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Derek R. Lovley - One of the best experts on this subject based on the ideXlab platform.
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Expressing the Geobacter Metallireducens PilA in Geobacter sulfurreducens Yields Pili with Exceptional Conductivity
Mbio, 2017Co-Authors: Yang Tan, Nikhil S. Malvankar, Kelly P Nevin, Ramesh Y. Adhikari, Joy E. Ward, Trevor L. Woodard, Derek R. LovleyAbstract:The electrically conductive pili (e-pili) of Geobacter sulfurreducens serve as a model for a novel strategy for long-range extracellular electron transfer. e-pili are also a new class of bioelectronic materials. However, the only other Geobacter pili previously studied, which were from G. uraniireducens, were poorly conductive. In order to obtain more information on the range of pili conductivities in Geobacter species, the pili of G. Metallireducens were investigated. Heterologously expressing the PilA gene of G. Metallireducens in G. sulfurreducens yielded a G. sulfurreducens strain, designated strain MP, that produced abundant pili. Strain MP exhibited phenotypes consistent with the presence of e-pili, such as high rates of Fe(III) oxide reduction and high current densities on graphite anodes. Individual pili prepared at physiologically relevant pH 7 had conductivities of 277 ± 18.9 S/cm (mean ± standard deviation), which is 5,000-fold higher than the conductivity of G. sulfurreducens pili at pH 7 and nearly 1 million-fold higher than the conductivity of G. uraniireducens pili at the same pH. A potential explanation for the higher conductivity of the G. Metallireducens pili is their greater density of aromatic amino acids, which are known to be important components in electron transport along the length of the pilus. The G. Metallireducens pili represent the most highly conductive pili found to date and suggest strategies for designing synthetic pili with even higher conductivities. e-pili are a remarkable electrically conductive material that can be sustainably produced without harsh chemical processes from renewable feedstocks and that contain no toxic components in the final product. Thus, e-pili offer an unprecedented potential for developing novel materials, electronic devices, and sensors for diverse applications with a new "green" technology. Increasing e-pili conductivity will even further expand their potential applications. A proven strategy is to design synthetic e-pili that contain tryptophan, an aromatic amino acid not found in previously studied e-pili. The studies reported here demonstrate that a productive alternative approach is to search more broadly in the microbial world. Surprisingly, even though G. Metallireducens and G. sulfurreducens are closely related, the conductivities of their e-pili differ by more than 3 orders of magnitude. The ability to produce e-pili with high conductivity without generating a genetically modified product enhances the attractiveness of this novel electronic material. Copyright © 2017 Tan et al.
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Expressing the Geobacter Metallireducens PilA in Geobacter sulfurreducens Yields Pili with Exceptional Conductivity
American Society for Microbiology, 2017Co-Authors: Yang Tan, Nikhil S. Malvankar, Kelly P Nevin, Derek R. Lovley, Ramesh Y. Adhikari, Joy E. Ward, Trevor L. Woodard, Eleftherios T. PapoutsakisAbstract:The electrically conductive pili (e-pili) of Geobacter sulfurreducens serve as a model for a novel strategy for long-range extracellular electron transfer. e-pili are also a new class of bioelectronic materials. However, the only other Geobacter pili previously studied, which were from G. uraniireducens, were poorly conductive. In order to obtain more information on the range of pili conductivities in Geobacter species, the pili of G. Metallireducens were investigated. Heterologously expressing the PilA gene of G. Metallireducens in G. sulfurreducens yielded a G. sulfurreducens strain, designated strain MP, that produced abundant pili. Strain MP exhibited phenotypes consistent with the presence of e-pili, such as high rates of Fe(III) oxide reduction and high current densities on graphite anodes. Individual pili prepared at physiologically relevant pH 7 had conductivities of 277 ± 18.9 S/cm (mean ± standard deviation), which is 5,000-fold higher than the conductivity of G. sulfurreducens pili at pH 7 and nearly 1 million-fold higher than the conductivity of G. uraniireducens pili at the same pH. A potential explanation for the higher conductivity of the G. Metallireducens pili is their greater density of aromatic amino acids, which are known to be important components in electron transport along the length of the pilus. The G. Metallireducens pili represent the most highly conductive pili found to date and suggest strategies for designing synthetic pili with even higher conductivities
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Promoting Interspecies Electron Transfer with Biochar
Scientific Reports, 2015Co-Authors: Shanshan Chen, Nikhil S. Malvankar, Amelia-elena Rotaru, Pravin Malla Shrestha, Kelly P Nevin, Fengshan Liu, Fanghua Liu, W. Fan, Derek R. LovleyAbstract:Biochar, a charcoal-like product of the incomplete combustion of organic materials, is an increasingly popular soil amendment designed to improve soil fertility. We investigated the possibility that biochar could promote direct interspecies electron transfer (DIET) in a manner similar to that previously reported for granular activated carbon (GAC). Although the biochars investigated were 1000 times less conductive than GAC, they stimulated DIET in co-cultures of Geobacter Metallireducens with Geobacter sulfurreducens or Methanosarcina barkeri in which ethanol was the electron donor. Cells were attached to the biochar, yet not in close contact, suggesting that electrons were likely conducted through the biochar, rather than biological electrical connections. The finding that biochar can stimulate DIET may be an important consideration when amending soils with biochar and can help explain why biochar may enhance methane production from organic wastes under anaerobic conditions.
