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Oxidation of phosphite (HPO32-) to phosphate (HPO42-) releases electrons at a very low redox potential (E0'= -690 mV) which renders phosphite an excellent electron donor for microbial energy metabolism. To date, two pure cultures of strictly anaerobic bacteria have been isolated that run their energy metabolism on the basis of phosphite oxidation, the Gram-negative Desulfotignum phosphitoxidans (DSM 13687) and the Gram-positive Phosphitispora fastidiosa (DSM 112739). Here, we describe the key enzyme for dissimilatory phosphite oxidation in these bacteria. The enzyme catalyzed phosphite oxidation in the presence of adenosine monophosphate (AMP) to form adenosine diphosphate (ADP), with concomitant reduction of oxidized nicotinamide adenine dinucleotide (NAD+) to reduced nicotinamide adenine dinucleotide (NADH). The enzyme of P. fastidiosa was heterologously expressed in Escherichia coli. It has a molecular mass of 35.2 kDa and a high affinity for phosphite and NAD+. Its activity was enhanced more than 100-fold by addition of ADP-consuming adenylate kinase (myokinase) to a maximal activity between 30 and 80 mU x mg protein-1. A similar NAD-dependent enzyme oxidizing phosphite to phosphate with concomitant phosphorylation of AMP to ADP is found in D. phosphitoxidans, but this enzyme could not be heterologously expressed. Based on sequence analysis, these phosphite-oxidizing enzymes are related to nucleotide-diphosphate-sugar epimerases and indeed represent AMP-dependent phosphite dehydrogenases (ApdA). A reaction mechanism is proposed for this unusual type of substrate-level phosphorylation reaction.

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Phosphite is a stable phosphorus compound that, together with phosphate, made up a substantial part of the total phosphorus content of the prebiotic Earth's crust. Oxidation of phosphite to phosphate releases electrons at an unusually low redox potential (-690 mV at pH 7.0). Numerous aerobic and anaerobic bacteria use phosphite as a phosphorus source and oxidise it to phosphate for synthesis of nucleotides and other phosphorus-containing cell constituents. Only two pure cultures of strictly anaerobic bacteria have been isolated so far that use phosphite as an electron donor in their energy metabolism, the Gram-positive Phosphitispora fastidiosa and the Gram-negative Desulfotignum phosphitoxidans. The key enzyme of this metabolism is an NAD+ -dependent phosphite dehydrogenase enzyme that phosphorylates AMP to ADP. These phosphorylating phosphite dehydrogenases were found to be related to nucleoside diphosphate sugar epimerases. The produced NADH is channelled into autotrophic CO2 fixation via the Wood-Ljungdahl (CO-DH) pathway, thus allowing for nearly complete assimilation of the substrate electrons into bacterial biomass. This extremely efficient type of electron flow connects energy and carbon metabolism directly through NADH and might have been important in the early evolution of life when phosphite was easily available on Earth.

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A new strictly anaerobic bacterium, strain DYL19T, was enriched and isolated with phosphite as the sole electron donor and CO2 as a single carbon source and electron acceptor from anaerobic sewage sludge sampled at a sewage treatment plant in Constance, Germany. It is a Gram-positive, spore-forming, slightly curved, rod-shaped bacterium which oxidizes phosphite to phosphate while reducing CO2 to biomass and small amounts of acetate. Optimal growth is observed at 30 °C, pH 7.2, with a doubling time of 3 days. Beyond phosphite, no further inorganic or organic electron donor can be used, and no other electron acceptor than CO2 is reduced. Sulphate inhibits growth with phosphite and CO2. The G+C content is 45.95 mol%, and dimethylmenaquinone-7 is the only quinone detectable in the cells. On the basis of 16S rRNA gene sequence analysis and other chemotaxonomic properties, strain DYL19T is described as the type strain of a new genus and species, Phosphitispora fastidiosa gen. nov., sp. nov.

