NirF. The periplasmic nitrous oxide reductase NosZ is linked via NosR. The nitrate transporter NarK2, the nitrate regulatory system NarXL, various nitrite reductase maturation proteins, NirEJMNQ, and the Nos assembly lipoproteins NosFL were also found to be attached. A number of proteins associated with energy generation, including electron-donating dehydrogenases, the complete ATP synthase, almost all enzymes of the tricarboxylic acid (TCA) cycle, and the Sec system of protein transport, among many other proteins, were found to interact with the denitrification proteins. This deduced nitrate respirasome is presumably only one part of an extensive cytoplasmic membrane-anchored protein network connecting cytoplasmic, inner membrane, and periplasmic proteins to mediate key activities occurring at the barrier/interface between the cytoplasm and the external environment.
  
  
  OBJECTIVE
  The processes of cellular energy generation are catalyzed by large multiprotein enzyme complexes. The molecular basis for the interaction of these complexes is poorly understood. We employed membrane interactomics and electron microscopy to determine the protein-protein interactions involved. The well-investigated enzyme complexes of denitrification of the pathogenic bacterium Pseudomonas aeruginosa served as a model. Denitrification is one essential step of the universal N cycle and provides the bacterium with an effective alternative to oxygen respiration. This process allows the bacterium to form biofilms, which create low-oxygen habitats and which are a key in the infection mechanism. Our results provide new insights into the molecular basis of respiration, as well as opening a new window into the infection strategies of this pathogen.
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Germany.
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Borrero-de Acuña
authors.affiliation: Central Facility for Microscopy, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Rohde
authors.affiliation: Department of Cellular Proteome Research, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Wissing
authors.affiliation: Department of Cellular Proteome Research, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Lothar
authors.initials: L
authors.lastname: Jänsch
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Germany.
authors.forename: Max
authors.initials: M
authors.lastname: Schobert
authors.affiliation: Central Facility for Microscopy, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Molinari
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Germany.
authors.forename: Kenneth N
authors.initials: KN
authors.lastname: Timmis
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, 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, Germany d.jahn@tu-bs.de.
authors.forename: Dieter
authors.initials: D
authors.lastname: Jahn
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D058440:Denitrification; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D050901:Nitrate Reductase / Q000235:genetics / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D060066:Protein Interaction Maps; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D009566:Nitrates; C075970:NosR protein, Bacteria; D010088:Oxidoreductases; C022763:nitrous oxide reductase; C034747:nitric-oxide reductase; D050901:Nitrate Reductase; D010100:Oxygen