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Bernhard Schink - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Degradation of Acetaldehyde and Its Precursors by Pelobacter carbinolicus and
2016Co-Authors: P. Acetylenicus, David Schleheck, Marco Frensch, Er Schmidt, Bernhard SchinkAbstract: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
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Degradation of Acetaldehyde and Its Precursors by Pelobacter carbinolicus and P. acetylenicus
PloS one, 2014Co-Authors: Alexander Schmidt, Bernhard Schink, Marco Frensch, David Schleheck, Nicolai MüllerAbstract: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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Specific enzyme activities (U per mg protein) in cytoplasmic fractions of Pelobacter carbinolicus cultivated with and without molybdate and tungstate. (n. ac. = non-acetylating, BV = benzyl viologen, MV = methyl viologen).
2014Co-Authors: Alexander Schmidt, Bernhard Schink, Marco Frensch, David Schleheck, Nicolai MüllerAbstract:Specific enzyme activities (U per mg protein) in cytoplasmic fractions of Pelobacter carbinolicus cultivated with and without molybdate and tungstate. (n. ac. = non-acetylating, BV = benzyl viologen, MV = methyl viologen).
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Specific enzyme activities (U per mg protein) in Pelobacter species. If not mentioned otherwise, all enzyme assays were done with the P. carbinolicus/M. hungatei strain M1h coculture. (DCPIP = 2,6-dichlorophenolindophenol, n. ac. = non-acetylatin
2014Co-Authors: Alexander Schmidt, Bernhard Schink, Marco Frensch, David Schleheck, Nicolai MüllerAbstract:Specific enzyme activities (U per mg protein) in Pelobacter species. If not mentioned otherwise, all enzyme assays were done with the P. carbinolicus/M. hungatei strain M1h coculture. (DCPIP = 2,6-dichlorophenolindophenol, n. ac. = non-acetylating, BV = benzyl viologen, MV = methyl viologen, M. arb. = Methanobrevibacter arboriphilus, n. d. = not determined).
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Crystallization and preliminary X-ray analysis of the tungsten-dependent acetylene hydratase from Pelobacter acetylenicus.
Acta crystallographica. Section F Structural biology and crystallization communications, 2005Co-Authors: Oliver Einsle, Bernhard Schink, Holger Niessen, Dietmar J Abt, Grazyna Seiffert, Robert Huber, Albrecht Messerschmidt, Peter M H KroneckAbstract:Acetylene hydratase is a tungsten-containing hydroxylase that converts acetylene to acetaldehyde in a unique reaction that requires a strong reductant. The subsequent disproportionation of acetaldehyde yields acetate and ethanol. Crystals of the tungsten/iron-sulfur protein acetylene hydratase from Pelobacter acetylenicus strain WoAcy 1 (DSM 3246) were grown by the vapour-diffusion method in an N2/H2 atmosphere using polyethylene glycol as precipitant. Growth of crystals suitable for X-ray analysis strictly depended on the presence of Ti(III) citrate or dithionite as reducing agents.
Derek R. Lovley - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Open Access
2013Co-Authors: Jun Sun, Shelley A. Haveman, Olivia Bui, Tom R Fahl, Derek R. LovleyAbstract:Background: Pelobacter species are commonly found in a number of subsurface environments, and are unique members of the Geobacteraceae family. They are phylogenetically intertwined with both Geobacter and Desulfuromonas species. Pelobacter species likely play important roles in the fermentative degradation of unusual organic matters and syntrophic metabolism in the natural environments, and are of interest for applications in bioremediation and microbial fuel cells. Results: In order to better understand the physiology of Pelobacter species, genome-scale metabolic models for Pelobacter carbinolicus and Pelobacter propionicus were developed. Model development was greatly aided by the availability of models of the closely related Geobacter sulfurreducens and G. metallireducens. The reconstructed P. carbinolicus model contains 741 genes and 708 reactions, whereas the reconstructed P. propionicus model contains 661 genes and 650 reactions. A total of 470 reactions are shared among the two Pelobacter models and the two Geobacter models. The different reactions between the Pelobacter and Geobacter models reflect some unique metabolic capabilities such as fermentative growth for both Pelobacter species. The reconstructed Pelobacter models were validated by simulating published growth conditions including fermentations, hydrogen production in syntrophic co-culture conditions, hydrogen utilization, and Fe(III) reduction. Simulation results matched well wit
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The genome of Pelobacter carbinolicus reveals surprising metabolic capabilities and physiological features
