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Institut für Nutztierwissenschaften

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    Adaptations of Prevotella bryantii B14 to short-chain fatty acids and monensin exposure
    (2023) Trautmann, Andrej; Seifert, Jana
    The rumen microbiome constitutes a complex ecosystem including a vast diversity of organisms that produce and consume short-chain fatty acids (SCFAs). It is of great interest to analyze these activities as they are of benefit for both, the microbiome and the host. This dissertation aims to display the proteome and metabolome of the predominant ruminal representative Prevotella bryantii B14 in presence of various SCFA and under exposure of the antibiotic monensin in pure and mixed culture (in vitro). Due to the strong contributing abundance of Prevotellaceae in the rumen microbiome, the representative P. bryantii B14 (DSM 11371) was chosen to investigate biochemical factors for the success of withstanding monensin and the impact of SCFA on their growth. The current work is composed of two effective publications. The formatting was aligned to the dissertation. The first publication, studying the supplementation of various SCFAs, showed SCFAs as growth promoting but not essential for P. bryantii B14. Pure cultures of P. bryantii B14 were grown in Hungate tubes under anaerobic conditions. Gas chromatography time of flight mass spectrometry (GC-ToF MS) was used to quantify long-chain fatty acid (LCFA) profiles of P. bryantii B14. Proteins of P. bryantii B14 were identified and quantified by using a mass spectrometry-based, label-free approach. Different growth behavior was observed depending on the supplemented SCFA. An implementation of SCFAs on LCFAs and the composition on membrane proteins became evident. Supplementing P. bryantii B14 with branched-chain fatty acids (BCFAs), in particular isovaleric acid, showed an increase of the 3-IPM pathway, which is part of the branched-chain amino acid (BCAA) metabolism. Findings point out that the structure similarity of isovaleric acid and valine is most likely enhancing the conversion of BCFA into BCAA. The required set of enzymes of the BCAA metabolism supported this perspective. The ionophore monensin has antibiotic properties which are used in cattle fattening but also for treating ketosis and acidosis in ruminants. In the second publication, P. bryantii B14 was exposed to different concentrations of monensin (0, 10, 20 and 50 uM) and to different exposure times (9, 24, 48 and 72 h) with and without monensin. Growth behavior, glucose and intracellular sodium concentration were determined. Proteins were analyzed by label-free quantification method using the same method as in the previous mentioned experiment. Fluorescence microscopy was used to observe extracellular polysaccharides (EPS) of P. bryantii B14. A progressing monensin exposure triggered disconnection between P. bryantii B14 cells to the sacrificial EPS layer by increasing its number and amount of carbohydrate active enzymes (CAZymes). Simultaneously, an increase of extracellular glucose was monitored. Reduction of intracellular sodium was likely to be performed by increasing the abundance of ion-transporters and an increased activity of Na+-translocating NADH:quinone oxidoreductase under monensin supplementation. The role of monensin supplemented Prevotella in a mixed culture of the rumen microbiome was described. Extracted rumen fluid from cows was incubated anaerobically by using the rumen simulation technique (Rusitec). Proteomics of the solid phase was applied by using a similar approach as in the previous related studies. Metabolomics of the liquid phase from the Rusitec content was performed by using 1H-nuclear magnetic resonance (NMR) spectroscopy. Further parameters such as pH, gas and methane production were monitored over time. The experiment was constituted out of three phases starting with an adaptation phase of 7 days. A subsequent treatment phase followed, where monensin was supplemented via the daily introduced total mixed ration (TMR) for further 7 days. The elution phase was the final phase when monensin supplementation was stopped and monitoring was continued for further 3 days. Metabolomics and proteomics showed that members of the genus Prevotella remained most abundant under monensin supplementation. Furthermore, shifting the ruminal metabolism to an increased production of propionate by shifting the metabolism of Prevotella sp. to an enhanced succinate production. The current work shows the impact of SCFAs on various metabolic functions of P. bryantii B14. Diverse defence mechanisms of Prevotella sp., in particular P. bryantii B14, were shown to avoid the antibiotic effects of monensin.