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A rhamnose-degrading bacterium, strain BoRhaAT, was isolated from profundal sediment of Lake Constance in agar dilution series with l-rhamnose as substrate and with a background lawn of Methanospirillum hungatei. The isolated strain was a motile rod that stained Gram positive. Growth was observed within a pH range of 4.0-7.5 and a temperature range of 15-30°C. Fermentation products of rhamnose or glucose were acetate, propionate, ethanol, butyrate, and 1-propanol. The G+C content was 40.6% G+C. The dominant fatty acids are C16:1ω9c, i-C13:03OH, C16:0 and C17:1ω8c with 8-21% relative abundance. Polar lipids were glycolipids, phosphatidylethanolamine, phosphoaminolipid and other lipids, of which phosphatidylethanolamine was most abundant. The sequence of the 16S rRNA gene of the new isolate matches the sequence of its closest relative Anaerosporomusa subterranea to 92.4%. A comparison of the genome with this strain showed 60.2% genome-wide average amino acid identity (AAI), comparisons with other type strains showed a maximum of 62.7% AAI. Thus, the definition of a new genus is justified for which we propose the name Pelorhabdus. For strain BoRhaAT, we propose the name Pelorhabdus rhamnosifermentans gen. nov., sp. nov., with strain BoRhaAT (DSM 111565T = JCM 39158T) as the type strain.

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The platform chemicals n-caproate and iso-butyrate can be produced by anaerobic fermentation from agro-industrial residues in a process known as microbial chain elongation. Few lactate-consuming chain-elongating species have been isolated and knowledge on their shared genetic features is still limited. Recently we isolated three novel clostridial strains (BL-3, BL-4, and BL-6) that convert lactate to n-caproate and iso-butyrate. Here, we analyzed the genetic background of lactate-based chain elongation in these isolates and other chain-elongating species by comparative genomics. The three strains produced n-caproate, n-butyrate, iso-butyrate, and acetate from lactate, with the highest proportions of n-caproate (18%) for BL-6 and of iso-butyrate (23%) for BL-4 in batch cultivation at pH 5.5. They show high genomic heterogeneity and a relatively small core-genome size. The genomes contain highly conserved genes involved in lactate oxidation, reverse ß-oxidation, hydrogen formation and either of two types of energy conservation systems (Rnf and Ech). Including genomes of another eleven experimentally validated chain-elongating strains, we found that the chain elongation-specific core-genome encodes the pathways for reverse ß-oxidation, hydrogen formation and energy conservation, while displaying substantial genome heterogeneity. Metabolic features of these isolates are important for biotechnological applications in n-caproate and iso-butyrate production.

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The critical role of bacterial biofilms in chronic human infections calls for novel anti-biofilm strategies targeting the regulation of biofilm development. However, the regulation of biofilm development is very complex and can include multiple, highly interconnected signal transduction/response pathways, which are incompletely understood. We demonstrated previously that in the opportunistic, human pathogen P. aeruginosa, the PP2C-like protein phosphatase SiaA and the di-guanylate cyclase SiaD control the formation of macroscopic cellular aggregates, a type of suspended biofilms, in response to surfactant stress. In this study, we demonstrate that the SiaABC proteins represent a signal response pathway that functions through a partner switch mechanism to control biofilm formation. We also demonstrate that SiaABCD functionality is dependent on carbon substrate availability for a variety of substrates, and that upon carbon starvation, SiaB mutants show impaired dispersal, in particular with the primary fermentation product ethanol. This suggests that carbon availability is at least one of the key environmental cues integrated by the SiaABCD system. Further, our biochemical, physiological and crystallographic data reveals that the phosphatase SiaA and its kinase counterpart SiaB balance the phosphorylation status of their target protein SiaC at threonine 68 (T68). Crystallographic analysis of the SiaA-PP2C domain shows that SiaA is present as a dimer. Dynamic modelling of SiaA with SiaC suggested that SiaA interacts strongly with phosphorylated SiaC and dissociates rapidly upon dephosphorylation of SiaC. Further, we show that the known phosphatase inhibitor fumonisin inhibits SiaA mediated phosphatase activity in vitro. In conclusion, the present work improves our understanding of how P. aeuruginosa integrates specific environmental conditions, such as carbon availability and surfactant stress, to regulate cellular aggregation and biofilm formation. With the biochemical and structural characterization of SiaA, initial data on the catalytic inhibition of SiaA, and the interaction between SiaA and SiaC, our study identifies promising targets for the development of biofilm-interference drugs to combat infections of this aggressive opportunistic pathogen.

