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.

Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H2-Consuming and H2-Producing Reactions in Escherichia coli

Escherichia coliuptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H2to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochromebmembrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H2evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanidem-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H2evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H2-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H2oxidation or H2evolution in whole cells, reversible H2-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H2evolution mode. Our findings suggest that Hyd-2 switches between H2-consuming and H2-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H2evolution from glycerol requires reverse electron transport.

Shewanella spp. Use Acetate as an Electron Donor for Denitrification but Not Ferric Iron or Fumarate Reduction

ABSTRACTLactate but not acetate oxidation was reported to support electron acceptor reduction byShewanellaspp. under anoxic conditions. We demonstrate that the denitrifiersShewanella loihicastrain PV-4 andShewanella denitrificansOS217 utilize acetate as an electron donor for denitrification but not for fumarate or ferric iron reduction.

Genetic Characterization of a Single Bifunctional Enzyme for Fumarate Reduction and Succinate Oxidation in Geobacter sulfurreducens and Engineering of Fumarate Reduction in Geobacter metallireducens

ABSTRACT The mechanism of fumarate reduction in Geobacter sulfurreducens was investigated. The genome contained genes encoding a heterotrimeric fumarate reductase, FrdCAB, with homology to the fumarate reductase of Wolinella succinogenes and the succinate dehydrogenase of Bacillus subtilis. Mutation of the putative catalytic subunit of the enzyme resulted in a strain that lacked fumarate reductase activity and was unable to grow with fumarate as the terminal electron acceptor. The mutant strain also lacked succinate dehydrogenase activity and did not grow with acetate as the electron donor and Fe(III) as the electron acceptor. The mutant strain could grow with acetate as the electron donor and Fe(III) as the electron acceptor if fumarate was provided to alleviate the need for succinate dehydrogenase activity in the tricarboxylic acid cycle. The growth rate of the mutant strain under these conditions was faster and the cell yields were higher than for wild type grown under conditions requiring succinate dehydrogenase activity, suggesting that the succinate dehydrogenase reaction consumes energy. An orthologous frdCAB operon was present in Geobacter metallireducens, which cannot grow with fumarate as the terminal electron acceptor. When a putative dicarboxylic acid transporter from G. sulfurreducens was expressed in G. metallireducens, growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms, G. sulfurreducens and possibly G. metallireducens use the same enzyme for both fumarate reduction and succinate oxidation in vivo.

Preferential Reduction of Fe(III) over Fumarate by Geobacter sulfurreducens

ABSTRACT The presence of Fe(III), but not that of Fe(II), resulted in ca. 20-fold-lower levels of mRNA for fumarate reductase, inhibiting fumarate reduction and favoring utilization of fumarate as an electron donor in chemostat cultures of Geobacter sulfurreducens, despite the fact that growth yield with fumarate was 3-fold higher than with Fe(III).