BMC genomics, 2012Co-Authors: Muktak Aklujkar, Shelley A. Haveman, Raymond J. Didonato, Olga Chertkov, Cliff Han, Miriam Land, Peter Brown, Derek R. LovleyAbstract:The bacterium Pelobacter carbinolicus is able to grow by fermentation, syntrophic hydrogen/formate transfer, or electron transfer to sulfur from short-chain alcohols, hydrogen or formate; it does not oxidize acetate and is not known to ferment any sugars or grow autotrophically. The genome of P. carbinolicus was sequenced in order to understand its metabolic capabilities and physiological features in comparison with its relatives, acetate-oxidizing Geobacter species. Pathways were predicted for catabolism of known substrates: 2,3-butanediol, acetoin, glycerol, 1,2-ethanediol, ethanolamine, choline and ethanol. Multiple isozymes of 2,3-butanediol dehydrogenase, ATP synthase and [FeFe]-hydrogenase were differentiated and assigned roles according to their structural properties and genomic contexts. The absence of asparagine synthetase and the presence of a mutant tRNA for asparagine encoded among RNA-active enzymes suggest that P. carbinolicus may make asparaginyl-tRNA in a novel way. Catabolic glutamate dehydrogenases were discovered, implying that the tricarboxylic acid (TCA) cycle can function catabolically. A phosphotransferase system for uptake of sugars was discovered, along with enzymes that function in 2,3-butanediol production. Pyruvate:ferredoxin/flavodoxin oxidoreductase was identified as a potential bottleneck in both the supply of oxaloacetate for oxidation of acetate by the TCA cycle and the connection of glycolysis to production of ethanol. The P. carbinolicus genome was found to encode autotransporters and various appendages, including three proteins with similarity to the geopilin of electroconductive nanowires. Several surprising metabolic capabilities and physiological features were predicted from the genome of P. carbinolicus, suggesting that it is more versatile than anticipated.
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Constraint-based modeling analysis of the metabolism of two Pelobacter species
BMC Systems Biology, 2010Co-Authors: Shelley A. Haveman, Tom R Fahland, Derek R. LovleyAbstract:Background Pelobacter species are commonly found in a number of subsurface environments, and are unique members of the Geobacteraceae family. They are phylogenetically intertwined with both Geobacter and Desulfuromonas species. Pelobacter species likely play important roles in the fermentative degradation of unusual organic matters and syntrophic metabolism in the natural environments, and are of interest for applications in bioremediation and microbial fuel cells.
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Interference with Histidyl-tRNA Synthetase by a CRISPR Spacer Sequence as a Factor in the
2010Co-Authors: Evolution Of Pelobacter Carbinolicus, Muktak Aklujkar, Derek R. LovleyAbstract:Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicu
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Evolution from a respiratory ancestor to fill syntrophic and fermentative niches: comparative genomics of six Geobacteraceae species
BMC genomics, 2009Co-Authors: Jessica E. Butler, Nelson D. Young, Derek R. LovleyAbstract:The anaerobic degradation of organic matter in natural environments, and the biotechnical use of anaerobes in energy production and remediation of subsurface environments, both require the cooperative activity of a diversity of microorganisms in different metabolic niches. The Geobacteraceae family contains members with three important anaerobic metabolisms: fermentation, syntrophic degradation of fermentation intermediates, and anaerobic respiration. In order to learn more about the evolution of anaerobic microbial communities, the genome sequences of six Geobacteraceae species were analyzed. The results indicate that the last common Geobacteraceae ancestor contained sufficient genes for anaerobic respiration, completely oxidizing organic compounds with the reduction of external electron acceptors, features that are still retained in modern Geobacter and Desulfuromonas species. Evolution of specialization for fermentative growth arose twice, via distinct lateral gene transfer events, in Pelobacter carbinolicus and Pelobacter propionicus. Furthermore, P. carbinolicus gained hydrogenase genes and genes for ferredoxin reduction that appear to permit syntrophic growth via hydrogen production. The gain of new physiological capabilities in the Pelobacter species were accompanied by the loss of several key genes necessary for the complete oxidation of organic compounds and the genes for the c-type cytochromes required for extracellular electron transfer. The results suggest that Pelobacter species evolved parallel strategies to enhance their ability to compete in environments in which electron acceptors for anaerobic respiration were limiting. More generally, these results demonstrate how relatively few gene changes can dramatically transform metabolic capabilities and expand the range of environments in which microorganisms can compete.
