• 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.

      Borrero-de Acuña, José Manuel; Molinari, Gabriella; Rohde, Manfred; Dammeyer, Thorben; Wissing, Josef; Jänsch, Lothar; Arias, Sagrario; Jahn, Martina; Schobert, Max; Timmis, Kenneth N; et al. (2015-10-01)
      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.
    • The Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus.

      Borrero-de Acuña, José Manuel; Rohde, Manfred; Wissing, Josef; Jänsch, Lothar; Schobert, Max; Molinari, Gabriella; Timmis, Kenneth N; Jahn, Martina; Jahn, Dieter; Helmholtz Centre for infection research (HZI), Inhoffenstraße 7, 38124 Braunschweig, Germany. (2016-02-22)
      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 co-localization 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 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.