Crystal structure of glutathione reductase Glr1 from the yeast Saccharomyces cerevisiae

Yeast glutathione (GSH) reductase Glr1 is a dimeric flavo-oxidoreductase involved in cytoplasmic and mitochondrial redox regulatory systems. It reduces the oxidized GSH GSSG to the reduced form, GSH with NADPH as electron donor and FAD as coenzyme. Crystal structures and enzymatic mechanisms of GSH reductases from Escherichia coli and Homo sapiens have been well investigated, whereas the structural properties of yeast Glr1 remain unknown. Herein, we overexpressed Saccharomyces cerevisiae Glr1 in Pichia pastoris GS115 and determined its crystal structure at 2.40 A resolution. Although the overall structure and the active site are much conserved, obvious variety was found at the interface of Glr1 monomers when superimposed against the homolog from E. coli or human. The nonconserved C239 is exposed to the solvent and accessible to GSH or GSSG enriched in a microenvironment around the Glr1 molecules, leading to the partial and transient glutathionylation, as primarily identified from the 2Fo-Fc electron density map and further confirmed by biochemical assays. Meanwhile N278 at the vicinity of NADP-binding pocket was artificially glycosylated when heterogeneously overexpressed in P. pastoris. The highly motile oligosaccharide chain linked to N278 of the recombinant Glr1 interferes with the entry of NADPH, which results in a dramatic increase of Km for NAPDH and a significant decrease of turnover number, when compared with the native protein.     

Antiadhesive property of microalgal polysaccharide extract on the binding of Helicobacter pylori to gastric mucin

The emergence of antibiotic-resistant Helicobacter pylori is of concern in the treatment of H. pylori-associated gastroduodenal diseases. As the organism was reported to bind gastric mucin, we used porcine gastric mucin as substrate to assess the antiadhesive property of polysaccharides derived from Spirulina (PS), a commercially available microalga, against the binding of H. pylori to gastric mucin. Results show that polysaccharides prevented H. pylori from binding to gastric mucin optimally at pH 2.0, without affecting the viability of either bacteria or gastric epithelial cells, thus favouring its antiadhesive action in a gastric environment. Using ligand overlay analysis, polysaccharide was demonstrated to bind H. pylori alkyl hydroperoxide reductase (AhpC) and urease, which have shown here to possess mucin-binding activity. An in vivo study demonstrated that bacteria load was reduced by >90% in BALB/c mice treated with either Spirulina or polysaccharides. It is thus suggested that polysaccharides may function as a potential antiadhesive agent against H. pylori colonization of gastric mucin.     

Density functional theory studies of model complexes for molybdenum-dependent nitrate reductase active sites

Molybdenum and tungsten complexes as models for the active sites of assimilatory or dissimilatory nitrate reductases (NR) were computed at the CPCM-B98/SDDp//B3LYP/Lanl2DZp* plus zero point energy level of density functional theory. The ligands were chosen on the basis of available experimental protein or small chemical model structures. A water molecule is found to bind to assimilatory NR models [(Me₂C₂S₂)MO(YMe)]⁻ (−11.5 kcal mol⁻¹ for M is Mo, Y is S) and may be replaced by nitrate (−4.5 kcal mol⁻¹) (but a hydroxy group may not). Nature’s choice of M is Mo and Y is S for NR has the largest activation energy for protein-free models (13.3 kcal mol⁻¹) and the least exothermic reaction energy for the nitrate reduction (−14.9 kcal mol⁻¹) compared with M is W and Y is O or Se alternatives. Water binding to dissimilatory NR model complexes [(Me₂C₂S₂)₂M(YR)]⁻ is considerably endothermic (10.3 kcal mol⁻¹); nitrate binding is only slightly so (1.5 kcal mol⁻¹ for RY⁻ is MeS⁻). The exchange of an oxo ligand (assimilatory NR) for a dithiolato ligand (dissimilatory NR model) reduces the exothermicity (−8.6 kcal mol⁻¹ relative to the fivefold-coordinate reduced complex) and raises the barrier for oxygen atom transfer (OAT) in the nitrate complex (19.2 kcal mol⁻¹). Not for the mono but only for the bisdithiolato complexes hydrogen bonding involving the coordinated substrate may significantly lower the OAT barrier as shown by explicitly adding water molecules. Substitution of tungsten for molybdenum generally lowers OAT activation energies and makes nitrate reduction reaction energies more negative. Bidentate carboxylato binding identified in Escherichia coli NarGHI is the preferred binding mode also for an acetato model. However, one dithiolato ligand folds when the Mo^VI center is bare of a good π-donor ligand, e.g., an oxo group. Computations on [(mnt)₂Mo^IV(YR)(PPh₃)]⁻ [mnt is (CN)₂C₂S₂ ²⁻] gave a smaller nitrate reduction activation energy for RY⁻ is Cl⁻, compared with RY⁻ is PhS⁻, although experimentally only the phenyl thiolato complex and not the chloro complex was found to be a functional NR model. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II (cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.     