Peter M H Kroneck - One of the best experts on this subject based on the ideXlab platform.
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Crystallization and preliminary X-ray analysis of the tungsten-dependent acetylene hydratase from Pelobacter acetylenicus.
Acta crystallographica. Section F Structural biology and crystallization communications, 2005Co-Authors: Oliver Einsle, Bernhard Schink, Holger Niessen, Dietmar J Abt, Grazyna Seiffert, Robert Huber, Albrecht Messerschmidt, Peter M H KroneckAbstract:Acetylene hydratase is a tungsten-containing hydroxylase that converts acetylene to acetaldehyde in a unique reaction that requires a strong reductant. The subsequent disproportionation of acetaldehyde yields acetate and ethanol. Crystals of the tungsten/iron-sulfur protein acetylene hydratase from Pelobacter acetylenicus strain WoAcy 1 (DSM 3246) were grown by the vapour-diffusion method in an N2/H2 atmosphere using polyethylene glycol as precipitant. Growth of crystals suitable for X-ray analysis strictly depended on the presence of Ti(III) citrate or dithionite as reducing agents.
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Crystallization and preliminary X-ray analysis of the molybdenum-dependent pyrogallol-phloroglucinol transhydroxylase of Pelobacter acidigallici
Acta Crystallographica Section D Biological Crystallography, 2002Co-Authors: Dietmar J Abt, Bernhard Schink, Oliver Einsle, Holger Niessen, Albrecht Messerschmidt, Robert Krieger, Peter M H KroneckAbstract:Crystals of the molybdo-/iron–sulfur protein pyrogallol:phloroglucinol hydroxyltransferase (transhydroxylase; EC 1.97.1.2) from Pelobacter acidigallici were grown by vapour diffusion in an N2/H2 atmosphere using polyethylene glycol as a precipitant. In this microorganism, transhydroxylase converts pyrogallol to phloroglucinol in a unique reaction without oxygen transfer from water. Growth of crystals suitable for X-ray analysis was strongly dependent on the presence of dithionite as a reducing agent. The crystals belonged to space group P1 and MAD data were collected on the iron K edge to resolutions higher than 2.5 A.
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a structural comparison of molybdenum cofactor containing enzymes
Fems Microbiology Reviews, 1998Co-Authors: Caroline Kisker, Dietmar Baas, János Rétey, Hermann Schindelin, Rainer U Meckenstock, Peter M H KroneckAbstract:This work gives an overview of the recent achievements which have contributed to the understanding of the structure and function of molybdenum and tungsten enzymes. Known structures of molybdo-pterin cofactor-containing enzymes will be described briefly and the structural differences between representatives of the same and different families will be analyzed. This comparison will show that the molybdo-pterin cofactor-containing enzymes represent a very heterogeneous group with differences in overall enzyme structure, cofactor composition and stoichiometry, as well as differences in the immediate molybdenum environment. Two recently discovered molybdo-pterin cofactor-containing enzymes will be described with regard to molecular and EPR spectroscopic properties, pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici and acetylene hydratase from Pelobacter acetylenicus. On the basis of its amino acid sequence, transhydroxylase can be classified as a member of the dimethylsulfoxide reductase family, whereas classification of the tungsten/molybdenum-containing acetylene hydratase has to await the determination of its amino acid sequence.
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A structural comparison of molybdenum cofactor-containing enzymes
FEMS microbiology reviews, 1998Co-Authors: Caroline Kisker, Dietmar Baas, János Rétey, Hermann Schindelin, Rainer U Meckenstock, Peter M H KroneckAbstract:This work gives an overview of the recent achievements which have contributed to the understanding of the structure and function of molybdenum and tungsten enzymes. Known structures of molybdo-pterin cofactor-containing enzymes will be described briefly and the structural differences between representatives of the same and different families will be analyzed. This comparison will show that the molybdo-pterin cofactor-containing enzymes represent a very heterogeneous group with differences in overall enzyme structure, cofactor composition and stoichiometry, as well as differences in the immediate molybdenum environment. Two recently discovered molybdo-pterin cofactor-containing enzymes will be described with regard to molecular and EPR spectroscopic properties, pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici and acetylene hydratase from Pelobacter acetylenicus. On the basis of its amino acid sequence, transhydroxylase can be classified as a member of the dimethylsulfoxide reductase family, whereas classification of the tungsten/molybdenum-containing acetylene hydratase has to await the determination of its amino acid sequence.