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The anaerobic degradation of aniline was studied in the sulfate-reducing bacterium Desulfatiglans anilini. Our aim was to identify the genes and their proteins that are required for the initial activation of aniline as well as to characterize intermediates of this reaction. Aniline-induced genes were revealed by comparison of the proteomes of D. anilini grown with different substrates (aniline, 4-aminobenzoate, phenol, and benzoate). Most genes encoding proteins that were highly abundant in aniline- or 4-aminobenzoate-grown D. anilini cells but not in phenol- or benzoate-grown cells were located in the putative gene clusters ani (aniline degradation), hcr (4-hydroxybenzoyl-CoA reductase) and phe (phenol degradation). Of these putative gene clusters, only the phe gene cluster has been studied previously. Based on the differential proteome analysis, four candidate genes coding for kinase subunits and carboxylase subunits were suspected to be responsible for the initial conversion of aniline to 4-aminobenzoate. These genes were cloned and overproduced in E. coli. The recombinant proteins were obtained in inclusion bodies but could be refolded successfully. Two subunits of phenylphosphoamidate synthase and two carboxylase subunits converted aniline to 4-aminobenzoate with phenylphosphoamidate as intermediate under consumption of ATP. Only when both carboxylase subunits, one from gene cluster ani and the other from gene cluster phe, were combined, phenylphosphoamidate was converted to 4-aminobenzoate in vitro, with Mn2+, K+, and FMN as co-factors. Thus, aniline is degraded by the anaerobic bacterium D. anilini only by recruiting genes for the enzymatic machinery from different gene clusters. We conclude, that D. anilini carboxylates aniline to 4-aminobenzoate via phenylphosphoamidate as an energy rich intermediate analogous to the degradation of phenol to 4-hydroxybenzoate via phenylphosphate.

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The thermophilic acetogen Thermacetogenium phaeum uses the Wood-Ljungdahl pathway (WLP) in both directions, either for the production of acetate from various compounds or for the oxidation of acetate in syntrophic cooperation with methanogens. In this study, energy-conserving enzyme systems in T. phaeum were investigated in both metabolic directions. A gene cluster containing a membrane-bound periplasmically oriented formate dehydrogenase directly adjacent to putative menaquinone synthesis genes was identified in the genome. The protein products of these genes were identified by total proteome analysis, and menaquinone MK-7 had been found earlier as the dominant quinone in the membrane. Enzyme assays with membrane preparations and anthraquinone-2,6-disulfonate as electron acceptor verified the presence of a quinone-dependent formate dehydrogenase. A quinone-dependent methylene-THF reductase is active in the soluble fraction and in the membrane fraction. From these results we conclude a reversed electron transport system from methyl-THF oxidation to CO2 reduction yielding formate as reduced product which is transferred to the methanogenic partner. The redox potential difference between methyl-THF (Eo' = -200 mV) and formate (Eo' = -432 mV) does not allow electron transfer through syntrophic formate removal alone. We postulate that part of the ATP conserved by substrate-level phosphorylation has to be invested into the generation of a transmembrane proton gradient by ATPase. This proton gradient could drive the endergonic oxidation of methyl-THF in an enzyme reaction similar to the membrane-bound reversed electron transport system previously observed in the syntrophically butyrate-oxidizing bacterium Syntrophomonas wolfei. To balance the overall ATP budget in acetate oxidation, we postulate that acetate is activated through an ATP-independent path via aldehyde:ferredoxin oxidoreductase (AOR) and subsequent oxidation of acetaldehyde to acetyl-CoA.

