@article {3051, title = {Direct microbial electron uptake as a mechanism for stainless steel corrosion in aerobic environments.}, journal = {Water Res}, volume = {219}, year = {2022}, month = {2022 Jul 01}, pages = {118553}, abstract = {

Shewanella oneidensis MR-1 is an attractive model microbe for elucidating the biofilm-metal interactions that contribute to the billions of dollars in corrosion damage to industrial applications each year. Multiple mechanisms for S. oneidensis-enhanced corrosion have been proposed, but none of these mechanisms have previously been rigorously investigated with methods that rule out alternative routes for electron transfer. We found that S. oneidensis grown under aerobic conditions formed thick biofilms (\~{}50~{\textmu}m) on stainless steel coupons, accelerating corrosion over sterile controls. H and flavins were ruled out as intermediary electron carriers because stainless steel did not reduce riboflavin and previous studies have demonstrated stainless does not generate H. Strain ∆mtrCBA, in which the genes for the most abundant porin-cytochrome conduit in S. oneidensis were deleted, corroded stainless steel substantially less than wild-type in aerobic cultures. Wild-type biofilms readily reduced nitrate with stainless steel as the sole electron donor under anaerobic conditions, but strain ∆mtrCBA did not. These results demonstrate that S. oneidensis can directly consume electrons from iron-containing metals and illustrate how direct metal-to-microbe electron transfer can be an important route for corrosion, even in aerobic environments.

}, keywords = {Biofilms, Corrosion, Electron Transport, Electrons, Metals, Oxidation-Reduction, Stainless Steel, Steel}, issn = {1879-2448}, doi = {10.1016/j.watres.2022.118553}, author = {Zhou, Enze and Li, Feng and Zhang, Dawei and Xu, Dake and Li, Zhong and Jia, Ru and Jin, Yuting and Song, Hao and Li, Huabing and Wang, Qiang and Wang, Jianjun and Li, Xiaogang and Gu, Tingyue and Homborg, Axel M and Mol, Johannes M C and Smith, Jessica A and Wang, Fuhui and Lovley, Derek R} } @article {3063, title = {Correlation of Key Physiological Properties of Isolates with Environment of Origin.}, journal = {Appl Environ Microbiol}, volume = {87}, year = {2021}, month = {2021 Jun 11}, pages = {e0073121}, abstract = {

It is known that the physiology of species can differ significantly, but the ecological impact of these differences is unclear. We recovered two strains of from two different ecosystems with a similar enrichment and isolation method. Both strains had the same ability to metabolize organic substrates and participate in direct interspecies electron transfer but also had major physiological differences. Strain DH-1, which was isolated from an anaerobic digester, used H as an electron donor. Genome analysis indicated that it lacks an Rnf complex and conserves energy from acetate metabolism via intracellular H cycling. In contrast, strain DH-2, a subsurface isolate, lacks hydrogenases required for H uptake and cycling and has an Rnf complex for energy conservation when growing on acetate. Further analysis of the genomes of previously described isolates, as well as phylogenetic and metagenomic data on uncultured in anaerobic digesters and diverse soils and sediments, revealed a physiological dichotomy that corresponded with environment of origin. The physiology of type I revolves around H production and consumption. In contrast, type II species eschew H and have genes for an Rnf complex and the multiheme, membrane-bound -type cytochrome MmcA, shown to be essential for extracellular electron transfer. The distribution of species in diverse environments suggests that the type I H-based physiology is well suited for high-energy environments, like anaerobic digesters, whereas type II Rnf/cytochrome-based physiology is an adaptation to the slower, steady-state carbon and electron fluxes common in organic-poor anaerobic soils and sediments. Biogenic methane is a significant greenhouse gas, and the conversion of organic wastes to methane is an important bioenergy process. species play an important role in methane production in many methanogenic soils and sediments as well as anaerobic waste digesters. The studies reported here emphasize that the genus is composed of two physiologically distinct groups. This is important to recognize when interpreting the role of in methanogenic environments, especially regarding H metabolism. Furthermore, the finding that type I species predominate in environments with high rates of carbon and electron flux and that type II species predominate in lower-energy environments suggests that evaluating the relative abundance of type I and type II may provide further insights into rates of carbon and electron flux in methanogenic environments.

}, keywords = {Acetates, Anaerobiosis, Bioreactors, Ecosystem, Electron Transport, Ethanol, Genome, Archaeal, Hydrogen, Methane, Methanosarcina, Phylogeny}, issn = {1098-5336}, doi = {10.1128/AEM.00731-21}, author = {Zhou, Jinjie and Holmes, Dawn E and Tang, Hai-Yan and Lovley, Derek R} } @article {3056, title = {Mechanisms for Electron Uptake by Methanosarcina acetivorans during Direct Interspecies Electron Transfer.}, journal = {mBio}, volume = {12}, year = {2021}, month = {2021 Oct 26}, pages = {e0234421}, abstract = {

Direct interspecies electron transfer (DIET) between bacteria and methanogenic archaea appears to be an important syntrophy in both natural and engineered methanogenic environments. However, the electrical connections on the outer surface of methanogens and the subsequent processing of electrons for carbon dioxide reduction to methane are poorly understood. Here, we report that the genetically tractable methanogen Methanosarcina acetivorans can grow via DIET in coculture with Geobacter metallireducens serving as the electron-donating partner. Comparison of gene expression patterns in grown in coculture versus pure-culture growth on acetate revealed that transcripts for the outer-surface multiheme type cytochrome MmcA were higher during DIET-based growth. Deletion of inhibited DIET. The high aromatic amino acid content of archaellins suggests that they might assemble into electrically conductive archaella. A mutant that could not express archaella was deficient in DIET. However, this mutant grew in DIET-based coculture as well as the archaellum-expressing parental strain in the presence of granular activated carbon, which was previously shown to serve as a substitute for electrically conductive pili as a conduit for long-range interspecies electron transfer in other DIET-based cocultures. Transcriptomic data suggesting that the membrane-bound Rnf, Fpo, and HdrED complexes also play a role in DIET were incorporated into a charge-balanced model illustrating how electrons entering the cell through MmcA can yield energy to support growth from carbon dioxide reduction. The results are the first genetics-based functional demonstration of likely outer-surface electrical contacts for DIET in a methanogen. The conversion of organic matter to methane plays an important role in the global carbon cycle and is an effective strategy for converting wastes to a useful biofuel. The reduction of carbon dioxide to methane accounts for approximately a third of the methane produced in anaerobic soils and sediments as well as waste digesters. Potential electron donors for carbon dioxide reduction are H or electrons derived from direct interspecies electron transfer (DIET) between bacteria and methanogens. Elucidating the relative importance of these electron donors has been difficult due to a lack of information on the electrical connections on the outer surfaces of methanogens and how they process the electrons received from DIET. Transcriptomic patterns and gene deletion phenotypes reported here provide insight into how a group of organisms that play an important role in methane production in soils and sediments participate in DIET.

}, keywords = {Archaeal Proteins, Electron Transport, Electrons, Methane, Methanosarcina, Transcriptome}, issn = {2150-7511}, doi = {10.1128/mBio.02344-21}, author = {Holmes, Dawn E and Zhou, Jinjie and Ueki, Toshiyuki and Woodard, Trevor and Lovley, Derek R} } @article {3064, title = {Solvent-Induced Assembly of Microbial Protein Nanowires into Superstructured Bundles.}, journal = {Biomacromolecules}, volume = {22}, year = {2021}, month = {2021 Mar 08}, pages = {1305-1311}, abstract = {

Protein-based electronic biomaterials represent an attractive alternative to traditional metallic and semiconductor materials due to their environmentally benign production and purification. However, major challenges hindering further development of these materials include (1) limitations associated with processing proteins in organic solvents and (2) difficulties in forming higher-order structures or scaffolds with multilength scale control. This paper addresses both challenges, resulting in the formation of one-dimensional bundles composed of electrically conductive protein nanowires harvested from the microbes and . Processing these bionanowires from common organic solvents, such as hexane, cyclohexane, and DMF, enabled the production of multilength scale structures composed of distinctly visible pili. Transmission electron microscopy revealed striking images of bundled protein nanowires up to 10 μm in length and with widths ranging from 50-500 nm (representing assembly of tens to hundreds of nanowires). Conductive atomic force microscopy confirmed the presence of an appreciable nanowire conductivity in their bundled state. These results greatly expand the possibilities for fabricating a diverse array of protein nanowire-based electronic device architectures.

}, keywords = {Electric Conductivity, Electron Transport, Geobacter, Nanowires, Solvents}, issn = {1526-4602}, doi = {10.1021/acs.biomac.0c01790}, author = {Sun, Yun-Lu and Montz, Brian J and Selhorst, Ryan and Tang, Hai-Yan and Zhu, Jiaxin and Nevin, Kelly P and Woodard, Trevor L and Ribbe, Alexander E and Russell, Thomas P and Nonnenmann, Stephen S and Lovley, Derek R and Emrick, Todd} } @article {3069, title = {Bioinspired bio-voltage memristors.}, journal = {Nat Commun}, volume = {11}, year = {2020}, month = {2020 Apr 20}, pages = {1861}, abstract = {

Memristive devices are promising candidates to emulate biological computing. However, the typical switching voltages (0.2-2 V) in previously described devices are much higher than the amplitude in biological counterparts. Here we demonstrate a type of diffusive memristor, fabricated from the protein nanowires harvested from the bacterium Geobacter sulfurreducens, that functions at the biological voltages of 40-100 mV. Memristive function at biological voltages is possible because the protein nanowires catalyze metallization. Artificial neurons built from these memristors not only function at biological action potentials (e.g., 100 mV, 1 ms) but also exhibit temporal integration close to that in biological neurons. The potential of using the memristor to directly process biosensing signals is also demonstrated.

}, keywords = {Action Potentials, Biosensing Techniques, Electricity, Electronics, Equipment Design, Geobacter, Humans, Molecular Dynamics Simulation, Nanotechnology, Nanowires, Neural Networks, Computer, Neurons, Synapses, Wearable Electronic Devices}, issn = {2041-1723}, doi = {10.1038/s41467-020-15759-y}, author = {Fu, Tianda and Liu, Xiaomeng and Gao, Hongyan and Ward, Joy E and Liu, Xiaorong and Yin, Bing and Wang, Zhongrui and Zhuo, Ye and Walker, David J F and Joshua Yang, J and Chen, Jianhan and Lovley, Derek R and Yao, Jun} } @article {3067, title = { Capable of Direct Interspecies Electron Transfer.}, journal = {Environ Sci Technol}, volume = {54}, year = {2020}, month = {2020 Dec 01}, pages = {15347-15354}, abstract = {

Direct interspecies electron transfer (DIET) from bacteria to methanogens is a revolutionary concept for syntrophic metabolism in methanogenic soils/sediments and anaerobic digestion. Previous studies have indicated that the potential for DIET is limited to methanogens in the , leading to the assumption that an abundance of other types of methanogens, such as species, indicates a lack of DIET. We report here on a strain of , designated strain YSL, that grows via DIET in defined cocultures with . The cocultures formed aggregates, in which cells of strain YSL and were uniformly dispersed throughout. This close association of the two species is the likely explanation for the ability of a strain of that could not express electrically conductive pili to grow in coculture with strain YSL. Granular activated carbon promoted the initial formation of the DIET-based cocultures. The discovery of DIET in , the genus of methanogens that has been the exemplar for interspecies electron transfer H, suggests that the capacity for DIET is much more broadly distributed among methanogens than previously considered. More innovative approaches to microbial isolation and characterization are needed in order to better understand how methanogenic communities function.

}, keywords = {Electron Transport, Electrons, Geobacter, Methane, Methanobacterium}, issn = {1520-5851}, doi = {10.1021/acs.est.0c05525}, author = {Zheng, Shiling and Liu, Fanghua and Wang, Bingchen and Zhang, Yuechao and Lovley, Derek R} } @article {3066, title = {Sparking Anaerobic Digestion: Promoting Direct Interspecies Electron Transfer to Enhance Methane Production.}, journal = {iScience}, volume = {23}, year = {2020}, month = {2020 Dec 18}, pages = {101794}, abstract = {

Anaerobic digestion was one of the first bioenergy strategies developed, yet the interactions of the microbial community that is responsible for the production of methane are still poorly understood. For example, it has only recently been recognized that the bacteria that oxidize organic waste components can forge electrical connections with methane-producing microbes through biologically produced, protein-based, conductive circuits. This direct interspecies electron transfer (DIET) is faster than interspecies electron exchange via diffusive electron carriers, such as H. DIET is also more resilient to perturbations such as increases in organic load inputs or toxic compounds. However, with current digester practices DIET rarely predominates. Improvements in anaerobic digestion associated with the addition of electrically conductive materials have been attributed to increased DIET, but experimental verification has been lacking. This deficiency may soon be overcome with improved understanding of the diversity of microbes capable of DIET, which is leading to molecular tools for determining the extent of DIET. Here we review the microbiology of DIET, suggest molecular strategies for monitoring DIET in anaerobic digesters, and propose approaches for re-engineering digester design and practices to encourage DIET.

