Browsing by Person "Schleicher, Lena"

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Central carbon metabolism, sodium-motive electron ransfer, and ammonium formation by the vaginal pathogen Prevotella bivia
(2021) Schleicher, Lena; Herdan, Sebastian; Fritz, Günter; Trautmann, Andrej; Seifert, Jana; Steuber, Julia
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.
Energy conservation in anaerobic Prevotella bryantii and Prevotella bivia : the role of membrane bound electron transfer complexes
(2022) Schleicher, Lena; Fritz-Steuber, Julia
Members of the family Prevotellaceae are Gram-negative, obligate anaerobic bacteria found in animal and human microbiomes, where they participate in the degradation of carbohydrates and peptides. Some Prevotella species are also opportunistic pathogens. In this study, growth requirements and central catabolic reactions of two different Prevotella strains were characterized. First, the energy conservation by Prevotella bryantii was analyzed. P. bryantii is a dominant species in the ruminal microbiome. It was demonstrated, that P. bryantii ferments glucose mainly to acetate and succinate. Furthermore, enzymatic and biochemical studies revealed that P. bryantii membranes harbor fully functional Na+ -translocating NADH:quinone oxidoreductase and quinol:fumarate reductase. It was shown, that electron transfer between these two enzymes occurs in native membranes. The enzymatic activities increased significantly by anoxic membrane preparations. Electron transfer in membrane vesicles was coupled to the build-up of a sodium motive force in P. bryantii. A respiratory chain composed of NQR and QFR in P. bryantii was proposed, which links succinate formation to NADH oxidation and SMF formation. Thus, P. bryantii does not rely solely on substrate-level phosphorylation for energy conservation, but gains additional energy utilizing the Na+-pump, NQR. This increases the overall yield of ATP per consumed glucose molecule. By gel electrophoresis and size exclusion chromatography, the existence of a supercomplex composed of NQR and QFR in P. bryantii membranes was demonstrated, which operates as sodium-translocating NADH:fumarate oxidoreductase. The understanding of the catabolic reactions in the rumen by the ruminal microbiota is important for the optimal nutrition of the ruminant. Our results indicate that P. bryantii plays an important role in the ruminal microbiota. P. bryantii extrudes mainly acetate and succinate as fermentative end-products into the rumen. The latter can be used by other organisms of the ruminal microbiome to metabolize propionate, which is an important nutrient for the ruminant since it enters the pathway of gluconeogenesis, yielding glucose. Prevotella bivia is considered to act as causative agent of human bacterial vaginosis. Growth of P. bivia on glucose was dependent on CO2 and resulted in the production of succinate, malate and acetate. With the help of optical spectroscopy and enzymatic measurements, the presence and activity of NQR and QFR in P. bivia were demonstrated. Electron transfer in membrane vesicles of P. bivia resulted in the build-up of a SMF. Similar to P. bryantii, P. bivia operates NQR and QFR for energy conservation in its membrane, resulting in succinate formation and SMF generation. P. bivia also exhibits high L-asparaginase and aspartate ammonia lyase activities in vitro, catalyzing the conversion of L-asparagine to fumarate and NH4+. These results were confirmed in vivo by growth experiments. Additional L-asparagine in the growth medium led to an elevated production of NH4+ and succinate from fumarate obtained during degradation of L-asparagine. At the same time an inhibitory effect of NH4+ on growth of P. bivia was observed. It is proposed, that amino acid degradation by P. bivia in microbial consortia associated with BV depends on the consumption of ammonium by Gardnerella vaginalis, another typical pathogen found in BV. At the same time, G. vaginalis could provide L-asparagine to P. bivia, strengthening their symbiotic relationship and triggering BV
Na+-coupled respiration and reshaping of extracellular polysaccharide layer counteract monensin-induced cation permeability in Prevotella bryantii B14
(2021) Trautmann, Andrej; Schleicher, Lena; Pfirrmann, Jana; Boldt, Christin; Steuber, Julia; Seifert, Jana
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 shift towards succinate‐producing Prevotella in the ruminal microbiome challenged with monensin
(2022) Trautmann, Andrej; Schleicher, Lena; Koch, Ariane; Günther, Johannes; Steuber, Julia; Seifert, Jana
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.