A shift towards succinate-producing Prevotella in the ruminal microbiome challenged with monensin.

The time-resolved impact of monensin on the active rumen microbiome was studied in a rumen-simulating technique (Rusitec) with metaproteomic and metabolomic approaches. Monensin treatment caused a decreased fibre degradation potential that was observed by the reduced abundance of proteins assigned to fibrolytic bacteria and glycoside hydrolases, sugar transporters and carbohydrate metabolism. Decreased proteolytic activities resulted in reduced amounts of ammonium as well as branched-chain fatty acids. The family Prevotellaceae exhibited increased resilience in the presence of monensin, with a switch of the metabolism from acetate to succinate production. Prevotella species harbour a membrane-bound electron transfer complex, which drives the reduction of fumarate to succinate, which is the substrate for propionate production in the rumen habitat. Besides the increased succinate production, a concomitant depletion of methane concentration was observed upon monensin exposure. Our study demonstrates that Prevotella sp. shifts its metabolism successfully in response to monensin exposure and Prevotellaceae represents the key bacterial family stabilizing the rumen microbiota during exposure to monensin.

The low mutational flexibility of the EPSP synthase in Bacillus subtilis is due to a higher demand for shikimate pathway intermediates.

Glyphosate (GS) inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that is required for aromatic amino acid, folate and quinone biosynthesis in Bacillus subtilis and Escherichia coli. The inhibition of the EPSP synthase by GS depletes the cell of these metabolites, resulting in cell death. Here, we show that like the laboratory B. subtilis strains also environmental and undomesticated isolates adapt to GS by reducing herbicide uptake. Although B. subtilis possesses a GS-insensitive EPSP synthase, the enzyme is strongly inhibited by GS in the native environment. Moreover, the B. subtilis EPSP synthase mutant was only viable in rich medium containing menaquinone, indicating that the bacteria require a catalytically efficient EPSP synthase under nutrient-poor conditions. The dependency of B. subtilis on the EPSP synthase probably limits its evolvability. In contrast, E. coli rapidly acquires GS resistance by target modification. However, the evolution of a GS-resistant EPSP synthase under non-selective growth conditions indicates that GS resistance causes fitness costs. Therefore, in both model organisms, the proper function of the EPSP synthase is critical for the cellular viability. This study also revealed that the uptake systems for folate precursors, phenylalanine and tyrosine need to be identified and characterized in B. subtilis.

Functionality of the Na+-translocating NADH:quinone oxidoreductase and quinol:fumarate reductase from Prevotella bryantii inferred from homology modeling.

Members of the family Prevotellaceae are Gram-negative, obligate anaerobic bacteria found in animal and human microbiota. In Prevotella bryantii, the Na+-translocating NADH:quinone oxidoreductase (NQR) and quinol:fumarate reductase (QFR) interact using menaquinone as electron carrier, catalyzing NADH:fumarate oxidoreduction. P. bryantii NQR establishes a sodium-motive force, whereas P. bryantii QFR does not contribute to membrane energization. To elucidate the possible mode of function, we present 3D structural models of NQR and QFR from P. bryantii to predict cofactor-binding sites, electron transfer routes and interaction with substrates. Molecular docking reveals the proposed mode of menaquinone binding to the quinone site of subunit NqrB of P. bryantii NQR. A comparison of the 3D model of P. bryantii QFR with experimentally determined structures suggests alternative pathways for transmembrane proton transport in this type of QFR. Our findings are relevant for NADH-dependent succinate formation in anaerobic bacteria which operate both NQR and QFR.

Fast IMAC purification of non-tagged S100A8/A9 (calprotectin) from Homo sapiens and Sus scrofa.