}, issn = {2589-0042}, doi = {10.1016/j.isci.2020.101794}, author = {Zhao, Zhiqiang and Li, Yang and Zhang, Yaobin and Lovley, Derek R} } @article {3072, title = {Syntrophus conductive pili demonstrate that common hydrogen-donating syntrophs can have a direct electron transfer option.}, journal = {ISME J}, volume = {14}, year = {2020}, month = {2020 Mar}, pages = {837-846}, abstract = {

Syntrophic interspecies electron exchange is essential for the stable functioning of diverse anaerobic microbial communities. Hydrogen/formate interspecies electron transfer (HFIT), in which H and/or formate function as diffusible electron carriers, has been considered to be the primary mechanism for electron transfer because most common syntrophs were thought to lack biochemical components, such as electrically conductive pili (e-pili), necessary for direct interspecies electron transfer (DIET). Here we report that Syntrophus aciditrophicus, one of the most intensively studied microbial models for HFIT, produces e-pili and can grow via DIET. Heterologous expression of the putative S. aciditrophicus type IV pilin gene in Geobacter sulfurreducens yielded conductive pili of the same diameter (4 nm) and conductance of the native S. aciditrophicus pili and enabled long-range electron transport in G. sulfurreducens. S. aciditrophicus lacked abundant c-type cytochromes often associated with DIET. Pilin genes likely to yield e-pili were found in other genera of hydrogen/formate-producing syntrophs. The finding that DIET is a likely option for diverse syntrophs that are abundant in many anaerobic environments necessitates a reexamination of the paradigm that HFIT is the predominant mechanism for syntrophic electron exchange within anaerobic microbial communities of biogeochemical and practical significance.

}, keywords = {Deltaproteobacteria, Electric Conductivity, Electron Transport, Electrons, Fimbriae Proteins, Fimbriae, Bacterial, Formates, Geobacter, Hydrogen}, issn = {1751-7370}, doi = {10.1038/s41396-019-0575-9}, author = {Walker, David J F and Nevin, Kelly P and Holmes, Dawn E and Rotaru, Amelia-Elena and Ward, Joy E and Woodard, Trevor L and Zhu, Jiaxin and Ueki, Toshiyuki and Nonnenmann, Stephen S and McInerney, Michael J and Lovley, Derek R} } @article {3079, title = {The Archaellum of Methanospirillum hungatei Is Electrically Conductive.}, journal = {mBio}, volume = {10}, year = {2019}, month = {2019 Apr 16}, abstract = {

Microbially produced electrically conductive protein filaments are of interest because they can function as conduits for long-range biological electron transfer. They also show promise as sustainably produced electronic materials. Until now, microbially produced conductive protein filaments have been reported only for bacteria. We report here that the archaellum of is electrically conductive. This is the first demonstration that electrically conductive protein filaments have evolved in Furthermore, the structure of the archaellum was previously determined (N. Poweleit, P. Ge, H. N. Nguyen, R. R. O. Loo, et al., Nat Microbiol 2:16222, 2016, https://doi.org/10.1038/nmicrobiol.2016.222). Thus, the archaellum of is the first microbially produced electrically conductive protein filament for which a structure is known. We analyzed the previously published structure and identified a core of tightly packed phenylalanines that is one likely route for electron conductance. The availability of the archaellum structure is expected to substantially advance mechanistic evaluation of long-range electron transport in microbially produced electrically conductive filaments and to aid in the design of "green" electronic materials that can be microbially produced with renewable feedstocks. Microbially produced electrically conductive protein filaments are a revolutionary, sustainably produced, electronic material with broad potential applications. The design of new protein nanowires based on the known archaellum structure could be a major advance over the current empirical design of synthetic protein nanowires from electrically conductive bacterial pili. An understanding of the diversity of outer-surface protein structures capable of electron transfer is important for developing models for microbial electrical communication with other cells and minerals in natural anaerobic environments. Extracellular electron exchange is also essential in engineered environments such as bioelectrochemical devices and anaerobic digesters converting wastes to methane. The finding that the archaellum of is electrically conductive suggests that some archaea might be able to make long-range electrical connections with their external environment.

}, keywords = {Electric Conductivity, Electricity, Electron Transport, Flagella, Methanospirillum, Phenylalanine}, issn = {2150-7511}, doi = {10.1128/mBio.00579-19}, author = {Walker, David J F and Martz, Eric and Holmes, Dawn E and Zhou, Zimu and Nonnenmann, Stephen S and Lovley, Derek R} } @article {3074, title = {A Membrane-Bound Cytochrome Enables To Conserve Energy from Extracellular Electron Transfer.}, journal = {mBio}, volume = {10}, year = {2019}, month = {2019 Aug 20}, abstract = {

Extracellular electron exchange in species and closely related plays an important role in the global carbon cycle and enhances the speed and stability of anaerobic digestion by facilitating efficient syntrophic interactions. Here, we grew with methanol provided as the electron donor and the humic analogue, anthraquione-2,6-disulfonate (AQDS), provided as the electron acceptor when methane production was inhibited with bromoethanesulfonate. AQDS was reduced with simultaneous methane production in the absence of bromoethanesulfonate. Transcriptomics revealed that expression of the gene for the transmembrane, multiheme, -type cytochrome MmcA was higher in AQDS-respiring cells than in cells performing methylotrophic methanogenesis. A strain in which the gene for MmcA was deleted failed to grow via AQDS reduction but grew with the conversion of methanol or acetate to methane, suggesting that MmcA has a specialized role as a conduit for extracellular electron transfer. Enhanced expression of genes for methanol conversion to methyl-coenzyme M and the Rnf complex suggested that methanol is oxidized to carbon dioxide in AQDS-respiring cells through a pathway that is similar to methyl-coenzyme M oxidation in methanogenic cells. However, during AQDS respiration the Rnf complex and reduced methanophenazine probably transfer electrons to MmcA, which functions as the terminal reductase for AQDS reduction. Extracellular electron transfer may enable the survival of methanogens in dynamic environments in which oxidized humic substances and Fe(III) oxides are intermittently available. The availability of tools for genetic manipulation of makes it an excellent model microbe for evaluating -type cytochrome-dependent extracellular electron transfer in The discovery of a methanogen that can conserve energy to support growth solely from the oxidation of organic carbon coupled to the reduction of an extracellular electron acceptor expands the possible environments in which methanogens might thrive. The potential importance of -type cytochromes for extracellular electron transfer to syntrophic bacterial partners and/or Fe(III) minerals in some was previously proposed, but these studies with provide the first genetic evidence for cytochrome-based extracellular electron transfer in The results suggest parallels with Gram-negative bacteria, such as and species, in which multiheme outer-surface -type cytochromes are an essential component for electrical communication with the extracellular environment. offers an unprecedented opportunity to study mechanisms for energy conservation from the anaerobic oxidation of one-carbon organic compounds coupled to extracellular electron transfer in with implications not only for methanogens but possibly also for that anaerobically oxidize methane.

}, keywords = {Acetates, Anthraquinones, Cytochromes, Electron Transport, Electrons, Ferric Compounds, Gene Expression Regulation, Archaeal, Gram-Negative Bacteria, Membranes, Mesna, Methane, Methanol, Methanosarcina, Oxidation-Reduction, Oxidoreductases, Transcriptome}, issn = {2150-7511}, doi = {10.1128/mBio.00789-19}, author = {Holmes, Dawn E and Ueki, Toshiyuki and Tang, Hai-Yan and Zhou, Jinjie and Smith, Jessica A and Chaput, Gina and Lovley, Derek R} } @article {3078, title = {A pilin chaperone required for the expression of electrically conductive Geobacter sulfurreducens pili.}, journal = {Environ Microbiol}, volume = {21}, year = {2019}, month = {2019 Jul}, pages = {2511-2522}, abstract = {

Mechanisms controlling the expression of the electrically conductive pili (e-pili) of Geobacter species are of interest because of the important role of e-pili in diverse biogeochemical processes, anaerobic digestion and electromicrobiological applications. We investigated the function of the protein, designated Spc (short pilin chaperone), encoded by the gene immediately downstream from the gene for PilA, the monomer that assembles into e-pili. Multiple lines of evidence suggest that Spc forms an oligomer that is associated with the inner membrane. Mutating the start codon of spc to prevent translation increased the transcript abundance of pilA but greatly diminished the abundance of PilA, and e-pili could no longer be detected. Cross-linking, protein capture and two-hybrid studies demonstrated that Spc and PilA interacted. Two sites in PilA for electrostatic interaction with Spc were identified. The results demonstrate that Spc is required for PilA stability prior to incorporation into e-pili, suggesting that Spc has a chaperone function that may be specific to the relatively short PilA monomers that assemble into e-pili. These results are important for identifying microorganisms likely to express e-pili from (meta)genomic data and for the construction of microbial strains expressing e-pili.

}, keywords = {Electric Conductivity, Fimbriae Proteins, Fimbriae, Bacterial, Geobacter, Molecular Chaperones}, issn = {1462-2920}, doi = {10.1111/1462-2920.14638}, author = {Liu, Xing and Zhan, Ji and Jing, Xianyue and Zhou, Shungui and Lovley, Derek R} } @article {3099, title = {Enhancing anaerobic digestion of complex organic waste with carbon-based conductive materials.}, journal = {Bioresour Technol}, volume = {220}, year = {2016}, month = {2016 Nov}, pages = {516-522}, abstract = {

The aim of this work was to study the methanogenic metabolism of dog food, a food waste surrogate, in laboratory-scale reactors with different carbon-based conductive materials. Carbon cloth, carbon felt, and granular activated carbon all permitted higher organic loading rates and promoted faster recovery of soured reactors than the control reactors. Microbial community analysis revealed that specific and substantial enrichments of Sporanaerobacter and Methanosarcina were present on the carbon cloth surface. These results, and the known ability of Sporanaerobacter species to transfer electrons to elemental sulfur, suggest that Sporanaerobacter species can participate in direct interspecies electron transfer with Methanosarcina species when carbon cloth is available as an electron transfer mediator.

}, keywords = {Anaerobiosis, Animals, Bacteria, Bioreactors, Carbon, Carbon Fiber, Charcoal, Dogs, Electric Conductivity, Fatty Acids, Volatile, Hydrogen-Ion Concentration, Methane, Organic Chemicals, Waste Products}, issn = {1873-2976}, doi = {10.1016/j.biortech.2016.08.114}, author = {Dang, Yan and Holmes, Dawn E and Zhao, Zhiqiang and Woodard, Trevor L and Zhang, Yaobin and Sun, Dezhi and Wang, Li-Ying and Nevin, Kelly P and Lovley, Derek R} } @article {3104, title = {Potential enhancement of direct interspecies electron transfer for syntrophic metabolism of propionate and butyrate with biochar in up-flow anaerobic sludge blanket reactors.}, journal = {Bioresour Technol}, volume = {209}, year = {2016}, month = {2016 Jun}, pages = {148-56}, abstract = {

Promoting direct interspecies electron transfer (DIET) to enhance syntrophic metabolism may be a strategy for accelerating the conversion of organic wastes to methane, but microorganisms capable of metabolizing propionate and butyrate via DIET under methanogenic conditions have yet to be identified. In an attempt to establish methanogenic communities metabolizing propionate or butyrate with DIET, enrichments were initiated with up-flow anaerobic sludge blanket (UASB), similar to those that were previously reported to support communities that metabolized ethanol with DIET that relied on direct biological electrical connections. In the absence of any amendments, microbial communities enriched were dominated by microorganisms closely related to pure cultures that are known to metabolize propionate or butyrate to acetate with production of H2. When biochar was added to the reactors there was a substantial enrichment on the biochar surface of 16S rRNA gene sequences closely related to Geobacter and Methanosaeta species known to participate in DIET.

