@article {1207, title = {Complete genome sequence of the hyperthermophilic archaeon Pyrococcus sp. strain ST04, isolated from a deep-sea hydrothermal sulfide chimney on the Juan de Fuca Ridge.}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 Aug}, pages = {4434-5}, abstract = {

Pyrococcus sp. strain ST04 is a hyperthermophilic, anaerobic, and heterotrophic archaeon isolated from a deep-sea hydrothermal sulfide chimney on the Endeavour Segment of the Juan de Fuca Ridge in the northeastern Pacific Ocean. To further understand the distinct characteristics of this archaeon at the genome level (polysaccharide utilization at high temperature and ATP generation by a Na(+) gradient), the genome of strain ST04 was completely sequenced and analyzed. Here, we present the complete genome sequence analysis results of Pyrococcus sp. ST04 and report the major findings from the genome annotation, with a focus on its saccharolytic and metabolite production potential.

}, keywords = {Adenosine Triphosphate, Anaerobiosis, DNA, Archaeal, Genome, Archaeal, Heterotrophic Processes, Hydrothermal Vents, Molecular Sequence Data, Pacific Ocean, Polysaccharides, Pyrococcus, Seawater, Sequence Analysis, DNA, Sodium Chloride, Sulfides}, issn = {1098-5530}, doi = {10.1128/JB.00824-12}, author = {Jung, Jong-Hyun and Lee, Ju-Hoon and Holden, James F and Seo, Dong-Ho and Shin, Hakdong and Kim, Hae-Yeong and Kim, Wooki and Ryu, Sangryeol and Park, Cheon-Seok} } @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 {476, title = {Geobacter sulfurreducens strain engineered for increased rates of respiration.}, journal = {Metab Eng}, volume = {10}, year = {2008}, month = {2008 Sep}, pages = {267-75}, abstract = {Geobacter species are among the most effective microorganisms known for the bioremediation of radioactive and toxic metals in contaminated subsurface environments and for converting organic compounds to electricity in microbial fuel cells. However, faster rates of electron transfer could aid in optimizing these processes. Therefore, the Optknock strain design methodology was applied in an iterative manner to the constraint-based, in silico model of Geobacter sulfurreducens to identify gene deletions predicted to increase respiration rates. The common factor in the Optknock predictions was that each resulted in a predicted increase in the cellular ATP demand, either by creating ATP-consuming futile cycles or decreasing the availability of reducing equivalents and inorganic phosphate for ATP biosynthesis. The in silico model predicted that increasing the ATP demand would result in higher fluxes of acetate through the TCA cycle and higher rates of NADPH oxidation coupled with decreases in flux in reactions that funnel acetate toward biosynthetic pathways. A strain of G. sulfurreducens was constructed in which the hydrolytic, F(1) portion of the membrane-bound F(0)F(1) (H(+))-ATP synthase complex was expressed when IPTG was added to the medium. Induction of the ATP drain decreased the ATP content of the cell by more than half. The cells with the ATP drain had higher rates of respiration, slower growth rates, and a lower cell yield. Genome-wide analysis of gene transcript levels indicated that when the higher rate of respiration was induced transcript levels were higher for genes involved in energy metabolism, especially in those encoding TCA cycle enzymes, subunits of the NADH dehydrogenase, and proteins involved in electron acceptor reduction. This was accompanied by lower transcript levels for genes encoding proteins involved in amino acid biosynthesis, cell growth, and motility. Several changes in gene expression that involve processes not included in the in silico model were also detected, including increased expression of a number of redox-active proteins, such as c-type cytochromes and a putative multicopper outer-surface protein. The results demonstrate that it is possible to genetically engineer increased respiration rates in G. sulfurreducens in accordance with predictions from in silico metabolic modeling. To our knowledge, this is the first report of metabolic engineering to increase the respiratory rate of a microorganism.}, keywords = {Adenosine Triphosphate, Bacterial Proteins, Biodegradation, Environmental, Citric Acid Cycle, Electron Transport, Geobacter, Metals, Models, Biological, NADH Dehydrogenase, NADP, Oxygen Consumption, Phosphates, Proton-Translocating ATPases, Radioactive Pollutants}, issn = {1096-7184}, doi = {10.1016/j.ymben.2008.06.005}, author = {Izallalen, Mounir and Mahadevan, Radhakrishnan and Burgard, Anthony and Postier, Bradley and DiDonato, Raymond and Sun, Jun and Schilling, Christopher H and Lovley, Derek R} } @article {812, title = {RecA-mediated SOS induction requires an extended filament conformation but no ATP hydrolysis.}, journal = {Mol Microbiol}, volume = {69}, year = {2008}, month = {2008 Sep}, pages = {1165-79}, abstract = {The Escherichia coli SOS response to DNA damage is modulated by the RecA protein, a recombinase that forms an extended filament on single-stranded DNA and hydrolyzes ATP. The RecA K72R (recA2201) mutation eliminates the ATPase activity of RecA protein. The mutation also limits the capacity of RecA to form long filaments in the presence of ATP. Strains with this mutation do not undergo SOS induction in vivo. We have combined the K72R variant of RecA with another mutation, RecA E38K (recA730). In vitro, the double mutant RecA E38K/K72R (recA730,2201) mimics the K72R mutant protein in that it has no ATPase activity. The double mutant protein will form long extended filaments on ssDNA and facilitate LexA cleavage almost as well as wild-type, and do so in the presence of ATP. Unlike recA K72R, the recA E38K/K72R double mutant promotes SOS induction in vivo after UV treatment. Thus, SOS induction does not require ATP hydrolysis by the RecA protein, but does require formation of extended RecA filaments. The RecA E38K/K72R protein represents an improved reagent for studies of the function of ATP hydrolysis by RecA in vivo and in vitro.}, keywords = {Adenosine Triphosphate, Amino Acid Substitution, Bacterial Proteins, DNA, Bacterial, DNA, Single-Stranded, Escherichia coli, Escherichia coli Proteins, Hydrolysis, Rec A Recombinases, Serine Endopeptidases, SOS Response (Genetics), Ultraviolet Rays}, issn = {1365-2958}, doi = {10.1111/j.1365-2958.2008.06341.x}, author = {Gruenig, Marielle C and Renzette, Nicholas and Long, Edward and Chitteni-Pattu, Sindhu and Inman, Ross B and Cox, Michael M and Sandler, Steven J} } @article {813, title = {Requirements for ATP binding and hydrolysis in RecA function in Escherichia coli.}, journal = {Mol Microbiol}, volume = {67}, year = {2008}, month = {2008 Mar}, pages = {1347-59}, abstract = {RecA is essential for recombination, DNA repair and SOS induction in Escherichia coli. ATP hydrolysis is known to be important for RecA{\textquoteright}s roles in recombination and DNA repair. In vitro reactions modelling SOS induction minimally require ssDNA and non-hydrolyzable ATP analogues. This predicts that ATP hydrolysis will not be required for SOS induction in vivo. The requirement of ATP binding and hydrolysis for SOS induction in vivo is tested here through the study of recA4159 (K72A) and recA2201 (K72R). RecA4159 is thought to have reduced affinity for ATP. RecA2201 binds, but does not hydrolyse ATP. Neither mutant was able to induce SOS expression after UV irradiation. RecA2201, unlike RecA4159, could form filaments on DNA and storage structures as measured with RecA-GFP. RecA2201 was able to form hybrid filaments and storage structures and was either recessive or dominant to RecA(+), depending on the ratio of the two proteins. RecA4159 was unable to enter RecA(+) filaments on DNA or storage structures and was recessive to RecA(+). It is concluded that ATP hydrolysis is essential for SOS induction. It is proposed that ATP binding is essential for storage structure formation and ability to interact with other RecA proteins in a filament.}, keywords = {Adenosine Triphosphate, Chromosomes, Bacterial, Escherichia coli, Escherichia coli Proteins, Hydrolysis, Mutation, Protein Binding, Rec A Recombinases, Ultraviolet Rays}, issn = {1365-2958}, doi = {10.1111/j.1365-2958.2008.06130.x}, author = {Renzette, Nicholas and Sandler, Steven J} } @article {1199, title = {Phosphoenolpyruvate synthetase from the hyperthermophilic archaeon Pyrococcus furiosus.}, journal = {J Bacteriol}, volume = {183}, year = {2001}, month = {2001 Jan}, pages = {709-15}, abstract = {

