Model-based quantification of metabolic interactions from dynamic microbial-community data.

An important challenge in microbial ecology is to infer metabolic-exchange fluxes between growing microbial species from community-level data, concerning species abundances and metabolite concentrations. Here we apply a model-based approach to integrate such experimental data and thereby infer metabolic-exchange fluxes. We designed a synthetic anaerobic co-culture of Clostridium acetobutylicum and Wolinella succinogenes that interact via interspecies hydrogen transfer and applied different environmental conditions for which we expected the metabolic-exchange rates to change. We used stoichiometric models of the metabolism of the two microorganisms that represents our current physiological understanding and found that this understanding - the model - is sufficient to infer the identity and magnitude of the metabolic-exchange fluxes and it suggested unexpected interactions. Where the model could not fit all experimental data, it indicates specific requirement for further physiological studies. We show that the nitrogen source influences the rate of interspecies hydrogen transfer in the co-culture. Additionally, the model can predict the intracellular fluxes and optimal metabolic exchange rates, which can point to engineering strategies. This study therefore offers a realistic illustration of the strengths and weaknesses of model-based integration of heterogenous data that makes inference of metabolic-exchange fluxes possible from community-level experimental data.

An unconventional anaerobic membrane protein production system based on Wolinella succinogenes.

In cases where membrane protein production attempts in more conventional Escherichia coli-based systems have failed, a solution is to resort to a system based on the nonpathogenic epsilon-proteobacterium Wolinella succinogenes. This approach has been demonstrated to be successful for structural and mechanistic analyses not only for homologous production of W. succinogenes membrane proteins but also for the heterologous production of membrane protein complexes from the human pathogens Helicobacter pylori and Campylobacter jejuni. The procedure to establish a system for the production of native and variant enzymes in W. succinogenes is presented in detail for the examples of the quinol:fumarate reductase and the SdhABE complexes of W. succinogenes. Subsequently, further projects using W. succinogenes as expression host are covered.

Role of individual nap gene cluster products in NapC-independent nitrate respiration of Wolinella succinogenes.

Bacterial nap gene clusters, encoding periplasmic nitrate reductase (NapA), are complex and diverse, and the composition of the electron transport chain donating electrons to NapA is poorly characterized in most organisms. Exceptionally, Wolinella succinogenes transfers electrons from formate via the menaquinone pool to NapA independently of a membrane-bound c-type cytochrome of the NapC family. The role of individual ORFs of the W. succinogenes napAGHBFLD gene cluster is assessed here by characterizing in-frame gene inactivation mutants. The ability of the mutants to grow by nitrate respiration was tested and their NapA content and specific nitrate reductase activity were determined. The napB and napD gene products proved to be essential for nitrate respiration, with NapD being required for the production of mature NapA. Inactivation of either subunit of the putative membrane-bound menaquinol dehydrogenase complex NapGH almost abolished growth by nitrate respiration. Substitution of the twin-arginine sequence of NapG had the same effect as absence of NapG. Phenotypes of mutants lacking either NapF or NapL suggest that both proteins function in NapA assembly and/or export. The data substantiate the current model of the composition of the NapC-independent electron transport chain as well as of NapA maturation, and indicate the presence of an alternative electron transport pathway to NapA.

The hydE gene is essential for the formation of Wolinella succinogenes NiFe-hydrogenase.

Wolinella succinogenes grows by anaerobic respiration using hydrogen gas as electron donor. The hydE gene is located on the genome downstream of the structural genes encoding the membrane-bound NiFe-hydrogenase complex (HydABC) and a putative protease (HydD) possibly involved in hydrogenase maturation. Homologs of hydE are found in the vicinity of NiFe-hydrogenase-encoding genes on the genomes of several other proteobacteria. A hydE deletion mutant of W. succinogenes does not catalyze hydrogen oxidation with various electron acceptors. The hydrogenase iron-sulfur subunit HydA is absent in mutant cells whereas the apparently processed NiFe subunit (HydB) is located exclusively in the soluble cell fraction. It is suggested that HydE is involved in the maturation and/or stability of HydA or the HydAB complex in some, but not all bacteria containing NiFe-hydrogenases.

