Identification of a Compound in Chamaecyparis taiwanensis Inhibiting the Ice-nucleating Activity of Pseudomonas fluorescens KUIN-1

Inactivation of the ice-nucleating activity of Pseudomonas fluorescens KUIN-1 by compounds in the leaves from coniferous trees were investigated, and the inactivated material was identified. Intact cells of the strain KUIN-1 and the acetone or methanol extracts of leaves of various coniferous trees were allowed to react for 30 min at 18°C. Antinucleation compounds were obtained from Chamaecyparis taiwanensis. When the acetone extract from the leaves of coniferous trees was added to the cell suspension (about 10⁶ cells/ml) in 50 mM potassium phosphate buffer (pH 7.0), the ice nucleating temperature, T₅₀, was significantly decreased (T₅₀<-5°C). This inhibitor was isolated by using TLC, then identified as hinokitiol based on UV-VIS, IR, and mass spectral data. When intact cells of the strain KUIN-1 were incubated with hinokitiol, limonene, and α-pinene of the principal constituent of the leaves of coniferous trees in 50 mM potassium phosphate buffer (pH 7.0), the ice-nucleating activity decreased, but not in α-terpinene. Furthermore, the ice-nucleating activities from other ice-nucleating bacteria also decreased in the presence of hinokitiol. This inhibition was proportional to the concentration of hinokitinol. The pH and thermal stabilities of the ice-nucleating activity of the cells were changed by the addition of hinokitiol (10 mM).     

Synthesis of Several Fructo-oligosaccharides by Asparagus Fructosyltransferases

Nine fructo-oligosaccharides, synthesized in vitro from sucrose by an enzyme preparation from asparagus roots, were isolated and their structures were elucidated to be 1^F (1-β-fructofuranosyl)_n sucrose [n = 1 (1-kestose), 2 (nystose) and 3], 6^G (1-β-fructofuranosyl)_n sucrose [n=1 (neokestose), 2 and 3] and 1^F (1-β-fructofuranosyl)_m-6^G (1-β-fructofuranosyl)_n sucrose [m=1, n=1; m=2, n =1; and m =1, n=2]. These saccharides are all known to occur naturally in asparagus roots, but 6^G (1-β-fructofuranosyl)₃ sucrose and 1^F (1-β-fructofuranosyl)_m-6^G-(1-β-fructofuranosyl)_n sucrose (m=1, n =1; and m=1, n=2) were the first saccharides enzymatically synthesized in vitro. Also three types of fructosyltransferases were presumed to be involved in the biosynthesis of these oligosaccharides in asparagus roots.     

Cloning,Expression, and Identification of Genes Involved in the Conversion of DL-2-Amino-Δ2-thiazoline-4-carboxylic Acid to L-Cysteine via S-Carbamyl-L-cysteine Pathway in Pseudomonas sp. TS1138

Two novel genes (tsB, tsC) involved in the conversion of DL-2-amino-Δ²-thiazoline-4-carboxylic acid (DL-ATC) to L-cysteine through S-carbamyl-L-cysteine (L-SCC) pathway were cloned from the genomic DNA library of Pseudomonas sp. TS1138. The recombinant proteins of these two genes were expressed in Escherichia coli BL21, and their enzymatic activity assays were performed in vitro. It was found that the tsB gene encoded an L-ATC hydrolase, which catalyzed the conversion of L-ATC to L-SCC, while the tsC gene encoded an L-SCC amidohydrolase, which showed the catalytic ability to convert L-SCC to L-cysteine. These results suggest that tsB and tsC play important roles in the L-SCC pathway and L-cysteine biosynthesis in Pseudomonas sp. TS1138, and that they have potential applications in the industrial production of L-cysteine.     

Production of P(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate) Terpolymers Using a Chimeric PHA Synthase in Recombinant Ralstonia eutropha and Pseudomonas putida

Recombinant strains of Ralstonia eutropha and Pseudomonas putida harboring a chimeric polyhydroxyalkanoate (PHA) synthase, which consisted of PHA synthases of Aeromonas caviae and R. eutropha, produced 3-hydroxybutyrate (3HB)-based PHA copolymers comprised of 3-hydroxyhexanoate and 3-hydroxyoctanoate units from dodecanoate (87–97 mol % 3HB), indicating that the chimeric PHA synthase possesses desirable substrate specificity leading to the production of 3HB-rich copolymers.     

