Relationships among three Japanese <Emphasis Type="Italic">Laetiporus</Emphasis> taxa based on phylogenetic analysis and incompatibility tests

Relationships among three Japanese Laetiporus taxa (“L. sulphureus var. sulphureus” auct. jap., L. sulphureus var. miniatus, and L. versisporus) were assessed with phylogenetic analysis and incompatibility tests. Gene phylogenies inferred from the internal transcribed spacer region of nuclear ribosomal DNA, elongation factor 1α, and β-tubulin gene regions suggested that Japanese Laetiporus was divided into four groups: the yellow pore form of L. sulphureus var. miniatus, the white pore form of L. sulphureus var. miniatus, and two “L. sulphureus var. sulphureus”/ L. versisporus groups. A morphologically distinct species, Laetiporus versisporus, sharing a clade with “L. sulphureus var. sulphureus” auct. jap., was proved to be an anamorphic form of “L. sulphureus var. sulphureus” auct. jap. The “sulphureus/versisporus” isolates showed two divergent sequence types in each region. Some isolates had intraindividual polymorphism assigned to both sequence types. This finding suggests that speciation via hybridization is ongoing in the “sulphureus/versisporus” group. Single spore isolates from the “sulphureus/versisporus” group, white pore group, and yellow pore group were incompatible with each other. Our results provided strong support for the new recognition of three Laetiporus taxa in Japan.     

Evolution of binding sites for zinc and calcium ions playing structural roles

The geometry of metal coordination by proteins is well understood, but the evolution of metal binding sites has been less studied. Here we present a study on a small number of well-documented structural calcium and zinc binding sites, concerning how the geometry diverges between relatives, how often nonrelatives converge towards the same structure, and how often these metal binding sites are lost in the course of evolution. Both calcium and zinc binding site structure is observed to be conserved; structural differences between those atoms directly involved in metal binding in related proteins are typically less than 0.5 A root mean square deviation, even in distant relatives. Structural templates representing these conserved calcium and zinc binding sites were used to search the Protein Data Bank for cases where unrelated proteins have converged upon the same residue selection and geometry for metal binding. This allowed us to identify six "archetypal" metal binding site structures: two archetypal zinc binding sites, both of which had independently evolved on a large number of occasions, and four diverse archetypal calcium binding sites, where each had evolved independently on only a handful of occasions. We found that it was common for distant relatives of metal-binding proteins to lack metal-binding capacity. This occurred for 13 of the 18 metal binding sites we studied, even though in some of these cases the original metal had been classified as "essential for protein folding." For most of the calcium binding sites studied (seven out of eleven cases), the lack of metal binding in relatives was due to point mutation of the metal-binding residues, whilst for zinc binding sites, lack of metal binding in relatives always involved more extensive changes, with loss of secondary structural elements or loops around the binding site.     

Diurnal fluctuations of dissolved nitrous oxide (N2O) concentrations and estimates of N2O emissions from a spring-fed river: implications for IPCC methodology

There is uncertainty in the estimates of indirect nitrous oxide (N₂O) emissions as defined by the Intergovernmental Panel on Climate Change (IPCC). The uncertainty is due to the challenge and dearth of in situ measurements. Recent work in a subtropical stream system has shown the potential for diurnal variability to influence the downstream N transfer, N form, and estimates of in‐stream N₂O production. Studies in temperate stream systems have also shown diurnal changes in stream chemistry. The objectives of this study were to measure N₂O fluxes and dissolved N₂O concentrations from a spring‐fed temperate river to determine if diurnal cycles were occurring. The study was performed during a 72 h period, over a 180 m reach, using headspace chamber methodology. Significant diurnal cycles were observed in radiation, river temperature and chemistry including dissolved N₂O‐N concentrations. These data were used to further assess the IPCC methodology and experimental methodology used. River NO₃‐N and N₂O‐N concentrations averaged 3.0 mg L⁻¹ and 1.6 μg L⁻¹, respectively, with N₂O saturation reaching a maximum of 664%. The N₂O‐N fluxes, measured using chamber methodology, ranged from 52 to 140 μg m⁻² h⁻¹ while fluxes predicted using the dissolved N₂O concentration ranged from 13 to 25 μg m⁻² h⁻¹. The headspace chamber methodology may have enhanced the measured N₂O flux and this is discussed. Diurnal cycles in N₂O% saturation were not large enough to influence downstream N transfer or N form with variability in measured N₂O fluxes greater and more significant than diurnal variability in N₂O% saturation. The measured N₂O fluxes, extrapolated over the study reach area, represented only 6 × 10⁻⁴% of the NO₃‐N that passed through the study reach over a 72 h period. This is only 0.1% of the IPCC calculated flux.     

