Distribution of major fatty acid in total and different lipid classes in strain MAR441^T when grown in marine broth at 10°C.

<p>Distribution of major fatty acid in total and different lipid classes in strain MAR441^T when grown in marine broth at 10°C.</p

Change in average chain length (ACL, open diamonds) and relative proportion of whole cell FAs in strain MAR441^T and its NTG mutants (A4 and A13) grown at 4, 15 and 25°C.

<p>Straight chain fatty acids (SCFAs, filled circles), branched chain fatty acids (BCFAs, filled boxes), monounsaturated fatty acids (MUFAs, open boxes), polyunsaturated fatty acids (PUFAs, upside down triangles) and eicosapentaenoic acid (EPA, open circles). The experiments were carried out in triplicate and values are means of three samples based on <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188081#pone.0188081.s007" target="_blank">S5 Table</a></b>.</p

Lipid composition of strain MAR441^T when grown in marine broth at 10°C.^a.

<p>Lipid composition of strain MAR441^T when grown in marine broth at 10°C.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188081#t001fn001" target="_blank">^a</a>.</p

Enhanced eicosapentaenoic acid production by a new deep-sea marine bacterium <i>Shewanella electrodiphila</i> MAR441^T

<div><p>Omega-3 fatty acids are products of secondary metabolism, essential for growth and important for human health. Although there are numerous reports of bacterial production of omega-3 fatty acids, less information is available on the biotechnological production of these compounds from bacteria. The production of eicosapentaenoic acid (EPA, 20:5ω3) by a new species of marine bacteria <i>Shewanella electrodiphila</i> MAR441^T was investigated under different fermentation conditions. This strain produced a high percentage (up to 26%) of total fatty acids and high yields (mg / g of biomass) of EPA at or below the optimal growth temperature. At higher growth temperatures these values decreased greatly. The amount of EPA produced was affected by the carbon source, which also influenced fatty acid composition. This strain required Na⁺ for growth and EPA synthesis and cells harvested at late exponential or early stationary phase had a higher EPA content. Both the highest amounts (20 mg g^-1) and highest percent EPA content (18%) occurred with growth on L-proline and (NH₄)₂SO₄. The addition of cerulenin further enhanced EPA production to 30 mg g^-1. Chemical mutagenesis using NTG allowed the isolation of mutants with improved levels of EPA content (from 9.7 to 15.8 mg g^-1) when grown at 15°C. Thus, the yields of EPA could be substantially enhanced without the need for recombinant DNA technology, often a commercial requirement for food supplement manufacture.</p></div

Effects of interaction with sulphur compounds and free volume in imidazolium-based ionic liquid on desulphurisation: a molecular dynamics study

<p>Interaction energy with sulphur compounds and free volume in imidazolium-based ionic liquid were calculated by molecular dynamics (MD) simulations to examine their effects on desulphurisation. From microstructure analysis and energy contribution calculation, it is found that an increasing fractional free volume in ionic liquid and an enhancement of interaction with solute by tuning the structure of ionic liquid or oxidising sulphur compounds are favourable for desulphurisation, which allows more efficient packing of sulphur compounds in ionic liquids and more easily extraction of sulphur compounds from fuel. The MD results are in good agreement with experimental desulphurisation performance.</p

Enhanced Electricity Production by Use of Reconstituted Artificial Consortia of Estuarine Bacteria Grown as Biofilms

Microbial fuel cells (MFCs) can convert organic compounds directly into electricity by catalytic oxidation, and although MFCs have attracted considerable interest, there is little information on the electricity-generating potential of artificial bacterial biofilms. We have used acetate-fed MFCs inoculated with sediment, with two-chamber bottles and carbon cloth electrodes to deliver a maximum power output of ∼175 mW·m^–2 and a stable power output of ∼105 mW·m^–2. Power production was by direct transfer of electrons to the anode from bacterial consortia growing on the anode, as confirmed by cyclic voltammetry (CV) and scanning electron microscopy (SEM). Twenty different species (74 strains) of bacteria were isolated from the consortium under anaerobic conditions and cultured in the laboratory, of which 34% were found to be exoelectrogens in single-species studies. Exoelectrogenesis by members of the genera Vibrio, Enterobacter, and Citrobacter and by Bacillus stratosphericus was confirmed, by use of culture-based methods, for the first time. An MFC with a natural bacterial consortium showed higher power densities than those obtained with single strains. In addition, the maximum power output could be further increased to ∼200 mW·m^–2 when an artificial consortium consisting of the best 25 exoelectrogenic isolates was used, demonstrating the potential for increased performance and underlying the importance of artificial biofilms for increasing power output

Hypothetical model for the role of the CTD/HisRS domain interaction in stabilizing the inhibitory CTD/KD interaction.