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carbon cloth stimulates direct interspecies electron transfer in syntrophic co cultures
Bioresource Technology, 2014Co-Authors: Shanshan Chen, Amelia-elena Rotaru, Kelly P Nevin, Fanghua Liu, Jo Philips, Trevor L. Woodard, Derek R. LovleyAbstract:This study investigated the possibility that the electrical conductivity of carbon cloth accelerates direct interspecies electron transfer (DIET) in co-cultures. Carbon cloth accelerated metabolism of DIET co-cultures (Geobacter Metallireducens-Geobacter sulfurreducens and G. Metallireducens-Methanosarcina barkeri) but did not promote metabolism of co-cultures performing interspecies H-2 transfer (Desulfovibrio vulgaris-G. sulfurreducens). On the other hand, DIET co-cultures were not stimulated by poorly conductive cotton cloth. Mutant strains lacking electrically conductive pili, or pili-associated cytochromes participated in DIET only in the presence of carbon cloth. In co-cultures promoted by carbon cloth, cells were primarily associated with the cloth although the syntrophic partners were too far apart for cell-to-cell biological electrical connections to be feasible. Carbon cloth seemingly mediated interspecies electron transfer between the distant syntrophic partners. These results suggest that the ability of carbon cloth to accelerate DIET should be considered in anaerobic digester designs that incorporate carbon cloth. (C) 2014 Elsevier Ltd. All rights reserved.
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direct interspecies electron transfer between Geobacter Metallireducens and methanosarcina barkeri
Applied and Environmental Microbiology, 2014Co-Authors: Amelia-elena Rotaru, Pravin Malla Shrestha, Beatrice Markovaite, Kelly P Nevin, Shanshan Chen, Derek R. LovleyAbstract:Direct interspecies electron transfer (DIET) is potentially an effective form of syntrophy in methanogenic communities, but little is known about the diversity of methanogens capable of DIET. The ability of Methanosarcina barkeri to participate in DIET was evaluated in coculture with Geobacter Metallireducens. Cocultures formed aggregates that shared electrons via DIET during the stoichiometric conversion of ethanol to methane. Cocultures could not be initiated with a pilin-deficient G. Metallireducens strain, suggesting that long-range electron transfer along pili was important for DIET. Amendments of granular activated carbon permitted the pilin-deficient G. Metallireducens isolates to share electrons with M. barkeri, demonstrating that this conductive material could substitute for pili in promoting DIET. When M. barkeri was grown in coculture with the H-2-producing Pelobacter carbinolicus, incapable of DIET, M. barkeri utilized H-2 as an electron donor but metabolized little of the acetate that P. carbinolicus produced. This suggested that H-2, but not electrons derived from DIET, inhibited acetate metabolism. P. carbinolicus-M. barkeri cocultures did not aggregate, demonstrating that, unlike DIET, close physical contact was not necessary for interspecies H-2 transfer. M. barkeri is the second methanogen found to accept electrons via DIET and the first methanogen known to be capable of using either H-2 or electrons derived from DIET for CO2 reduction. Furthermore, M. barkeri is genetically tractable, making it a model organism for elucidating mechanisms by which methanogens make biological electrical connections with other cells.
Kelly P Nevin - One of the best experts on this subject based on the ideXlab platform.