Enzymology and bioenergetics of respiratory nitrite ammonification.

Nitrite is widely used by bacteria as an electron acceptor under anaerobic conditions. In respiratory nitrite ammonification an electrochemical proton potential across the membrane is generated by electron transport from a non-fermentable substrate like formate or H(2) to nitrite. The corresponding electron transport chain minimally comprises formate dehydrogenase or hydrogenase, a respiratory quinone and cytochrome c nitrite reductase. The catalytic subunit of the latter enzyme (NrfA) catalyzes nitrite reduction to ammonia without liberating intermediate products. This review focuses on recent progress that has been made in understanding the enzymology and bioenergetics of respiratory nitrite ammonification. High-resolution structures of NrfA proteins from different bacteria have been determined, and many nrf operons sequenced, leading to the prediction of electron transfer pathways from the quinone pool to NrfA. Furthermore, the coupled electron transport chain from formate to nitrite of Wolinella succinogenes has been reconstituted by incorporating the purified enzymes into liposomes. The NrfH protein of W. succinogenes, a tetraheme c-type cytochrome of the NapC/NirT family, forms a stable complex with NrfA in the membrane and serves in passing electrons from menaquinol to NrfA. Proteins similar to NrfH are predicted by open reading frames of several bacterial nrf gene clusters. In gamma-proteobacteria, however, NrfH is thought to be replaced by the nrfBCD gene products. The active site heme c group of NrfA proteins from different bacteria is covalently bound via the cysteine residues of a unique CXXCK motif. The lysine residue of this motif serves as an axial ligand to the heme iron thus replacing the conventional histidine residue. The attachment of the lysine-ligated heme group requires specialized proteins in W. succinogenes and Escherichia coli that are encoded by accessory nrf genes. The proteins predicted by these genes are unrelated in the two bacteria but similar to proteins of the respective conventional cytochrome c biogenesis systems.

Modification of heme c binding motifs in the small subunit (NrfH) of the Wolinella succinogenes cytochrome c nitrite reductase complex.

The two multiheme c-type cytochromes NrfH and NrfA form a membrane-bound complex that catalyzes menaquinol oxidation by nitrite during respiratory nitrite ammonification of Wolinella succinogenes. Each cysteine residue of the four NrfH heme c binding motifs was individually replaced by serine. Of the resulting eight W. succinogenes mutants, only one is able to grow by nitrite respiration although its electron transport activity from formate to nitrite is decreased. NrfH from this mutant was shown by matrix-assisted laser desorption/ionization mass spectrometry to carry four covalently bound heme groups like wild-type NrfH indicating that the cytochrome c biogenesis system II organism W. succinogenes is able to attach heme to an SXXCH motif.

Cysteine-mediated electron transfer in syntrophic acetate oxidation by cocultures of Geobacter sulfurreducens and Wolinella succinogenes.

Syntrophic cocultures of Geobacter sulfurreducens and Wolinella succinogenes oxidize acetate with nitrate as terminal electron acceptor. It has been postulated earlier that electrons are transferred in these cocultures not via hydrogen, but via a different carrier, e.g., a small c-type cytochrome that is detected in the supernatant of growing cultures. In the present study, L -cysteine, which was provided as a reducing agent, was found to mediate the electron transfer between the two partners. Low concentrations of L -cysteine or L -cystine (10-100 microM) supported syntrophic growth, and no acetate oxidation was observed in the absence of cysteine or cystine. Cell suspensions of G. sulfurreducens or coculture cell suspensions reduced cystine to cysteine, and suspensions of W. succinogenes or coculture suspensions oxidized cysteine with nitrate, as measured by the formation or depletion of free thiol groups. Added cysteine was rapidly oxidized by the coculture during growth, but the formed cystine was not entirely rereduced even under acceptor-limited conditions. The redox potential prevailing in acetate-oxidizing cocultures was -160 to -230 mV. Sulfide at low concentrations supported syntrophic growth as well and could replace cysteine. Neither growth nor acetate degradation was found with D-cysteine, homocysteine, cysteamine, 3-mercaptopropionate, dithiothreithol, thioglycolate, glutathione, coenzyme M, dimethylsulfoxide, trimethylamine- N-oxide, anthraquinone-2,6-disulfonate, or ascorbate.