<p>(A) (<i>left</i>) In nonstarved cells, Gcn2 exists as an inactive dimer with interactions between the CTD (light and dark blue), HisRS domain (light and dark orange for HisRS_N and cyan for HisRS_C) and KD (light and dark green) of each protomer. The KDs assume the anti-parallel mode of dimerization seen in the crystal structure of the inactive KD [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004991#pgen.1004991.ref012" target="_blank">12</a>], and the HisRS domains dimerize as observed in crystal structures of authentic HisRS, as shown for <i>T</i>. <i>cruzi</i> HisRS in panel B. The YKD domain (light and dark purple) is not engaged with the KD owing to the inhibitory KD/CTD interaction, which is stabilized by CTD interaction with the HisRS domain. YKD/CTD interactions might also stabilize this conformation but were omitted for simplicity. (<i>right</i>) In amino acid-starved cells, uncharged tRNA binds to the HisRS domains and possibly also the CTD [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004991#pgen.1004991.ref017" target="_blank">17</a>] (tRNA binding to only one protomer is depicted for simplicity), evoking a conformational change in the CTD-binding surface of the HisRS domain that triggers dissociation of the CTD/HisRS interaction, which in turn weakens the CTD/KD interaction to allow the stimulatory YKD/KD interaction to prevail. The KDs dimerize in the active back-to-back conformation observed in active PKR dimers [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004991#pgen.1004991.ref023" target="_blank">23</a>]. (<b>B</b>) Two views of the crystal structure of the <i>T</i>. <i>cruzi</i> HisRS dimer complexed with HAM (PDB: 1KMM) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004991#pgen.1004991.ref051" target="_blank">51</a>] with modeled uncharged tRNA. HisRS residues Cys-67 to Arg-156, corresponding to the HisRS-N segment of Gcn2 (residues 1028–1120), are colored orange or brown in each protomer; and residues Lys-332 to Iso-380, corresponding to the Gcn2 HisRS-C segment (residues 1315–1383), colored light or dark cyan in each protomer. Arg-341 and Asp-343, corresponding to Gcn2 regulatory residues Arg-1325 and Asp-1327, are colored red. Modeled tRNAs are colored salmon (omitted for clarity in <i>left</i> panel) and magenta. (<b>C</b>) Two views of the proposed interaction of the Gcn2 CTD with the HisRS-C segment (1) and interaction of the hinge region of the KD with the HisRS-N segment (2), enabling cooperative binding between the KD and CTD (3), which is important for autoinhibition of kinase function in non-starvation conditions. Dimerization of the KDs (4) that occurs in the antiparallel arrangement observed in the crystal structure of the inactive conformation of the Gcn2 KD is indicated by a bidirectional red arrowhead [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004991#pgen.1004991.ref012" target="_blank">12</a>].</p

Summary of domain interactions in Gcn2 that couple binding of uncharged tRNA to activation of kinase function in starved cells.