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Desulfatiglans anilini is a sulfate-reducing bacterium (SRB) capable of oxidizing aniline, although growth and aniline turnover rates are slow, making it difficult to analyze the metabolism of the strain. Therefore, this study was designed to investigate the effect of sulfide on growth of D. anilini cultures, in order to improve its growth and aniline turnover rates, and study the biochemical mechanisms of sulfide inhibition. Hydrogen sulfide was found to inhibit growth of D. anilini, regardless of whether the strain was grown with aniline or phenol, and complete inhibition was observed at 20mM hydrogen sulfide. For improving the growth of D. anilini with aniline, the sulfide-consuming phototrophic bacterium Thiocapsa roseopersicina was co-cultured in a synthetic microbial community with D. anilini using a co-cultivation device that continuously removed hydrogen sulfide from the culture. The doubling time of D. anilini with aniline was 15 days in the co-cultivation device, compared to 26 days in the absence of a sulfide-oxidizing partner. Moreover, the aniline degradation rate was significantly increased by a factor of 2.66 during co-cultivation of D. anilini with T. roseopersicina. The initial carboxylation reaction during aniline degradation was measured in cell-free extracts of D. anilini with carbon dioxide (CO2) as a co-substrate in the presence of aniline and ATP. The effects of hydrogen sulfide on this aniline carboxylating system and on phenylphosphate synthase activity for phenol activation were studied, and it was concluded that hydrogen sulfide severely inhibited these enzyme activities.

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Growth of the anaerobic thermophile Thermacetogenium phaeum with methanol, ethanol, ethanolamine, and acetate was investigated in axenic cultures and in syntrophic cultures with Methanothermobacter thermautotrophicus. Microcompartment genes were identified in the T. phaeum genome, and presence of microcompartments was confirmed by transmission electron microscopy and proteome analysis. These genes were expressed only during growth with ethanolamine. Proteome data were compared after growth with all four substrates, and activities of key enzymes of the Wood-Ljungdahl pathway and of enzyme systems leading to production or degradation of acetaldehyde such as alcohol dehydrogenase, aldehyde:ferredoxin oxidoreductase, acetate kinase, and phosphate acetyltransferase were measured in cytoplasmic fractions. Accounting of fermentation stoichiometries and growth yields with all four substrates showed that ethanol and methanol oxidation follow the same stoichiometries as in Acetobacterium woodii. On the other hand, the pathways of ethanol and methanol degradations vary between both organisms. Growth yields of T. phaeum were substantially lower than reported for A. woodii. Since T. phaeum has no Rnf complex encoded in its genome, the mechanisms of ATP synthesis have to be different from those of A. woodii. In addition to the central degradation pathways also found in A. woodii, T. phaeum maintains enzyme systems that compensate for the absence of an Rnf-complex but which on the other hand cause a loss of energy. On the basis of our data, pathways of methanol and ethanol degradation in T. phaeum are discussed.