Diversity and ubiquity of bacteria capable of utilizing humic substances as electron donors for anaerobic respiration.

Previous studies have demonstrated that reduced humic substances (HS) can be reoxidized by anaerobic bacteria such as Geobacter, Geothrix, and Wolinella species with a suitable electron acceptor; however, little is known of the importance of this metabolism in the environment. Recently we investigated this metabolism in a diversity of environments including marine and aquatic sediments, forest soils, and drainage ditch soils. Most-probable-number enumeration studies were performed using 2,6-anthrahydroquinone disulfonate (AHDS), an analog for reduced HS, as the electron donor with nitrate as the electron acceptor. Anaerobic organisms capable of utilizing reduced HS as an electron donor were found in all environments tested and ranged from a low of 2.31 x 10(1) in aquifer sediments to a high of 9.33 x 10(6) in lake sediments. As part of this study we isolated six novel organisms capable of anaerobic AHDS oxidation. All of the isolates coupled the oxidation of AHDS to the reduction of nitrate with acetate (0.1 mM) as the carbon source. In the absence of cells, no AHDS oxidation was apparent, and in the absence of AHDS, no cell density increase was observed. Generally, nitrate was reduced to N(2). Analysis of the AHDS and its oxidized form, 2,6-anthraquinone disulfonate (AQDS), in the medium during growth revealed that the anthraquinone was not being biodegraded as a carbon source and was simply being oxidized as an energy source. Determination of the AHDS oxidized and nitrate reduced accounted for 109% of the theoretical electron transfer. In addition to AHDS, all of these isolates could also couple the oxidation of reduced humic substances to the reduction of nitrate. No HS oxidation occurred in the absence of cells and in the absence of a suitable electron acceptor, demonstrating that these organisms were capable of utilizing natural HS as an energy source and that AHDS serves as a suitable analog for studying this metabolism. Alternative electron donors included simple volatile fatty acids such as propionate, butyrate, and valerate as well as simple organic acids such as lactate and pyruvate. Analysis of the complete sequences of the 16S rRNA genes revealed that the isolates were not closely related to each other and were phylogenetically diverse, with members in the alpha, beta, gamma, and delta subdivisions of the PROTEOBACTERIA: Most of the isolates were closely related to known genera not previously recognized for their ability to couple growth to HS oxidation, while one of the isolates represented a new genus in the delta subclass of the PROTEOBACTERIA: The results presented here demonstrate that microbial oxidation of HS is a ubiquitous metabolism in the environment. This study represents the first description of HS-oxidizing isolates and demonstrates that microorganisms capable of HS oxidation are phylogenetically diverse.

Periplasmic methacrylate reductase activity in Wolinella succinogenes.