<p><b>(A)</b> Schematic diagram of the domain structure of yeast Gcn2 and interdomain interactions controlling kinase activity. The amino acid coordinates of the RWD/GI domain (pdb 2yz0), pseudokinase domain (YKD) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004991#pgen.1004991.ref021" target="_blank">21</a>], kinase domain (KD) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004991#pgen.1004991.ref012" target="_blank">12</a>], HisRS-like domain [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004991#pgen.1004991.ref014" target="_blank">14</a>], and CTD [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004991#pgen.1004991.ref026" target="_blank">26</a>] are derived from crystal structures (RWD, KD, CTD) or multiple sequence alignments (YKD and HisRS-like) of the respective domains. Stimulatory interactions are depicted with arrows, and involve domain interactions with Gcn1/Gcn20 (RWD) or uncharged tRNA (HisRS-like); or dimerization and ribosome-binding activities (CTD). The CTD also mediates autoinhibition of kinase activity, depicted with a bar. <b>(B)</b> Schematic summary of conformational rearrangements in the KD evoked by uncharged tRNA binding to the HisRS domain in amino acid-starved cells. The mode of dimerization and disposition of helix αC, β strand-3 (β3), and the activation loop, as well as key residues in the KD controlling ATP binding and catalysis (Lys-628, Asn-793, Glu-643, Arg-834, and Leu-856) are depicted for the inactive and active states of the Gcn2 KD that prevail in nonstarved or amino acid-starved cells, respectively. It is assumed that, in both states, Gcn2 dimerizes through self-interaction of the CTD and the schematic depicts only the disposition of the KDs within the full-length dimer. At low levels of uncharged tRNA in nonstarved cells (<i>left</i>), the KD exists in an equilibrium between two inactive conformations, with monomeric KDs (<i>upper</i>) or the KDs in an antiparallel dimer (<i>lower</i>). In both, ATP binding is hindered by a closed conformation of the N- and C-lobes and by Gln-793 (N793), which forms a flap over the ATP-binding pocket; and catalysis is blocked by hinge rigidity and rotation of αC to a nonproductive orientation that is stabilized by L856 and the inhibitory E643-R834 salt bridge. CTD/KD domain interaction also promotes this inactive conformation by an unknown mechanism. In amino acid-starved cells, binding of uncharged tRNA to the HisRS domain, along with stimulatory contributions of the RWD domain (engaged with Gcn1/Gcn20), the YKD domain, and the CTD, evokes a parallel mode of KD dimerization with αC properly oriented to form the stimulatory K628-E643 salt-bridge, and with ATP binding enhanced by a reduction in hinge rigidity that facilitates an open conformation of the N- and C-lobes and displaces the inhibitory N793 flap. Autophosphorylation of the activation loop ensues to produce a fully functional kinase locked into the active conformation. (See text for additional details; modified from Garriz et al [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004991#pgen.1004991.ref013" target="_blank">13</a>].)</p

Mutations of conserved surface-exposed residues of the Gcn2 HisRS domain that impair activation of Gcn2 in vivo.

<p><b>(A)</b> Growth phenotypes of transformants of <i>gcn2Δ</i> strain H1149 containing the indicated plasmid-borne <i>GCN2</i> alleles were analyzed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004991#pgen.1004991.g002" target="_blank">Fig. 2A</a>. <b>(B)</b> Cultures of strains from panel A were analyzed for levels of eIF2α-P as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004991#pgen.1004991.g002" target="_blank">Fig. 2B</a>. <b>(C)</b><i>HIS4-lacZ</i> expression was analyzed in strains from (A) as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004991#pgen.1004991.g002" target="_blank">Fig. 2C</a>.</p

A subset of Gcn^- substitutions of conserved surface-exposed residues in the HisRS domain impair kinase activity but not tRNA binding by purified Gcn2 in vitro.

<p><b>(A, B and C)</b> The indicated purified Gcn2 proteins were incubated with [³²P]-labeled total yeast tRNA in 20 μL of GMSA buffer. The Gcn2-tRNA complexes were resolved by electrophoresis through a 1% agarose gel in 1×MOPS buffer (1.5h, 100 V), transferred to a nitrocellulose membrane and visualized by autoradiography. Unbound [³²P]-tRNA, which has a higher mobility, was present at essentially identical amounts in each lane at levels ~15-fold higher than the WT Gcn2/tRNA complexes formed at 4μM. The purified Gcn2 proteins used in the assays were visualized by staining with Coomassie brilliant blue following separation by SDS-PAGE (images labelled Coomassie). <b>(D)</b> The indicated Gcn2 proteins (ca. 0.25 μg) were incubated with 3 μCi of [γ-³²P]ATP (6000 Ci/mmol, Amersham), 1 μg of recombinant eIF2α−ΔC purified from <i>E</i>. <i>coli</i>, and 0.5 μg of bovine serum albumin in 20 μL of kinase assay buffer at 30^°C for the indicated times. Samples were resolved by 8%–16% SDS—PAGE and subjected to autoradiography. Positions of autophosphorylated Gcn2 (Gcn2-P) and phosphorylated eIF2α−ΔC (eIF2α-P) are indicated.</p