Differences in the degree of inhibition of NDP reductase by chemical inactivation and by the thermosensitive mutation <Emphasis Type="Italic">nrdA101</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> suggest an effect on chromosome segregation

NDP reductase activity can be inhibited either by treatment with hydroxyurea or by incubation of an nrdA _ts mutant strain at the non-permissive temperature. Both methods inhibit replication, but experiments on these two types of inhibition yielded very different results. The chemical treatment immediately inhibited DNA synthesis but did not affect the cell and nucleoid appearance, while the incubation of an nrdA101 mutant strain at the non-permissive temperature inhibited DNA synthesis after more than 50 min, and resulted in aberrant chromosome segregation, long filaments, and a high frequency of anucleate cells. These phenotypes are not induced by SOS. In view of these results, we suggest there is an indirect relationship between NDP reductase and the chromosome segregation machinery through the maintenance of the proposed replication hyperstructure.     

Production of methanethiol and volatile sulfur compounds by the archaeon “Ferroplasma acidarmanus”

Acidophiles are typically isolated from sulfate-rich ecological niches yet the role of sulfur metabolism in their growth and survival is poorly defined. Studies of heterotrophically grown “Ferroplasma acidarmanus” showed that its growth requires a minimum of 100 mM of a sulfate-containing salt. Headspace gas analyses by GC/MS determined that the volatile sulfur compound emitted by active “F. acidarmanus” cultures is methanethiol. In “F. acidarmanus” cultures grown either heterotrophically or chemolithotrophically, methanethiol was produced constitutively. Radiotracer studies with ³⁵S-labeled methionine, cysteine, and sulfate showed that all three were used in methanethiol production. Additionally, ³H-labeled methionine was incorporated into methanethiol and was probably used as a methyl-group donor. Methanethiol production in whole cell lysates supplied with SO₃²⁻ indicated that NADPH-dependant sulfite reductase and methyltransferase activities were present. Cell lysates also contained enzymatic activity for methionine-γ-lyase that cleaved the side chain of either methionine to form methanethiol or cysteine to produce H₂S. Since methanethiol was detected from the degradation of cysteine, it is likely that sulfide was methylated by a thiol methyltransferase. Collectively, these data demonstrate that “F. acidarmanus” produces methanethiol through the metabolism of methionine, cysteine, or sulfate. This is the first report of a methanethiol-producing acidophile, thus identifying a new contributor to the global sulfur cycle.     