}, keywords = {Acetates, Bioreactors, Butyric Acid, Charcoal, Electron Transport, Geobacter, Methane, Microbial Consortia, Propionates, RNA, Ribosomal, 16S, Sewage, Waste Disposal, Fluid}, issn = {1873-2976}, doi = {10.1016/j.biortech.2016.03.005}, author = {Zhao, Zhiqiang and Zhang, Yaobin and Holmes, Dawn E and Dang, Yan and Woodard, Trevor L and Nevin, Kelly P and Lovley, Derek R} } @article {3109, title = {Enhancing syntrophic metabolism in up-flow anaerobic sludge blanket reactors with conductive carbon materials.}, journal = {Bioresour Technol}, volume = {191}, year = {2015}, month = {2015 Sep}, pages = {140-5}, abstract = {

Syntrophic metabolism of alcohols and fatty acids is a critical step in anaerobic digestion, which if enhanced can better stabilize the process and enable shorter retention times. Direct interspecies electron transfer (DIET) has recently been recognized as an alternative route to hydrogen interspecies transfer as a mechanism for interspecies syntrophic electron exchange. Therefore, the possibility of accelerating syntrophic metabolism of ethanol in up-flow anaerobic sludge blanket (UASB) reactors by incorporating conductive materials in reactor design was investigated. Graphite, biochar, and carbon cloth all immediately enhanced methane production and COD removal. As the hydraulic retention time was decreased the increased effectiveness of treatment in reactors with conductive materials increased versus the control reactor. When these conductive materials were removed from the reactors rates of syntrophic metabolism declined to rates comparable to the control reactor. These results suggest that incorporating conductive materials in the design of UASB reactors may enhance digester effectiveness.

}, keywords = {Anaerobiosis, Bioreactors, Carbon, Charcoal, Electron Transport, Graphite, Hydrogen, Methane, Sewage, Waste Disposal, Fluid}, issn = {1873-2976}, doi = {10.1016/j.biortech.2015.05.007}, author = {Zhao, Zhiqiang and Zhang, Yaobin and Woodard, T L and Nevin, K P and Lovley, D R} } @article {3127, title = {Constraint-based modeling of carbon fixation and the energetics of electron transfer in Geobacter metallireducens.}, journal = {PLoS Comput Biol}, volume = {10}, year = {2014}, month = {2014 Apr}, pages = {e1003575}, abstract = {

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.

}, keywords = {Carbon, Electron Transport, Energy Metabolism, Genome, Bacterial, Geobacter, Models, Biological}, issn = {1553-7358}, doi = {10.1371/journal.pcbi.1003575}, author = {Feist, Adam M and Nagarajan, Harish and Rotaru, Amelia-Elena and Tremblay, Pier-Luc and Zhang, Tian and Nevin, Kelly P and Lovley, Derek R and Zengler, Karsten} } @article {3122, title = {Identification of genes specifically required for the anaerobic metabolism of benzene in Geobacter metallireducens.}, journal = {Front Microbiol}, volume = {5}, year = {2014}, month = {2014}, pages = {245}, abstract = {

Although the biochemical pathways for the anaerobic degradation of many of the hydrocarbon constituents in petroleum reservoirs have been elucidated, the mechanisms for anaerobic activation of benzene, a very stable molecule, are not known. Previous studies have demonstrated that Geobacter metallireducens can anaerobically oxidize benzene to carbon dioxide with Fe(III) as the sole electron acceptor and that phenol is an intermediate in benzene oxidation. In an attempt to identify enzymes that might be involved in the conversion of benzene to phenol, whole-genome gene transcript abundance was compared in cells metabolizing benzene and cells metabolizing phenol. Eleven genes had significantly higher transcript abundance in benzene-metabolizing cells. Five of these genes had annotations suggesting that they did not encode proteins that could be involved in benzene metabolism and were not further studied. Strains were constructed in which one of the remaining six genes was deleted. The strain in which the monocistronic gene Gmet 0232 was deleted metabolized phenol, but not benzene. Transcript abundance of the adjacent monocistronic gene, Gmet 0231, predicted to encode a zinc-containing oxidoreductase, was elevated in cells metabolizing benzene, although not at a statistically significant level. However, deleting Gmet 0231 also yielded a strain that could metabolize phenol, but not benzene. Although homologs of Gmet 0231 and Gmet 0232 are found in microorganisms not known to anaerobically metabolize benzene, the adjacent localization of these genes is unique to G. metallireducens. The discovery of genes that are specifically required for the metabolism of benzene, but not phenol in G. metallireducens is an important step in potentially identifying the mechanisms for anaerobic benzene activation.

}, issn = {1664-302X}, doi = {10.3389/fmicb.2014.00245}, author = {Zhang, Tian and Tremblay, Pier-Luc and Chaurasia, Akhilesh K and Smith, Jessica A and Bain, Timothy S and Lovley, Derek R} } @article {1417, title = {Land use change alters functional gene diversity, composition and abundance in Amazon forest soil microbial communities.}, journal = {Mol Ecol}, volume = {23}, year = {2014}, month = {2014 Jun}, pages = {2988-99}, abstract = {

Land use change in the Amazon rainforest alters the taxonomic structure of soil microbial communities, but whether it alters their functional gene composition is unknown. We used the highly parallel microarray technology GeoChip 4.0, which contains 83,992 probes specific for genes linked nutrient cycling and other processes, to evaluate how the diversity, abundance and similarity of the targeted genes responded to forest-to-pasture conversion. We also evaluated whether these parameters were reestablished with secondary forest growth. A spatially nested scheme was employed to sample a primary forest, two pastures (6 and 38 years old) and a secondary forest. Both pastures had significantly lower microbial functional genes richness and diversity when compared to the primary forest. Gene composition and turnover were also significantly modified with land use change. Edaphic traits associated with soil acidity, iron availability, soil texture and organic matter concentration were correlated with these gene changes. Although primary and secondary forests showed similar functional gene richness and diversity, there were differences in gene composition and turnover, suggesting that community recovery was not complete in the secondary forest. Gene association analysis revealed that response to ecosystem conversion varied significantly across functional gene groups, with genes linked to carbon and nitrogen cycling mostly altered. This study indicates that diversity and abundance of numerous environmentally important genes respond to forest-to-pasture conversion and hence have the potential to affect the related processes at an ecosystem scale.

}, keywords = {Agriculture, Carbon Cycle, Ecosystem, Genes, Bacterial, Genes, Fungal, Genetic Variation, Metagenome, Multigene Family, Nitrogen Cycle, Oligonucleotide Array Sequence Analysis, Soil Microbiology, Trees, Tropical Climate}, issn = {1365-294X}, doi = {10.1111/mec.12786}, author = {Paula, Fabiana S and Rodrigues, Jorge L M and Zhou, Jizhong and Wu, Liyou and Mueller, Rebecca C and Mirza, Babur S and Bohannan, Brendan J M and N{\"u}sslein, Klaus and Deng, Ye and Tiedje, James M and Pellizari, Vivian H} } @article {3135, title = {Sulfur oxidation to sulfate coupled with electron transfer to electrodes by Desulfuromonas strain TZ1.}, journal = {Microbiology (Reading)}, volume = {160}, year = {2014}, month = {2014 Jan}, pages = {123-129}, abstract = {

Microbial oxidation of elemental sulfur with an electrode serving as the electron acceptor is of interest because this may play an important role in the recovery of electrons from sulfidic wastes and for current production in marine benthic microbial fuel cells. Enrichments initiated with a marine sediment inoculum, with elemental sulfur as the electron donor and a positively poised (+300 mV versus Ag/AgCl) anode as the electron acceptor, yielded an anode biofilm with a diversity of micro-organisms, including Thiobacillus, Sulfurimonas, Pseudomonas, Clostridium and Desulfuromonas species. Further enrichment of the anode biofilm inoculum in medium with elemental sulfur as the electron donor and Fe(III) oxide as the electron acceptor, followed by isolation in solidified sulfur/Fe(III) medium yielded a strain of Desulfuromonas, designated strain TZ1. Strain TZ1 effectively oxidized elemental sulfur to sulfate with an anode serving as the sole electron acceptor, at rates faster than Desulfobulbus propionicus, the only other organism in pure culture previously shown to oxidize S{\textdegree} with current production. The abundance of Desulfuromonas species enriched on the anodes of marine benthic fuel cells has previously been interpreted as acetate oxidation driving current production, but the results presented here suggest that sulfur-driven current production is a likely alternative.

}, keywords = {Bioelectric Energy Sources, Desulfuromonas, DNA, Bacterial, Electricity, Electrodes, Geologic Sediments, Molecular Sequence Data, Oxidation-Reduction, Sequence Analysis, DNA, Sulfates, Sulfur}, issn = {1465-2080}, doi = {10.1099/mic.0.069930-0}, author = {Zhang, Tian and Bain, Timothy S and Barlett, Melissa A and Dar, Shabir A and Snoeyenbos-West, Oona L and Nevin, Kelly P and Lovley, Derek R} } @article {3136, title = {Anaerobic benzene oxidation via phenol in Geobacter metallireducens.}, journal = {Appl Environ Microbiol}, volume = {79}, year = {2013}, month = {2013 Dec}, pages = {7800-6}, abstract = {

Anaerobic activation of benzene is expected to represent a novel biochemistry of environmental significance. Therefore, benzene metabolism was investigated in Geobacter metallireducens, the only genetically tractable organism known to anaerobically degrade benzene. Trace amounts (<0.5 μM) of phenol accumulated in cultures of Geobacter metallireducens anaerobically oxidizing benzene to carbon dioxide with the reduction of Fe(III). Phenol was not detected in cell-free controls or in Fe(II)- and benzene-containing cultures of Geobacter sulfurreducens, a Geobacter species that cannot metabolize benzene. The phenol produced in G. metallireducens cultures was labeled with (18)O during growth in H2(18)O, as expected for anaerobic conversion of benzene to phenol. Analysis of whole-genome gene expression patterns indicated that genes for phenol metabolism were upregulated during growth on benzene but that genes for benzoate or toluene metabolism were not, further suggesting that phenol was an intermediate in benzene metabolism. Deletion of the genes for PpsA or PpcB, subunits of two enzymes specifically required for the metabolism of phenol, removed the capacity for benzene metabolism. These results demonstrate that benzene hydroxylation to phenol is an alternative to carboxylation for anaerobic benzene activation and suggest that this may be an important metabolic route for benzene removal in petroleum-contaminated groundwaters, in which Geobacter species are considered to play an important role in anaerobic benzene degradation.

}, keywords = {Anaerobiosis, Benzene, Carbon Dioxide, Gene Deletion, Gene Expression Profiling, Geobacter, Iron, Metabolic Networks and Pathways, Oxidation-Reduction, Phenol, Water}, issn = {1098-5336}, doi = {10.1128/AEM.03134-13}, author = {Zhang, Tian and Tremblay, Pier-Luc and Chaurasia, Akhilesh Kumar and Smith, Jessica A and Bain, Timothy S and Lovley, Derek R} } @article {3133, title = {Characterization and modelling of interspecies electron transfer mechanisms and microbial community dynamics of a syntrophic association.}, journal = {Nat Commun}, volume = {4}, year = {2013}, month = {2013}, pages = {2809}, abstract = {

Syntrophic associations are central to microbial communities and thus have a fundamental role in the global carbon cycle. Despite biochemical approaches describing the physiological activity of these communities, there has been a lack of a mechanistic understanding of the relationship between complex nutritional and energetic dependencies and their functioning. Here we apply a multi-omic modelling workflow that combines genomic, transcriptomic and physiological data with genome-scale models to investigate dynamics and electron flow mechanisms in the syntrophic association of Geobacter metallireducens and Geobacter sulfurreducens. Genome-scale modelling of direct interspecies electron transfer reveals insights into the energetics of electron transfer mechanisms. While G. sulfurreducens adapts to rapid syntrophic growth by changes at the genomic and transcriptomic level, G. metallireducens responds only at the transcriptomic level. This multi-omic approach enhances our understanding of adaptive responses and factors that shape the evolution of syntrophic communities.

}, keywords = {Adaptation, Biological, Biological Evolution, Electron Transport, Genome, Bacterial, Geobacter, Microbial Interactions, Models, Biological, Symbiosis, Transcriptome}, issn = {2041-1723}, doi = {10.1038/ncomms3809}, author = {Nagarajan, Harish and Embree, Mallory and Rotaru, Amelia-Elena and Shrestha, Pravin M and Feist, Adam M and Palsson, Bernhard {\O} and Lovley, Derek R and Zengler, Karsten} } @article {3132, title = {Characterizing acetogenic metabolism using a genome-scale metabolic reconstruction of Clostridium ljungdahlii.}, journal = {Microb Cell Fact}, volume = {12}, year = {2013}, month = {2013 Nov 25}, pages = {118}, abstract = {

BACKGROUND: The metabolic capabilities of acetogens to ferment a wide range of sugars, to grow autotrophically on H2/CO2, and more importantly on synthesis gas (H2/CO/CO2) make them very attractive candidates as production hosts for biofuels and biocommodities. Acetogenic metabolism is considered one of the earliest modes of bacterial metabolism. A thorough understanding of various factors governing the metabolism, in particular energy conservation mechanisms, is critical for metabolic engineering of acetogens for targeted production of desired chemicals.