The cell homogenate and the soluble cell fraction of Wolinella succinogenes grown with formate and fumarate catalyzed the oxidation of benzyl viologen radical by methacrylate [apparent Km=0.23 mM, Vmax=1.0 U (mg cell protein) -1] or acrylate [apparent Km=0.50 mM, Vmax=0.77 U (mg cell protein) -1]. Crotonate did not serve as an oxidant. A mutant of W. succinogenes lacking the fccABC operon was unable to catalyze methacrylate or acrylate reduction. In contrast, the inactivation of fccC alone had no effect on these activities. Methacrylate reduction by benzyl viologen radical was not catalyzed by fumarate reductase isolated from the membrane of W. succinogenes. Cells grown with formate and fumarate did not catalyze methacrylate reduction by formate, and W. succinogenes did not grow with formate and methacrylate as catabolic substrates. The results suggest that the reduction of methacrylate or acrylate by benzyl viologen radical is most likely catalyzed either by the periplasmic flavoprotein FccA or by a complex consisting of FccA and the predicted c-type cytochrome FccB. The metabolic function of the fccABC operon remains unknown.

A third crystal form of Wolinella succinogenes quinol:fumarate reductase reveals domain closure at the site of fumarate reduction.

Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone. Previously, the crystal structure of QFR from Wolinella succinogenes was determined based on two different crystal forms, and the site of fumarate binding in the flavoprotein subunit A of the enzyme was located between the FAD-binding domain and the capping domain [Lancaster, C.R.D., Kröger, A., Auer, M., & Michel, H. (1999) Nature 402, 377--385]. Here we describe the structure of W. succinogenes QFR based on a third crystal form and refined at 3.1 A resolution. Compared with the previous crystal forms, the capping domain is rotated in this structure by approximately 14 degrees relative to the FAD-binding domain. As a consequence, the topology of the dicarboxylate binding site is much more similar to those of membrane-bound and soluble fumarate reductase enzymes from other organisms than to that found in the previous crystal forms of W. succinogenes QFR. This and the effects of the replacement of Arg A301 by Glu or Lys by site-directed mutagenesis strongly support a common mechanism for fumarate reduction in this superfamily of enzymes.

Oxidation of acetate through reactions of the citric acid cycle by Geobacter sulfurreducens in pure culture and in syntrophic coculture.

Geobacter sulfurreducens strain PCA oxidized acetate to CO2 via citric acid cycle reactions during growth with acetate plus fumarate in pure culture, and with acetate plus nitrate in coculture with Wolinella succinogenes. Acetate was activated by succinyl-CoA:acetate CoA-transferase and also via acetate kinase plus phosphotransacetylase. Citrate was formed by citrate synthase. Soluble isocitrate and malate dehydrogenases NADP+ and NAD+, respectively. Oxidation of 2-oxoglutarate was measured as benzyl viologen reduction and strictly CoA-dependent; a low activity was also observed with NADP+. Succinate dehydrogenase and fumarate ductase both were membrane-bound. Succinate oxidation was coupled to NADP+ reduction whereas fumarate reduction was coupled to NADPH and NADH Coupling of succinate oxidation to NADP+ or cytochrome(s) reduction required an ATP-dependent reversed electron transport. Net ATP synthesis proceeded exclusively through electron transport phosphorylation. During fumarate reduction, both NADPH and NADH delivered reducing equivalents into the electron transport chain, which contained a menaquinone. Overall, acetate oxidation with fumarate proceeded through an open loop of citric acid cycle reactions, excluding succinate dehydrogenase, with fumarate reductase as the key reaction for electron delivery, whereas acetate oxidation in the syntrophic coculture required the complete citric acid cycle.

Essential role of Glu-C66 for menaquinol oxidation indicates transmembrane electrochemical potential generation by Wolinella succinogenes fumarate reductase.

Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalyzed by the related enzyme succinate:quinone reductase (succinate dehydrogenase). In the previously determined structure of QFR from Wolinella succinogenes, the site of fumarate reduction in the flavoprotein subunit A of the enzyme was identified, but the site of menaquinol oxidation was not. In the crystal structure, the acidic residue Glu-66 of the membrane spanning, diheme-containing subunit C lines a cavity that could be occupied by the substrate menaquinol. Here we describe that, after replacement of Glu-C66 with Gln by site-directed mutagenesis, the resulting mutant is unable to grow on fumarate and the purified enzyme lacks quinol oxidation activity. X-ray crystal structure analysis of the Glu-C66-->Gln variant enzyme at 3.1-A resolution rules out any major structural changes compared with the wild-type enzyme. The oxidation-reduction potentials of the heme groups are not significantly affected. We conclude that Glu-C66 is an essential constituent of the menaquinol oxidation site. Because Glu-C66 is oriented toward a cavity leading to the periplasm, the release of two protons on menaquinol oxidation is expected to occur to the periplasm, whereas the uptake of two protons on fumarate reduction occurs from the cytoplasm. Thus our results indicate that the reaction catalyzed by W. succinogenes QFR generates a transmembrane electrochemical potential.

Fermentative toluene degradation in anaerobic defined syntrophic cocultures.

A syntrophic coculture of a new sulfate-reducing isolate, strain TRM1, with Wolinella succinogenes degraded toluene with either fumarate or NO3- as the terminal electron acceptor. Neither strain TRM1 nor W. succinogenes could metabolise toluene under these conditions in pure culture. Syntrophic degradation was 2-3 times slower than toluene utilisation by strain TRM1 in pure culture with sulfate as electron acceptor. The culture did not produce benzoate or fatty acids like acetate or propionate in detectable amounts. An increase in biomass of the syntrophic toluene-degrading culture was shown in a growth curve with nitrate as the terminal electron acceptor. Both partner organisms were detected microscopically at the end of the growth experiment. Syntrophic degradation of toluene with W. succinogenes and fumarate as the terminal electron acceptor was also demonstrated with the iron reducer Geobacter metallireducens. The results provide the first example of a fermentative oxidation of an aromatic hydrocarbon in a defined coculture.

Identification and characterization of IS1302, a novel insertion element from Wolinella succinogenes belonging to the IS3 family.

A new insertion sequence (IS) designated IS1302 was identified in Wolinella succinogenes. IS1302 is 1,306 bp in size with 36-bp imperfect terminal inverted repeats. It contains only one open reading frame (tnpA), which encodes a putative transposase whose sequence is similar to that of transposases of various IS elements of the IS3 family. IS1302 was identified in the genome of a W. succinogenes fumarate reductase deletion mutant in which the frd operon had been replaced by the kan gene. The insertion of IS1302 occurred when the mutant was propagated in the presence of a high concentration of kanamycin. Two different target sites of IS1302 were found immediately upstream of the kan gene, where the insertion of IS1302 resulted in a duplication of 3 bp of the target DNA. Upon insertion of IS1302, new possible promoter structures of the kan gene were created, which might lead to a stimulated transcription of the kan gene and result in a selective advantage of cells containing IS1302 at one of the two target sites. Southern blot analysis suggested the presence of at least 13 copies of IS1302 in the genome of W. succinogenes. This is the first IS element discovered in W. succinogenes.

Two membrane anchors of Wolinella succinogenes hydrogenase and their function in fumarate and polysulfide respiration.

Wolinella succinogenes can grow by anaerobic respiration with fumarate or polysulfide as the terminal electron acceptor, and H2 or formate as the electron donor. A DeltahydABC mutant lacking the hydrogenase structural genes did not grow with H2 and either fumarate or polysulfide. In contrast to the wild-type strain, the mutant grown with fumarate and with formate instead of H2 did not catalyze the reduction of fumarate, polysulfide, dimethylnaphthoquinone, or benzyl viologen by H2. Growth and enzymic activities were restored upon integration of a plasmid carrying hydABC into the genome of the DeltahydABC mutant. The DeltahydABC mutant was complemented with hydABC operons modified by artificial stop codons in hydA (StopA) or at the 5'-end of hydC (StopC). The StopC mutant lacked HydC, and the hydrophobic C-terminus of HydA was missing in the hydrogenase of the StopA mutant. The two mutants catalyzed benzyl viologen reduction by H2. The enzyme activity was located in the membrane of the mutants. A mutant with both modifications (StopAC) contained the activity in the periplasm. The three mutants did not grow with H2 and either fumarate or polysulfide, and did not catalyze dimethylnaphthoquinone reduction by H2. We conclude that the same hydrogenase serves in the anaerobic respiration with fumarate and with polysulfide. HydC and the C-terminus of HydA appear to be required for both routes of electron transport and for dimethylnaphthoquinone reduction by H2. The hydrogenase is anchored in the membrane by HydC and by the C-terminus of HydA. The catalytic subunit HydB is oriented towards the periplasmic side of the membrane.