Crystal structure and substrate specificity of ExoY,a unique T3SS mediated secreted nucleotidyl cyclase toxin from Pseudomonas aeruginosa

Background

The nucleotidyl cyclase toxin ExoY is an important virulence determinant of Pseudomonas aeruginosa that causes severe acute and chronic infections in immune-compromised individuals. Additionally, this unique T3SS effector shows a striking preference for cUMP, a newly identified non-canonical secondary messenger. Thereby, ExoY is also considered as a potential tool to study unexplored cUMP signaling pathways.

Taxonomic reassessment of Chondrus verrucosus (Rhodophyta,Gigartinales), with a description of Chondrus retortus sp. nov

Phylogenetic and morphological reinvestigation of Chondrus verrucosus Mikami was conducted in the Pacific coast of southeastern Honshu, Japan, including the holotype specimen and samples near the type locality. Two entities were recognized by phylogenetic analyses of plastid rbcL and nuclear EF2 sequences. In morphological analyses, the two entities were distinguished by: (i) thallus size and form, and (ii) cell shape of the medullary layer surrounding young carposporangia: one has larger thalli, is regularly canaliculated, with linear medullary cells (large‐type); the other is smaller, canaliculated with crispation and has roundish medullary cells (small‐type). The large‐type was common in southern to eastern Boso Peninsula including near the type locality; the small‐type was typical in the southwest Boso Peninsula and Sagami Bay. The large‐type was shown to be equivalent to C. verrucosus by examination of the holotype specimen. The small‐type is described as a new species, C. retortus Matsumoto et Shimada.     

Solution structures of polcalcin Phl p 7 in three ligation states: Apo‐, hemi‐Mg2+‐bound,and fully Ca2+‐bound

Polcalcins are small EF‐hand proteins believed to assist in regulating pollen‐tube growth. Phl p 7, from timothy grass (Phleum pratense), crystallizes as a domain‐swapped dimer at low pH. This study describes the solution structures of the recombinant protein in buffered saline at pH 6.0, containing either 5.0 mM EDTA, 5.0 mM Mg²⁺, or 100 μM Ca²⁺. Phl p 7 is monomeric in all three ligation states. In the apo‐form, both EF‐hand motifs reside in the closed conformation, with roughly antiparallel N‐ and C‐terminal helical segments. In 5.0 mM Mg²⁺, the divalent ion is bound by EF‐hand 2, perturbing interhelical angles and imposing more regular helical structure. The structure of Ca²⁺‐bound Phl p 7 resembles that previously reported for Bet v 4—likewise exposing apolar surface to the solvent. Occluded in the apo‐ and Mg²⁺‐bound forms, this surface presumably provides the docking site for Phl p 7 targets. Unlike Bet v 4, EF‐hand 2 in Phl p 7 includes five potential anionic ligands, due to replacement of the consensus serine residue at –x (residue 55 in Phl p 7) with aspartate. In the Phl p 7 crystal structure, D55 functions as a helix cap for helix D. In solution, however, D55 apparently serves as a ligand to the bound Ca²⁺. When Mg²⁺ resides in site 2, the D55 carboxylate withdraws to a distance consistent with a role as an outer‐sphere ligand. ¹⁵N relaxation data, collected at 600 MHz, indicate that backbone mobility is limited in all three ligation states. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

