122,240 research outputs found
Bimuria D. Hawksw., Chea & Sheridan, N. Z. Jl Bot.
No full text<i>Bimuria</i> D. Hawksw., Chea & Sheridan, N.Z. Jl Bot. 17(3): 267 (1979) <i>amend</i>. <p> <i>Saprobic</i> in terrestrial habitats. <b>Sexual morph:</b> See Hawksworth <i>et al</i>. (1979) and Ariyawansa <i>et al</i>. (2014a). <b>Asexual morph:</b> Coelomycetous. <i>Conidiomata</i> pycnidial, arise on mycelia as black spore mass, aggregated clusters are scattered, irregular and superficial to semi-immersed. <i>Conidiomatal wall</i> composed of thick walled, pale to dark brown cells of <i>textura angularis</i>. <i>Conidiogenous cells</i> enteroblastic, phialidic, ampulliform or short cylindrical, determinate, sometimes cylindrical, elongated neck, rough and hyaline. <i>Conidia</i> oblong to cylindrical, 1-septate, smooth and thin-walled, pale brown to hyaline.</p> <p> Type species:— <i>Bimuria novae -zelandiae</i> D. Hawksw., Chea & Sheridan, N.Z. Jl Bot. 17(3): 268 (1979)</p> <p> Notes:—Hawksworth <i>et al</i>. (1979)introduced <i>Bimuria</i> and it was placed in Pleosporaceae based on its sexual morphology. Based on phylogenetic analysis of SSU, LSU, RPB2 and TEF1-α sequence data, Schoch <i>et al</i>. (2009) and Ariyawansa <i>et al</i>. (2014a) confirmed that the <i>Bimuria novae-zelandiae</i> (CBS 107.79) should be placed in Montagnulaceae (= Didymosphaeriaceae) and related to <i>Tremateia</i>. In this current study, we observed that our novel strain (SQUCC 15280) clusters with <i>Bimuria novae-zelandiae</i> with strong bootstrap support in our phylogenetic analyses (Fig. 1). Therefore, we conclude that it is appropriate to consider our isolate as a species in <i>Bimuria</i>. <i>Bimuria</i> was only known from its sexual morph and we amend <i>Bimuria</i> in order to accommodate its coelomycetous asexual morph from our novel taxonomic account.</p>Published as part of <i>Wijesinghe, Subodini N., Wanasinghe, Dhanushka N., Maharachchikumbura, Sajeewa S. N., Wang, Yong, Al-Sadi, Abdullah M. & Hyde, Kevin D., 2020, Bimuria omanensis sp. nov. (Didymosphaeriaceae, Pleosporales) from Oman, pp. 97-108 in Phytotaxa 449 (2)</i> on pages 103-104, DOI: 10.11646/phytotaxa.449.2.1, <a href="http://zenodo.org/record/5585894">http://zenodo.org/record/5585894</a>
Les excuses de Frère N°2 (Nuon Chea): "Je ne cherche pas à fuir mes responsabilités"
No full text[ndlr] La presse française relaie les regrets tardifs de Nuon Chea, l'idéologue des Khmers rouges jugé à Phnom Penh pour crimes contre l'humanité. Extraits. 31 Aug 2011:Nuon Chea during the third day of Trial Chamber's preliminary hearing on fitness to stand trial © 2011 ECCC Khmers rouges/génocide: l'idéologue assume L'idéologue du régime des Khmers rouges, Nuon Chea, jugé pour génocide, crimes de guerre et crimes contre l'humanité, a admis pour la première fois jeudi sa responsabilité d..
Identification of intramolecular phosphoryl transfer within CheA<sub>3</sub> and CheS<sub>3</sub>.