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Expressing the Geobacter Metallireducens PilA in Geobacter sulfurreducens Yields Pili with Exceptional Conductivity
Mbio, 2017Co-Authors: Yang Tan, Nikhil S. Malvankar, Kelly P Nevin, Ramesh Y. Adhikari, Joy E. Ward, Trevor L. Woodard, Derek R. LovleyAbstract:The electrically conductive pili (e-pili) of Geobacter sulfurreducens serve as a model for a novel strategy for long-range extracellular electron transfer. e-pili are also a new class of bioelectronic materials. However, the only other Geobacter pili previously studied, which were from G. uraniireducens, were poorly conductive. In order to obtain more information on the range of pili conductivities in Geobacter species, the pili of G. Metallireducens were investigated. Heterologously expressing the PilA gene of G. Metallireducens in G. sulfurreducens yielded a G. sulfurreducens strain, designated strain MP, that produced abundant pili. Strain MP exhibited phenotypes consistent with the presence of e-pili, such as high rates of Fe(III) oxide reduction and high current densities on graphite anodes. Individual pili prepared at physiologically relevant pH 7 had conductivities of 277 ± 18.9 S/cm (mean ± standard deviation), which is 5,000-fold higher than the conductivity of G. sulfurreducens pili at pH 7 and nearly 1 million-fold higher than the conductivity of G. uraniireducens pili at the same pH. A potential explanation for the higher conductivity of the G. Metallireducens pili is their greater density of aromatic amino acids, which are known to be important components in electron transport along the length of the pilus. The G. Metallireducens pili represent the most highly conductive pili found to date and suggest strategies for designing synthetic pili with even higher conductivities. e-pili are a remarkable electrically conductive material that can be sustainably produced without harsh chemical processes from renewable feedstocks and that contain no toxic components in the final product. Thus, e-pili offer an unprecedented potential for developing novel materials, electronic devices, and sensors for diverse applications with a new "green" technology. Increasing e-pili conductivity will even further expand their potential applications. A proven strategy is to design synthetic e-pili that contain tryptophan, an aromatic amino acid not found in previously studied e-pili. The studies reported here demonstrate that a productive alternative approach is to search more broadly in the microbial world. Surprisingly, even though G. Metallireducens and G. sulfurreducens are closely related, the conductivities of their e-pili differ by more than 3 orders of magnitude. The ability to produce e-pili with high conductivity without generating a genetically modified product enhances the attractiveness of this novel electronic material. Copyright © 2017 Tan et al.
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Expressing the Geobacter Metallireducens PilA in Geobacter sulfurreducens Yields Pili with Exceptional Conductivity
American Society for Microbiology, 2017Co-Authors: Yang Tan, Nikhil S. Malvankar, Kelly P Nevin, Derek R. Lovley, Ramesh Y. Adhikari, Joy E. Ward, Trevor L. Woodard, Eleftherios T. PapoutsakisAbstract:The electrically conductive pili (e-pili) of Geobacter sulfurreducens serve as a model for a novel strategy for long-range extracellular electron transfer. e-pili are also a new class of bioelectronic materials. However, the only other Geobacter pili previously studied, which were from G. uraniireducens, were poorly conductive. In order to obtain more information on the range of pili conductivities in Geobacter species, the pili of G. Metallireducens were investigated. Heterologously expressing the PilA gene of G. Metallireducens in G. sulfurreducens yielded a G. sulfurreducens strain, designated strain MP, that produced abundant pili. Strain MP exhibited phenotypes consistent with the presence of e-pili, such as high rates of Fe(III) oxide reduction and high current densities on graphite anodes. Individual pili prepared at physiologically relevant pH 7 had conductivities of 277 ± 18.9 S/cm (mean ± standard deviation), which is 5,000-fold higher than the conductivity of G. sulfurreducens pili at pH 7 and nearly 1 million-fold higher than the conductivity of G. uraniireducens pili at the same pH. A potential explanation for the higher conductivity of the G. Metallireducens pili is their greater density of aromatic amino acids, which are known to be important components in electron transport along the length of the pilus. The G. Metallireducens pili represent the most highly conductive pili found to date and suggest strategies for designing synthetic pili with even higher conductivities
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Promoting Interspecies Electron Transfer with Biochar
Scientific Reports, 2015Co-Authors: Shanshan Chen, Nikhil S. Malvankar, Amelia-elena Rotaru, Pravin Malla Shrestha, Kelly P Nevin, Fengshan Liu, Fanghua Liu, W. Fan, Derek R. LovleyAbstract:Biochar, a charcoal-like product of the incomplete combustion of organic materials, is an increasingly popular soil amendment designed to improve soil fertility. We investigated the possibility that biochar could promote direct interspecies electron transfer (DIET) in a manner similar to that previously reported for granular activated carbon (GAC). Although the biochars investigated were 1000 times less conductive than GAC, they stimulated DIET in co-cultures of Geobacter Metallireducens with Geobacter sulfurreducens or Methanosarcina barkeri in which ethanol was the electron donor. Cells were attached to the biochar, yet not in close contact, suggesting that electrons were likely conducted through the biochar, rather than biological electrical connections. The finding that biochar can stimulate DIET may be an important consideration when amending soils with biochar and can help explain why biochar may enhance methane production from organic wastes under anaerobic conditions.