RESULTS: Here, we present the genome-scale metabolic network of Clostridium ljungdahlii, the first such model for an acetogen. This genome-scale model (iHN637) consisting of 637 genes, 785 reactions, and 698 metabolites captures all the major central metabolic and biosynthetic pathways, in particular pathways involved in carbon fixation and energy conservation. A combination of metabolic modeling, with physiological and transcriptomic data provided insights into autotrophic metabolism as well as aided the characterization of a nitrate reduction pathway in C. ljungdahlii. Analysis of the iHN637 metabolic model revealed that flavin based electron bifurcation played a key role in energy conservation during autotrophic growth and helped identify genes for some of the critical steps in this mechanism.

CONCLUSIONS: iHN637 represents a predictive model that recapitulates experimental data, and provides valuable insights into the metabolic response of C. ljungdahlii to genetic perturbations under various growth conditions. Thus, the model will be instrumental in guiding metabolic engineering of C. ljungdahlii for the industrial production of biocommodities and biofuels.

}, keywords = {Acetates, Biofuels, Clostridium, Genome, Metabolic Engineering, Metabolic Networks and Pathways}, issn = {1475-2859}, doi = {10.1186/1475-2859-12-118}, author = {Nagarajan, Harish and Sahin, Merve and Nogales, Juan and Latif, Haythem and Lovley, Derek R and Ebrahim, Ali and Zengler, Karsten} } @article {3140, title = {Characterizing the interplay between multiple levels of organization within bacterial sigma factor regulatory networks.}, journal = {Nat Commun}, volume = {4}, year = {2013}, month = {2013}, pages = {1755}, abstract = {

Bacteria contain multiple sigma factors, each targeting diverse, but often overlapping sets of promoters, thereby forming a complex network. The layout and deployment of such a sigma factor network directly impacts global transcriptional regulation and ultimately dictates the phenotype. Here we integrate multi-omic data sets to determine the topology, the operational, and functional states of the sigma factor network in Geobacter sulfurreducens, revealing a unique network topology of interacting sigma factors. Analysis of the operational state of the sigma factor network shows a highly modular structure with σ(N) being the major regulator of energy metabolism. Surprisingly, the functional state of the network during the two most divergent growth conditions is nearly static, with sigma factor binding profiles almost invariant to environmental stimuli. This first comprehensive elucidation of the interplay between different levels of the sigma factor network organization is fundamental to characterize transcriptional regulatory mechanisms in bacteria.

}, keywords = {Energy Metabolism, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Gene Regulatory Networks, Genes, Bacterial, Geobacter, Models, Biological, Regulon, Sigma Factor}, issn = {2041-1723}, doi = {10.1038/ncomms2743}, author = {Qiu, Yu and Nagarajan, Harish and Embree, Mallory and Shieu, Wendy and Abate, Elisa and Ju{\'a}rez, Katy and Cho, Byung-Kwan and Elkins, James G and Nevin, Kelly P and Barrett, Christian L and Lovley, Derek R and Palsson, Bernhard O and Zengler, Karsten} } @article {3139, title = {Improved cathode for high efficient microbial-catalyzed reduction in microbial electrosynthesis cells.}, journal = {Phys Chem Chem Phys}, volume = {15}, year = {2013}, month = {2013 Sep 14}, pages = {14290-4}, abstract = {

Microbial electrosynthesis cells (MECs) are devices wherein microorganisms can electrochemically interact with electrodes, directly donating or accepting electrons from electrode surfaces. Here, we developed a novel cathode by using nickel nanowires anchored to graphite for the improvement of microbial-catalyzed reduction in MEC cathode chamber. This porous nickel-nanowire-network-coated graphite electrode increased the interfacial area and interfacial interactions between the cathode surface and the microbial biofilm. A 2.3 fold increase in bio-reduction rate over the untreated graphite was observed. Around 282 mM day(-1) m(-2) of acetate resulting from the bio-reduction of carbon dioxide by Sporomusa was produced with 82 {\textpm} 14\% of the electrons consumed being recovered in acetate.

}, keywords = {Biocatalysis, Bioelectric Energy Sources, Biofilms, Carbon Dioxide, Electrochemical Techniques, Electrodes, Graphite, Nanowires, Nickel, Oxidation-Reduction, Veillonellaceae}, issn = {1463-9084}, doi = {10.1039/c3cp52697f}, author = {Nie, Huarong and Zhang, Tian and Cui, Mengmeng and Lu, Haiyun and Lovley, Derek R and Russell, Thomas P} } @article {3155, title = {Anaerobic benzene oxidation by Geobacter species.}, journal = {Appl Environ Microbiol}, volume = {78}, year = {2012}, month = {2012 Dec}, pages = {8304-10}, abstract = {

The abundance of Geobacter species in contaminated aquifers in which benzene is anaerobically degraded has led to the suggestion that some Geobacter species might be capable of anaerobic benzene degradation, but this has never been documented. A strain of Geobacter, designated strain Ben, was isolated from sediments from the Fe(III)-reducing zone of a petroleum-contaminated aquifer in which there was significant capacity for anaerobic benzene oxidation. Strain Ben grew in a medium with benzene as the sole electron donor and Fe(III) oxide as the sole electron acceptor. Furthermore, additional evaluation of Geobacter metallireducens demonstrated that it could also grow in benzene-Fe(III) medium. In both strain Ben and G. metallireducens the stoichiometry of benzene metabolism and Fe(III) reduction was consistent with the oxidation of benzene to carbon dioxide with Fe(III) serving as the sole electron acceptor. With benzene as the electron donor, and Fe(III) oxide (strain Ben) or Fe(III) citrate (G. metallireducens) as the electron acceptor, the cell yields of strain Ben and G. metallireducens were 3.2 {\texttimes} 10(9) and 8.4 {\texttimes} 10(9) cells/mmol of Fe(III) reduced, respectively. Strain Ben also oxidized benzene with anthraquinone-2,6-disulfonate (AQDS) as the sole electron acceptor with cell yields of 5.9 {\texttimes} 10(9) cells/mmol of AQDS reduced. Strain Ben serves as model organism for the study of anaerobic benzene metabolism in petroleum-contaminated aquifers, and G. metallireducens is the first anaerobic benzene-degrading organism that can be genetically manipulated.

}, keywords = {Anaerobiosis, Benzene, Carbon Dioxide, Cluster Analysis, Culture Media, DNA, Bacterial, DNA, Ribosomal, Ferric Compounds, Geobacter, Groundwater, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, Sequence Analysis, DNA}, issn = {1098-5336}, doi = {10.1128/AEM.02469-12}, author = {Zhang, Tian and Bain, Timothy S and Nevin, Kelly P and Barlett, Melissa A and Lovley, Derek R} } @article {410, title = {The design of long-term effective uranium bioremediation strategy using a community metabolic model.}, journal = {Biotechnol Bioeng}, volume = {109}, year = {2012}, month = {2012 Oct}, pages = {2475-83}, abstract = {Acetate amendment at uranium contaminated sites in Rifle, CO. leads to an initial bloom of Geobacter accompanied by the removal of U(VI) from the groundwater, followed by an increase of sulfate-reducing bacteria (SRBs) which are poor reducers of U(VI). One of the challenges associated with bioremediation is the decay in Geobacter abundance, which has been attributed to the depletion of bio-accessible Fe(III), motivating the investigation of simultaneous amendments of acetate and Fe(III) as an alternative bioremediation strategy. In order to understand the community metabolism of Geobacter and SRBs during artificial substrate amendment, we have created a genome-scale dynamic community model of Geobacter and SRBs using the previously described Dynamic Multi-species Metabolic Modeling framework. Optimization techniques are used to determine the optimal acetate and Fe(III) addition profile. Field-scale simulation of acetate addition accurately predicted the in situ data. The simulations suggest that batch amendment of Fe(III) along with continuous acetate addition is insufficient to promote long-term bioremediation, while continuous amendment of Fe(III) along with continuous acetate addition is sufficient to promote long-term bioremediation. By computationally minimizing the acetate and Fe(III) addition rates as well as the difference between the predicted and target uranium concentration, we showed that it is possible to maintain the uranium concentration below the environmental safety standard while minimizing the cost of chemical additions. These simulations show that simultaneous addition of acetate and Fe(III) has the potential to be an effective uranium bioremediation strategy. They also show that computational modeling of microbial community is an important tool to design effective strategies for practical applications in environmental biotechnology. Biotechnol. Bioeng. 2012; 109: 2475-2483. {\textcopyright} 2012 Wiley Periodicals, Inc.}, issn = {1097-0290}, doi = {10.1002/bit.24528}, author = {Zhuang, K and Ma, E and Lovley, Derek R and Mahadevan, Radhakrishnan} } @article {413, title = {Microbial functional gene diversity with a shift of subsurface redox conditions during In Situ uranium reduction.}, journal = {Appl Environ Microbiol}, volume = {78}, year = {2012}, month = {2012 Apr}, pages = {2966-72}, abstract = {To better understand the microbial functional diversity changes with subsurface redox conditions during in situ uranium bioremediation, key functional genes were studied with GeoChip, a comprehensive functional gene microarray, in field experiments at a uranium mill tailings remedial action (UMTRA) site (Rifle, CO). The results indicated that functional microbial communities altered with a shift in the dominant metabolic process, as documented by hierarchical cluster and ordination analyses of all detected functional genes. The abundance of dsrAB genes (dissimilatory sulfite reductase genes) and methane generation-related mcr genes (methyl coenzyme M reductase coding genes) increased when redox conditions shifted from Fe-reducing to sulfate-reducing conditions. The cytochrome genes detected were primarily from Geobacter sp. and decreased with lower subsurface redox conditions. Statistical analysis of environmental parameters and functional genes indicated that acetate, U(VI), and redox potential (E(h)) were the most significant geochemical variables linked to microbial functional gene structures, and changes in microbial functional diversity were strongly related to the dominant terminal electron-accepting process following acetate addition. The study indicates that the microbial functional genes clearly reflect the in situ redox conditions and the dominant microbial processes, which in turn influence uranium bioreduction. Microbial functional genes thus could be very useful for tracking microbial community structure and dynamics during bioremediation.}, keywords = {Biodegradation, Environmental, Biota, Environmental Microbiology, Environmental Pollutants, Genetic Variation, Microarray Analysis, Oxidation-Reduction, Uranium}, issn = {1098-5336}, doi = {10.1128/AEM.06528-11}, author = {Liang, Yuting and Van Nostrand, Joy D and N{\textquoteright}guessan, Lucie A and Peacock, Aaron D and Deng, Ye and Long, Philip E and Resch, C Tom and Wu, Liyou and He, Zhili and Li, Guanghe and Hazen, Terry C and Lovley, Derek R and Zhou, Jizhong} } @article {3148, title = {The Rnf complex of Clostridium ljungdahlii is a proton-translocating ferredoxin:NAD+ oxidoreductase essential for autotrophic growth.}, journal = {mBio}, volume = {4}, year = {2012}, month = {2012 Dec 26}, pages = {e00406-12}, abstract = {

UNLABELLED: It has been predicted that the Rnf complex of Clostridium ljungdahlii is a proton-translocating ferredoxin:NAD(+) oxidoreductase which contributes to ATP synthesis by an H(+)-translocating ATPase under both autotrophic and heterotrophic growth conditions. The recent development of methods for genetic manipulation of C. ljungdahlii made it possible to evaluate the possible role of the Rnf complex in energy conservation. Disruption of the C. ljungdahlii rnf operon inhibited autotrophic growth. ATP synthesis, proton gradient, membrane potential, and proton motive force collapsed in the Rnf-deficient mutant with H(2) as the electron source and CO(2) as the electron acceptor. Heterotrophic growth was hindered in the absence of a functional Rnf complex, as ATP synthesis, proton gradient, and proton motive force were significantly reduced with fructose as the electron donor. Growth of the Rnf-deficient mutant was also inhibited when no source of fixed nitrogen was provided. These results demonstrate that the Rnf complex of C. ljungdahlii is responsible for translocation of protons across the membrane to elicit energy conservation during acetogenesis and is a multifunctional device also implicated in nitrogen fixation.

IMPORTANCE: Mechanisms for energy conservation in the acetogen Clostridium ljungdahlii are of interest because of its potential value as a chassis for the production of biocommodities with novel electron donors such as carbon monoxide, syngas, and electrons derived from electrodes. Characterizing the components implicated in the chemiosmotic ATP synthesis during acetogenesis by C. ljungdahlii is a prerequisite for the development of highly productive strains. The Rnf complex has been considered the prime candidate to be the pump responsible for the formation of an ion gradient coupled with ATP synthesis in multiple acetogens. However, experimental evidence for a proton-pumping Rnf complex has been lacking. This study establishes the C. ljungdahlii Rnf complex as a proton-translocating ferredoxin:NAD(+) oxidoreductase and demonstrates that C. ljungdahlii has the potential of becoming a model organism to study proton translocation, electron transport, and other functions of the Rnf complex in energy conservation or other processes.