Properties of a Wolinella succinogenes mutant lacking periplasmic sulfide dehydrogenase (Sud).

A delta sud deletion mutant of Wolinella succinogenes that lacked the periplasmic sulfide dehydrogenase (Sud) was constructed using homologous recombination. The mutant grew with sulfide and fumarate, indicating that Sud was not a component of the electron transport chain that catalyzed fumarate respiration with sulfide as an electron donor. Likewise, growth with formate and either polysulfide or sulfur was not affected by the deletion. Removal of Sud from wild-type W. succinogenes by spheroplast formation did not decrease the activity of electron transport to polysulfide. The delta psr deletion mutant that lacks polysulfide reductase (Psr) grew by fumarate respiration with sulfide as an electron donor, indicating that Psr is not required for this activity.

The function of Wolinella succinogenes psr genes in electron transport with polysulphide as the terminal electron acceptor.

The membrane-integrated polysulphide reductase (Psr) of Wolinella succinogenes is part of the electron transport chain catalyzing polysulphide reduction by formate or hydrogen. The isolated enzyme catalyzes sulphide oxidation by dimethylnaphthoquinone. The two hydrophilic subunits, PsrA and PsrB of the enzyme, are encoded by genes that form an apparent operon psrABC together with a third gene. Using homologous recombination, three deletion mutants of W. succinogenes were constructed that lack psrC, psrBC or the whole psr operon. The mutants grown with formate and fumarate were fractionated, and the cell fractions were analyzed for the presence of PsrA and enzyme activity. It was concluded that: (a) polysulphide reductase is a constituent of the wild-type chain catalyzing electron transport from formate to polysulphide; (b) the gene psrC encodes a subunit that anchors the enzyme in the membrane and is required for electron transport; (c) PsrA which probably carries the substrate site, is exposed to the bacterial periplasm; (d) PsrA and PsrB are required for the activity of sulphide oxidation with 2,3-dimethyl-1,4-naphthoquinone. Surprisingly, the delta psrABC mutant could grow with formate and polysulphide. The membrane fraction of the mutant grown under these conditions contained an enzyme that replaced polysulphide reductase in electron transport, and catalyzed sulphide oxidation with 2,3-dimethyl-1,4-naphthoquinone.

DMSO respiration by the anaerobic rumen bacterium Wolinella succinogenes.

The anaerobic rumen bacterium Wolinella succinogenes was able to grow by respiration with dimethylsulphoxide (DMSO) as electron acceptor and formate or H2 as electron donors. The growth yield amounted to 6.7 g and 6.4 g dry cells/mol DMSO with formate or H2 as the donors, respectively. This suggested an ATP yield of about 0.7 mol ATP/mol DMSO. Cell homogenates and the membrane fraction contained DMSO reductase activity with a high Km (43 mM) for DMSO. The electron transport from H2 to DMSO in the membranes was inhibited by 2-(heptyl)-4-hydroxyquinoline N-oxide, indicating the participation of menaquinone. Formation of DMSO reductase activity occurred only during growth on DMSO, presence of other electron acceptors (fumarate, nitrate, nitrite, N2O, and sulphur) repressed the DMSO reductase activity. DMSO can therefore be used by W. succinogenes as an acceptor for phosphorylative electron transport, but other electron acceptors are used preferentially.