No full text<p>(A) CheA<sub>3</sub>∼P is acid- and alkaline-labile, whereas the REC mutant CheA<sub>3</sub>:D663A∼P is acid-labile and base-resistant. (B) Both CheS<sub>3</sub>∼P and its REC mutant CheS<sub>3</sub>:D54A are acid-labile and alkaline-stable. (C) CheA<sub>3</sub>:D663A∼P phosphorylates CheA<sub>3</sub>-REC truncation protein in Buffer 15 containing K<sup>+</sup> and 18 mM Mg<sup>2+</sup>. (D) Phosphoryl transfer from CheS<sub>3</sub>:D54A∼P to CheS<sub>3</sub>-REC1 truncation protein was not observed in Buffer 15.</p
Sinorhizobium meliloti CheA Complexed with CheS Exhibits Enhanced Binding to CheY1, Resulting in Accelerated CheY1 Dephosphorylation
No full textRetrophosphorylation of the histidine kinase CheA in the chemosensory transduction chain is a widespread mechanism for efficient dephosphorylation of the activated response regulator. First discovered in Sinorhizobium meliloti, the main response regulator CheY2-P shuttles its phosphoryl group back to CheA, while a second response regulator, CheY1, serves as a sink for surplus phosphoryl groups from CheA-P. We have identified a new component in this phospho-relay system, a small 97-amino-acid protein named CheS. CheS has no counterpart in enteric bacteria but revealed distinct similarities to proteins of unknown function in other members of the a subgroup of proteobacteria. Deletion of cheS causes a phenotype similar to that of a cheY1 deletion strain. Fluorescence microscopy revealed that CheS is part of the polar chemosensory cluster and that its cellular localization is dependent on the presence of CheA. In vitro binding, as well as coexpression and copurification studies, gave evidence of CheA/CheS complex formation. Using limited proteolysis coupled with mass spectrometric analyses, we defined CheA(163-256) to be the CheS binding domain, which overlaps with the N-terminal part of the CheY2 binding domain (CheA(174-316)). Phosphotransfer experiments using isolated CheA-P showed that dephosphorylation of CheY1-P but not CheY2-P is increased in the presence of CheS. As determined by surface plasmon resonance spectroscopy, CheY1 binds similar to 100-fold more strongly to CheA/CheS than to CheA. We propose that CheS facilitates signal termination by enhancing the interaction of CheY1 and CheA, thereby promoting CheY1-P dephosphorylation, which results in a more efficient drainage of the phosphate sink
In vitro reconstitution of the TlpD, CheW, and CheA chemotaxis signaling complex.
No full text(A) Kinetics of HpCheA with varying concentrations of ATP are shown. Experiments were conducted in triplicate with 4 μM CheA and varying amounts of ATP in 50 mM Tris (pH 7.5), 100 mM NaCl, and 10 mM MgCl2. The average kobs for three replicate time courses are shown at various concentrations of ATP (black dots), error bars are the sample standard deviation, and these measurements are fit to the Michaelis-Menten curve (black line). (B) Representative analytical ultracentrifugation data (black axes) are shown for TlpD at 1 μM (black dotted black line) in PBS buffer (pH 7) with 1 mM TCEP. At this concentration, peaks corresponding to the TlpD monomer and dimer occur near 3.5 [S] and 5.2 [S], respectively. The average dimer KD for the recombinant TlpD construct calculated across various protein concentrations was found to be 188 nM (S1 Table). Shown in red on secondary axes are fluorescence anisotropy data for a titration of TlpD under identical conditions with experiments run in triplicate. See also S1 Fig. for a comparison of the TlpD dimer KD with the N-terminal His tag present or cleaved off and simulated data showing the expected monomer and dimer populations expected based on measured KD values. (C) Shown on top are representative raw data from radio-ATP labeling experiments of 15-minute reactions with CheA alone (“A”) and additions of CheW (“AW”) and TlpD (“AWD”). Below are reactions of 1 mM ATP and 4 μM CheA (black circles), +8 μM CheW (blue triangles), and +8 μM CheW; 24 μM TlpD (red squares) run in triplicate and fit to a pseudo–first-order reaction curve (solid lines). (D) CheW was titrated against 4 μM CheA and resulting kobs measurements were fit to a binding isotherm to estimate a kinetically-defined KD of 14.6 μM for the CheA↔CheW interaction. (E) TlpD was titrated against 4 μM CheA and 40 μM CheW and fit to a binding isotherm as in panel C to approximate the thermodynamics of the CheA, CheW↔TlpD interaction to have a KD of 15.2 μM. (F) A titration of TlpD against 4 μM CheA shows no activation (gray squares). For CheA in the presence of saturating [CheW], a 2.7-fold activation occurs (blue line), and with saturating [CheW] and [TlpD], this is increased to a 14.6-fold activation (red line) over CheA alone (black line). See S1 Table for a summary of reaction parameters and statistics. CheA, chemotaxis protein A; CheA-Pi, phosphorylated CheA; CheW, chemotaxis protein W; HpCheA, Helicobacter pylori CheA; S, Sedverg; TCEP, tris(2-carboxyethyl)phosphine; TlpD, transducer-like protein D.</p