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Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is produced by plants and other phototrophs and its biodegradation is a relevant component of the biogeochemical carbon and sulfur cycles. SQ is known to be degraded by aerobic bacterial consortia in two tiers via C3-organosulfonates as transient intermediates to CO2, water and sulfate. In this study, we present a first laboratory model for anaerobic degradation of SQ by bacterial consortia in two tiers to acetate and hydrogen sulfide (H2S). For the first tier, SQ-degrading Escherichia coli K-12 was used. It catalyzes the fermentation of SQ to 2,3-dihydroxypropane-1-sulfonate (DHPS), succinate, acetate and formate, thus, a novel type of mixed-acid fermentation. It employs the characterized SQ Embden-Meyerhof-Parnas pathway, as confirmed by mutational and proteomic analyses. For the second tier, a DHPS-degrading Desulfovibrio sp. isolate from anaerobic sewage sludge was used, strain DF1. It catalyzes another novel fermentation, of the DHPS to acetate and H2S. Its DHPS desulfonation pathway was identified by differential proteomics and demonstrated by heterologously produced enzymes: DHPS is oxidized via 3-sulfolactaldehyde to 3-sulfolactate (SL) by two NAD+-dependent dehydrogenases (DhpA, SlaB); the SL is cleaved by an SL sulfite-lyase known from aerobic bacteria (SuyAB) to pyruvate and sulfite. The pyruvate is oxidized to acetate, while the sulfite is used as electron acceptor in respiration and reduced to H2S. In conclusion, anaerobic sulfidogenic SQ degradation was demonstrated as a novel link in the biogeochemical sulfur cycle. SQ is also a constituent of the green-vegetable diet of herbivores and omnivores and H2S production in the intestinal microbiome has many recognized and potential contributions to human health and disease. Hence, it is important to examine bacterial SQ degradation also in the human intestinal microbiome, in relation to H2S production, dietary conditions and human health.

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Lysine degradation has remained elusive in many organisms including Escherichia coli. Here we report catabolism of lysine to succinate in E. coli involving glutarate and L-2-hydroxyglutarate as intermediates. We show that CsiD acts as an α-ketoglutarate-dependent dioxygenase catalysing hydroxylation of glutarate to L-2-hydroxyglutarate. CsiD is found widespread in bacteria. We present crystal structures of CsiD in complex with glutarate, succinate, and the inhibitor N-oxalyl-glycine, demonstrating strong discrimination between the structurally related ligands. We show that L-2-hydroxyglutarate is converted to α-ketoglutarate by LhgO acting as a membrane-bound, ubiquinone-linked dehydrogenase. Lysine enters the pathway via 5-aminovalerate by the promiscuous enzymes GabT and GabD. We demonstrate that repression of the pathway by CsiR is relieved upon glutarate binding. In conclusion, lysine degradation provides an important link in central metabolism. Our results imply the gut microbiome as a potential source of glutarate and L-2-hydroxyglutarate associated with human diseases such as cancer and organic acidurias.

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BACKGROUND: The sulfate-reducing bacterium Desulfatiglans anilini can grow with phenol as sole source of carbon and energy under strictly anaerobic, sulfate-reducing conditions. In the nitrate-reducing bacterium Thauera aromatica, the enzymes involved in phenol degradation have been well elucidated, whereas the anaerobic phenol degradation pathway by D. anilini was not studied in detail yet. RESULTS: The pathway of anaerobic phenol degradation by the sulfate-reducing bacterium Desulfatiglans anilini was studied by identification of genes coding for phenylphosphate synthase (encoded by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) in the genome of D. anilini, by analysis of the transcription and translation of pps-ppc genes, and by measurement of phenylphosphate synthase activity in cell-free extracts of phenol-grown cells. The majority of genes involved in phenol degradation were found to be organized in one gene cluster. The gene cluster contained genes ppsα (phenylphosphate synthase alpha subunit), ppsß (phenylphosphate synthase beta subunit), ppcß (phenylphosphate carboxylase beta subunit), as well as 4-hydroxybenzoyl-CoA ligase and 4-hydroxylbenzoyl-CoA reductase-encoding genes. The genes ppsγ (phenylphosphate synthase gamma subunit), ppcα (phenylphosphate carboxylase alpha subunit) and ppcδ (phenylphosphate carboxylase delta subunit) were located elsewhere in the genome of D. anilini, and no obvious homologue of ppcγ (phenylphosphate carboxylase gamma subunit) was found in the genome. Induction of genes pps and ppc during growth on phenol was confirmed by reverse transcription polymerase chain reaction. Total proteome analysis revealed that the abundance of enzymes encoded by the gene cluster under study was much higher in phenol-grown cells than that in benzoate-grown cells. In in-vitro enzyme assays with cell-free extracts of phenol-grown cells, phenylphosphate was formed from phenol in the presence of ATP, Mg2+, Mn2+, K+ as co-factors. CONCLUSIONS: The genes coding for enzymes involved in the anaerobic phenol degradation pathway were identified in the sulfate-reducing bacterium D. anilini. The results indicate that the first steps of anaerobic phenol degradation in D. anilini are phosphorylation of phenol to phenylphosphate by phenylphosphate synthase and carboxylation of phenylphosphate by phenylphosphate carboxylase.

