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

Abstract

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 rumen microbiota belong to the family of Prevotellaceae which ferment sugars to organic acids such as acetate, propionate as well as 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 S odium-translocating N ADH: F umarate oxido R eductase (SNFR) supercomplex was enriched by 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 (1200 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 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 build-up 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 sp. 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 S odium-translocating N ADH: F umarate oxido R eductase (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.

The enzyme Rnf completes the pathway for forming propionate during fermentation inPrevotella

“…5B). The membrane-bound proteins Nqr (EC 7.2.1.1) and fumarate reductase (EC 1.3.5.1) in our bacteria have also been detected in Prevotella bryantii B 1 4 (28), where they have been characterized. Together, these enzymes form a pathway where Rnf is needed to regenerate NAD red and Fd ox .…”

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

“…This raised the question for the redox enzyme providing quinol to QFR. The related P. bryantii operates the Na + -translocating NADH:quinone oxidoreductase (NQR), feeding redox equivalents from NADH to the quinone pool [ 14 , 21 ]. The NQR is a membrane-bound protein complex composed of six subunits (NqrABCDEF) harboring one FAD, two iron–sulfur centers, one riboflavin, and two covalently bound FMNs [ 22 , 23 ].…”

“…The molecular mass of NqrC’ was 25.38 kDa. Proteolysis of proteins separated by SDS-PAGE, followed by mass spectrometric analysis of the peptides, was performed as described previously [ 21 ].…”

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

“…Proteins contributing to glycolysis were also lowered, meaning that either a switch to hibernation or an alternative energy generation via anaerobic respiration is made as described for P. bryantii B 1 4 [44]. Reduced glycolysis induced by monensin was also described in studies with P. bryantii [43] and Streptococcus faecalis [45], which reveal a reduction of the whole ruminal carbohydrate degenerative system.…”

“…Prevotella was able to consolidate its ecological niche within the monensin environment and was able to utilize the provided nutrients for acetate and succinate biosynthesis. In P. bryantii B 1 4, succinate is the product of a respiratory supercomplex which oxidizes NADH and reduces fumarate under formation of an electrochemical Na + ‐gradient [44]. Furthermore, less sugar is assimilated in Prevotella under monensin [43], which point to an increased metabolic shift to succinate production in order to maintain its concentration.…”