Structural and chemical requirements for histidine phosphorylation by the chemotaxis kinase CheA
No full textThe CheA histidine kinase initiates the signal transduction pathway of bacterial chemotaxis by autophosphorylating a conserved histidine on its phosphotransferase domain (P1). Site-directed mutations of neighboring conserved P1 residues (Glu-67, Lys-48, and His-64) show that a hydrogen-bonding network controls the reactivity of the phospho-accepting His (His-45) in Thermotoga maritima CheA. In particular, the conservative mutation E67Q dramatically reduces phospho-transfer to P1 without significantly affecting the affinity of P1 for the CheA ATP-binding domain. High resolution crystallographic studies revealed that although all mutants disrupt the hydrogen-bonding network to varying degrees, none affect the conformation of His-45. N-15-NMR chemical shift studies instead showed that Glu-67 functions to stabilize the unfavored (NH)-H-delta 1 tautomer of His-45, thereby rendering the N-epsilon 2 imidazole unprotonated and well positioned for accepting the ATP phosphoryl group
Mutational analysis of the P1 phosphorylation domain in E. coli CheA, the signaling kinase for chemotaxis
No full textpre-printThe histidine autokinase CheA functions as the central processing unit in the Escherichia coli chemotaxis signaling machinery. CheA receives autophosphorylation control inputs from chemoreceptors and in turn regulates the flux of signaling phosphates to the CheY and CheB response regulator proteins. Phospho-CheY changes the direction of flagellar rotation; phospho-CheB covalently modifies receptor molecules during sensory adaptation. The CheA phosphorylation site, His-48, lies in the N-terminal P1 domain, which must engage the CheA ATP-binding domain, P4, to initiate an autophosphorylation reaction cycle. The docking determinants for the P1-P4 interaction have not been experimentally identified. We devised mutant screens to isolate P1 domains with impaired autophosphorylation or phosphotransfer activities. One set of P1 mutants identified amino acid replacements at surface-exposed residues, distal to His-48. These lesions reduced the rate of P1 transphosphorylation by P4. However, once phosphorylated, the mutant P1 domains transferred phosphate to CheY at the wild-type rate. Thus, these P1 mutants appear to define interaction determinants for P1-P4 docking during the CheA autophosphorylation reaction
Chemotactic signaling by an Escherichia coli CheA mutant that lacks the binding domain for phosphoacceptor partners
No full textCheA is a multidomain histidine kinase for chemotaxis in Escherichia coli. CheA autophosphorylates through interaction of its N-terminal phosphorylation site domain (P1) with its central dimerization (P3) and ATP-binding (P4) domains. This activity is modulated through the C-terminal P5 domain, which couples CheA to chemoreceptor control. CheA phosphoryl groups are donated to two response regulators, CheB and CheY, to control swimming behavior. The phosphorylated forms of CheB and CheY turn over rapidly, enabling receptor signaling complexes to elicit fast behavioral responses by regulating the production and transmission of phosphoryl groups from CheA. To promote rapid phosphotransfer reactions, CheA contains a phosphoacceptor-binding domain (P2) that serves to increase CheB and CheY concentrations in the vicinity of the adjacent P1 phosphodonor domain. To determine whether the P2 domain is crucial to CheA's signaling specificity, we constructed CheADeltaP2 deletion mutants and examined their signaling properties in vitro and in vivo. We found that CheADeltaP2 autophosphorylated and responded to receptor control normally but had reduced rates of phosphotransfer to CheB and CheY. This defect lowered the frequency of tumbling episodes during swimming and impaired chemotactic ability. However, expression of additional P1 domains in the CheADeltaP2 mutant raised tumbling frequency, presumably by buffering the irreversible loss of CheADeltaP2-generated phosphoryl groups from CheB and CheY, and greatly improved its chemotactic ability. These findings suggest that P2 is not crucial for CheA signaling specificity and that the principal determinants that favor appropriate phosphoacceptor partners, or exclude inappropriate ones, most likely reside in the P1 domain.This work was supported by research grant GM19559 from the National Institutes of Health. K.J. was supported by a Feodor Lynen postdoctoral fellowship from the Alexander von Humboldt Foundation. A.G. was supported by a postdoctoral fellowship from the Spanish Ministry of Education under the auspices of the Fulbright Program. The Protein-DNA Core Facility at the University of Utah receives support from National Cancer Institute grant CA42014 to the Huntsman Cancer Institute.Peer reviewe
Further insights into the mechanism of function of the response regulator CheY from crystallographic studies of the CheY--CheA(124--257) complex.