S100A8/A9 (calprotectin) is a damage-associated molecular pattern molecule (DAMP) that plays a key role in the innate immune response of mammalia. S100A8/A9 is therefore widely used as a biomarker in human and veterinary medicine, but diagnostic tools for the detection of S100A8/A9 are rarely optimised for the specific organism, since the corresponding S100A8/A9 is often not available. There is need for an easy, reliable protocol for the production of recombinant, highly pure S100A8/A9 from various mammalia. Here we describe the expression and purification of recombinant human and porcine S100A8/A9 by immobilized metal affinity chromatography (IMAC), which takes advantage of the intrinsic, high-affinity binding of native un-tagged S100A8/A9 to metal ions. Highly pure S100A8/A9 is obtained by a combination of IMAC, ion exchange and size exclusion chromatographic steps. Considering the high sequence homology and conservation of the metal ion coordinating residues of S100A8/A9 metal binding sites, the protocol is presumably applicable to S100A8/A9 of various mammalia.

Intravenous Infusion of Cortisol, Adrenaline, or Noradrenaline Alters Porcine Immune Cell Numbers and Promotes Innate over Adaptive Immune Functionality.

Despite the importance of pigs (Sus scrofa domestica) in livestock production and their increasing role as a model organism for human physiology, knowledge about the porcine immune system under the influence of stress hormones is fragmentary. Exceptionally little is known about the effects of catecholamines. Therefore, the aim of this study was to examine the in vivo effects of adrenaline, noradrenaline, and cortisol on number and functionality of porcine blood immune cells. Castrated male pigs (n = 34) were treated with physiological doses of either adrenaline, noradrenaline, or cortisol via i.v. infusion for 48 h. Blood samples were collected before treatment (-24 h, -22 h, 0 h), during treatment (+2 h, +24 h, +48 h), and at 72 h postinfusion. Immune cell numbers and phagocytic activity were evaluated by flow cytometry and lymphocyte proliferation by 3H-thymidine incorporation. Total IgG and IgM Ab levels were determined via ELISA. Pigs receiving cortisol showed strongly decreased adaptive immune cell numbers and increased neutrophils, accompanied by hampered lymphocyte proliferation but increased monocyte phagocytosis. Catecholamine effects on immune cell numbers were mostly similar to cortisol in direction but smaller in intensity and duration. Lymphocyte proliferation was inhibited after 2 h of noradrenaline infusion, and both catecholamines promoted monocyte and neutrophil phagocytosis. These findings indicate a shift from adaptive to innate immunity in stressful situations. This study is the first (to our knowledge) to systematically investigate specific glucocorticoid and catecholamine actions on the porcine immune system in this level of detail and confirms many similarities to humans, thus strengthening the pig as a human model in psychoneuroimmunology.

A Sodium-Translocating Module Linking Succinate Production to Formation of Membrane Potential in Prevotella bryantii.

Ruminants such as cattle and sheep depend on the breakdown of carbohydrates from plant-based feedstuff, which is accomplished by the microbial community in the rumen. Roughly 40% of the members of the rumen microbiota belong to the family Prevotellaceae, which ferments sugars to organic acids such as acetate, propionate, and succinate. These substrates are important nutrients for the ruminant. In a metaproteome analysis of the rumen of cattle, proteins that are homologous to the Na+-translocating NADH:quinone oxidoreductase (NQR) and the quinone:fumarate reductase (QFR) were identified in different Prevotella species. Here, we show that fumarate reduction to succinate in anaerobically growing Prevotella bryantii is coupled to chemiosmotic energy conservation by a supercomplex composed of NQR and QFR. This sodium-translocating NADH:fumarate oxidoreductase (SNFR) supercomplex was enriched by blue native PAGE (BN-PAGE) and characterized by in-gel enzyme activity staining and mass spectrometry. High NADH oxidation (850 nmol min-1 mg-1), quinone reduction (490 nmol min-1 mg-1), and fumarate reduction (1,200 nmol min-1 mg-1) activities, together with high expression levels, demonstrate that SNFR represents a charge-separating unit in P. bryantii. Absorption spectroscopy of SNFR exposed to different substrates revealed intramolecular electron transfer from the flavin adenine dinucleotide (FAD) cofactor in NQR to heme b cofactors in QFR. SNFR catalyzed the stoichiometric conversion of NADH and fumarate to NAD+ and succinate. We propose that the regeneration of NAD+ in P. bryantii is intimately linked to the buildup of an electrochemical gradient which powers ATP synthesis by electron transport phosphorylation. IMPORTANCE Feeding strategies for ruminants are designed to optimize nutrient efficiency for animals and to prevent energy losses like enhanced methane production. Key to this are the fermentative reactions of the rumen microbiota, dominated by Prevotella spp. We show that succinate formation by P. bryantii is coupled to NADH oxidation and sodium gradient formation by a newly described supercomplex consisting of Na+-translocating NADH:quinone oxidoreductase (NQR) and fumarate reductase (QFR), representing the sodium-translocating NADH:fumarate oxidoreductase (SNFR) supercomplex. SNFR is the major charge-separating module, generating an electrochemical sodium gradient in P. bryantii. Our findings offer clues to the observation that use of fumarate as feed additive does not significantly increase succinate production, or decrease methanogenesis, by the microbial community in the rumen.

