@article {3094, title = {Expressing the Geobacter metallireducens PilA in Geobacter sulfurreducens Yields Pili with Exceptional Conductivity.}, journal = {mBio}, volume = {8}, year = {2017}, month = {2017 Jan 17}, abstract = {

UNLABELLED: The electrically conductive pili (e-pili) of Geobacter sulfurreducens serve as a model for a novel strategy for long-range extracellular electron transfer. e-pili are also a new class of bioelectronic materials. However, the only other Geobacter pili previously studied, which were from G.~uraniireducens, were poorly conductive. In order to obtain more information on the range of pili conductivities in Geobacter species, the pili of G.~metallireducens were investigated. Heterologously expressing the PilA gene of G.~metallireducens in G.~sulfurreducens yielded a G.~sulfurreducens strain, designated strain MP, that produced abundant pili. Strain MP exhibited phenotypes consistent with the presence of e-pili, such as high rates of Fe(III) oxide reduction and high current densities on graphite anodes. Individual pili prepared at physiologically relevant pH~7 had conductivities of 277 {\textpm} 18.9 S/cm (mean {\textpm} standard deviation), which is 5,000-fold higher than the conductivity of G.~sulfurreducens pili at pH~7 and nearly 1 million-fold higher than the conductivity of G.~uraniireducens pili at the same pH. A potential explanation for the higher conductivity of the G.~metallireducens pili is their greater density of aromatic amino acids, which are known to be important components in electron transport along the length of the pilus. The G.~metallireducens pili represent the most highly conductive pili found to date and suggest strategies for designing synthetic pili with even higher conductivities.

IMPORTANCE: e-pili are a remarkable electrically conductive material that can be sustainably produced without harsh chemical processes from renewable feedstocks and that contain no toxic components in the final product. Thus, e-pili offer an unprecedented potential for developing novel materials, electronic devices, and sensors for diverse applications with a new "green" technology. Increasing e-pili conductivity will even further expand their potential applications. A proven strategy is to design synthetic e-pili that contain tryptophan, an aromatic amino acid not found in previously studied e-pili. The studies reported here demonstrate that a productive alternative approach is to search more broadly in the microbial world. Surprisingly, even though G.~metallireducens and G.~sulfurreducens are closely related, the conductivities of their e-pili differ by more than 3 orders of magnitude. The ability to produce e-pili with high conductivity without generating a genetically modified product enhances the attractiveness of this novel electronic material.

}, keywords = {Electric Conductivity, Electrodes, Electron Transport, Ferric Compounds, Fimbriae Proteins, Gene Expression, Geobacter, Oxidation-Reduction, Recombinant Proteins}, issn = {2150-7511}, doi = {10.1128/mBio.02203-16}, author = {Tan, Yang and Adhikari, Ramesh Y and Malvankar, Nikhil S and Ward, Joy E and Woodard, Trevor L and Nevin, Kelly P and Lovley, Derek R} } @article {3115, title = {Converting carbon dioxide to butyrate with an engineered strain of Clostridium ljungdahlii.}, journal = {mBio}, volume = {5}, year = {2014}, month = {2014 Oct 21}, pages = {e01636-14}, abstract = {

Microbial conversion of carbon dioxide to organic commodities via syngas metabolism or microbial electrosynthesis is an attractive option for production of renewable biocommodities. The recent development of an initial genetic toolbox for the acetogen Clostridium ljungdahlii has suggested that C. ljungdahlii may be an effective chassis for such conversions. This possibility was evaluated by engineering a strain to produce butyrate, a valuable commodity that is not a natural product of C. ljungdahlii metabolism. Heterologous genes required for butyrate production from acetyl-coenzyme A (CoA) were identified and introduced initially on plasmids and in subsequent strain designs integrated into the C. ljungdahlii chromosome. Iterative strain designs involved increasing translation of a key enzyme by modifying a ribosome binding site, inactivating the gene encoding the first step in the conversion of acetyl-CoA to acetate, disrupting the gene which encodes the primary bifunctional aldehyde/alcohol dehydrogenase for ethanol production, and interrupting the gene for a CoA transferase that potentially represented an alternative route for the production of acetate. These modifications yielded a strain in which ca. 50 or 70\% of the carbon and electron flow was diverted to the production of butyrate with H2 or CO as the electron donor, respectively. These results demonstrate the possibility of producing high-value commodities from carbon dioxide with C. ljungdahlii as the catalyst. Importance: The development of a microbial chassis for efficient conversion of carbon dioxide directly to desired organic products would greatly advance the environmentally sustainable production of biofuels and other commodities. Clostridium ljungdahlii is an effective catalyst for microbial electrosynthesis, a technology in which electricity generated with renewable technologies, such as solar or wind, powers the conversion of carbon dioxide and water to organic products. Other electron donors for C. ljungdahlii include carbon monoxide, which can be derived from industrial waste gases or the conversion of recalcitrant biomass to syngas, as well as hydrogen, another syngas component. The finding that carbon and electron flow in C. ljungdahlii can be diverted from the production of acetate to butyrate synthesis is an important step toward the goal of renewable commodity production from carbon dioxide with this organism.