No full textInternational audienceNew crystallographic structures of the response regulator CheY in association with CheA(124--257), its binding domain in the kinase CheA, have been determined. In all crystal forms, the molecular interactions at the heterodimer interface are identical. Soaking experiments have been performed on the crystals using acetyl phosphate as phosphodonor to CheY. No phosphoryl group attached to Asp57 of CheY is visible from the electron density, but the response regulator in the CheY-CheA(124--257) complex may have undergone a phosphorylation-dephosphorylation process. The distribution of water molecules and the geometry of the active site have changed and are now similar to those of isolated CheY. In a second soaking experiment, imido-diphosphate, an inhibitor of the phosphorylation reaction, was used. This compound binds in the vicinity of the active site, close to the N-terminal part of the first alpha-helix. Together, these results suggest that the binding of CheY to CheA(124--257) generates a geometry of the active site that favours phosphorylation and that imido-diphosphate interferes with phosphorylation by precluding structural changes in this region.New crystallographic structures of the response regulator CheY in association with CheA(124--257), its binding domain in the kinase CheA, have been determined. In all crystal forms, the molecular interactions at the heterodimer interface are identical. Soaking experiments have been performed on the crystals using acetyl phosphate as phosphodonor to CheY. No phosphoryl group attached to Asp57 of CheY is visible from the electron density, but the response regulator in the CheY-CheA(124--257) complex may have undergone a phosphorylation-dephosphorylation process. The distribution of water molecules and the geometry of the active site have changed and are now similar to those of isolated CheY. In a second soaking experiment, imido-diphosphate, an inhibitor of the phosphorylation reaction, was used. This compound binds in the vicinity of the active site, close to the N-terminal part of the first alpha-helix. Together, these results suggest that the binding of CheY to CheA(124--257) generates a geometry of the active site that favours phosphorylation and that imido-diphosphate interferes with phosphorylation by precluding structural changes in this region
Intermolecular phosphoryl transfer events assayed among CheS<sub>3</sub>, CheA<sub>3</sub>, and CheY<sub>3</sub>.
No full text<p>2–5 µM CheS<sub>3</sub>, CheA<sub>3</sub>, or their REC mutant forms were autophosphorylated in 200 µM ATP for 30 min before 1/10 volumes of 65 mM CheY<sub>3</sub> or REC domain truncations were added. (A, B) Neither CheA<sub>3</sub>∼P nor CheA<sub>3</sub>:D663A∼P are able to phosphorylate CheY<sub>3</sub> in Buffer 9 containing K<sup>+</sup> and 6 mM Ca<sup>2+</sup>. (C, D) CheS<sub>3</sub>∼P and CheS<sub>3</sub>:D54A∼P phosphorylates CheY<sub>3</sub> within 15 sec of CheY<sub>3</sub> addition in Buffer 5 containing Na<sup>+</sup>, 3 mM Ca<sup>2+</sup>, and 3 mM Mg<sup>2+</sup>. (E) Intermolecular phosphoryl transfer analyses of CheA<sub>3</sub> and CheS<sub>3</sub>. (A) CheA<sub>3</sub>:D663A∼P phosphorylates CheS<sub>3</sub>-REC1 in Buffer 15 containing K<sup>+</sup> and 18 mM Mg<sup>2+</sup>. (F) CheS<sub>3</sub> is unable to phosphorylate CheA<sub>3</sub>-REC in Buffer 15.</p
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