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.

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.

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.

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.

Short-Chain Fatty Acids Modulate Metabolic Pathways and Membrane Lipids in Prevotella bryantii B14.

Short-chain fatty acids (SCFAs) are bacterial products that are known to be used as energy sources in eukaryotic hosts, whereas their role in the metabolism of intestinal microbes is rarely explored. In the present study, acetic, propionic, butyric, isobutyric, valeric, and isovaleric acid, respectively, were added to a newly defined medium containing Prevotella bryantii B14 cells. After 8 h and 24 h, optical density, pH and SCFA concentrations were measured. Long-chain fatty acid (LCFA) profiles of the bacterial cells were analyzed via gas chromatography-time of flight-mass spectrometry (GC-ToF MS) and proteins were quantified using a mass spectrometry-based, label-free approach. Cultures supplemented with single SCFAs revealed different growth behavior. Structural features of the respective SCFAs were identified in the LCFA profiles, which suggests incorporation into the bacterial membranes. The proteomes of cultures supplemented with acetic and valeric acid differed by an increased abundance of outer membrane proteins. The proteome of the isovaleric acid supplementation showed an increase of proteins in the amino acid metabolism. Our findings indicate a possible interaction between SCFAs, the lipid membrane composition, the abundance of outer membrane proteins, and a modulation of branched chain amino acid biosynthesis by isovaleric acid.