Dataset Information

Occurrence and Function of the Na+-Translocating NADH:Quinone Oxidoreductase in Prevotella spp.

ABSTRACT: Strictly anaerobic Prevotella spp. are characterized by their vast metabolic potential. As members of the Prevotellaceae family, they represent the most abundant organisms in the rumen and are typically found in monogastrics such as pigs and humans. Within their largely anoxic habitats, these bacteria are considered to rely primarily on fermentation for energy conservation. A recent study of the rumen microbiome identified multiple subunits of the Na+-translocating NADH:quinone oxidoreductase (NQR) belonging to different Prevotella spp. Commonly, the NQR is associated with biochemical energy generation by respiration. The existence of this Na+ pump in Prevotella spp. may indicate an important role for electrochemical Na+ gradients in their anaerobic metabolism. However, detailed information about the potential activity of the NQR in Prevotella spp. is not available. Here, the presence of a functioning NQR in the strictly anaerobic model organism P. bryantii B14 was verified by conducting mass spectrometric, biochemical, and kinetic experiments. Our findings propose that P. bryantii B14 and other Prevotella spp. retrieved from the rumen operate a respiratory NQR together with a fumarate reductase which suggests that these ruminal bacteria utilize a sodium motive force generated during respiratory NADH:fumarate oxidoreduction.

SUBMITTER: Deusch S

PROVIDER: S-EPMC6560451 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Occurrence and Function of the Na+-Translocating NADH:Quinone Oxidoreductase in Prevotella spp.

Deusch Simon S Bok Eva E Schleicher Lena L Seifert Jana J Steuber Julia J

Microorganisms 20190427 5

Strictly anaerobic Prevotella spp. are characterized by their vast metabolic potential. As members of the Prevotellaceae family, they represent the most abundant organisms in the rumen and are typically found in monogastrics such as pigs and humans. Within their largely anoxic habitats, these bacteria are considered to rely primarily on fermentation for energy conservation. A recent study of the rumen microbiome identified multiple subunits of the Na+-translocating NADH:quinone ...[more]

PMID: 31035603

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Project description:UnlabelledNa(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) catalyzes electron transfer from NADH to ubiquinone in the bacterial respiratory chain, coupled with Na(+) translocation across the membrane. Na(+)-NQR maturation involves covalent attachment of flavin mononucleotide (FMN) residues, catalyzed by flavin transferase encoded by the nqr-associated apbE gene. Analysis of complete bacterial genomes has revealed another putative gene (duf539, here renamed nqrM) that usually follows the apbE gene and is present only in Na(+)-NQR-containing bacteria. Expression of the Vibrio harveyi nqr operon alone or with the associated apbE gene in Escherichia coli, which lacks its own Na(+)-NQR, resulted in an enzyme incapable of Na(+)-dependent NADH or reduced nicotinamide hypoxanthine dinucleotide (dNADH) oxidation. However, fully functional Na(+)-NQR was restored when these genes were coexpressed with the V. harveyi nqrM gene. Furthermore, nqrM lesions in Klebsiella pneumoniae and V. harveyi prevented production of functional Na(+)-NQR, which could be recovered by an nqrM-containing plasmid. The Na(+)-NQR complex isolated from the nqrM-deficient strain of V. harveyi lacks several subunits, indicating that nqrM is necessary for Na(+)-NQR assembly. The protein product of the nqrM gene, NqrM, contains a single putative transmembrane α-helix and four conserved Cys residues. Mutating one of these residues (Cys33 in V. harveyi NqrM) to Ser completely prevented Na(+)-NQR maturation, whereas mutating any other Cys residue only decreased the yield of the mature protein. These findings identify NqrM as the second specific maturation factor of Na(+)-NQR in proteobacteria, which is presumably involved in the delivery of Fe to form the (Cys)4[Fe] center between subunits NqrD and NqrE.ImportanceNa(+)-translocating NADH:quinone oxidoreductase complex (Na(+)-NQR) is a unique primary Na(+) pump believed to enhance the vitality of many bacteria, including important pathogens such as Vibrio cholerae, Vibrio parahaemolyticus, Haemophilus influenzae, Neisseria gonorrhoeae, Pasteurella multocida, Porphyromonas gingivalis, Enterobacter aerogenes, and Yersinia pestis. Production of Na(+)-NQR in bacteria requires Na(+)-NQR-specific maturation factors. We earlier identified one such factor (ApbE) that covalently attaches flavin residues to Na(+)-NQR. Here we identify the other protein factor, designated NqrM, and show that NqrM and ApbE suffice to produce functional Na(+)-NQR from the Vibrio harveyi nqr operon. NqrM may be involved in Fe delivery to a unique Cys4[Fe] center during Na(+)-NQR assembly. Besides highlighting Na(+)-NQR biogenesis, these findings suggest a novel drug target to combat Na(+)-NQR-containing bacteria.

Project description: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.

Project description: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 ?mol superoxide min(-1) mg(-1) membrane protein) compared to membranes from the mutant lacking Na(+)-NQR (0.18 ± 0.01 ?mol 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 ?mol 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.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.

Project description:UnlabelledThe proton-translocating NADH:quinone oxidoreductase (complex I) is a multisubunit integral membrane enzyme found in the respiratory chains of both bacteria and eukaryotic organelles. Although much research has focused on the enzyme's central role in the mitochondrial respiratory chain, comparatively little is known about its role in the diverse energetic lifestyles of different bacteria. Here, we used a phylogenomic approach to better understand the distribution of complex I across bacteria, the evolution of this enzyme, and its potential roles in shaping the physiology of different bacterial groups. By surveying 970 representative bacterial genomes, we predict complex I to be present in ~50% of bacteria. While this includes bacteria with a wide range of energetic schemes, the presence of complex I is associated with specific lifestyles, including aerobic respiration and specific types of phototrophy (bacteria with only a type II reaction center). A phylogeny of bacterial complex I revealed five main clades of enzymes whose evolution is largely congruent with the evolution of the bacterial groups that encode complex I. A notable exception includes the gammaproteobacteria, whose members encode one of two distantly related complex I enzymes predicted to participate in different types of respiratory chains (aerobic versus anaerobic). Comparative genomic analyses suggest a broad role for complex I in reoxidizing NADH produced from various catabolic reactions, including the tricarboxylic acid (TCA) cycle and fatty acid beta-oxidation. Together, these findings suggest diverse roles for complex I across bacteria and highlight the importance of this enzyme in shaping diverse physiologies across the bacterial domain.ImportanceLiving systems use conserved energy currencies, including a proton motive force (PMF), NADH, and ATP. The respiratory chain enzyme, complex I, connects these energy currencies by using NADH produced during nutrient breakdown to generate a PMF, which is subsequently used for ATP synthesis. Our goal is to better understand the role of complex I in bacteria, whose energetic diversity allows us to view its function in a range of biological contexts. We analyzed sequenced bacterial genomes to predict the presence, evolution, and function of complex I in bacteria. We identified five main classes of bacterial complex I and predict that different classes participate in different types of respiratory chains (aerobic and anaerobic). We also predict that complex I helps maintain a cellular redox state by reoxidizing NADH produced from central metabolism. Our findings suggest diverse roles for complex I in bacterial physiology, highlighting the need for future laboratory-based studies.

Dataset Information

Occurrence and Function of the Na+-Translocating NADH:Quinone Oxidoreductase in Prevotella spp.

Publications

Occurrence and Function of the Na<sup>+</sup>-Translocating NADH:Quinone Oxidoreductase in <i>Prevotella</i> spp.

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