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 a 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 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.

Atividade antimicrobiana in vitro de uma combinação de óleos vegetais de caju e mamona e de óleos essenciais de cravo, eugenol, timol e vanilina contra bactérias Gram-negativas e Gram-positivas no rúmen de bovinos

A preocupação pública com o uso rotineiro de antibióticos e ionóforos na dieta de ruminantes aumentou devido ao surgimento de bactérias resistentes aos antibióticos que podem representar riscos à saúde humana. Assim, esforços têm sido empregados para o desenvolvimento de compostos alternativos para substituir antibióticos e ionóforos na dieta de ruminantes. Este estudo foi realizado para avaliar os efeitos in vitro de uma mistura contendo óleos vegetais de caju e mamona e óleos essenciais de cravo-da-índia, eugenol, timol e vanilina sobre a atividade das bactérias Gram-negativas e Gram-positivas presentes no rúmen. O experimento foi elaborado de forma que cada bactéria fosse exposta às doses de 1,5, 3, 5 e 6,0 mg/mL da mistura, com seis repetições. As bactérias foram cultivadas em meio M2 de Hobson em tubos Hungate. A atividade antimicrobiana foi avaliada em espectrofotômetro a 600 nm. As leituras foram realizadas 0, 8, 12 e 24 horas após a inoculação a 39º C. As quatro concentrações (1,5, 3,0, 4,5 e 6,0 mg/mL) da mistura de óleos vegetais e essenciais inibiram o crescimento da Prevotella albensis, Prevotella bryantii, Prevotella ruminicola e Anaerovibrio lipolyticus 8 e 12 horas após a incubação. Para Ruminococcus albus e Ruminococcus flavefaciens a adição da mistura de óleos essenciais nas concentrações de 3,0 e 4,5 mg/mL resultou em maior impacto na dinâmica de crescimento, com redução na densidade óptica após 12 h de incubação. Em conclusão, os resultados mostram que a ação combinada dos óleos vegetais e essenciais influencia o crescimento de bactérias gram-negativas e gram-positivas e pode ser usado como um modulador ruminal. Deste modo, o estudo contribui com novas informações sobre a ação combinada dos óleos vegetais e essenciais como agentes antimicrobianos na produção de ruminantes.

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.

PSXI-19 Levels of a blend of clove, castor and cashew oils and microencapsulated active ingredients (eugenol, thymol and vanillin) against some ruminal Prevotella

This study was conducted to evaluate the effect of a blend of vegetable oils and active ingredients (Ruminatus® - [email protected]) on ruminal population density of Prevotella albensis, Prevotella bryantii and Prevotella ruminicola. The blend contained clove (Eugenia caryophyllata), castor (Ricinus communis) and cashew (Anacardium occidentale) oils and microencapsulated active ingredients (eugenol, thymol and vanillin). The experiments were designed using each bacteria exposed to doses of 1.5; 3.0; 4.5 and 6.0 mg/ml of the product and having six replicates. Bacteria were cultivated on Hobson’s M2 medium in Hungate tubes. Cultures cultivated without the addition of any oils were considered as control. Bacteria were cultivated overnight and freshly inoculated into new tubes containing medium. The antimicrobial activity was evaluated using a Spectrophotometer Evolution 201 UV-visible (Thermo Scientific) at 600 nm. Readings were performed at 0, 8, 12 and 24 hours after inoculation. Data were analyzed using the linear mixed models and the NLME Procedure in R software. The use of the blend reduced (P < 0.05) the population density of P. albensis and P. bryantii in comparison to the control treatment. P. ruminicola population only decreased (P < 0.05) when 3.0 mg/ml of the blend was used. Growth impairment of Prevotellaceae prevent excessive degradation of dietary protein, increasing protein digestibility. In addition, there is a reduction on ammonia synthesis, the end product of proteolysis, which is excreted in animal waste. Therefore, the combined action of clove, castor and castor essential oils and microencapsulated active ingredients influences the growth of Gram-negative bacteria and can be used as a rumen modulator.