Browsing by Subject "Vibrio cholerae"

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    Powerful proteins : structure and function of catalytic subunits of electrogenic NADH:quinone oxidoreductases
    (2013) Steffen, Wojtek; Fritz-Steuber, Julia
    Electrogenic NADH:quinone oxidoreductases are large, membrane-embedded enzyme complexes found in the respiratory chain of prokaryotes and the mitochondria of eukaryotes. They represent the first module of the oxidative phosphorylation system which converts the energy from nutrients into an electrochemical gradient by coupling redox reactions to the translocation of cations across membranes. A long chain of events, such as the synthesis of ATP, ion homeostasis, reactive oxygen species production and bacterial motility depend on the activity of these complexes. Complex I consists of up to 45 subunits and can be found in the inner mitochondrial membrane of eukaryotes and in prokaryotes, where it is called NDH I. We investigated the isolated, hydrophobic ND5 subunit, which shows homologies to cation/proton antiporters, from human or Yarrowia lipolytica complex I. In vivo and biochemical analyses provided data on the cation translocation function and the alteration of function by disease-associated mutations. Taken together with the recently published 3D structure of bacterial complex I, these data allowed us to demonstrate that the ND5 subunit could possibly act as an antiporter module of mitochondrial complex I. Sodium ion translocating NADH:quinone oxidoreductase (Na+-NQR) is an enzyme found in many pathogenic bacteria. It consists of six subunits (NqrA - NqrF) whose 3D structures and enzymatic mechanisms were not known in detail at the time this project was initiated. By using high-resolution X-ray structures and site-directed mutagenesis, combined with biochemical studies, we proposed a model for catalysis and substrate selectivity on the atomic level of the electron input module of the complex, the NADH oxidizing domain of subunit NqrF. Furthermore, we analyzed the binding of silver ions to a cysteine residue in the NADH binding pocket and found that it leads to the inhibition of the Na+-NQR and to the killing of Vibrio cholerae in the nanomolar range. Subunit NqrA forms part of the quinone reductase module. By the use of physicochemical and biochemical methods we identified the herbicide 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) as a quinone antagonist and inhibitor of the Na+-NQR complex and discovered two adjacent quinone binding sites on NqrA.
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    Stressful environments : motility and catecholamine response in Vibrio cholerae
    (2014) Halang, Petra; Fritz-Steuber, Julia
    The human pathogen Vibrio cholerae is able to inhabit a variety of environments. These include especially aquatic ecosystems, but the human intestine as well. V. cholerae is thus tolerant to a wide range of salinity and pH. Motility is achieved by a sodium driven polar flagellum. The affinity for Na+ to run the flagellum is determined by the stator complex PomAB, which is embedded in the cell membrane within the flagellar motor. A critical aminoacid residue for the binding of Na+ is aspartate 23 within the transmembrane helix of PomB. A mutation of this aminoacid residue leads to an immotile phenotype of V. cholerae. It was thus of interest to investigate if other polar or acidic aminoacid residues within PomB are important for the passage of Na+. Two potential candidates are serine at position 26 and aspartate at position 42 of PomB, both aminoacid residues are conserved within sodium driven flagellar stator complexes. To characterize the pathway of Na+ through the PomAB channel, the influence of chloride salts (Na+ and K+) and the pH on the motility of V. cholerae was studied. Motility decreased at elevated pH but increased if a chaotropic chloride salt was added, which excludes a direct Na+ and H+ competition in the process of binding to the conserved PomB D23 residue. Cells expressing the PomB S26A/T or D42N variants lost motility at low Na+ concentrations but regained motility in the presence of 170mM chloride. The swimming speeds of individual cells were also analyzed and revealed that S26 located within the membrane helix of PomB is required to promote very fast swimming of V. cholerae. Loss of hypermotility was observed with the S26T variant of PomB which was partially restored by lowering the pH of the external medium. Modification of PomA and PomB by N,N’-dicyclohexylcarbodiimide indicates the presence of protonated carboxyl groups in the hydrophobic regions of the two proteins. Na+ did not protect PomA and PomB from this modification. It could be demonstrated that the motility of V. cholerae is influenced by the pH and osmolality of the medium and thus, the aminoacid residues – S26 and D42 together with D23 – of PomB have a function in the passage of Na+ into the cell. The H+ rather than the Na+ concentration determines the efficiency of the motor, indicating the presence of a catalytical important hydrogen bond network in the motor channel. It is proposed that D23, S26 and D42 of PomB are part of an ion-conducting pathway formed by the PomAB stator complex. As mentioned above, V. cholerae is a pathogen which settles the human intestine. As other pathogens are able to respond specifically to the stress associated mammalian hormones epinephrine and norepinephrine it was of an interest to investigate the influence of these hormones on growth and motility of V. cholerae. The response to epinephrine and norepinephrine is mediated by the QseC sensor protein. The genome of V. cholerae comprises a gene which is homolog to qseC from E. coli. Growth and swarming of V. cholerae was enhanced in the presence of 0.1mM epinephrine or norepinephrine. qRT-PCR experiments revealed increased expression of the genes encoding the putative sensor kinase qseC and pomB, a component of the flagellar motor complex under the influence of catecholates. HPLC measurements of bacterial supernatant revealed that norepinephrine is completely degraded or metabolized after 48 h in the presence of V. cholerae, concomitant with the appearance of another, unidentified compound. On the other hand, V. cholerae seemed to stabilize epinephrine. After 48 h, 0.46% of the epinephrine added at the beginning of the growth experiment was retained. Again, a yet unidentified compound was detected. The experiments conducted in this work strongly indicate the presence of a catecholate receptor in V. cholerae.