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carbon cloth stimulates direct interspecies electron transfer in syntrophic co cultures
Bioresource Technology, 2014Co-Authors: Shanshan Chen, Amelia-elena Rotaru, Kelly P Nevin, Fanghua Liu, Jo Philips, Trevor L. Woodard, Derek R. LovleyAbstract:This study investigated the possibility that the electrical conductivity of carbon cloth accelerates direct interspecies electron transfer (DIET) in co-cultures. Carbon cloth accelerated metabolism of DIET co-cultures (Geobacter Metallireducens-Geobacter sulfurreducens and G. Metallireducens-Methanosarcina barkeri) but did not promote metabolism of co-cultures performing interspecies H-2 transfer (Desulfovibrio vulgaris-G. sulfurreducens). On the other hand, DIET co-cultures were not stimulated by poorly conductive cotton cloth. Mutant strains lacking electrically conductive pili, or pili-associated cytochromes participated in DIET only in the presence of carbon cloth. In co-cultures promoted by carbon cloth, cells were primarily associated with the cloth although the syntrophic partners were too far apart for cell-to-cell biological electrical connections to be feasible. Carbon cloth seemingly mediated interspecies electron transfer between the distant syntrophic partners. These results suggest that the ability of carbon cloth to accelerate DIET should be considered in anaerobic digester designs that incorporate carbon cloth. (C) 2014 Elsevier Ltd. All rights reserved.
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direct interspecies electron transfer between Geobacter Metallireducens and methanosarcina barkeri
Applied and Environmental Microbiology, 2014Co-Authors: Amelia-elena Rotaru, Pravin Malla Shrestha, Beatrice Markovaite, Kelly P Nevin, Shanshan Chen, Derek R. LovleyAbstract:Direct interspecies electron transfer (DIET) is potentially an effective form of syntrophy in methanogenic communities, but little is known about the diversity of methanogens capable of DIET. The ability of Methanosarcina barkeri to participate in DIET was evaluated in coculture with Geobacter Metallireducens. Cocultures formed aggregates that shared electrons via DIET during the stoichiometric conversion of ethanol to methane. Cocultures could not be initiated with a pilin-deficient G. Metallireducens strain, suggesting that long-range electron transfer along pili was important for DIET. Amendments of granular activated carbon permitted the pilin-deficient G. Metallireducens isolates to share electrons with M. barkeri, demonstrating that this conductive material could substitute for pili in promoting DIET. When M. barkeri was grown in coculture with the H-2-producing Pelobacter carbinolicus, incapable of DIET, M. barkeri utilized H-2 as an electron donor but metabolized little of the acetate that P. carbinolicus produced. This suggested that H-2, but not electrons derived from DIET, inhibited acetate metabolism. P. carbinolicus-M. barkeri cocultures did not aggregate, demonstrating that, unlike DIET, close physical contact was not necessary for interspecies H-2 transfer. M. barkeri is the second methanogen found to accept electrons via DIET and the first methanogen known to be capable of using either H-2 or electrons derived from DIET for CO2 reduction. Furthermore, M. barkeri is genetically tractable, making it a model organism for elucidating mechanisms by which methanogens make biological electrical connections with other cells.
Carlos A Salgueiro - One of the best experts on this subject based on the ideXlab platform.
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a unique aromatic residue modulates the redox range of a periplasmic multiheme cytochrome from Geobacter Metallireducens
Journal of Biological Chemistry, 2021Co-Authors: Pilar C. Portela, Marta A Silva, Liliana R Teixeira, Carlos A SalgueiroAbstract:Geobacter bacteria are able to transfer electrons to the exterior of the cell and reduce extracellular electron acceptors including toxic/radioactive metals and electrode surfaces, with potential applications in bioremediation or electricity harvesting. The triheme c-type cytochrome PpcA from Geobacter Metallireducens plays a crucial role in bridging the electron transfer from the inner to the outer membrane, ensuring an effective extracellular electron transfer. This cytochrome shares 80% identity with PpcA from Geobacter sulfurreducens, but their redox properties are markedly different, thus determining the distinctive working redox potential ranges in the two bacteria. PpcA from G. Metallireducens possesses two extra aromatic amino acids (Phe-6 and Trp-45) in its hydrophobic heme core, whereas PpcA from G. sulfurreducens has a leucine and a methionine in the equivalent positions. Given the different nature of these residues in the two cytochromes, we have hypothesized that the extra aromatic amino acids could be partially responsible for the observed functional differences. In this work, we have replaced Phe-6 and Trp-45 residues by their nonaromatic counterparts in PpcA from G. sulfurreducens. Using redox titrations followed by UV–visible and NMR spectroscopy we observed that residue Trp-45 shifted the redox potential range 33% toward that of PpcA from G. sulfurreducens, whereas Phe-6 produced a negligible effect. For the first time, it is shown that the inclusion of an aromatic residue at the heme core can modulate the working redox range in abundant periplasmic proteins, paving the way to engineer bacterial strains for optimal microbial bioelectrochemical applications.