János Rétey - One of the best experts on this subject based on the ideXlab platform.
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Investigation of the mechanism of action of pyrogallol-phloroglucinol transhydroxylase by using putative intermediates.
Chemistry (Weinheim an der Bergstrasse Germany), 2007Co-Authors: Csaba Paizs, Ulrike Bartlewski-hof, János RéteyAbstract:Pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici, a molybdopterin-containing enzyme, catalyzes a key reaction in the anaerobic degradation of aromatic compounds. In vitro, the enzymatic reaction requires 1,2,3,5-tetrahydroxybenzene as a cocatalyst and the transhydroxylation occurs without exchange with hydroxy groups from water. To test our previous proposal that the transfer of the hydroxy group occurs via 2,4,6,3',4',5'-hexahydroxydiphenyl ether as an intermediate, we synthesized this compound and investigated its properties. We also describe the synthesis and characterization of 3,4,5,3',4',5'-hexahydroxydiphenyl ether. Both compounds could substitute for the cocatalyst in vitro. This indicates that the diphenyl ethers can intrude into the active site and initiate the catalytic cycle. Recently, the X-ray crystal structure of the transhydroxylase (TH) was published [16] and it supports the proposed mechanism of hydroxy-group transfer.
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Cloning, sequencing and heterologous expression of pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici.
European journal of biochemistry, 1999Co-Authors: Dietmar Baas, János RéteyAbstract:A genomic λ-library of Pelobacter acidigallici has been established. Proteolytic digestion of homogeneous pyrogallol-phloroglucinol transhydroxylase from the same microorganism afforded polypeptide fragments whose N-terminal sequences allowed the generation of oligonucleotide primers. Together with primers deduced from the known N-terminal sequences of the two intact subunits these were used in PCR experiments to obtain three amplificates. Screening the λ-library with the three amplificates led eventually to clones containing the whole gene coding for the transhydroxylase. Sequencing the gene revealed two open reading frames coding for 875 and 275 amino acids which correspond to the α- and β-subunits of THL, respectively. The two subunits are separated by a 48-bp noncoding region. Comparison of the sequence with those of other molybdopterin cofactor (MoCo)-enzymes places THL in the dimethylsulfoxide reductase family. Possible contact sites to the MoCo and to the iron-sulphur clusters were spotted. Using the expression vectors pQE 30 and pT 7-7 three constructs harbouring the THL gene were created. One of them carried a His6-tag at the N-terminus of the α-subunit, another at the C-terminus of the β-subunit. Immunoblot analysis showed high expression of THL, but the inclusion bodies could not be refolded to active enzyme.
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a structural comparison of molybdenum cofactor containing enzymes
Fems Microbiology Reviews, 1998Co-Authors: Caroline Kisker, Dietmar Baas, János Rétey, Hermann Schindelin, Rainer U Meckenstock, Peter M H KroneckAbstract:This work gives an overview of the recent achievements which have contributed to the understanding of the structure and function of molybdenum and tungsten enzymes. Known structures of molybdo-pterin cofactor-containing enzymes will be described briefly and the structural differences between representatives of the same and different families will be analyzed. This comparison will show that the molybdo-pterin cofactor-containing enzymes represent a very heterogeneous group with differences in overall enzyme structure, cofactor composition and stoichiometry, as well as differences in the immediate molybdenum environment. Two recently discovered molybdo-pterin cofactor-containing enzymes will be described with regard to molecular and EPR spectroscopic properties, pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici and acetylene hydratase from Pelobacter acetylenicus. On the basis of its amino acid sequence, transhydroxylase can be classified as a member of the dimethylsulfoxide reductase family, whereas classification of the tungsten/molybdenum-containing acetylene hydratase has to await the determination of its amino acid sequence.