Na+-Coupled Respiration and Reshaping of Extracellular Polysaccharide Layer Counteract Monensin-Induced Cation Permeability in Prevotella bryantii B14.

Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B14 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P.bryantii, as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P.bryantii B14 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na+-translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P.bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.

Central Carbon Metabolism, Sodium-Motive Electron Transfer, and Ammonium Formation by the Vaginal Pathogen Prevotella bivia.

Replacement of the Lactobacillus dominated vaginal microbiome by a mixed bacterial population including Prevotella bivia is associated with bacterial vaginosis (BV). To understand the impact of P. bivia on this microbiome, its growth requirements and mode of energy production were studied. Anoxic growth with glucose depended on CO2 and resulted in succinate formation, indicating phosphoenolpyruvate carboxylation and fumarate reduction as critical steps. The reductive branch of fermentation relied on two highly active, membrane-bound enzymes, namely the quinol:fumarate reductase (QFR) and Na+-translocating NADH:quinone oxidoreductase (NQR). Both enzymes were characterized by activity measurements, in-gel fluorography, and VIS difference spectroscopy, and the Na+-dependent build-up of a transmembrane voltage was demonstrated. NQR is a potential drug target for BV treatment since it is neither found in humans nor in Lactobacillus. In P. bivia, the highly active enzymes L-asparaginase and aspartate ammonia lyase catalyze the conversion of asparagine to the electron acceptor fumarate. However, the by-product ammonium is highly toxic. It has been proposed that P. bivia depends on ammonium-utilizing Gardnerella vaginalis, another typical pathogen associated with BV, and provides key nutrients to it. The product pattern of P. bivia growing on glucose in the presence of mixed amino acids substantiates this notion.

Impact of Na+-Translocating NADH:Quinone Oxidoreductase on Iron Uptake and nqrM Expression in Vibrio cholerae.

The Na+ ion-translocating NADH:quinone oxidoreductase (NQR) from Vibrio cholerae is a membrane-bound respiratory enzyme which harbors flavins and Fe-S clusters as redox centers. The NQR is the main producer of the sodium motive force (SMF) and drives energy-dissipating processes such as flagellar rotation, substrate uptake, ATP synthesis, and cation-proton antiport. The NQR requires for its maturation, in addition to the six structural genes nqrABCDEF, a flavin attachment gene, apbE, and the nqrM gene, presumably encoding a Fe delivery protein. We here describe growth studies and quantitative real-time PCR for the V. cholerae O395N1 wild-type (wt) strain and its mutant Δnqr and ΔubiC strains, impaired in respiration. In a comparative proteome analysis, FeoB, the membrane subunit of the uptake system for Fe2+ (Feo), was increased in V. choleraeΔnqr In this study, the upregulation was confirmed on the mRNA level and resulted in improved growth rates of V. choleraeΔnqr with Fe2+ as an iron source. We studied the expression of feoB on other respiratory enzyme deletion mutants such as the ΔubiC mutant to determine whether iron transport is specific to the absence of NQR resulting from impaired respiration. We show that the nqr operon comprises, in addition to the structural nqrABCDEF genes, the downstream apbE and nqrM genes on the same operon and demonstrate induction of the nqr operon by iron in V. cholerae wt. In contrast, expression of the nqrM gene in V. choleraeΔnqr is repressed by iron. The lack of functional NQR has a strong impact on iron homeostasis in V. cholerae and demonstrates that central respiratory metabolism is interwoven with iron uptake and regulation.IMPORTANCE Investigating strategies of iron acquisition, storage, and delivery in Vibrio cholerae is a prerequisite to understand how this pathogen thrives in hostile, iron-limited environments such as the human host. In addition to highlighting the maturation of the respiratory complex NQR, this study points out the influence of NQR on iron metabolism, thereby making it a potential drug target for antibiotics.