New insights into the interactions of the translation initiation factor 2 from archaea with guanine nucleotides and initiator tRNA

Heterotrimeric a/eIF2alphabetagamma (archaeal homologue of the eukaryotic translation initiation factor 2 with alpha, beta and gamma subunits) delivers charged initiator tRNA (tRNAi) to the small ribosomal subunit. In this work, we determined the structures of aIF2gamma from the archaeon Sulfolobus solfataricus in the nucleotide-free and GDP-bound forms. Comparison of the free, GDP and Gpp(NH)p-Mg2+ forms of aIF2gamma revealed a sequence of conformational changes upon GDP and GTP binding. Our results show that the affinity of GDP to the G domain of the gamma subunit is higher than that of Gpp(NH)p. In analyzing a pyrophosphate molecule binding to domain II of the gamma subunit, we found a cleft that is very suitable for the acceptor stem of tRNA accommodation. It allows the suggestion of an alternative position for Met-tRNA i Met on the alphagamma intersubunit dimer, at variance with a recently published one. In the model reported here, the acceptor stem of the tRNAi is approximately perpendicular to that of tRNA in the ternary complex elongation factor Tu-Gpp(NH)p-tRNA. According to our analysis, the elbow and T stem of Met-tRNA i Met in this position should make extensive contact with the alpha subunit of aIF2. Thus, this model is in good agreement with experimental data showing that the alpha subunit of aIF2 is necessary for the stable interaction of aIF2gamma with Met-tRNA i Met.     

The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain

Efficient protein synthesis in bacteria requires initiation factor 2 (IF2), elongation factors Tu (EF-Tu) and G (EF-G), and release factor 3 (RF3), each of which catalyzes a major step of translation in a GTP-dependent fashion. Previous reports have suggested that recruitment of factors to the ribosome and subsequent GTP hydrolysis involve the dimeric protein L12, which forms a flexible "stalk" on the ribosome. Using heteronuclear NMR spectroscopy we demonstrate that L12 binds directly to the factors IF2, EF-Tu, EF-G, and RF3 from Escherichia coli, and map the region of L12 involved in these interactions. Factor-dependent chemical shift changes show that all four factors bind to the same region of the C-terminal domain of L12. This region includes three strictly conserved residues, K70, L80, and E82, and a set of highly conserved residues, including V66, A67, V68 and G79. Upon factor binding, all NMR signals from the C-terminal domain become broadened beyond detection, while those from the N-terminal domain are virtually unaffected, implying that the C-terminal domain binds to the factor, while the N-terminal domain dimer retains its rotational freedom mediated by the flexible hinge between the two domains. Factor-dependent variations in linewidths further reveal that L12 binds to each factor with a dissociation constant in the millimolar range in solution. These results indicate that the L12-factor complexes will be highly populated on the ribosome, because of the high local concentration of ribosome-bound factor with respect to L12.     

Structural and functional self-organization of Photosystem II in grana thylakoids

The biogenesis of the well-ordered macromolecular protein arrangement of photosystem (PS)II and light harvesting complex (LHC)II in grana thylakoid membranes is poorly understood and elusive. In this study we examine the capability of self organization of this arrangement by comparing the PSII distribution and antenna organization in isolated untreated stacked thylakoids with restacked membranes after unstacking. The PS II distribution was deduced from freeze-fracture electron microscopy. Furthermore, changes in the antenna organization and in the oligomerization state of photosystem II were monitored by chlorophyll a fluorescence parameters and size analysis of exoplasmatic fracture face particles. Low-salt induced unstacking leads to a randomization and intermixing of the protein complexes. In contrast, macromolecular PSII arrangement as well as antenna organization in thylakoids after restacking by restoring the original solvent composition is virtually identical to stacked control membranes. This indicates that the supramolecular protein arrangement in grana thylakoids is a self-organized process.