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thermodynamic properties of triheme cytochrome ppcf from Geobacter Metallireducens reveal unprecedented functional mechanism
Biochimica et Biophysica Acta, 2020Co-Authors: Marisa R Ferreira, Tomas M Fernandes, Carlos A SalgueiroAbstract:Abstract The bacterium Geobacter Metallireducens is highly efficient in long-range extracellular electron transfer, a process that relies on an efficient bridging between the cytoplasmic electron donors and the extracellular acceptors. The periplasmic triheme cytochromes are crucial players in these processes and thus the understanding of their functional mechanism is crucial to elucidate the extracellular electron transfer processes in this microorganism. The triheme cytochrome PpcF from G. Metallireducens has the lowest amino acid sequence identity with the remaining cytochromes from the PpcA-family of G. sulfurreducens and G. Metallireducens, making it an interesting target for structural and functional studies. In this work, we performed a detailed functional and thermodynamic characterization of cytochrome PpcF by the complementary usage of NMR and visible spectroscopic techniques. The results obtained show that the heme reduction potentials are negative, different from each other and are also modulated by the redox and redox-Bohr interactions that assure unprecedented mechanistic features to the protein. The results showed that the order of oxidation of the hemes in cytochrome PpcF is maintained in the entire physiological pH range. The considerable separation of the hemes' redox potential values facilitates a sequential transfer within the chain of redox centers in PpcF, thus assuring electron transfer directionality to the electron acceptors.
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Backbone, side chain and heme resonance assignment of the triheme cytochrome PpcA from Geobacter Metallireducens in the oxidized state
Biomolecular NMR Assignments, 2019Co-Authors: Pilar C. Portela, Joana M. Dantas, Carlos A SalgueiroAbstract:The bacterium Geobacter Metallireducens is capable of transferring electrons to the cell exterior, a process designated extracellular electron transfer. This mechanism allows the microorganism to reduce extracellular acceptors such as Fe(III) (hydr)oxides and water toxic and/or radioactive contaminants including Cr(VI) and U(VI). It is also capable of oxidizing waste water aromatic organic compounds being an important microorganism for bioremediation of polluted waters. Extracellular electron transfer also allows electricity harvesting from microbial fuel cells, a promising sustainable form of energy production. However, extracellular electron transfer processes in this microorganism are still poorly characterized. The triheme c -type cytochrome PpcA from G. Metallireducens is abundant in the periplasm and is crucial for electron transfer between the cytoplasm and the cell’s exterior. In this work, we report near complete assignment of backbone, side chain and heme resonances for PpcA in the oxidized state that will permit its structure determination and identification of interactions with physiological redox partners.
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determination of the magnetic properties and orientation of the heme axial ligands of ppca from Geobacter Metallireducens by paramagnetic nmr
Journal of Inorganic Biochemistry, 2019Co-Authors: Tomas M Fernandes, Carlos A Salgueiro, Leonor Morgado, David L TurnerAbstract:The rising interest in the use of Geobacter bacteria for biotechnological applications demands a deep understanding of how these bacteria are able to thrive in a variety of environments and perform extracellular electron transfer. The Geobacter Metallireducens bacterium can couple the oxidation of a wide range of compounds to the reduction of several extracellular acceptors, including heavy metals, toxic organic compounds or electrode surfaces. The periplasmic c-type cytochrome PpcA from this bacterium is a member of a family composed of five periplasmic triheme cytochromes, which are important to bridge the electron transfer between the cytoplasm and the extracellular environment. To better understand the functional mechanism of PpcA it is essential to obtain structural data for this cytochrome. In this work, the geometry of the heme axial ligands, as well as the magnetic properties of the hemes were determined for the oxidized form of the cytochrome, using the 13C NMR chemical shifts of the heme α-substituents. The results were further compared with those previously obtained for the homologous cytochrome from Geobacter sulfurreducens. The orientations of the axial histidine planes and the magnetic properties of the hemes are conserved in both proteins. Overall, the results obtained allowed the definition of the orientation of the magnetic axes of PpcA from G. Metallireducens, which will be used as constraints to assist the solution structure determination of the cytochrome in the oxidized form.