Anoxic cell rupture of Prevotella bryantii by high-pressure homogenization protects the Na+-translocating NADH:quinone oxidoreductase from oxidative damage.

Respiratory NADH oxidation in the rumen bacterium Prevotella bryantii is catalyzed by the Na+-translocating NADH:quinone oxidoreductase (NQR). A method for cell disruption and membrane isolation of P. bryantii under anoxic conditions using the EmulisFlex-C3 homogenizer is described. We compared NQR activity and protein yield after oxic and anoxic cell disruption by the EmulsiFlex, by ultrasonication, and by glass beads treatment. With an overall membrane protein yield of 50 mg L-1 culture and a NADH oxidation activity of 0.8 µmol min-1 mg-1, the EmulsiFlex was the most efficient method. Anoxic preparation yielded fourfold higher NQR activity compared to oxic preparation. P. bryantii lacks genes coding for superoxide dismutases and cell extracts do not exhibit superoxide dismutase activity. We propose that inactivation of NQR during oxic cell rupture is caused by superoxide, which accumulates in P. bryantii extracts exposed to air. Anoxic cell rupture is indispensable for the preparation of redox-active proteins and enzymes such as NQR from P. bryantii.

Structure of the V. cholerae Na+-pumping NADH:quinone oxidoreductase.

NADH oxidation in the respiratory chain is coupled to ion translocation across the membrane to build up an electrochemical gradient. The sodium-translocating NADH:quinone oxidoreductase (Na(+)-NQR), a membrane protein complex widespread among pathogenic bacteria, consists of six subunits, NqrA, B, C, D, E and F. To our knowledge, no structural information on the Na(+)-NQR complex has been available until now. Here we present the crystal structure of the Na(+)-NQR complex at 3.5 Å resolution. The arrangement of cofactors both at the cytoplasmic and the periplasmic side of the complex, together with a hitherto unknown iron centre in the midst of the membrane-embedded part, reveals an electron transfer pathway from the NADH-oxidizing cytoplasmic NqrF subunit across the membrane to the periplasmic NqrC, and back to the quinone reduction site on NqrA located in the cytoplasm. A sodium channel was localized in subunit NqrB, which represents the largest membrane subunit of the Na(+)-NQR and is structurally related to urea and ammonia transporters. On the basis of the structure we propose a mechanism of redox-driven Na(+) translocation where the change in redox state of the flavin mononucleotide cofactor in NqrB triggers the transport of Na(+) through the observed channel.

Respiratory Membrane Protein Complexes Convert Chemical Energy.