}, keywords = {Adenosine Triphosphate, Autotrophic Processes, Clostridium, Energy Metabolism, Fructose, Gene Knockout Techniques, Genes, Essential, Nitrogen, Operon, Oxidoreductases, Proton-Motive Force}, issn = {2150-7511}, doi = {10.1128/mBio.00406-12}, author = {Tremblay, Pier-Luc and Zhang, Tian and Dar, Shabir A and Leang, Ching and Lovley, Derek R} } @article {441, title = {A c-type cytochrome and a transcriptional regulator responsible for enhanced extracellular electron transfer in Geobacter sulfurreducens revealed by adaptive evolution.}, journal = {Environ Microbiol}, volume = {13}, year = {2011}, month = {2011 Jan}, pages = {13-23}, abstract = {The stimulation of subsurface microbial metabolism often associated with engineered bioremediation of groundwater contaminants presents subsurface microorganisms, which are adapted for slow growth and metabolism in the subsurface, with new selective pressures. In order to better understand how Geobacter species might adapt to selective pressure for faster metal reduction in the subsurface, Geobacter sulfurreducens was put under selective pressure for rapid Fe(III) oxide reduction. The genomes of two resultant strains with rates of Fe(III) oxide reduction that were 10-fold higher than those of the parent strain were resequenced. Both strains contain either a single base-pair change or a 1 nucleotide insertion in a GEMM riboswitch upstream of GSU1761, a gene coding for the periplasmic c-type cytochrome designated PgcA. GSU1771, a gene coding for a SARP regulator, was also mutated in both strains. Introduction of either of the GEMM riboswitch mutations upstream of pgcA in the wild-type increased the abundance of pgcA transcripts, consistent with increased expression of pgcA in the adapted strains. One of the mutations doubled the rate of Fe(III) oxide reduction. Interruption of GSU1771 doubled the Fe(III) oxide reduction rate. This was associated with an increased in expression of pilA, the gene encoding the structural protein for the pili thought to function as microbial nanowires. The combination of the GSU1771 interruption with either of the pgcA mutations resulted in a strain that reduced Fe(III) as fast as the comparable adapted strain. These results suggest that the accumulation of a small number of beneficial mutations under selective pressure, similar to that potentially present during bioremediation, can greatly enhance the capacity for Fe(III) oxide reduction in G. sulfurreducens. Furthermore, the results emphasize the importance of the c-type cytochrome PgcA and pili in Fe(III) oxide reduction and demonstrate how adaptive evolution studies can aid in the elucidation of complex mechanisms, such as extracellular electron transfer.}, keywords = {Adaptation, Physiological, Biodegradation, Environmental, Cytochrome c Group, DNA, Bacterial, Electron Transport, Evolution, Molecular, Ferric Compounds, Gene Expression Profiling, Genes, Bacterial, Genome, Bacterial, Geobacter, Mutagenesis, Insertional, Mutation, Oligonucleotide Array Sequence Analysis, Oxidation-Reduction, Riboswitch, Sequence Analysis, DNA}, issn = {1462-2920}, doi = {10.1111/j.1462-2920.2010.02302.x}, author = {Tremblay, Pier-Luc and Summers, Zarath M and Glaven, Richard H and Nevin, Kelly P and Zengler, Karsten and Barrett, Christian L and Qiu, Yu and Palsson, Bernhard O and Lovley, Derek R} } @article {438, title = {Genome-scale dynamic modeling of the competition between Rhodoferax and Geobacter in anoxic subsurface environments.}, journal = {ISME J}, volume = {5}, year = {2011}, month = {2011 Feb}, pages = {305-16}, abstract = {The advent of rapid complete genome sequencing, and the potential to capture this information in genome-scale metabolic models, provide the possibility of comprehensively modeling microbial community interactions. For example, Rhodoferax and Geobacter species are acetate-oxidizing Fe(III)-reducers that compete in anoxic subsurface environments and this competition may have an influence on the in situ bioremediation of uranium-contaminated groundwater. Therefore, genome-scale models of Geobacter sulfurreducens and Rhodoferax ferrireducens were used to evaluate how Geobacter and Rhodoferax species might compete under diverse conditions found in a uranium-contaminated aquifer in Rifle, CO. The model predicted that at the low rates of acetate flux expected under natural conditions at the site, Rhodoferax will outcompete Geobacter as long as sufficient ammonium is available. The model also predicted that when high concentrations of acetate are added during in situ bioremediation, Geobacter species would predominate, consistent with field-scale observations. This can be attributed to the higher expected growth yields of Rhodoferax and the ability of Geobacter to fix nitrogen. The modeling predicted relative proportions of Geobacter and Rhodoferax in geochemically distinct zones of the Rifle site that were comparable to those that were previously documented with molecular techniques. The model also predicted that under nitrogen fixation, higher carbon and electron fluxes would be diverted toward respiration rather than biomass formation in Geobacter, providing a potential explanation for enhanced in situ U(VI) reduction in low-ammonium zones. These results show that genome-scale modeling can be a useful tool for predicting microbial interactions in subsurface environments and shows promise for designing bioremediation strategies.}, keywords = {Acetates, Anaerobiosis, Biodegradation, Environmental, Biomass, Comamonadaceae, Genome, Genome, Bacterial, Geobacter, Models, Biological, Nitrogen Fixation, Quaternary Ammonium Compounds, RNA, Ribosomal, 16S, Uranium, Water Microbiology, Water Pollutants, Radioactive}, issn = {1751-7370}, doi = {10.1038/ismej.2010.117}, author = {Zhuang, Kai and Izallalen, Mounir and Mouser, Paula and Richter, Hanno and Risso, Carla and Mahadevan, Radhakrishnan and Lovley, Derek R} } @article {415, title = {Geobacter: the microbe electric{\textquoteright}s physiology, ecology, and practical applications.}, journal = {Adv Microb Physiol}, volume = {59}, year = {2011}, month = {2011}, pages = {1-100}, abstract = {Geobacter species specialize in making electrical contacts with extracellular electron acceptors and other organisms. This permits Geobacter species to fill important niches in a diversity of anaerobic environments. Geobacter species appear to be the primary agents for coupling the oxidation of organic compounds to the reduction of insoluble Fe(III) and Mn(IV) oxides in many soils and sediments, a process of global biogeochemical significance. Some Geobacter species can anaerobically oxidize aromatic hydrocarbons and play an important role in aromatic hydrocarbon removal from contaminated aquifers. The ability of Geobacter species to reductively precipitate uranium and related contaminants has led to the development of bioremediation strategies for contaminated environments. Geobacter species produce higher current densities than any other known organism in microbial fuel cells and are common colonizers of electrodes harvesting electricity from organic wastes and aquatic sediments. Direct interspecies electron exchange between Geobacter species and syntrophic partners appears to be an important process in anaerobic wastewater digesters. Functional and comparative genomic studies have begun to reveal important aspects of Geobacter physiology and regulation, but much remains unexplored. Quantifying key gene transcripts and proteins of subsurface Geobacter communities has proven to be a powerful approach to diagnose the in situ physiological status of Geobacter species during groundwater bioremediation. The growth and activity of Geobacter species in the subsurface and their biogeochemical impact under different environmental conditions can be predicted with a systems biology approach in which genome-scale metabolic models are coupled with appropriate physical/chemical models. The proficiency of Geobacter species in transferring electrons to insoluble minerals, electrodes, and possibly other microorganisms can be attributed to their unique "microbial nanowires," pili that conduct electrons along their length with metallic-like conductivity. Surprisingly, the abundant c-type cytochromes of Geobacter species do not contribute to this long-range electron transport, but cytochromes are important for making the terminal electrical connections with Fe(III) oxides and electrodes and also function as capacitors, storing charge to permit continued respiration when extracellular electron acceptors are temporarily unavailable. The high conductivity of Geobacter pili and biofilms and the ability of biofilms to function as supercapacitors are novel properties that might contribute to the field of bioelectronics. The study of Geobacter species has revealed a remarkable number of microbial physiological properties that had not previously been described in any microorganism. Further investigation of these environmentally relevant and physiologically unique organisms is warranted.}, keywords = {Biotechnology, Ecology, Environmental Remediation, Ferric Compounds, Geobacter}, issn = {0065-2911}, doi = {10.1016/B978-0-12-387661-4.00004-5}, author = {Lovley, Derek R and Ueki, Toshiyuki and Zhang, Tian and Malvankar, Nikhil S and Shrestha, Pravin M and Flanagan, Kelly A and Aklujkar, Muktak and Butler, Jessica E and Giloteaux, Ludovic and Rotaru, Amelia-Elena and Holmes, Dawn E and Franks, Ashley E and Orellana, Roberto and Risso, Carla and Nevin, Kelly P} } @article {381, title = {Silencing of a putative inner arm dynein heavy chain results in flagellar immotility in Trypanosoma brucei.}, journal = {Mol Biochem Parasitol}, volume = {175}, year = {2011}, month = {2011 Jan}, pages = {68-75}, abstract = {The Trypanosoma brucei flagellum controls motility and is crucial for cell polarity and division. Unique features of trypanosome motility suggest that flagellar beat regulation in this organism is unusual and worthy of study. The flagellar axoneme, required for motility, has a structure that is highly conserved among eukaryotes. Of the several dyneins in the axonemal inner arm complex, dynein f is thought to control flagellar waveform shape. A T. brucei gene predicted to encode the dynein f alpha heavy chain, TbDNAH10, was silenced using RNA interference in procyclic T. brucei cells. This resulted in immotile flagella, showing no movement except for occasional slight twitches at the tips. Cell growth slowed dramatically and cells were found in large clusters. Microscopic analysis of silenced cultures showed many cells with detached flagella, sometimes entangled between multiple cells. DAPI staining showed an increased frequency of mis-positioned kinetoplasts and multinucleate cells, suggesting that these cells experience disruption at an early cell cycle stage, probably secondary to the motility defect. TEM images showed apparently normal axonemes and no discernable defects in inner arm structure. This study demonstrates the use of RNAi as an effective method to study very large genes such as dynein heavy chains (HCs), and the immotility phenotype of these dynein knockdowns suggests that an intact inner arm is necessary for flagellar beating in T. brucei. Since analogous mutants in Chlamydomonas reinhardtii retain motility, this phenotype likely reflects differences in requirements for motility and/or dynein assembly between the two organisms and these comparative studies will help elucidate the mechanisms of flagellar beat regulation.}, keywords = {Cell Nucleus, Dyneins, Flagella, Locomotion, Microscopy, Electron, Transmission, Organelles, Protozoan Proteins, RNA Interference, Trypanosoma brucei brucei}, issn = {1872-9428}, doi = {10.1016/j.molbiopara.2010.09.005}, author = {Springer, Amy L and Bruhn, David F and Kinzel, Kathryn W and Rosenthal, No{\"e}l F and Zukas, Randi and Klingbeil, Michele M} } @article {443, title = {De Novo assembly of the complete genome of an enhanced electricity-producing variant of Geobacter sulfurreducens using only short reads.}, journal = {PLoS One}, volume = {5}, year = {2010}, month = {2010}, pages = {e10922}, abstract = {State-of-the-art DNA sequencing technologies are transforming the life sciences due to their ability to generate nucleotide sequence information with a speed and quantity that is unapproachable with traditional Sanger sequencing. Genome sequencing is a principal application of this technology, where the ultimate goal is the full and complete sequence of the organism of interest. Due to the nature of the raw data produced by these technologies, a full genomic sequence attained without the aid of Sanger sequencing has yet to be demonstrated.We have successfully developed a four-phase strategy for using only next-generation sequencing technologies (Illumina and 454) to assemble a complete microbial genome de novo. We applied this approach to completely assemble the 3.7 Mb genome of a rare Geobacter variant (KN400) that is capable of unprecedented current production at an electrode. Two key components of our strategy enabled us to achieve this result. First, we integrated the two data types early in the process to maximally leverage their complementary characteristics. And second, we used the output of different short read assembly programs in such a way so as to leverage the complementary nature of their different underlying algorithms or of their different implementations of the same underlying algorithm.The significance of our result is that it demonstrates a general approach for maximizing the efficiency and success of genome assembly projects as new sequencing technologies and new assembly algorithms are introduced. The general approach is a meta strategy, wherein sequencing data are integrated as early as possible and in particular ways and wherein multiple assembly algorithms are judiciously applied such that the deficiencies in one are complemented by another.}, keywords = {Algorithms, Electricity, Genome, Bacterial, Geobacter, Polymerase Chain Reaction}, issn = {1932-6203}, doi = {10.1371/journal.pone.0010922}, author = {Nagarajan, Harish and Butler, Jessica E and Klimes, Anna and Qiu, Yu and Zengler, Karsten and Ward, Joy and Young, Nelson D and Meth{\'e}, Barbara A and Palsson, Bernhard {\O} and Lovley, Derek R and Barrett, Christian L} } @article {454, title = {Modeling and sensitivity analysis of electron capacitance for Geobacter in sedimentary environments.