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Anaerobium acetethylicum strain GluBS11T belongs to the family Lachnospiraceae within the order Clostridiales. It is a Gram-positive, non-motile and strictly anaerobic bacterium isolated from biogas slurry that was originally enriched with gluconate as carbon source (Patil, et al., Int J Syst Evol Microbiol 65:3289-3296, 2015). Here we describe the draft genome sequence of strain GluBS11T and provide a detailed insight into its physiological and metabolic features. The draft genome sequence generated 4,609,043 bp, distributed among 105 scaffolds assembled using the SPAdes genome assembler method. It comprises in total 4,132 genes, of which 4,008 were predicted to be protein coding genes, 124 RNA genes and 867 pseudogenes. The G + C content was 43.51 mol %. The annotated genome of strain GluBS11T contains putative genes coding for the pentose phosphate pathway, the Embden-Meyerhoff-Parnas pathway, the Entner-Doudoroff pathway and the tricarboxylic acid cycle. The genome revealed the presence of most of the necessary genes required for the fermentation of glucose and gluconate to acetate, ethanol, and hydrogen gas. However, a candidate gene for production of formate was not identified.

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Hydrogen and formate are important electron carriers in methanogenic degradation in anoxic environments such as sediments, sewage sludge digestors and biogas reactors. Especially in the terminal steps of methanogenesis, they determine the energy budgets of secondary (syntrophically) fermenting bacteria and their methanogenic partners. The literature provides considerable data on hydrogen pool sizes in such habitats, but little data exist for formate concentrations due to technical difficulties in formate determination at low concentration. Recent evidence from biochemical and molecular biological studies indicates that several secondary fermenters can use both hydrogen and formate for electron release, and may do so even simultaneously. Numerous strictly anaerobic bacteria contain enzymes which equilibrate hydrogen and formate pools to energetically equal values, and recent measurements in sewage digestors and biogas reactors indicate that - beyond occasional fluctuations - the pool sizes of hydrogen and formate are indeed energetically nearly equivalent. Nonetheless, a thermophilic archaeon from a submarine hydrothermal vent, Thermococcus onnurineus, can obtain ATP from the conversion of formate to hydrogen plus bicarbonate at 80°C, indicating that at least in this extreme environment the pools of formate and hydrogen are likely to be sufficiently different to support such an unusual type of energy conservation.

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Growth of biodiesel industries resulted in increased coproduction of crude glycerol which is therefore becoming a waste product instead of a valuable 'coproduct'. Glycerol can be used for the production of valuable chemicals, e.g. biofuels, to reduce glycerol waste disposal. In this study, a novel bacterial strain is described which converts glycerol mainly to ethanol and hydrogen with very little amounts of acetate, formate and 1,2-propanediol as coproducts. The bacterium offers certain advantages over previously studied glycerol-fermenting microorganisms. Anaerobium acetethylicum during growth with glycerol produces very little side products and grows in the presence of maximum glycerol concentrations up to 1500 mM and in the complete absence of complex organic supplements such as yeast extract or tryptone. The highest observed growth rate of 0.116 h-1 is similar to that of other glycerol degraders, and the maximum concentration of ethanol that can be tolerated was found to be about 60 mM (2.8 g l-1 ) and further growth was likely inhibited due to ethanol toxicity. Proteome analysis as well as enzyme assays performed in cell-free extracts demonstrated that glycerol is degraded via glyceraldehyde-3-phosphate, which is further metabolized through the lower part of glycolysis leading to formation of mainly ethanol and hydrogen. In conclusion, fermentation of glycerol to ethanol and hydrogen by this bacterium represents a remarkable option to add value to the biodiesel industries by utilization of surplus glycerol.