The invention of a biological membrane which is used as energy storage system to drive the metabolism of a primordial, unicellular organism represents a key event in the evolution of life. The innovative, underlying principle of this key event is respiration. In respiration, a lipid bilayer with insulating properties is chosen as the site for catalysis of an exergonic redox reaction converting substrates offered from the environment, using the liberated Gibbs free energy (ΔG) for the build-up of an electrochemical H+ (proton motive force, PMF) or Na+ gradient (sodium motive force, SMF) across the lipid bilayer. Very frequently , several redox reactions are performed in a consecutive manner, with the first reaction delivering a product which is used as substrate for the second redox reaction, resulting in a respiratory chain. From today's perspective, the (mostly) unicellular bacteria and archaea seem to be much simpler and less evolved when compared to multicellular eukaryotes. However, they are overwhelmingly complex with regard to the various respiratory chains which permit survival in very different habitats of our planet, utilizing a plethora of substances to drive metabolism. This includes nitrogen, sulfur and carbon compounds which are oxidized or reduced by specialized, respiratory enzymes of bacteria and archaea which lie at the heart of the geochemical N, S and C-cycles. This chapter gives an overview of general principles of microbial respiration considering thermodynamic aspects, chemical reactions and kinetic restraints. The respiratory chains of Escherichia coli and Vibrio cholerae are discussed as models for PMF- versus SMF-generating processes, respectively. We introduce main redox cofactors of microbial respiratory enzymes, and the concept of intra-and interelectron transfer. Since oxygen is an electron acceptor used by many respiratory chains, the formation and removal of toxic oxygen radicals is described. Promising directions of future research are respiratory enzymes as novel bacterial targets, and biotechnological applications relying on respiratory complexes.

Phytate degradation, intestinal microbiota, microbial metabolites and immune values are changed in growing pigs fed diets with varying calcium-phosphorus concentration and fermentable substrates.

The present study assessed effects of diets containing varying calcium-phosphorus (CaP) concentration and fermentable substrates on digestibility of diets, intestinal microbiota and immune system using 32 crossbred pigs (initial BW 54.7 kg). In a 2 × 2 factorial arrangement, pigs were fed either a corn-soybean meal (CSB) or corn-field pea (CFP) diet with either low [-] (4.4 g Ca/kg; 4.2 g total P/kg) or high [+] (8.3 g Ca/kg; 7.5 g total P/kg; supplemented with monocalcium phosphate) CaP content for a period of 9 weeks. In week 8, blood samples were taken, and at the end of the trial, all pigs were euthanized to collect digesta and mesenteric lymphoid tissue. Apparent total tract digestibility (ATTD) of P was greater (p < 0.05) for pigs fed the CaP+ and CFP diets than CaP- and CSB diets. The myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP6 ) concentration in jejunal digesta was higher (p < 0.05) for CaP+ than in CaP- fed pigs. In addition, caecal and faecal InsP5 isomer concentration were greater (p < 0.05) for CSB than CFP diets. In the caecum, gene copy numbers of saccharolytic bacteria, such as Eubacterium rectale and Roseburia spp., as well as SCFA concentration were higher (p < 0.05) for CaP+ than CaP- diets. In particular, innate immune cell numbers, such as natural killer cells, dendritic cells, monocytes and neutrophils, were greater (p < 0.05) for CaP+ than CaP- fed pigs. Diets high in CaP resulted in higher abundance of potential beneficial bacteria and might promote the first line of defence enhancing the activation of the cellular adaptive immune response, thereby possibly decreasing the risk for intestinal disturbances. These results strongly suggest that both, CaP supply and dietary ingredients differing in fermentability, may beneficially affect gut health through increase in SCFA-producing bacteria and/or bacteria with anti-inflammatory properties.

Metabolic Reprogramming of Vibrio cholerae Impaired in Respiratory NADH Oxidation Is Accompanied by Increased Copper Sensitivity.