}, journal = {J Contam Hydrol}, volume = {112}, year = {2010}, month = {2010 Mar 1}, pages = {30-44}, abstract = {In situ stimulation of the metabolic activity of Geobacter species through acetate amendment has been shown to be a promising bioremediation strategy to reduce and immobilize hexavalent uranium [U(VI)] as insoluble U(IV). Although Geobacter species are reducing U(VI), they primarily grow via Fe(III) reduction. Unfortunately, the biogeochemistry and the physiology of simultaneous reduction of multiple metals are still poorly understood. A detailed model is therefore required to better understand the pathways leading to U(VI) and Fe(III) reduction by Geobacter species. Based on recent experimental evidence of temporary electron capacitors in Geobacter we propose a novel kinetic model that physically distinguishes planktonic cells into electron-loaded and -unloaded states. Incorporation of an electron load-unload cycle into the model provides insight into U(VI) reduction efficiency, and elucidates the relationship between U(VI)- and Fe(III)-reducing activity and further explains the correlation of high U(VI) removal with high fractions of planktonic cells in subsurface environments. Global sensitivity analysis was used to determine the level of importance of geochemical and microbial processes controlling Geobacter growth and U(VI) reduction, suggesting that the electron load-unload cycle and the resulting repartition of the microbes between aqueous and attached phases are critical for U(VI) reduction. As compared with conventional Monod modeling approaches without inclusion of the electron capacitance, the new model attempts to incorporate a novel cellular mechanism that has a significant impact on the outcome of in situ bioremediation.}, keywords = {Biodegradation, Environmental, Electric Capacitance, Geobacter, Kinetics, Models, Biological, Uranium}, issn = {1873-6009}, doi = {10.1016/j.jconhyd.2009.10.002}, author = {Zhao, Jiao and Fang, Yilin and Scheibe, Timothy D and Lovley, Derek R and Mahadevan, R} } @article {442, title = {Production of pilus-like filaments in Geobacter sulfurreducens in the absence of the type IV pilin protein PilA.}, journal = {FEMS Microbiol Lett}, volume = {310}, year = {2010}, month = {2010 Sep 1}, pages = {62-8}, abstract = {The pili of Geobacter sulfurreducens are of interest because of the apparent importance of the type IV pili in extracellular electron transfer. A strain of G. sulfurreducens, designated strain MA, produced many more pili than the previously studied DL-1 strain even though genome resequencing indicated that the MA and DL-1 genome sequences were identical. Filaments that looked similar to type IV pili in transmission electron micrographs were abundant even after the gene encoding PilA, the structural pilin protein, was deleted. The results of proteinase K treatment indicated that the filaments were proteinaceous. The simultaneous deletion of several genes encoding homologues of type II pseudopilins was required before the filaments were significantly depleted. The pilA-deficient MA strain attached to glass as well as the wild-type MA did, but strains in which three or four pseudopilin genes were deleted in addition to pilA had impaired attachment capabilities. These results demonstrate that there are several proteins that can yield pilin-like filaments in G. sulfurreducens and that some means other than microscopic observation is required before the composition of filaments can be unambiguously specified.}, keywords = {Bacterial Adhesion, Fimbriae Proteins, Fimbriae, Bacterial, Gene Deletion, Geobacter, Glass, Microscopy, Electron, Transmission}, issn = {1574-6968}, doi = {10.1111/j.1574-6968.2010.02046.x}, author = {Klimes, Anna and Franks, Ashley E and Glaven, Richard H and Tran, Hoa and Barrett, Christian L and Qiu, Yu and Zengler, Karsten and Lovley, Derek R} } @article {448, title = {Stimulating the anaerobic degradation of aromatic hydrocarbons in contaminated sediments by providing an electrode as the electron acceptor.}, journal = {Environ Microbiol}, volume = {12}, year = {2010}, month = {2010 Apr}, pages = {1011-20}, abstract = {The possibility that electrodes might serve as an electron acceptor to simulate the degradation of aromatic hydrocarbons in anaerobic contaminated sediments was investigated. Initial studies with Geobacter metallireducens demonstrated that although toluene was rapidly adsorbed onto the graphite electrodes it was rapidly oxidized to carbon dioxide with the electrode serving as the sole electron acceptor. Providing graphite electrodes as an electron acceptor in hydrocarbon-contaminated sediments significantly stimulated the removal of added toluene and benzene. Rates of toluene and benzene removal accelerated with continued additions of toluene and benzene. [(14)C]-Toluene and [(14)C]-benzene were quantitatively recovered as [(14)C]-CO(2), demonstrating that even though the graphite adsorbed toluene and benzene they were degraded. Introducing an electrode as an electron acceptor also accelerated the loss of added naphthalene and [(14)C]-naphthalene was converted to [(14)C]-CO(2). The results suggest that graphite electrodes can serve as an electron acceptor for the degradation of aromatic hydrocarbon contaminants in sediments, co-localizing the contaminants, the degradative organisms and the electron acceptor. Once in position, they provide a permanent, low-maintenance source of electron acceptor. Thus, graphite electrodes may offer an attractive alternative for enhancing contaminant degradation in anoxic environments.}, keywords = {Anaerobiosis, Benzene, Carbon Dioxide, Electrodes, Geobacter, Geologic Sediments, Graphite, Naphthalenes, Oxidation-Reduction, Toluene, Water Pollutants, Chemical}, issn = {1462-2920}, doi = {10.1111/j.1462-2920.2009.02145.x}, author = {Zhang, Tian and Gannon, Sarah M and Nevin, Kelly P and Franks, Ashley E and Lovley, Derek R} } @article {456, title = {Genome-scale comparison and constraint-based metabolic reconstruction of the facultative anaerobic Fe(III)-reducer Rhodoferax ferrireducens.}, journal = {BMC Genomics}, volume = {10}, year = {2009}, month = {2009}, pages = {447}, abstract = {BACKGROUND: Rhodoferax ferrireducens is a metabolically versatile, Fe(III)-reducing, subsurface microorganism that is likely to play an important role in the carbon and metal cycles in the subsurface. It also has the unique ability to convert sugars to electricity, oxidizing the sugars to carbon dioxide with quantitative electron transfer to graphite electrodes in microbial fuel cells. In order to expand our limited knowledge about R. ferrireducens, the complete genome sequence of this organism was further annotated and then the physiology of R. ferrireducens was investigated with a constraint-based, genome-scale in silico metabolic model and laboratory studies. RESULTS: The iterative modeling and experimental approach unveiled exciting, previously unknown physiological features, including an expanded range of substrates that support growth, such as cellobiose and citrate, and provided additional insights into important features such as the stoichiometry of the electron transport chain and the ability to grow via fumarate dismutation. Further analysis explained why R. ferrireducens is unable to grow via photosynthesis or fermentation of sugars like other members of this genus and uncovered novel genes for benzoate metabolism. The genome also revealed that R. ferrireducens is well-adapted for growth in the subsurface because it appears to be capable of dealing with a number of environmental insults, including heavy metals, aromatic compounds, nutrient limitation and oxidative stress. CONCLUSION: This study demonstrates that combining genome-scale modeling with the annotation of a new genome sequence can guide experimental studies and accelerate the understanding of the physiology of under-studied yet environmentally relevant microorganisms.}, keywords = {Comamonadaceae, Comparative Genomic Hybridization, DNA, Bacterial, Ferric Compounds, Genome, Bacterial, Genomics, Models, Biological, Oxidation-Reduction, Sequence Analysis, DNA}, issn = {1471-2164}, doi = {10.1186/1471-2164-10-447}, author = {Risso, Carla and Sun, Jun and Zhuang, Kai and Mahadevan, Radhakrishnan and DeBoy, Robert and Ismail, Wael and Shrivastava, Susmita and Huot, Heather and Kothari, Sagar and Daugherty, Sean and Bui, Olivia and Schilling, Christophe H and Lovley, Derek R and Meth{\'e}, Barbara A} } @article {479, title = {Power output and columbic efficiencies from biofilms of Geobacter sulfurreducens comparable to mixed community microbial fuel cells.}, journal = {Environ Microbiol}, volume = {10}, year = {2008}, month = {2008 Oct}, pages = {2505-14}, abstract = {It has been previously noted that mixed communities typically produce more power in microbial fuel cells than pure cultures. If true, this has important implications for the design of microbial fuel cells and for studying the process of electron transfer on anode biofilms. To further evaluate this, Geobacter sulfurreducens was grown with acetate as fuel in a continuous flow {\textquoteright}ministack{\textquoteright} system in which the carbon cloth anode and cathode were positioned in close proximity, and the cation-selective membrane surface area was maximized in order to overcome some of the electrochemical limitations that were inherent in fuel cells previously employed for the study of pure cultures. Reducing the size of the anode in order to eliminate cathode limitation resulted in maximum current and power densities per m(2) of anode surface of 4.56 A m(-2) and 1.88 W m(-2) respectively. Electron recovery as current from acetate oxidation was c. 100\% when oxygen diffusion into the system was minimized. This performance is comparable to the highest levels previously reported for mixed communities in similar microbial fuel cells and slightly higher than the power output of an anaerobic sludge inoculum in the same ministack system. Minimizing the volume of the anode chamber yielded a volumetric power density of 2.15 kW m(-3), which is the highest power density per volume yet reported for a microbial fuel cell. Geobacter sulfurreducens formed relatively uniform biofilms 3-18 mum thick on the carbon cloth anodes. When graphite sticks served as the anode, the current density (3.10 A m(-2)) was somewhat less than with the carbon cloth anodes, but the biofilms were thicker (c. 50 mum) with a more complex pillar and channel structure. These results suggest that the previously observed disparity in power production in pure and mixed culture microbial fuel cell systems can be attributed more to differences in the fuel cell designs than to any inherent superior capability of mixed cultures to produce more power than pure cultures.}, keywords = {Acetic Acid, Biofilms, Carbon, Electricity, Electrodes, Geobacter, Oxidation-Reduction}, issn = {1462-2920}, doi = {10.1111/j.1462-2920.2008.01675.x}, author = {Nevin, K P and Richter, H and Covalla, S F and Johnson, J P and Woodard, T L and Orloff, A L and Jia, H and Zhang, M and Lovley, D R} } @article {502, title = {Genomic and microarray analysis of aromatics degradation in Geobacter metallireducens and comparison to a Geobacter isolate from a contaminated field site.}, journal = {BMC Genomics}, volume = {8}, year = {2007}, month = {2007}, pages = {180}, abstract = {BACKGROUND: Groundwater and subsurface environments contaminated with aromatic compounds can be remediated in situ by Geobacter species that couple oxidation of these compounds to reduction of Fe(III)-oxides. Geobacter metallireducens metabolizes many aromatic compounds, but the enzymes involved are not well known. RESULTS: The complete G. metallireducens genome contained a 300 kb island predicted to encode enzymes for the degradation of phenol, p-cresol, 4-hydroxybenzaldehyde, 4-hydroxybenzoate, benzyl alcohol, benzaldehyde, and benzoate. Toluene degradation genes were encoded in a separate region. None of these genes was found in closely related species that cannot degrade aromatic compounds. Abundant transposons and phage-like genes in the island suggest mobility, but nucleotide composition and lack of synteny with other species do not suggest a recent transfer. The inferred degradation pathways are similar to those in species that anaerobically oxidize aromatic compounds with nitrate as an electron acceptor. In these pathways the aromatic compounds are converted to benzoyl-CoA and then to 3-hydroxypimelyl-CoA. However, in G. metallireducens there were no genes for the energetically-expensive dearomatizing enzyme. Whole-genome changes in transcript levels were identified in cells oxidizing benzoate. These supported the predicted pathway, identified induced fatty-acid oxidation genes, and identified an apparent shift in the TCA cycle to a putative ATP-yielding succinyl-CoA synthase. Paralogs to several genes in the pathway were also induced, as were several putative molybdo-proteins. Comparison of the aromatics degradation pathway genes to the genome of an isolate from a contaminated field site showed very similar content, and suggested this strain degrades many of the same compounds. This strain also lacked a classical dearomatizing enzyme, but contained two copies of an eight-gene cluster encoding redox proteins that was 30-fold induced during benzoate oxidation. CONCLUSION: G. metallireducens appears to convert aromatic compounds to benzoyl-CoA, then to acetyl-CoA via fatty acid oxidation, and then to carbon dioxide via the TCA cycle. The enzyme responsible for dearomatizing the aromatic ring may be novel, and energetic investments at this step may be offset by a change in succinate metabolism. Analysis of a field isolate suggests that the pathways inferred for G. metallireducens may be applicable to modeling in situ bioremediation.}, keywords = {Biodegradation, Environmental, Citric Acid Cycle, Gene Expression Regulation, Bacterial, Genomics, Geobacter, Hydrocarbons, Aromatic, Microarray Analysis, Models, Chemical, Models, Genetic, Multigene Family, Oligonucleotide Array Sequence Analysis, Oxidation-Reduction, Phylogeny, Soil Pollutants, Water Pollutants, Chemical}, issn = {1471-2164}, doi = {10.1186/1471-2164-8-180}, author = {Butler, Jessica E and He, Qiang and Nevin, Kelly P and He, Zhili and Zhou, Jizhong and Lovley, Derek R} } @article {495, title = {Steady state protein levels in Geobacter metallireducens grown with iron (III) citrate or nitrate as terminal electron acceptor.}, journal = {Proteomics}, volume = {7}, year = {2007}, month = {2007 Nov}, pages = {4148-57}, abstract = {Geobacter species predominate in aquatic sediments and submerged soils where organic carbon sources are oxidized with the reduction of Fe(III). The natural occurrence of Geobacter in some waste sites suggests this microorganism could be useful for bioremediation if growth and metabolic activity can be regulated. 2-DE was used to monitor the steady state protein levels of Geobacter metallireducens grown with either Fe(III) citrate or nitrate to elucidate metabolic differences in response to different terminal electron acceptors present in natural environments populated by Geobacter. Forty-six protein spots varied significantly in abundance (p<0.05) between the two growth conditions; proteins were identified by tryptic peptide mass and peptide sequence determined by MS/MS. Enzymes involved in pyruvate metabolism and the tricarboxylic acid (TCA) cycle were more abundant in cells grown with Fe(III) citrate, while proteins associated with nitrate metabolism and sensing cellular redox status along with several proteins of unknown function were more abundant in cells grown with nitrate. These results indicate a higher level of flux through the TCA cycle in the presence of Fe(III) compared to nitrate. The oxidative stress response observed in previous studies of Geobacter sulfurreducens grown with Fe(III) citrate was not seen in G. metallireducens.