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A structural comparison of molybdenum cofactor-containing enzymes
FEMS microbiology reviews, 1998Co-Authors: Caroline Kisker, Dietmar Baas, János Rétey, Hermann Schindelin, Rainer U Meckenstock, Peter M H KroneckAbstract:This work gives an overview of the recent achievements which have contributed to the understanding of the structure and function of molybdenum and tungsten enzymes. Known structures of molybdo-pterin cofactor-containing enzymes will be described briefly and the structural differences between representatives of the same and different families will be analyzed. This comparison will show that the molybdo-pterin cofactor-containing enzymes represent a very heterogeneous group with differences in overall enzyme structure, cofactor composition and stoichiometry, as well as differences in the immediate molybdenum environment. Two recently discovered molybdo-pterin cofactor-containing enzymes will be described with regard to molecular and EPR spectroscopic properties, pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici and acetylene hydratase from Pelobacter acetylenicus. On the basis of its amino acid sequence, transhydroxylase can be classified as a member of the dimethylsulfoxide reductase family, whereas classification of the tungsten/molybdenum-containing acetylene hydratase has to await the determination of its amino acid sequence.
Karl B. Haase - One of the best experts on this subject based on the ideXlab platform.
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Detection of Diazotrophy in the Acetylene-Fermenting Anaerobe Pelobacter sp. Strain SFB93.
Applied and environmental microbiology, 2017Co-Authors: Denise M. Akob, Shaun M. Baesman, John M. Sutton, Janna L. Fierst, Adam C. Mumford, Yesha Shrestha, Amisha T. Poret-peterson, Stacy Bennett, Darren S. Dunlap, Karl B. HaaseAbstract:Acetylene (C2H2) is a trace constituent of the present Earth's oxidizing atmosphere, reflecting a mixture of terrestrial and marine emissions from anthropogenic, biomass-burning, and unidentified biogenic sources. Fermentation of acetylene was serendipitously discovered during C2H2 block assays of N2O reductase, and Pelobacter acetylenicus was shown to grow on C2H2 via acetylene hydratase (AH). AH is a W-containing, catabolic, low-redox-potential enzyme that, unlike nitrogenase (N2ase), is specific for acetylene. Acetylene fermentation is a rare metabolic process that is well characterized only in P. acetylenicus DSM3246 and DSM3247 and Pelobacter sp. strain SFB93. To better understand the genetic controls for AH activity, we sequenced the genomes of the three acetylene-fermenting Pelobacter strains. Genome assembly and annotation produced three novel genomes containing gene sequences for AH, with two copies being present in SFB93. In addition, gene sequences for all five compulsory genes for iron-molybdenum N2ase were also present in the three genomes, indicating the cooccurrence of two acetylene transformation pathways. Nitrogen fixation growth assays showed that DSM3426 could ferment acetylene in the absence of ammonium, but no ethylene was produced. However, SFB93 degraded acetylene and, in the absence of ammonium, produced ethylene, indicating an active N2ase. Diazotrophic growth was observed under N2 but not in experimental controls incubated under argon. SFB93 exhibits acetylene fermentation and nitrogen fixation, the only known biochemical mechanisms for acetylene transformation. Our results indicate complex interactions between N2ase and AH and suggest novel evolutionary pathways for these relic enzymes from early Earth to modern days.IMPORTANCE Here we show that a single Pelobacter strain can grow via acetylene fermentation and carry out nitrogen fixation, using the only two enzymes known to transform acetylene. These findings provide new insights into acetylene transformations and adaptations for nutrient (C and N) and energy acquisition by microorganisms. Enhanced understanding of acetylene transformations (i.e., extent, occurrence, and rates) in modern environments is important for the use of acetylene as a potential biomarker for extraterrestrial life and for degradation of anthropogenic contaminants.
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Discovery of Two Biological Mechanisms for Acetylene Metabolism in a Single Organism
2017Co-Authors: Denise M. Akob, Shaun M. Baesman, John M. Sutton, Janna L. Fierst, Adam C. Mumford, Yesha Shrestha, Amisha T. Poret-peterson, Stacy Bennett, Darren S. Dunlap, Karl B. HaaseAbstract:Acetylene fermentation assays, nitrogen fixation assays, and growth studies were performed with Pelobacter sp. strain SFB93 and Pelobacter acetylenicus DSM3246. Data includes concentrations of acetylene and ethylene over time, and growth measured with OD680 and cell counts.