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A novel strictly anaerobic, mesophilic bacterium was enriched and isolated with gluconate as sole substrate from a methanogenic sludge collected from a biogas reactor. Cells of strain GluBS11T stained Gram-positive and were non-motile, straight rods, measuring 3.0-4.5 × 0.8-1.2 µm. The temperature range for growth was 15-37 °C, with optimal growth at 30 °C, the pH range was 6.5-8.5, with optimal growth at pH 7, and the generation time under optimal conditions was 60 min. API Rapid 32A reactions were positive for α-galactosidase, α-glucosidase and ß-glucosidase and negative for catalase and oxidase. A broad variety of substrates was utilized, including gluconate, glucose, fructose, maltose, sucrose, lactose, galactose, melezitose, melibiose, mannitol, erythritol, glycerol and aesculin. Products of gluconate fermentation were ethanol, acetate, formate, H2 and CO2. Neither sulfate nor nitrate served as an electron acceptor. Predominant cellular fatty acids (>10 %) were C14 : 0, C16 : 0, C16 : 1ω7c/iso-C15 : 0 2-OH and C18 : 1ω7c. The DNA G+C content of strain GluBS11T was 44.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequence data revealed that strain GluBS11T is a member of subcluster XIVa within the order Clostridiales. The closest cultured relatives are Clostridium herbivorans (93.1 % similarity to the type strain), Clostridium populeti (93.3 %), Eubacterium uniforme (92.4 %) and Clostridium polysaccharolyticum (91.5 %). Based on this 16S rRNA gene sequence divergence (>6.5 %) as well as on chemotaxonomic and phenotypic differences from these taxa, strain GluBS11T is considered to represent a novel genus and species, for which the name Anaerobium acetethylicum gen. nov., sp. nov. is proposed. The type strain of Anaerobium acetethylicum is GluBS11T ( = LMG 28619T = KCTC 15450T = DSM 29698T).

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A novel type of anaerobic bacteria was previously isolated from profundal lake sediment by direct dilution of the sediment in mineral agar medium containing glucose and a background lawn of Methanospirillum hungatei as a syntrophic partner. The isolated bacteria grouped with aerobic Bacillus spp. according to their 16S rRNA gene sequence, and the most closely related species is Bacillus thioparans. Fermentative growth of the novel strain with glucose was possible only in the presence of syntrophic partners, and cocultures produced acetate and methane, in some cases also lactate and traces of succinate as fermentation products. In contrast, the closely related strains Bacillus jeotgali and Bacillus sp. strain PeC11 are able to grow with glucose axenically by mixed acid fermentation yielding lactate, acetate, formate, succinate, and ethanol as fermentation products. Alternatively, the isolated strain grew anaerobically in pure culture if pyruvate was added to glucose-containing media, and lactate, acetate and formate were the major fermentation products, but the strain never produced ethanol. Aerobic growth was found with a variety of organic substrates in the presence of partly reduced sulfur compounds. In the absence of sulfide and oxygen, nitrate served as an electron acceptor. Strain BoGlc83 was characterized as the type strain of a new species for which the name Bacillus stamsii sp. nov. (DSM 19598=JCM 30025) is proposed.

