Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria

<p>Abstract</p> <p>Background</p> <p>The validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll <it>a </it>and carry out photosynthetic water oxidation. These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of <it>Nostoc punctiforme </it>ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction.</p> <p>Results</p> <p>With this work we identify and explore strategies for improved and lower cost high quality RNA isolation from cyanobacteria. All the methods studied are suitable for RNA isolation and its use for downstream applications. We analyse different Trizol based protocols, introduce procedural changes and describe an alternative RNA extraction solution.</p> <p>Conclusion</p> <p>It was possible to improve purity of isolated RNA by modifying protocol procedures. Further improvements, both in RNA purity and experimental cost, were achieved by using a new extraction solution, PGTX.</p

the influence of phosphate on structure and activity

Two types of manganese oxides have been prepared by hydrolysis of tetranuclear Mn(III) complexes in the presence or absence of phosphate ions. The oxides have been characterized structurally using X-ray absorption spectroscopy and functionally by O2 evolution measurements. The structures of the oxides prepared in the absence of phosphate are dominated by di-μ-oxo bridged manganese ions that form layers with limited long-range order, consisting of edge-sharing MnO6 octahedra. The average manganese oxidation state is +3.5. The structure of these oxides is closely related to other manganese oxides reported as water oxidation catalysts. They show high oxygen evolution activity in a light-driven system containing [Ru(bpy)3]2+ and S2O82− at pH 7. In contrast, the oxides formed by hydrolysis in the presence of phosphate ions contain almost no di-μ-oxo bridged manganese ions. Instead the phosphate groups are acting as bridges between the manganese ions. The average oxidation state of manganese ions is +3. This type of oxide has much lower water oxidation activity in the light-driven system. Correlations between different structural motifs and the function as a water oxidation catalyst are discussed and the lower activity in the phosphate containing oxide is linked to the absence of protonable di-μ-oxo bridges

Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase

J Biol Inorg Chem (2007) 12:353–366 DOI 10.1007/s00775-006-0191-9Two arsenite-inhibited forms of each of the aldehyde oxidoreductases from Desulfovibrio gigas and Desulfovibrio desulfuricans have been studied by X-ray crystallography and electron paramagnetic resonance (EPR) spectroscopy. The molybdenum site of these enzymes shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. Arsenite addition to active as-prepared enzyme or to a reduced desulfo form yields two different species called A and B, respectively, which show different Mo(V) EPR signals. Both EPR signals show strong hyperfine and quadrupolar couplings with an arsenic nucleus, which suggests that arsenic interacts with molybdenum through an equatorial ligand. X-ray data of single crystals prepared from EPR-active samples show in both inhibited forms that the arsenic atom interacts with the molybdenum ion through an oxygen atom at the catalytic labile site and that the sulfido ligand is no longer present. EPR and X-ray data indicate that the main difference between both species is an equatorial ligand to molybdenum which was determined to be an oxo ligand in species A and a hydroxyl/water ligand in species B. The conclusion that the sulfido ligand is not essential to determine the EPR properties in both Mo-As complexes is achieved through EPR measurements on a substantial number of randomly oriented chemically reduced crystals immediately followed by X-ray studies on one of those crystals. EPR saturation studies show that the electron transfer pathway, which is essential for catalysis, is not modified upon inhibition

X-ray crystal structure and EPR spectra of "arsenite-inhibited" Desulfovibriogigas aldehyde dehydrogenase: a member of the xanthine oxidase family

J. Am. Chem. Soc., 2004, 126 (28), pp 8614–8615 DOI: 10.1021/ja0490222X-ray crystallography has been used to determine the structure of arsenite-inhibited aldehyde dehydrogenase from Desulfovibrio gigas, a member of the xanthine oxidase family of mononuclear molybdenum enzymes. The structure shows an AsO3 moiety bound to the molybdenum atom of the active site through one of the oxygen atoms. A reduced sample of arsenite-inhibited aldehyde dehydrogenase has a Mo(V) signal that shows anisotropic hyperfine and quadrupole coupling to one arsenic atom. This signal has a strong resemblance with a previously reported signal for arsenite-inhibited xanthine oxidase

Purification and Preliminary Characterization of Tetraheme Cytochrome c3 and Adenylylsulfate Reductase from the Peptidolytic Sulfate-Reducing Bacterium Desulfovibrio aminophilus DSM 12254