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the triheme cytochrome ppcf from Geobacter Metallireducens exhibits distinct redox properties
FEBS Open Bio, 2018Co-Authors: Marisa R Ferreira, Joana M. Dantas, Carlos A SalgueiroAbstract:Electrogenic bacteria, such as Geobacter, can couple the oxidation of carbon sources to the reduction of extracellular electron acceptors; such acceptors include toxic and radioactive metals, as well as electrode surfaces, making Geobacter a suitable candidate for applied use in bioremediation and bioenergy generation. Geobacter Metallireducens is more promising in this regard than the better studied Geobacter sulfurreducens, as it has more efficient Fe (III) reduction rates and can convert nitrate to ammonia. The operon responsible for nitrate reductase activity in G. Metallireducens includes the gene encoding the cytochrome PpcF, which was proposed to exchange electrons with nitrate reductase. In the present work, we perform a biochemical and a biophysical characterization of PpcF. Spectroscopic techniques, including circular dichroism (CD), UV-visible, and nuclear magnetic resonance (NMR), revealed that the cytochrome is very stable (T m > 85 °C), contains three low-spin hemes, and is diamagnetic (S = 0) and paramagnetic (S = 1/2) in the reduced and oxidized states, respectively. The NMR chemical shifts of the heme substituents were assigned and used to determine the heme core architecture of PpcF. Compared to the PpcA-family from G. sulfurreducens, the spatial disposition of the hemes is conserved, but the functional properties are clearly distinct. In fact, potentiometric titrations monitored by UV-visible absorption reveal that the reduction potential values of PpcF are significantly less negative (-56 and -64 mV, versus the normal hydrogen electrode at pH 7.0 and 8.0, respectively). NMR redox titrations showed that the order of oxidation of the hemes is IV-I-III, a feature not observed for G. sulfurreducens. The different redox properties displayed by PpcF, including the small redox-Bohr effect and low reduction potential value of heme IV, were structurally rationalized and attributed to the lower number of positively charged residues located in the vicinity of heme IV. Overall, the redox features of PpcF suggest that biotechnological applications of G. Metallireducens may require less negative working functional redox windows than those using by G. sulfurreducens.
Matthias Boll - One of the best experts on this subject based on the ideXlab platform.
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one megadalton metalloenzyme complex in Geobacter Metallireducens involved in benzene ring reduction beyond the biological redox window
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Simona G. Huwiler, Martin Von Bergen, Hans-joachim Stärk, Claudia Loffler, Sebastian E L Anselmann, Jennifer Flechsler, Reinhard Rachel, Matthias BollAbstract:Reversible biological electron transfer usually occurs between redox couples at standard redox potentials ranging from +0.8 to −0.5 V. Dearomatizing benzoyl-CoA reductases (BCRs), key enzymes of the globally relevant microbial degradation of aromatic compounds at anoxic sites, catalyze a biological Birch reduction beyond the negative limit of this redox window. The structurally characterized BamBC subunits of class II BCRs accomplish benzene ring reduction at an active-site tungsten cofactor; however, the mechanism and components involved in the energetic coupling of endergonic benzene ring reduction have remained hypothetical. We present a 1-MDa, membrane-associated, Bam[(BC) 2 DEFGHI] 2 complex from the anaerobic bacterium Geobacter Metallireducens harboring 4 tungsten, 4 zinc, 2 selenocysteines, 6 FAD, and >50 FeS cofactors. The results suggest that class II BCRs catalyze electron transfer to the aromatic ring, yielding a cyclic 1,5-dienoyl-CoA via two flavin-based electron bifurcation events. This work expands our knowledge of energetic couplings in biology by high-molecular-mass electron bifurcating machineries.
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Differential Membrane Proteome Analysis Reveals Novel Proteins Involved in the Degradation of Aromatic Compounds in
2016Co-Authors: Matthias BollAbstract:Aromatic compounds comprise a large class of natural and man-made compounds, many of which are of consid-erable concern for the environment and human health. In aromatic compound-degrading anaerobic bacteria the central intermediate of aromatic catabolism, benzoyl co-enzyme A, is attacked by dearomatizing benzoyl-CoA re-ductases (BCRs). An ATP-dependent BCR has been char-acterized in facultative anaerobes. In contrast, a previous analysis of the soluble proteome from the obligately an-aerobic model organism Geobacter Metallireducens iden-tified genes putatively coding for a completely different dearomatizing BCR. The corresponding BamBCDEFGHI complex is predicted to comprise soluble molybdenum or tungsten, selenocysteine, and FeS cluster-containing components. To elucidate key processes involved in th
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glutaryl coenzyme a dehydrogenase from Geobacter Metallireducens interaction with electron transferring flavoprotein and kinetic basis of unidirectional catalysis
FEBS Journal, 2014Co-Authors: Sebastian Estelmann, Matthias BollAbstract:Glutaryl-CoA dehydrogenases (GDHs) are FAD containing acyl-CoA dehydrogenases that usually catalyze the dehydrogenation and decarboxylation of glutaryl-CoA to crotonyl-CoA with an electron transferring flavoprotein (ETF) acting as natural electron acceptor. In anaerobic bacteria, GDHs play an important role in the benzoyl-CoA degradation pathway of monocyclic aromatic compounds. In the present study, we identified, purified and characterized the benzoate-induced BamOP as the electron accepting ETF of GDH (BamM) from the Fe(III)-respiring Geobacter Metallireducens. The BamOP heterodimer contained FAD and AMP as cofactors. In the absence of an artificial electron acceptor, at pH values above 8, the BamMOP-components catalyzed the expected glutaryl-CoA oxidation to crotonyl-CoA and CO2; however, at pH values below 7, the redox-neutral glutaryl-CoA conversion to butyryl-CoA and CO2 became the dominant reaction. This previously unknown, strictly ETF-dependent coupled glutaryl-CoA oxidation/crotonyl-CoA reduction activity was facilitated by an unexpected two-electron transfer between FAD(BamM) and FAD(BamOP), as well as by the similar redox potentials of the two FAD cofactors in the substrate-bound state. The strict order of electron/proton transfer and C-C-cleavage events including transient charge-transfer complexes did not allow an energetic coupling of electron transfer and decarboxylation. This explains why it was difficult to release the glutaconyl-CoA intermediate from reduced GDH. Moreover, it provides a kinetic rational for the apparent inability of BamM to catalyze the reverse reductive crotonyl-CoA carboxylation, even under thermodynamically favourable conditions. For this reason reductive crotonyl-CoA carboxylation, a key reaction in C2-assimilation via the ethylmalonyl-CoA pathway, is accomplished by a different crotonyl-CoA carboxylase/reductase via a covalent NADPH/ene-adduct.