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Pelobacter carbinolicus and P. acetylenicus oxidize ethanol in syntrophic cooperation with methanogens. Cocultures with Methanospirillum hungatei served as model systems for the elucidation of syntrophic ethanol oxidation previously done with the lost "Methanobacillus omelianskii" coculture. During growth on ethanol, both Pelobacter species exhibited NAD+-dependent alcohol dehydrogenase activity. Two different acetaldehyde-oxidizing activities were found: a benzyl viologen-reducing enzyme forming acetate, and a NAD+-reducing enzyme forming acetyl-CoA. Both species synthesized ATP from acetyl-CoA via acetyl phosphate. Comparative 2D-PAGE of ethanol-grown P. carbinolicus revealed enhanced expression of tungsten-dependent acetaldehyde: ferredoxin oxidoreductases and formate dehydrogenase. Tungsten limitation resulted in slower growth and the expression of a molybdenum-dependent isoenzyme. Putative comproportionating hydrogenases and formate dehydrogenase were expressed constitutively and are probably involved in interspecies electron transfer. In ethanol-grown cocultures, the maximum hydrogen partial pressure was about 1,000 Pa (1 mM) while 2 mM formate was produced. The redox potentials of hydrogen and formate released during ethanol oxidation were calculated to be EH2 = -358±12 mV and EHCOOH = -366±19 mV, respectively. Hydrogen and formate formation and degradation further proved that both carriers contributed to interspecies electron transfer. The maximum Gibbs free energy that the Pelobacter species could exploit during growth on ethanol was -35 to -28 kJ per mol ethanol. Both species could be cultivated axenically on acetaldehyde, yielding energy from its disproportionation to ethanol and acetate. Syntrophic cocultures grown on acetoin revealed a two-phase degradation: first acetoin degradation to acetate and ethanol without involvement of the methanogenic partner, and subsequent syntrophic ethanol oxidation. Protein expression and activity patterns of both Pelobacter spp. grown with the named substrates were highly similar suggesting that both share the same steps in ethanol and acetalydehyde metabolism. The early assumption that acetaldehyde is a central intermediate in Pelobacter metabolism was now proven biochemically.

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In syntrophic conversion of butyrate to methane and CO2, butyrate is oxidized to acetate by secondary fermenting bacteria such as Syntrophomonas wolfei in close cooperation with methanogenic partner organisms, e.g., Methanospirillum hungatei. This process involves an energetically unfavourable shift of electrons from the level of butyryl-CoA oxidation to the substantially lower redox potential of proton and/or CO2 reduction, in order to transfer these electrons to the methanogenic partner via hydrogen and/or formate. In the present study, all prominent membrane-bound and soluble proteins expressed in S. wolfei specifically during syntrophic growth with butyrate, in comparison to pure-culture growth with crotonate, were examined by one- and two-dimensional gel electrophoresis, and identified by peptide fingerprinting-mass spectrometry. A membrane-bound, externally oriented, quinone-linked formate dehydrogenase complex was expressed at high level specifically during syntrophic butyrate oxidation, comprising a selenocystein-linked catalytic subunit with a membrane-translocation pathway signal (TAT), a membrane-bound iron-sulfur subunit, and a membrane-bound cytochrome. Soluble hydrogenases were expressed at high levels specifically during growth with crotonate. The results were confirmed by native protein gel electrophoresis, by formate dehydrogenase and hydrogenase-activity staining, and by analysis of formate dehydrogenase and hydrogenase activities in intact cells and cell extracts. Furthermore, constitutive expression of a membrane-bound, internally oriented iron-sulfur oxidoreductase (DUF224) was confirmed, together with expression of soluble electron-transfer flavoproteins (EtfAB) and two previously identified butyryl-CoA dehydrogenases. Our findings allow to depict an electron flow scheme for syntrophic butyrate oxidation in S. wolfei. Electrons derived from butyryl-CoA are transferred through a membrane-bound EtfAB:quinone oxidoreductase (DUF224) to a menaquinone cycle and further via a b-type cytochrome to an externally oriented formate dehydrogenase. Hence, an ATP hydrolysis-driven proton-motive force across the cytoplasmatic membrane would provide the energy input for the electron potential shift necessary for formate formation.

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