转录因子cpcR同源基因cpcR-c1和cpcR-t的功能鉴定

苏云金芽胞杆菌（Bacillus thuringiensis,Bt） LM1212菌株与典型的Bt菌株表型不同,可分化形成芽胞、形成细胞和晶体产生细胞。在LM1212菌株中,转录因子CpcR不仅参与了细胞分化过程,而且能够激活晶体蛋白基因cry35-like的启动子(P₃₅)。【目的】筛选cpcR同源基因,验证其生物学功能。【方法】本研究克隆了2个cpcR同源基因,来源于蜡样芽胞杆菌的cpcR-c1和来源于东洋芽胞杆菌的cpcR-t,将cpcR及其同源基因分别构建在pHT304-P₃₅-gfp、pHT304-P₃₅-lacZ报告载体上,获得的重组质粒转入无cpcR基因且无晶体蛋白基因的Bt HD73^–菌株中。利用激光共聚焦显微镜观察重组菌HD^–(cpcR-c1-P₃₅-gfp)和HD^–(cpcR-t-P₃₅-gfp)的细胞表型并进行芽胞计数实验。测定HD^–(cpcR-c1-P_35<...     

Biological Control of Root-Knot Nematode <i>Meloidogyne incognita</i> in <i>Psoralea corylifolia</i> Plant by Enhancing the Biocontrol Efficacy of <i>Trichoderma harzianum</i> Using Press Mud

Meloidogyne incognita is a plant pathogen causing root-knot disease and loss of crop yield. The present study aimed to use Trichoderma harzianum as a biocontrol agent against plant-parasitic nematodes and used press mud, which is a solid waste by-product of sugarcane, as a biocontrol agent and biofertilizer. Therefore, the combined application of T. harzianum and press mud may enhance nematode control and plant growth. Elemental analysis of press mud using scanning electron microscopy (SEM) integrated with an Energy Dispersive X-ray (EDX) analyzer revealed the presence of different elements such as C, O, Mg, Si, P, K, Ca, Cu and Zn. In addition, a greenhouse study was conducted to investigate the combined effects of press mud and T. harzianum on M. incognita reproduction and growth and the biochemical features of Psoralea corylifolia. The results showed that plant length, dry biomass, leaf area, the number of seeds per plant, chlorophyll a, chl b, carotenoid content, nitrate reductase, carbonic anhydrase, and nitrogen content were significantly increased (P ≤ 0.05) in the T2 plants (plants were treated with 100 g of press mud + 50 mL T. harzianum before one week of M. incognita inoculation), over inoculated plants (IC). Antioxidant enzyme activity of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in the foliage of P. corylifolia was significantly increased when plants were treated with press mud + T. harzianum. A significant reduction in the number of egg masses, nematode population, and root-knot index (RKI) was found in plants with T2 plants. These results suggest that the combined application of T. harzianum and press mud has the potential to control the M. incognita infection and can be used as an environmentally safe alternative to chemical nematicides and also help in the removal of sugarcane waste that causes environmental pollution.     

First detection and molecular characterization of grapevine phytoplasmas in Kosovo

A search for phytoplasma-associated diseases was conducted for the first time in the main grapevine-growing localities of the Dukagjini plain in Kosovo. A total of 144 samples were collected from grapevine cultivars displaying leaf yellowing, reddening, discolouration and irregular wood ripening, and analysed using nested and quantitative PCR assays. These assays showed that 35.4% of samples belonging to eight cultivars were positive to the presence of phytoplasmas in the 16SrXII group. The 16S rDNA phytoplasma sequences obtained from 15 samples shared identity greater than 99.5% with ‘Candidatus Phytoplasma solani’. Sequence analysis of the tuf gene showed that the strains found in Kosovar grapevines are in the tuf-type b1 group, sharing 99.6% to 99.8% identity with ‘Ca. P. solani'-related strains associated with the “bois noir” grapevine disease in many European countries; the secY gene sequences, on the other hand, shared 100% identity with ‘Ca. P. solani' strains from Bosnia and Herzegovina, Serbia, Croatia and Turkey. This study constitutes the first report on the presence and molecular characterization of phytoplasmas in Kosovar vineyards. Based on these results, it is recommended that testing for phytoplasma be included in the certification program for grapevine in Kosovo.