}, keywords = {Acetyl Coenzyme A, Butyrates, Carbon Dioxide, Clostridium, Metabolic Engineering, Metabolic Flux Analysis, Metabolic Networks and Pathways, Recombinant Proteins}, issn = {2150-7511}, doi = {10.1128/mBio.01636-14}, author = {Ueki, Toshiyuki and Nevin, Kelly P and Woodard, Trevor L and Lovley, Derek R} } @article {1212, title = {Characterization of malate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum.}, journal = {Extremophiles}, volume = {11}, year = {2007}, month = {2007 Sep}, pages = {741-6}, abstract = {

Native and recombinant malate dehydrogenase (MDH) was characterized from the hyperthermophilic, facultatively autotrophic archaeon Pyrobaculum islandicum. The enzyme is a homotetramer with a subunit mass of 33 kDa. The activity kinetics of the native and recombinant proteins are the same. The apparent K ( m ) values of the recombinant protein for oxaloacetate (OAA) and NADH (at 80 degrees C and pH 8.0) were 15 and 86 microM, respectively, with specific activity as high as 470 U mg(-1). Activity decreased more than 90\% when NADPH was used. The catalytic efficiency of OAA reduction by P. islandicum MDH using NADH was significantly higher than that reported for any other archaeal MDH. Unlike other archaeal MDHs, specific activity of the P. islandicum MDH back-reaction also decreased more than 90\% when malate and NAD(+) were used as substrates and was not detected with NADP(+). A phylogenetic tree of 31 archaeal MDHs shows that they fall into 5 distinct groups separated largely along taxonomic lines suggesting minimal lateral mdh transfer between Archaea.