Two proteins were purified and preliminarily characterized from the soluble extract of cells (310 g, wet weight) of the aminolytic and peptidolytic sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254. The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5'-phosphosulfate, APS) reductase, a key enzyme in the microbial dissimilatory sulfate reduction, has been purified in three chromatographic steps (DEAE-Biogel A, Source 15, and Superdex 200 columns). It contains two different subunits with molecular masses of 75 and 18 kDa. The fraction after the last purification step had a purity index (A278nm / A388nm) of 5.34, which was used for further EPR spectroscopic studies. The D. aminophilus APS reductase is very similar to the homologous enzymes isolated from D. gigas and D. desulfuricans ATCC 27774. A tetraheme cytochrome c3 (His-heme iron-His) has been purified in three chromatographic steps (DEAE- Biogel A, Source 15, and Biogel-HTP columns) and preliminarily characterized. It has a purity index ([A553nm - A570nm]red / A280nm) of 2.9 and a molecular mass of around 15 kDa, and its spectroscopic characterization (NMR and EPR) has been carried out. This hemoprotein presents similarities with the tetraheme cytochrome c3 from Desulfomicrobium (Des.) norvegicum (NMR spectra, and N-terminal amino acid sequence)

Purification and Preliminary Characterization of Tetraheme Cytochrome and Adenylylsulf te Reductase from the Peptidolytic Sulfate-Reducing Bacterium Desulfovibrio aminophilus DSM 12254

Bioinorganic Chemistry and Applications Volume 3 (2005), Issue 1-2, Pages 81-91Two proteins were purified and preliminarily characterized from the soluble extract of cells (310 g, wet weight) of the aminolytic and peptidolytic sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254. The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5"-phosphosulfate, APS)reductase, a key enzyme in the microbial dissimilatory sulfate reduction, has been purified in three chromatographic steps(DEAE-Biogel A, Source 15, and Superdex 200 columns). It contains two different subunits with molecular masses of 75 and 18 kDa. The fraction after the last purification step had a purity index (A278 ,m/A388 nm) of 5.34, which was used for further EPR spectroscopic studies. The D. aminophilus APS reductase is very similar to the homologous enzymes isolated from D. gigas and D. desulfuricans ATCC 27774. A tetraheme cytochrome c3 (His-heme iron-His) has been purified in three chromatographic steps (DEAE- Biogel A,Source 15, and Biogel-HTP columns) and preliminarily characterized. It has a purity index ([A.s.s3 nm" A570 nm]rcd/m280nm ox) of 2.9 and a molecular mass of around 15 kDa, and its spectroscopic characterization(NMR and EPR) has been carried out. This hemoprotein presents similarities with the tetraheme cytochrome c3 from Desulfomicrobium (Des.) norvegicurn (NMR spectra, and N-terminal amino acid sequence)

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo-C Interaction in a Biological System

J. Am. Chem. Soc., 2009, 131 (23), pp 7990–7998 DOI: 10.1021/ja809448rAldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a member of the xanthine oxidase(XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V)EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerolinhibited enzyme, where the catalytically labile OH/OH2 ligand is lost and both alcohols coordinate the Mo site in a η2 fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system

Artificial Photosynthesis for Solar Fuels - an Evolving Research Field within AMPEA, a Joint Programme of the European Energy Research Alliance

On the path to an energy transition away from fossil fuels to sustainable sources, the European Union is for the moment keeping pace with the objectives of the Strategic Energy Technology-Plan. For this trend to continue after 2020, scientific breakthroughs must be achieved. One main objective is to produce solar fuels from solar energy and water in direct processes to accomplish the efficient storage of solar energy in a chemical form. This is a grand scientific challenge. One important approach to achieve this goal is Artificial Photosynthesis. The European Energy Research Alliance has launched the Joint Programme "Advanced Materials & Processes for Energy Applications” (AMPEA) to foster the role of basic science in Future Emerging Technologies. European researchers in artificial photosynthesis recently met at an AMPEA organized workshop to define common research strategies and milestones for the future. Through this work artificial photosynthesis became the first energy research sub-field to be organised into what is designated "an Application” within AMPEA. The ambition is to drive and accelerate solar fuels research into a powerful European field - in a shorter time and with a broader scope than possible for individual or national initiatives. Within AMPEA the Application Artificial Photosynthesis is inclusive and intended to bring together all European scientists in relevant fields. The goal is to set up a thorough and systematic programme of directed research, which by 2020 will have advanced to a point where commercially viable artificial photosynthetic devices will be under development in partnership with industr