Phosphoenolpyruvate synthetase (PpsA) was purified from the hyperthermophilic archaeon Pyrococcus furiosus. This enzyme catalyzes the conversion of pyruvate and ATP to phosphoenolpyruvate (PEP), AMP, and phosphate and is thought to function in gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and contains one calcium and one phosphorus atom per subunit. The active form has a molecular mass of 690+/-20 kDa and is assumed to be octomeric, while approximately 30\% of the protein is purified as a large ( approximately 1.6 MDa) complex that is not active. The apparent K(m) values and catalytic efficiencies for the substrates pyruvate and ATP (at 80 degrees C, pH 8.4) were 0.11 mM and 1.43 x 10(4) mM(-1). s(-1) and 0.39 mM and 3.40 x 10(3) mM(-1) x s(-1), respectively. Maximal activity was measured at pH 9.0 (at 80 degrees C) and at 90 degrees C (at pH 8.4). The enzyme also catalyzed the reverse reaction, but the catalytic efficiency with PEP was very low [k(cat)/K(m) = 32 (mM. s(-1)]. In contrast to several other nucleotide-dependent enzymes from P. furiosus, PpsA has an absolute specificity for ATP as the phosphate-donating substrate. This is the first PpsA from a nonmethanogenic archaeon to be biochemically characterized. Its kinetic properties are consistent with a role in gluconeogenesis, although its relatively high cellular concentration ( approximately 5\% of the cytoplasmic protein) suggests an additional function possibly related to energy spilling. It is not known whether interconversion between the smaller, active and larger, inactive forms of the enzyme has any functional role.

}, keywords = {Adenosine Monophosphate, Adenosine Triphosphate, Gluconeogenesis, Hydrogen-Ion Concentration, Phosphates, Phosphoenolpyruvate, Phosphotransferases (Paired Acceptors), Pyrococcus furiosus, Pyruvic Acid, Substrate Specificity}, issn = {0021-9193}, doi = {10.1128/JB.183.2.709-715.2001}, author = {Hutchins, A M and Holden, J F and Adams, M W} } @article {1707, title = {Citrate synthase.}, journal = {Curr Top Cell Regul}, volume = {10}, year = {1976}, month = {1976}, pages = {161-204}, keywords = {Adenosine Diphosphate, Adenosine Monophosphate, Adenosine Triphosphate, Amino Acids, Animals, Bacteria, Binding Sites, Cations, Divalent, Cations, Monovalent, Citrate (si)-Synthase, Dithionitrobenzoic Acid, Ketoglutaric Acids, Kinetics, Molecular Weight, NAD, NADP, Oxo-Acid-Lyases, Protein Binding, Species Specificity}, issn = {0070-2137}, author = {Weitzman, P D and Danson, M J} }