}, keywords = {Citric Acid Cycle, Hydrogen-Ion Concentration, Kinetics, Malate Dehydrogenase, Malates, Molecular Weight, NAD, NADP, Oxaloacetic Acid, Phylogeny, Protein Conformation, Protein Subunits, Pyrobaculum, Recombinant Proteins, Sequence Analysis, Protein, Substrate Specificity, Temperature}, issn = {1431-0651}, doi = {10.1007/s00792-007-0081-2}, author = {Yennaco, Lynda J and Hu, Yajing and Holden, James F} } @article {525, title = {Genetic characterization of a single bifunctional enzyme for fumarate reduction and succinate oxidation in Geobacter sulfurreducens and engineering of fumarate reduction in Geobacter metallireducens.}, journal = {J Bacteriol}, volume = {188}, year = {2006}, month = {2006 Jan}, pages = {450-5}, abstract = {The mechanism of fumarate reduction in Geobacter sulfurreducens was investigated. The genome contained genes encoding a heterotrimeric fumarate reductase, FrdCAB, with homology to the fumarate reductase of Wolinella succinogenes and the succinate dehydrogenase of Bacillus subtilis. Mutation of the putative catalytic subunit of the enzyme resulted in a strain that lacked fumarate reductase activity and was unable to grow with fumarate as the terminal electron acceptor. The mutant strain also lacked succinate dehydrogenase activity and did not grow with acetate as the electron donor and Fe(III) as the electron acceptor. The mutant strain could grow with acetate as the electron donor and Fe(III) as the electron acceptor if fumarate was provided to alleviate the need for succinate dehydrogenase activity in the tricarboxylic acid cycle. The growth rate of the mutant strain under these conditions was faster and the cell yields were higher than for wild type grown under conditions requiring succinate dehydrogenase activity, suggesting that the succinate dehydrogenase reaction consumes energy. An orthologous frdCAB operon was present in Geobacter metallireducens, which cannot grow with fumarate as the terminal electron acceptor. When a putative dicarboxylic acid transporter from G. sulfurreducens was expressed in G. metallireducens, growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms, G. sulfurreducens and possibly G. metallireducens use the same enzyme for both fumarate reduction and succinate oxidation in vivo.}, keywords = {Culture Media, Dicarboxylic Acids, Fumarates, Geobacter, Molecular Sequence Data, Operon, Oxidation-Reduction, Recombinant Proteins, Substrate Specificity, Succinate Dehydrogenase, Succinic Acid}, issn = {0021-9193}, doi = {10.1128/JB.188.2.450-455.2006}, author = {Butler, Jessica E and Glaven, Richard H and Esteve-N{\'u}{\~n}ez, Abraham and N{\'u}{\~n}ez, Cinthia and Shelobolina, Evgenya S and Bond, Daniel R and Lovley, Derek R} } @article {386, title = {Trypanosoma brucei has two distinct mitochondrial DNA polymerase beta enzymes.}, journal = {J Biol Chem}, volume = {278}, year = {2003}, month = {2003 Dec 5}, pages = {49095-101}, abstract = {In higher eukaryotes, DNA polymerase (pol) beta resides in the nucleus and participates primarily in DNA repair. The DNA polymerase beta from the trypanosomatid Crithidia fasciculata, however, was the first mitochondrial enzyme of this type described. Upon searching the nearly completed genome data base of the related parasite Trypanosoma brucei, we discovered genes for two pol beta-like proteins. One is approximately 70\% identical to the C. fasciculata pol beta and is likely the homolog of this enzyme. The other, although approximately 30\% identical within the polymerase region, has unusual structural features including a short C-terminal tail and a long N-terminal extension rich in prolines, alanines, and lysines. Both proteins, when expressed recombinantly, are active as DNA polymerases and deoxyribose phosphate lyases, but their polymerase activity optima differ with respect to pH and KCl and MgCl2 concentrations. Remarkably, green fluorescent protein fusion proteins and immunofluorescence demonstrate that both are mitochondrial, but their locations with respect to the mitochondrial DNA (kinetoplast DNA network) in this organism are strikingly different.}, keywords = {Animals, Base Sequence, Cell Line, DNA Polymerase beta, DNA Primers, Isoenzymes, Mitochondria, Recombinant Proteins, Trypanosoma brucei brucei}, issn = {0021-9258}, doi = {10.1074/jbc.M308565200}, author = {Saxowsky, Tina T and Choudhary, Gunjan and Klingbeil, Michele M and Englund, Paul T} } @article {832, title = {Diversity of radA genes from cultured and uncultured archaea: comparative analysis of putative RadA proteins and their use as a phylogenetic marker.}, journal = {J Bacteriol}, volume = {181}, year = {1999}, month = {1999 Feb}, pages = {907-15}, abstract = {Archaea-specific radA primers were used with PCR to amplify fragments of radA genes from 11 cultivated archaeal species and one marine sponge tissue sample that contained essentially an archaeal monoculture. The amino acid sequences encoded by the PCR fragments, three RadA protein sequences previously published (21), and two new complete RadA sequences were aligned with representative bacterial RecA proteins and eucaryal Rad51 and Dmc1 proteins. The alignment supported the existence of four insertions and one deletion in the archaeal and eucaryal sequences relative to the bacterial sequences. The sizes of three of the insertions were found to have taxonomic and phylogenetic significance. Comparative analysis of the RadA sequences, omitting amino acids in the insertions and deletions, shows a cladal distribution of species which mimics to a large extent that obtained by a similar analysis of archaeal 16S rRNA sequences. The PCR technique also was used to amplify fragments of 15 radA genes from uncultured natural sources. Phylogenetic analysis of the amino acid sequences encoded by these fragments reveals several clades with affinity, sometimes only distant, to the putative RadA proteins of several species of Crenarcheota. The two most deeply branching archaeal radA genes found had some amino acid deletion and insertion patterns characteristic of bacterial recA genes. Possible explanations are discussed. Finally, signature codons are presented to distinguish among RecA protein family members.}, keywords = {Amino Acid Sequence, Archaea, Archaeal Proteins, Bacteria, Bacterial Proteins, Cloning, Molecular, DNA Primers, DNA Repair, DNA-Binding Proteins, Evolution, Molecular, Humans, Molecular Sequence Data, Phylogeny, Polymerase Chain Reaction, Recombinant Proteins, RNA, Ribosomal, 16S, Saccharomyces cerevisiae, Sequence Alignment}, issn = {0021-9193}, author = {Sandler, S J and Hugenholtz, P and Schleper, C and DeLong, E F and Pace, N R and Clark, A J} } @article {833, title = {RadA protein is an archaeal RecA protein homolog that catalyzes DNA strand exchange.}, journal = {Genes Dev}, volume = {12}, year = {1998}, month = {1998 May 1}, pages = {1248-53}, abstract = {With the discovery that the Saccharomyces cerevisiae Rad51 protein is both structurally and functionally similar to the Escherichia coli RecA protein, the RecA paradigm for homologous recombination was extended to the Eucarya. The ubiquitous presence of RecA and Rad51 protein homologs raises the question of whether this archetypal protein exists within the third domain of life, the Archaea. Here we present the isolation of a Rad51/RecA protein homolog from the archaeon Sulfolobus solfataricus, and show that this protein, RadA, possesses the characteristics of a DNA strand exchange protein: The RadA protein is a DNA-dependent ATPase, forms a nucleoprotein filament on DNA, and catalyzes DNA pairing and strand exchange.}, keywords = {Adenosine Triphosphatases, Archaeal Proteins, Bacterial Proteins, DNA Helicases, DNA, Archaeal, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Genes, Archaeal, Nucleoproteins, Rad51 Recombinase, Rec A Recombinases, Recombinant Proteins, Recombination, Genetic, Sulfolobus}, issn = {0890-9369}, author = {Seitz, E M and Brockman, J P and Sandler, S J and Clark, A J and Kowalczykowski, S C} }