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Identification and Characterization of a Succinyl-Coenzyme A (CoA):Benzoate CoA Transferase in Geobacter Metallireducens
Journal of Bacteriology, 2012Co-Authors: Jana Oberender, Johannes W Kung, Martin Von Bergen, Jana Seifert, Matthias BollAbstract:ABSTRACT Geobacter Metallireducens is a Fe(III)-respiring deltaproteobacterium and serves as a model organism for aromatic compound-degrading, obligately anaerobic bacteria. In this study, a genetic system was established for G. Metallireducens using nitrate as an alternative electron acceptor. Surprisingly, disruption of the benzoate-induced bamY gene, encoding a benzoate coenzyme A (CoA) ligase, reproducibly showed an increased biomass yield in comparison to the wild type during growth with benzoate but not during growth with acetate. Complementation of bamY in trans converted the biomass yield back to the wild-type level. Growth of the bamY mutant with benzoate can be rationalized by the identification of a previously unknown succinyl-CoA:benzoate CoA transferase activity; it represents an additional, energetically less demanding mode of benzoate activation. The activity was highly enriched from extracts of cells grown on benzoate, yielding a 50-kDa protein band; mass spectrometric analysis identified the corresponding benzoate-induced gene annotated as a CoA transferase. It was heterologously expressed in Escherichia coli and characterized as a specific succinyl-CoA:benzoate CoA transferase. The newly identified enzyme in conjunction with a benzoate-induced succinyl-CoA synthetase links the tricarboxylic acid cycle to the upper benzoyl-CoA degradation pathway during growth on aromatic growth substrates.
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reversible biological birch reduction at an extremely low redox potential
Journal of the American Chemical Society, 2010Co-Authors: Johannes W Kung, Martin Von Bergen, Michael Müller, Sven Baumann, Peterleon Hagedoorn, Wilfred R Hagen, Matthias BollAbstract:The Birch reduction of aromatic rings to cyclohexadiene compounds is widely used in chemical synthesis and requires solvated electrons, the most potent reductants known in organic chemistry. Benzoyl-coenzyme A (CoA) reductases (BCR) are key enzymes in the anaerobic bacterial degradation of aromatic compounds and catalyze an analogous reaction under physiological conditions. Class I BCRs are FeS enzymes and couple the reductive dearomatization of benzoyl-CoA to cyclohexa-1,5-diene-1-carboxyl-CoA (dienoyl-CoA) to a stoichiometric ATP hydrolysis. Here, we report on a tungsten-containing class II BCR from Geobacter Metallireducens that catalyzed the fully reversible, ATP-independent dearomatization of benzoyl-CoA to dienoyl-CoA. BCR additionally catalyzed the disproportionation of dienoyl-CoA to benzoyl-CoA/monoenoyl-CoA and the four- and six-electron reduction of benzoyl-CoA in the presence of a reduced low-potential bridged 2,2′-bipyridyl redox dye. Reversible redox titration experiments in the presence of ...
Amelia-elena Rotaru - One of the best experts on this subject based on the ideXlab platform.
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Promoting Interspecies Electron Transfer with Biochar
Scientific Reports, 2015Co-Authors: Shanshan Chen, Nikhil S. Malvankar, Amelia-elena Rotaru, Pravin Malla Shrestha, Kelly P Nevin, Fengshan Liu, Fanghua Liu, W. Fan, Derek R. LovleyAbstract:Biochar, a charcoal-like product of the incomplete combustion of organic materials, is an increasingly popular soil amendment designed to improve soil fertility. We investigated the possibility that biochar could promote direct interspecies electron transfer (DIET) in a manner similar to that previously reported for granular activated carbon (GAC). Although the biochars investigated were 1000 times less conductive than GAC, they stimulated DIET in co-cultures of Geobacter Metallireducens with Geobacter sulfurreducens or Methanosarcina barkeri in which ethanol was the electron donor. Cells were attached to the biochar, yet not in close contact, suggesting that electrons were likely conducted through the biochar, rather than biological electrical connections. The finding that biochar can stimulate DIET may be an important consideration when amending soils with biochar and can help explain why biochar may enhance methane production from organic wastes under anaerobic conditions.