}, keywords = {Bacterial Proteins, Cell Proliferation, Electrons, Electrophoresis, Gel, Two-Dimensional, Ferric Compounds, Geobacter, Hydrogen-Ion Concentration, Nitrates, Oxidation-Reduction, Proteomics, Tandem Mass Spectrometry}, issn = {1615-9853}, doi = {10.1002/pmic.200600955}, author = {Ahrendt, Angela J and Tollaksen, Sandra L and Lindberg, Carl and Zhu, Wenhong and Yates, John R and Nevin, Kelly P and Babnigg, Gy{\"o}rgy and Lovley, Derek R and Giometti, Carol S} } @article {702, title = {Differential protein expression in the metal-reducing bacterium Geobacter sulfurreducens strain PCA grown with fumarate or ferric citrate.}, journal = {Proteomics}, volume = {6}, year = {2006}, month = {2006 Jan}, pages = {632-40}, abstract = {Geobacter sulfurreducens, generally considered to be a strict anaerobe, is a predominant microbe in subsurface environments, where it utilizes available metals as electron acceptors. To better understand the metabolic processes involved in the metal-reduction capability of this microbe, the proteins expressed by cells grown anaerobically with either fumarate or ferric citrate as electron acceptor were compared. Proteins were separated by 2-DE under denaturing or nondenaturing conditions, and proteins varying in abundance with a high level of statistical significance (p<0.0001) were identified by peptide mass analysis. Denaturing 2-DE revealed significant differences in the relative abundance of the membrane proteins OmpA and peptidoglycan-associated lipoprotein, several metabolic enzymes, and, in addition, superoxide dismutase and rubredoxin oxidoreductase. Nondenaturing 2-DE revealed elevated catalase in cells grown with ferric citrate. These results suggest that, in addition to adjustments in membrane transport and specific metabolic pathways in response to these two different electron acceptors, distinct differences exist in the oxidative environment within the cell when fumarate or soluble ferric citrate is provided as electron acceptor. Although an anaerobe, G. sulfurreducens appears to have alternate mechanisms for dealing with reactive oxygen species in response to increased intracellular soluble iron.}, keywords = {Anaerobiosis, Bacterial Proteins, Electrophoresis, Gel, Two-Dimensional, Ferric Compounds, Fumarates, Geobacter, Oxidation-Reduction, Proteomics, Reactive Oxygen Species, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization}, issn = {1615-9853}, doi = {10.1002/pmic.200500137}, author = {Khare, Tripti and Esteve-N{\'u}{\~n}ez, Abraham and Nevin, Kelly P and Zhu, Wenhong and Yates, John R and Lovley, Derek and Giometti, Carol S} } @article {523, title = {Harvesting energy from the marine sediment-water interface II. Kinetic activity of anode materials.}, journal = {Biosens Bioelectron}, volume = {21}, year = {2006}, month = {2006 May 15}, pages = {2058-63}, abstract = {Here, we report a comparative study on the kinetic activity of various anodes of a recently described microbial fuel cell consisting of an anode imbedded in marine sediment and a cathode in overlying seawater. Using plain graphite anodes, it was demonstrated that a significant portion of the anodic current results from oxidation of sediment organic matter catalyzed by microorganisms colonizing the anode and capable of directly reducing the anode without added exogenous electron-transfer mediators. Here, graphite anodes incorporating microbial oxidants are evaluated in the laboratory relative to plain graphite with the goal of increasing power density by increasing current density. Anodes evaluated include graphite modified by adsorption of anthraquinone-1,6-disulfonic acid (AQDS) or 1,4-naphthoquinone (NQ), a graphite-ceramic composite containing Mn2+ and Ni2+, and graphite modified with a graphite paste containing Fe3O4 or Fe3O4 and Ni2+. It was found that these anodes possess between 1.5- and 2.2-fold greater kinetic activity than plain graphite. Fuel cells were deployed in a coastal site near Tuckerton, NJ (USA) that utilized two of these anodes. These fuel cells generated ca. 5-fold greater current density than a previously characterized fuel cell equipped with a plain graphite anode, and operated at the same site.}, keywords = {Electrochemistry, Electrodes, Energy-Generating Resources, Ferumoxytol, Geologic Sediments, Kinetics, Oceans and Seas, Seawater}, issn = {0956-5663}, doi = {10.1016/j.bios.2006.01.033}, author = {Lowy, Daniel A and Tender, Leonard M and Zeikus, J Gregory and Park, Doo Hyun and Lovley, Derek R} } @article {518, title = {The proteome of dissimilatory metal-reducing microorganism Geobacter sulfurreducens under various growth conditions.}, journal = {Biochim Biophys Acta}, volume = {1764}, year = {2006}, month = {2006 Jul}, pages = {1198-206}, abstract = {The proteome of Geobacter sulfurreducens, a model for the Geobacter species that predominate in many Fe(III)-reducing subsurface environments, was characterized with ultra high-pressure liquid chromatography and mass spectrometry using accurate mass and time (AMT) tags as well as with more traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Cells were grown under six different growth conditions in order to enhance the potential that a wide range of genes would be expressed. The AMT tag approach was able to identify a much greater number of proteins than could be detected with the 2-D PAGE approach. With the AMT approach over 3,000 gene products were identified, representing about 90\% of the total predicted gene products in the genome. A high proportion of predicted proteins in most protein role categories were detected; the highest number of proteins was identified in the hypothetical protein role category. Furthermore, 91 c-type cytochromes of 111 predicted genes in the G. sulfurreducens genome were identified. Differences in the abundance of cytochromes and other proteins under different growth conditions provided information for future functional analysis of these proteins. These results demonstrate that a high percentage of the predicted proteins in the G. sulfurreducens genome are produced and that the AMT tag approach provides a rapid method for comparing differential expression of proteins under different growth conditions in this organism.}, keywords = {Bacterial Proteins, Bacteriological Techniques, Chromatography, High Pressure Liquid, Cytochrome c Group, Electrophoresis, Gel, Two-Dimensional, Ferric Compounds, Fumarates, Geobacter, Peptide Fragments, Proteome, Spectrometry, Mass, Electrospray Ionization}, issn = {0006-3002}, doi = {10.1016/j.bbapap.2006.04.017}, author = {Ding, Yan-Huai R and Hixson, Kim K and Giometti, Carol S and Stanley, Ann and Esteve-N{\'u}{\~n}ez, Abraham and Khare, Tripti and Tollaksen, Sandra L and Zhu, Wenhong and Adkins, Joshua N and Lipton, Mary S and Smith, Richard D and Mester, T{\"u}nde and Lovley, Derek R} } @article {861, title = {Poly(hydroxyalkanoic acid) Biosynthesis in Ectothiorhodospirashaposhnikovii: Characterization and Reactivity of a Type III PHA Synthase.}, journal = {Biomacromolecules}, volume = {5}, year = {2004}, month = {2004 Jan-Feb}, pages = {40-8}, abstract = {Ectothiorhodospira shaposhnikovii is able to accumulate polyhydroxybutyrate (PHB) photoautotrophically during nitrogen-limited growth. The activity of polyhydroxyalkanoate (PHA) synthase in the cells correlates with PHB accumulation. PHA synthase samples collected during the light period do not show a lag phase during in vitro polymerization. Synthase samples collected in the dark period displays a significant lag phase during in vitro polymerization. The lag phase can be eliminated by reacting the PHA synthase with the monomer, 3-hydroxybutyryl-CoA (3HBCoA). The PHA synthase genes (phaC and phaE) were cloned by screening a genomic library for PHA accumulation in E. coli cells. The PHA synthase expressed in the recombinant E. coli cells was purified to homogeneity. Both sequence analysis and biochemical studies indicated that this PHA synthase consists of two subunits, PhaE and PhaC and, therefore, belongs to the type III PHA synthases. Two major complexes were identified in preparations of purified PHA synthase. The large complex appears to be composed of 12 PhaC subunits and 12 PhaE subunits (dodecamer), whereas the small complex appears to be composed of 6 PhaC and 6 PhaE subunits (hexamer). In dilute aqueous solution, the synthase is predominantly composed of hexamer and has low activity accompanied with a significant lag period at the initial stage of reaction. The percentage of dodecameric complex increases with increasing salt concentration. The dodecameric complex has a greatly increased specific activity for the polymerization of 3HBCoA and a negligible lag period. The results from in vitro polymerizations of 3HBCoA suggest that the PHA synthase from E. shaposhnikovii may catalyze a living polymerization and demonstrate that two PhaC and two PhaE subunits comprise a single catalytic site in the synthase complex.}, keywords = {Acyltransferases, Cloning, Molecular, Ectothiorhodospira shaposhnikovii, Escherichia coli, Hydroxybutyrates, Kinetics, Molecular Weight, Polyesters}, issn = {1525-7797}, doi = {10.1021/bm034171i}, author = {Zhang, Shiming and Kolvek, Steven and Goodwin, Steve and Lenz, Robert W} } @article {562, title = {Genome of Geobacter sulfurreducens: metal reduction in subsurface environments.}, journal = {Science}, volume = {302}, year = {2003}, month = {2003 Dec 12}, pages = {1967-9}, abstract = {The complete genome sequence of Geobacter sulfurreducens, a delta-proteobacterium, reveals unsuspected capabilities, including evidence of aerobic metabolism, one-carbon and complex carbon metabolism, motility, and chemotactic behavior. These characteristics, coupled with the possession of many two-component sensors and many c-type cytochromes, reveal an ability to create alternative, redundant, electron transport networks and offer insights into the process of metal ion reduction in subsurface environments. As well as playing roles in the global cycling of metals and carbon, this organism clearly has the potential for use in bioremediation of radioactive metals and in the generation of electricity.}, keywords = {Acetates, Acetyl Coenzyme A, Aerobiosis, Anaerobiosis, Bacterial Proteins, Carbon, Chemotaxis, Chromosomes, Bacterial, Cytochromes c, Electron Transport, Energy Metabolism, Genes, Bacterial, Genes, Regulator, Genome, Bacterial, Geobacter, Hydrogen, Metals, Movement, Open Reading Frames, Oxidation-Reduction, Phylogeny}, issn = {1095-9203}, doi = {10.1126/science.1088727}, author = {Meth{\'e}, B A and Nelson, K E and Eisen, J A and Paulsen, I T and Nelson, W and Heidelberg, J F and Wu, D and Wu, M and Ward, N and Beanan, M J and Dodson, R J and Madupu, R and Brinkac, L M and Daugherty, S C and DeBoy, R T and Durkin, A S and Gwinn, M and Kolonay, J F and Sullivan, S A and Haft, D H and Selengut, J and Davidsen, T M and Zafar, N and White, O and Tran, B and Romero, C and Forberger, H A and Weidman, J and Khouri, H and Feldblyum, T V and Utterback, T R and Van Aken, S E and Lovley, D R and Fraser, C M} } @article {862, title = {Mechanism of the polymerization reaction initiated and catalyzed by the polyhydroxybutyrate synthase of Ralstonia eutropha.}, journal = {Biomacromolecules}, volume = {4}, year = {2003}, month = {2003 May-Jun}, pages = {504-9}, abstract = {Polyhydroxybutyrate (PHB) synthases (polymerases) catalyze the polymerization of the coenzyme A thioester of 3-hydroxybutyrate to PHB. The Ralstonia eutropha PHB synthase purified from recombinant E. coli cells exists in aqueous solution in both monomeric (single subunit) and homodimeric (two subunits) forms in equilibrium. Several lines of evidence suggest that the homodimer is the active form of the synthase. The initial mechanistic model for the polymerization reaction proposed that two different thiol groups form the catalytic site. The cysteine at 319 has been shown to provide one thiol group that is involved in the covalent catalysis, but a second thiol group on the same protein molecule has not yet been identified. It is suggested that cysteines at 319 from each of the two molecules of a homodimer synthase provide two identical thiol groups to jointly form a single catalytic site. To verify this model using the strategy of in vitro reconstitution, heterodimers composed of the wild-type subunit and of the C(319) mutated subunit were constructed and the activities at various ratios of the wild-type subunit to the mutated subunit were measured. The experimental results indicate that the homodimer is the active form of the enzyme, that the heterodimer containing the mutated subunit has no activity, and that a single cysteine is not sufficient for catalysis. Two identical thiol groups from C(319) residues on each subunit of the homodimer are required to form the catalytic site for the initiation and propagation reactions. We further demonstrate that a dimer synthase that has initiated the polymerization reaction (primed synthase) is significantly more stable against dissociation than the unprimed (unreacted) dimer synthase. These two properties explain the nature of lag phenomenon during the in vitro polymerization reaction catalyzed by this enzyme}, keywords = {Catalysis, Cupriavidus necator, Hydroxybutyrates, Polymers}, issn = {1525-7797}, doi = {10.1021/bm020099x}, author = {Zhang, Shiming and Kolvek, Steve and Lenz, Robert W and Goodwin, Steve} } @article {866, title = {Comparative study of the relationship between monomer structure and reactivity for two polyhydroxyalkanoate synthases.}, journal = {Appl Microbiol Biotechnol}, volume = {56}, year = {2001}, month = {2001 Jul}, pages = {131-6}, abstract = {Using organically synthesized hydroxyalkanoate coenzyme A thioesters, the activities of two short-chain polyhydroxalkanoate (PHA) synthases were investigated--Ralstonia eutropha PHA synthase (a type I PHA synthase) and Ectothiorhodospira shaposhnikovii PHA synthase (a type III synthase). The results indicate that the two synthases have similar activities towards most of the monomers tested. 3-Hydroxybutyryl CoA was found to be the most efficient substrate for both synthases. Changes in the side-chain length of the monomers affect monomer reactivity, with shortening of the side-chain length having the more severe effect. Hydrophobicity in the side chain appears to play an important role in the catalytic reaction. The configuration and the position of the hydroxyl group also affect the reactivity of a monomer. Monomers with the [S] configuration can not be recognized by either synthase. Moving the hydroxyl group from the beta carbon to the alpha carbon has a much more severe effect on the reactivity of the monomer than moving the hydroxyl group to the gamma carbon. The results demonstrate that the in vitro system can be used to prepare entirely novel polymers that may not be obtainable from living cells because of metabolic restrictions.}, keywords = {Acyl Coenzyme A, Acyltransferases, Structure-Activity Relationship, Substrate Specificity}, issn = {0175-7598}, author = {Zhang, S and Kamachi, M and Takagi, Y and Lenz, R W and Goodwin, S} } @article {1217, title = {Diversity among three novel groups of hyperthermophilic deep-sea Thermococcus species from three sites in the northeastern Pacific Ocean.}, journal = {FEMS Microbiol Ecol}, volume = {36}, year = {2001}, month = {2001 Jun}, pages = {51-60}, abstract = {