The electrogenic, sodium ion-translocating NADH:quinone oxidoreductase (NQR) from Vibrio cholerae is frequent in pathogenic bacteria and a potential target for antibiotics. NQR couples the oxidation of NADH to the formation of a sodium motive force (SMF) and therefore drives important processes, such as flagellar rotation, substrate uptake, and energy-dissipating cation-proton antiport. We performed a quantitative proteome analysis of V. cholerae O395N1 compared to its variant lacking the NQR using minimal medium with glucose as the carbon source. We found 84 proteins (regulation factor of ≥2) to be changed in abundance. The loss of NQR resulted in a decrease in the abundance of enzymes of the oxidative branch of the tricarboxylic acid (TCA) cycle and an increase in abundance of virulence factors AcfC and TcpA. Most unexpected, the copper resistance proteins CopA, CopG, and CueR were decreased in the nqr deletion strain. As a consequence, the mutant exhibited diminished resistance to copper compared to the reference strain, as confirmed in growth studies using either glucose or mixed amino acids as carbon sources. We propose that the observed adaptations of the nqr deletion strain represent a coordinated response which counteracts a drop in transmembrane voltage that challenges V. cholerae in its different habitats.IMPORTANCE The importance of the central metabolism for bacterial virulence has raised interest in studying catabolic enzymes not present in the host, such as NQR, as putative targets for antibiotics. Vibrio cholerae lacking the NQR, which is studied here, is a model to estimate the impact of specific NQR inhibitors on the phenotype of a pathogen. Our comparative proteomic study provides a framework to evaluate the chances of success of compounds directed against NQR with respect to their bacteriostatic or bactericidal action.

Role of the Na(+)-translocating NADH:quinone oxidoreductase in voltage generation and Na(+) extrusion in Vibrio cholerae.

For Vibrio cholerae, the coordinated import and export of Na(+) is crucial for adaptation to habitats with different osmolarities. We investigated the Na(+)-extruding branch of the sodium cycle in this human pathogen by in vivo (23)Na-NMR spectroscopy. The Na(+) extrusion activity of cells was monitored after adding glucose which stimulated respiration via the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR). In a V. cholerae deletion mutant devoid of the Na(+)-NQR encoding genes (nqrA-F), rates of respiratory Na(+) extrusion were decreased by a factor of four, but the cytoplasmic Na(+) concentration was essentially unchanged. Furthermore, the mutant was impaired in formation of transmembrane voltage (ΔΨ, inside negative) and did not grow under hypoosmotic conditions at pH8.2 or above. This growth defect could be complemented by transformation with the plasmid encoded nqr operon. In an alkaline environment, Na(+)/H(+) antiporters acidify the cytoplasm at the expense of the transmembrane voltage. It is proposed that, at alkaline pH and limiting Na(+) concentrations, the Na(+)-NQR is crucial for generation of a transmembrane voltage to drive the import of H(+) by electrogenic Na(+)/H(+) antiporters. Our study provides the basis to understand the role of the Na(+)-NQR in pathogenicity of V. cholerae and other pathogens relying on this primary Na(+) pump for respiration.

Strong pH dependence of coupling efficiency of the Na+ - translocating NADH:quinone oxidoreductase (Na+-NQR) of Vibrio cholerae.

The Na+-translocating NADH:quinone oxidoreductase (NQR) is the entry site for electrons into the respiratory chain of Vibrio cholerae, the causative agent of cholera disease. NQR couples the electron transfer from NADH to ubiquinone to the translocation of sodium ions across the membrane. We investigated the pH dependence of electron transfer and generation of a transmembrane voltage (ΔΨ) by NQR reconstituted in liposomes with Na+ or Li+ as coupling cation. ΔΨ formation was followed with the voltage-sensitive dye oxonol. With Na+, ΔΨ was barely influenced by pH (6.5-8.5), while Q reduction activity exhibited a maximum at pH 7.5-8.0. With Li+, ΔΨ was generally lower, and the pH profile of electron transfer activity did not reveal a pronounced maximum. We conclude that the coupling efficiency of NQR is influenced by the nature of the transported cation, and by the concentration of protons. The 3D structure of NQR reveals a transmembrane channel in subunit NqrB. It is proposed that partial uncoupling of the NQR observed with the smaller Li+, or with Na+ at pH 7.5-8.0, is caused by the backflow of the coupling cation through the channel in NqrB.

A miniaturized assay for kinetic characterization of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae.

We demonstrate the miniaturization of an enzymatic assay for the determination of NADH oxidation and quinone reduction by the Na+ -translocating NADH quinone oxidoreductase (NQR) in the 96-well plate format. The assay is based on the spectrophotometric detection of NADH consumption and quinol formation. We validated the new method with known inhibitors of the NQR and optimized conditions for high-throughput screening as demonstrated by excellent Z-factors well above the accepted threshold (≥0.5). Overall, the method allows the screening and identification of potential inhibitors of the NQR, and rapid characterization of NQR variants obtained by site-specific mutagenesis.