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carbon cloth stimulates direct interspecies electron transfer in syntrophic co cultures
Bioresource Technology, 2014Co-Authors: Shanshan Chen, Amelia-elena Rotaru, Kelly P Nevin, Fanghua Liu, Jo Philips, Trevor L. Woodard, Derek R. LovleyAbstract:This study investigated the possibility that the electrical conductivity of carbon cloth accelerates direct interspecies electron transfer (DIET) in co-cultures. Carbon cloth accelerated metabolism of DIET co-cultures (Geobacter Metallireducens-Geobacter sulfurreducens and G. Metallireducens-Methanosarcina barkeri) but did not promote metabolism of co-cultures performing interspecies H-2 transfer (Desulfovibrio vulgaris-G. sulfurreducens). On the other hand, DIET co-cultures were not stimulated by poorly conductive cotton cloth. Mutant strains lacking electrically conductive pili, or pili-associated cytochromes participated in DIET only in the presence of carbon cloth. In co-cultures promoted by carbon cloth, cells were primarily associated with the cloth although the syntrophic partners were too far apart for cell-to-cell biological electrical connections to be feasible. Carbon cloth seemingly mediated interspecies electron transfer between the distant syntrophic partners. These results suggest that the ability of carbon cloth to accelerate DIET should be considered in anaerobic digester designs that incorporate carbon cloth. (C) 2014 Elsevier Ltd. All rights reserved.
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direct interspecies electron transfer between Geobacter Metallireducens and methanosarcina barkeri
Applied and Environmental Microbiology, 2014Co-Authors: Amelia-elena Rotaru, Pravin Malla Shrestha, Beatrice Markovaite, Kelly P Nevin, Shanshan Chen, Derek R. LovleyAbstract:Direct interspecies electron transfer (DIET) is potentially an effective form of syntrophy in methanogenic communities, but little is known about the diversity of methanogens capable of DIET. The ability of Methanosarcina barkeri to participate in DIET was evaluated in coculture with Geobacter Metallireducens. Cocultures formed aggregates that shared electrons via DIET during the stoichiometric conversion of ethanol to methane. Cocultures could not be initiated with a pilin-deficient G. Metallireducens strain, suggesting that long-range electron transfer along pili was important for DIET. Amendments of granular activated carbon permitted the pilin-deficient G. Metallireducens isolates to share electrons with M. barkeri, demonstrating that this conductive material could substitute for pili in promoting DIET. When M. barkeri was grown in coculture with the H-2-producing Pelobacter carbinolicus, incapable of DIET, M. barkeri utilized H-2 as an electron donor but metabolized little of the acetate that P. carbinolicus produced. This suggested that H-2, but not electrons derived from DIET, inhibited acetate metabolism. P. carbinolicus-M. barkeri cocultures did not aggregate, demonstrating that, unlike DIET, close physical contact was not necessary for interspecies H-2 transfer. M. barkeri is the second methanogen found to accept electrons via DIET and the first methanogen known to be capable of using either H-2 or electrons derived from DIET for CO2 reduction. Furthermore, M. barkeri is genetically tractable, making it a model organism for elucidating mechanisms by which methanogens make biological electrical connections with other cells.
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constraint based modeling of carbon fixation and the energetics of electron transfer in Geobacter Metallireducens
PLOS Computational Biology, 2014Co-Authors: Adam M Feist, Amelia-elena Rotaru, Kelly P Nevin, Derek R. Lovley, Pier-luc Tremblay, Tian Zhang, Harish Nagarajan, Karsten ZenglerAbstract:Geobacter species are of great interest for environmental and biotechnology applications as they can carry out direct electron transfer to insoluble metals or other microorganisms and have the ability to assimilate inorganic carbon. Here, we report on the capability and key enabling metabolic machinery of Geobacter Metallireducens GS-15 to carry out CO2 fixation and direct electron transfer to iron. An updated metabolic reconstruction was generated, growth screens on targeted conditions of interest were performed, and constraint-based analysis was utilized to characterize and evaluate critical pathways and reactions in G. Metallireducens. The novel capability of G. Metallireducens to grow autotrophically with formate and Fe(III) was predicted and subsequently validated in vivo. Additionally, the energetic cost of transferring electrons to an external electron acceptor was determined through analysis of growth experiments carried out using three different electron acceptors (Fe(III), nitrate, and fumarate) by systematically isolating and examining different parts of the electron transport chain. The updated reconstruction will serve as a knowledgebase for understanding and engineering Geobacter and similar species.