Eight new strains of deep-sea hyperthermophilic sulfur reducers were isolated from hydrothermal vent fields at 9 degrees 50{\textquoteright}N East Pacific Rise (EPR) and at the Cleft and CoAxial segments along the Juan de Fuca Ridge (JdFR). 16S rRNA gene sequence analysis showed that each strain belongs to the genus Thermococcus. Restriction fragment length polymorphism patterns of the 16S/23S rRNA intergenic spacer region revealed that these isolates fell into three groups: those from the EPR, those from fluid and rock sources on the JdFR, and those isolated from Paralvinella spp. polychaete vent worms from the JdFR. The optimum-temperature specific growth rates and the temperature ranges for growth were significantly higher and broader for those strains isolated from worms relative to those isolated from low-temperature diffuse hydrothermal fluids. Furthermore, the worm-derived isolates generally produced a larger array of proteases and amylases based on zymogram analyses. The zymogram patterns also changed with growth temperature suggesting that these organisms alter their lytic protein suites in response to changes in temperature. This study suggests that there is significant phenotypic diversity in Thermococcus that is not apparent from their highly conserved 16S rRNA nucleotide sequences.

}, issn = {1574-6941}, author = {Holden, J F. and Takai, K and Summit, M and Bolton, S and Zyskowski, J and Baross, J A.} } @article {864, title = {In vitro polymerization and copolymerization of 3-hydroxypropionyl-CoA with the PHB synthase from Ralstonia eutropha.}, journal = {Biomacromolecules}, volume = {1}, year = {2000}, month = {2000 Fall}, pages = {433-9}, abstract = {The poly(3-hydroxybutyrate) (PHB) synthase of Ralstonia eutropha, which was produced by a recombinant strain of Escherichia coli and purified in one step with a methyl-HIC column to a purity of more than 90\%, was used to polymerize 3-hydroxypropionyl-CoA (3HPCoA) and to copolymerize 3HPCoA with 3-hydroxybutyryl-CoA (3HBCoA). A Km of 189 microM and a kcat of 10 s-1 were determined for the activity of the enzyme in the polymerization reaction of 3HPCoA based on the assumption that the dimer form of PHB synthase was the active form. Free coenzyme A was found to be a very effective competitive inhibitor for the polymerization of 3HPCoA with a Ki of 85 microM. The maximum degree of conversion of 3HPCoA to polymer was less than 40\%. In the simultaneous copolymerization reactions of these two monomers, both the turnover number for the copolymerization reaction and the maximum degree of conversion of 3HPCoA and 3HBCoA to copolymers increased with an increase in the amount of 3HBCoA in the monomer mixture. However, the maximum conversion of 3HPCoA to copolymer was always less than 35\%, regardless of the ratio of 3HPCoA to 3HBCoA. Block copolymers were obtained by the sequential copolymerization of the two monomers and these copolymers had a much narrower molecular weight distribution than those obtained by the simultaneous copolymerization for the same molar ratio of 3HPCoA to 3HBCoA.}, keywords = {Acyltransferases, Coenzyme A, Escherichia coli, Kinetics, Magnetic Resonance Spectroscopy, Molecular Weight}, issn = {1525-7797}, author = {Song, J J and Zhang, S and Lenz, R W and Goodwin, S} } @article {865, title = {Kinetic and mechanistic characterization of the polyhydroxybutyrate synthase from Ralstonia eutropha.}, journal = {Biomacromolecules}, volume = {1}, year = {2000}, month = {2000 Summer}, pages = {244-51}, abstract = {Purified Ralstonia eutropha polyhydroxybutyrate (PHB) synthase from recombinant cells can exist as monomer and dimer. The polymerization reaction catalyzed by this enzyme displays a lag phase, which causes difficulties for kinetic and mechanistic characterization of the enzymatic polymerization reaction. In this study, we developed a method to eliminate the lag phase of PHB synthase by physical means, i.e., adding multihydroxyl compounds to the enzyme solution. This method allows us to recognize the nature of the lag phase as a physical rather than a chemical process. With such lag-phase-free-enzyme, the kinetic properties of the enzyme were investigated. The results indicate that 3-hydroxybutyryl-CoA (3HBCoA) is the optimal substrate for the enzyme. A slower catalytic rate and lower binding ability account for a lower reactivity of 3-hydroxyvaleryl-CoA (3HVCoA) compared to that of 3HBCoA. The change of hydroxyl group from the beta to the gamma position causes dramatic decreases in the binding ability of 4-hydroxybutyryl-CoA (4HBCoA). By using a dilution strategy and size exclusion chromatographic technique, the active form of the enzyme was identified to be the dimeric form. The number of catalytic sites in the dimeric form of the enzyme was examined by comparing the molecular weight of polyhydroxybutyrate as a function of substrate-to-enzyme ratio. The results suggest that the dimeric enzyme has only one catalytic site. A revised model of polymerization reaction catalyzed by R. eutropha PHB synthase is described.}, keywords = {Acyltransferases, Catalysis, Chromatography, Gel, Coenzyme A, Culture Media, Hydrogen-Ion Concentration, Kinetics, Molecular Weight, Proteobacteria}, issn = {1525-7797}, author = {Zhang, S and Yasuo, T and Lenz, R W and Goodwin, S} } @article {830, title = {dnaC mutations suppress defects in DNA replication- and recombination-associated functions in priB and priC double mutants in Escherichia coli K-12.}, journal = {Mol Microbiol}, volume = {34}, year = {1999}, month = {1999 Oct}, pages = {91-101}, abstract = {PriA, PriB and PriC were originally discovered as proteins essential for the PhiX174 in vitro DNA replication system. Recent studies have shown that PriA mutants are poorly viable, have high basal levels of SOS expression (SOSH), are recombination deficient (Rec-), sensitive to UV irradiation (UVS) and sensitive to rich media. These data suggest that priA{\textquoteright}s role may be more complex than previously thought and may involve both DNA replication and homologous recombination. Based on the PhiX174 system, mutations in priB and priC should cause phenotypes like those seen in priA2:kan mutants. To test this, mutations in priB and priC were constructed. We found that, contrary to the PhiX174 model, del(priB)302 and priC303:kan mutants have almost wild-type phenotypes. Most unexpectedly, we then found that the priBC double mutant had very poor viability and/or a slow growth rate (even less than a priA2:kan mutant). This suggests that priB and priC have a redundant and important role in Escherichia coli. The priA2:kan suppressor, dnaC809, partially suppressed the poor viability/slow growth phenotype of the priBC double mutant. The resulting triple mutant (priBC dnaC809 ) had small colony size, recombination deficiency and levels of SOS expression similar to a priA2:kan mutant. The priBC dnaC809 mutant, however, was moderately UVR and had good viability, unlike a priA2:kan mutant. Additional mutations in the triple mutant were selected to suppress the slow growth phenotype. One suppressor restored all phenotypes tested to nearly wild-type levels. This mutation was identified as dnaC820 (K178N) [mapping just downstream of dnaC809 (E176G)]. Experiments suggest that dnaC820 makes dnaC809 suppression of priA and or priBC mutants priB and or priC independent. A model is proposed for the roles of these proteins in terms of restarting collapsed replication forks from recombinational intermediates.}, keywords = {Bacterial Proteins, Bacteriophage mu, Cell Division, DNA Replication, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Mutation, Phenotype, Recombination, Genetic, Replication Protein A, SOS Response (Genetics), Substrate Specificity, Suppression, Genetic}, issn = {0950-382X}, author = {Sandler, S J and Marians, K J and Zavitz, K H and Coutu, J and Parent, M A and Clark, A J} } @article {870, title = {Extracellular polymerization of 3-hydroxyalkanoate monomers with the polymerase of Alcaligenes eutrophus.}, journal = {Int J Biol Macromol}, volume = {25}, year = {1999}, month = {1999 Jun-Jul}, pages = {55-60}, abstract = {Previous investigations on the role of the polymerase in the synthesis of poly-3-hydroxybutyrate (PHB) are reviewed, and the results from earlier in vitro studies on the activity and selectivity of the polymerase of Alcaligenes eutrophus are discussed. In the present study the effect of glycerol on stabilizing the polymerase after purification and on eliminating the lag phase in in vitro polymerization reactions of 3-hydroxybutyl CoA (HBCoA), and 3-hydroxyvaleryl CoA (HVCoA) are described. K(M) values were determined for the activity of the polymerase with both HBCoA and HVCoA, and the rates of propagation for both monomers were estimated. With a racemic mixture of HBCoA, the enzyme polymerized only the [R] monomer.}, keywords = {Acyl Coenzyme A, Acyltransferases, Alcaligenes, Hydroxybutyrates, Kinetics, Polyesters, Substrate Specificity}, issn = {0141-8130}, author = {Lenz, R W and Farcet, C and Dijkstra, P J and Goodwin, S and Zhang, S} }