The Na+-Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae.

UNLABELLED: We searched for a source of reactive oxygen species (ROS) in the cytoplasm of the human pathogen Vibrio cholerae and addressed the mechanism of ROS formation using the dye 2',7'-dichlorofluorescein diacetate (DCFH-DA) in respiring cells. By comparing V. cholerae strains with or without active Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), this respiratory sodium ion redox pump was identified as a producer of ROS in vivo The amount of cytoplasmic ROS detected in V. cholerae cells producing variants of Na(+)-NQR correlated well with rates of superoxide formation by the corresponding membrane fractions. Membranes from wild-type V. cholerae showed increased superoxide production activity (9.8 ± 0.6 µmol superoxide min(-1) mg(-1) membrane protein) compared to membranes from the mutant lacking Na(+)-NQR (0.18 ± 0.01 µmol min(-1) mg(-1)). Overexpression of plasmid-encoded Na(+)-NQR in the nqr deletion strain resulted in a drastic increase in the formation of superoxide (42.6 ± 2.8 µmol min(-1) mg(-1)). By analyzing a variant of Na(+)-NQR devoid of quinone reduction activity, we identified the reduced flavin adenine dinucleotide (FAD) cofactor of cytoplasmic NqrF subunit as the site for intracellular superoxide formation in V. cholerae The impact of superoxide formation by the Na(+)-NQR on the virulence of V. cholerae is discussed. IMPORTANCE: In several studies, it was demonstrated that the Na(+)-NQR in V. cholerae affects virulence in a yet unknown manner. We identified the reduced FAD cofactor in the NADH-oxidizing NqrF subunit of the Na(+)-NQR as the site of superoxide formation in the cytoplasm of V. cholerae Our study provides the framework to understand how reactive oxygen species formed during respiration could participate in the regulated expression of virulence factors during the transition from aerobic to microaerophilic (intestinal) habitats. This hypothesis may turn out to be right for many other pathogens which, like V. cholerae, depend on the Na(+)-NQR as the sole electrogenic NADH dehydrogenase.

The Staphylococcus aureus NuoL-like protein MpsA contributes to the generation of membrane potential.

In aerobic microorganisms, the entry point of respiratory electron transfer is represented by the NADH:quinone oxidoreductase. The enzyme couples the oxidation of NADH with the reduction of quinone. In the type 1 NADH:quinone oxidoreductase (Ndh1), this reaction is accompanied by the translocation of cations, such as H(+) or Na(+). In Escherichia coli, cation translocation is accomplished by the subunit NuoL, thus generating membrane potential (Δψ). Some microorganisms achieve NADH oxidation by the alternative, nonelectrogenic type 2 NADH:quinone oxidoreductase (Ndh2), which is not cation translocating. Since these enzymes had not been described in Staphylococcus aureus, the goal of this study was to identify proteins operating in the NADH:quinone segment of its respiratory chain. We demonstrated that Ndh2 represents a NADH:quinone oxidoreductase in S. aureus. Additionally, we identified a hypothetical protein in S. aureus showing sequence similarity to the proton-translocating subunit NuoL of complex I in E. coli: the NuoL-like protein MpsA. Mutants with deletion of the nuoL-like gene mpsA and its corresponding operon, mpsABC (mps for membrane potential-generating system), exhibited a small-colony-variant-like phenotype and were severely affected in Δψ and oxygen consumption rates. The MpsABC proteins did not confer NADH oxidation activity. Using an Na(+)/H(+) antiporter-deficient E. coli strain, we could show that MpsABC constitute a cation-translocating system capable of Na(+) transport. Our study demonstrates that MpsABC represent an important functional system of the respiratory chain of S. aureus that acts as an electrogenic unit responsible for the generation of Δψ.