Modelling of molybdopterin-dependent enzymes

The thesis deals with models for molybdopterin-dependent enzymes. Several model systems containing molybdenum or tungsten have been prepared and characterised, and their reactivity with oxygen atom acceptors and donors have been investigated. Oxygen atom transfer reactions involving oxomolybdenum bis-dithiolene complexes have been modelled using density functional calculations. The first molybdenum(VI) complexes containing unperturbed cis-MoOS moieties, [MoOS(OSiPh3)2(L)] where L is bidentate nitrogen ligand, have been prepared as models for the xanthine oxidase family of mononuclear molybdenum enzymes. Spectroscopic measurements (1H-NMR, IR, XAS, and X-ray crystallography) confirm the integrity of the cis-MoOS moiety both in solution and in the solid state. When [MoOS(OSiPh3)2(Me4phen)] is reacted with PPh3, a good oxo acceptor, the complex does not undergo oxygen atom transfer but instead sulfur atom transfer to form Ph3PS and [MoVOCl(OSiPh3)2(Me4phen)]. Two dioxotungsten(VI) complexes, [WO2(tBuL-NS)2] (tBuL-NS- = bis(4-tert-butylphenyl)-2-pyridylmethanethiolate(1-)) and [WO2(tBuL-NO)2] (tBuL-NO- = bis(4-tert-butylphenyl)-2-pyridylmethanolate(1-)) have been prepared as models for mononuclear tungsten enzymes. They have been spectroscopically and structurally characterised. The dioxomolybdenum(VI) analogue of [WO2(tBuL-NS)2] can be reduced to a mono-oxomolybdenum(IV) complex by phosphines and the molybdenum(IV) complex can reduce a range of oxygen atom donors. In contrast [WO2(tBuL-NS)2] does not undergo the corresponding oxygen atom transfer chemistry with phoshines, which may be attributed to a larger thermodymic barrier for reduction of tungsten(VI) compared to molybdenum(VI). A dioxomolybdenum(VI) complex, [MoO2(L-O)]PF6 (L-OH = N-(2-hydroxybenzyl)-N,N-bis(2-pyridylmethyl)amine) has been synthesised as a functional model for molybdenum oxotransferases. When the complex is reacted with phosphines in methanol, phosphine oxides are formed together with a red, air-sensitive, molybdenum complex. The identity of the red complex is not established but it is proposed to be a Mo(V) complex. The red molybdenum complex can be oxidised to [MoO2(L-O)]+ by addition of oxygen atom donors such as DMSO or nitrate, thereby mimicking the activity of molybdenum oxotransferases. Attempts at isolating the red complex leads to the formation of a dark purple m-oxo-bridged Mo(V) dimer, [(L-O)OMo(m-O)MoO(L-O)]2+. Computer modelling of the reaction of [MoO2(mnt)2]2- (mnt2- = 1,2-dicyano-ethylene-1,2-dithiolate(2-)) with hydrogen sulfite shows that the reaction mechanism is likely to involve a direct attack of the sulfur lone pair on one of the oxo ligands. The reaction proceeds via an oxygen atom transfer reaction where the substrate is oxidised to hydrogen sulfate, this is in good agreement with proposed mechanisms from other model systems and with the proposed mechanism for sulfite oxidase itself. Density functional modelling of the reduction of Me3NO by [MoO(mnt)2]2- shows that the reaction proceeds via an intermediate containing coordinated Me3NO to formation of the products, [MoO2(mnt)2]2- and NMe3. In the final transition state, one of the Mo-S bonds of one mnt ligand is elongated due to the trans influence of the spectator oxo ligand. Modelling of the reduction of DMSO by [Mo(OCH3)(mnt)2]- shows that the methoxy group may be beneficial for the reaction in two ways: i) by lowering the energy of the products ([MoO(OCH3)(mnt)2]- and DMS) relative to the reactants, and ii) by offering an alternative reaction pathway with a twisted trigonal prismatic geometry in the transition state. This finding may have implications for the enzymes in the DMSO reductase family of mononuclear molybdenum enzymes where an amino acid residue (serine, cysteine or seleneocysteine) is found in the active site