Synthesis of the Enantiomers of (Z)-5-(1-Octenyl)oxacyclopentan-2-one,a Sex Pheromone of the Cupreous Chafer Beetle,Anomala cuprea Hope

Glutaminase (EC 3.5.1.2) was isolated from Pseudomonas nitroreducens IFO 12694 grown on 0.6% sodium glutamate as a nitrogen source (325-fold purification, 13% yield). The molecular weight of the enzyme was estimated to be 40,000 by gel filtration and SDS-gel electrophoresis. The enzyme hydro-lyzed glutamine optimally at pH 9, and its K_m was 6.5 mm. d-Glutamine, γ-glutamyl p-nitroanilide, γ-glutamylmethylamide, γ-glutamylethylamide (theanine), and glutathione showed respectively 107, 85, 78, 74, and 82% reactivity of glutamine. Zn²⁺, Ni²⁺, Cd²⁺, Co²⁺, Fe²⁺, and Cu²⁺ repressed the enzyme activity strongly.

Glutaminase formed γ-glutamylhydroxamate in the reaction mixture containing glutamine and hydroxylamine (transferring reaction). The optimum pH of the transferring reaction was 7–8, and the K_m for glutamine and hydroxylamine were 4 mm and 120 mm, respectively. γ-Glutamyl derivatives hydrolyzable by glutaminase showed reactivity for the transferring reaction. Methylamine or ethylamine was replaceable for hydroxylamine with 3 or 8% reactivity. The effect of divalent cations was not so striking as in the hydrolyzing reaction.     

Bacterial farming by the fungus Morchella crassipes

The interactions between bacteria and fungi, the main actors of the soil microbiome, remain poorly studied. Here, we show that the saprotrophic and ectomycorrhizal soil fungus Morchella crassipes acts as a bacterial farmer of Pseudomonas putida, which serves as a model soil bacterium. Farming by M. crassipes consists of bacterial dispersal, bacterial rearing with fungal exudates, as well as harvesting and translocation of bacterial carbon. The different phases were confirmed experimentally using cell counting and ¹³C probing. Common criteria met by other non-human farming systems are also valid for M. crassipes farming, including habitual planting, cultivation and harvesting. Specific traits include delocalization of food production and consumption and separation of roles in the colony (source versus sink areas), which are also found in human agriculture. Our study evidences a hitherto unknown mutualistic association in which bacteria gain through dispersal and rearing, while the fungus gains through the harvesting of an additional carbon source and increased stress resistance of the mycelium. This type of interaction between fungi and bacteria may play a key role in soils.     

The reproductive phenology of nine species of Rhodophyta in the subtidal region of the Isle of Man

Monthly samples of about 40 separate plants of each species were collected from 1 to 3 m below lowest astronomical tide on Port Erin breakwater, Isle of Man, Irish Sea. In three species growing on rock, Plocamium cartilagineum, Cryptopleura ramosa and Callophyllis laciniata, about 90% of the plants were fertile in late summer but less than 10% in spring although some fertile plants were always present. Delesseria sanguinea and Odonthalia dentata, also epilithic, had a winter sporing season, Odonthalia extending into late spring, and all plants were sterile in summer. Three species growing epiphytically, Palmaria palmata, Membranoptera alata and Phycodrys rubens, reproduced maximally in the first half of the year at the time when the stipes of the host species, Laminaria hyperborea, grow fastest. Only Palmaria had a sterile season, late summer. The encrusting Cruoria pellita showed little seasonality. The first three species, which reproduce mainly when the sea temperature is above average, are in the northern part of their geographical range. The remaining species (apart from Cruoria) reproduce mainly at low temperatures and are in the southern half of their ranges. Male plants appear to be in a minority in all species, presumably because they were manifest for a shorter period than carposporic plants. They appeared first after sterile periods and were absent as sporing declined. Plocamium and to a lesser extent Cryptopleura show an extremely high preponderance of tetrasporophytes in the population. This is attributed to perennation and some factor disallowing the survival of most of the tetraspores.     

Alternative oxidase impacts the plant response to biotic stress by influencing the mitochondrial generation of reactive oxygen species

Previously, we showed that inoculation of tobacco with Pseudomonas syringae incompatible pv. maculicola results in a rapid and persistent burst of superoxide (O₂^‐) from mitochondria, no change in amount of mitochondrial alternative oxidase (AOX) and induction of the hypersensitive response (HR). However, inoculation with incompatible pv. phaseolicola resulted in increased AOX, no O₂^‐ burst and no HR. Here, we show that in transgenic plants unable to induce AOX in response to pv. phaseolicola, there is now a strong mitochondrial O₂^‐ burst, similar to that normally seen only with pv. maculicola. This interaction did not however result in a HR. This indicates that AOX amount is a key determinant of the mitochondrial O₂^‐ burst but also that the burst itself is not sufficient to induce the HR. Surprisingly, the O₂^‐ burst normally seen towards pv. maculicola is delayed in plants lacking AOX. This delay is associated with a delayed HR, suggesting that the burst does promote the HR. A O₂^‐ burst can also be induced by the complex III inhibitor antimycin A (AA), but is again delayed in plants lacking AOX. The similar mitochondrial response induced by pv. maculicola and AA suggests that electron transport is a target during HR‐inducing biotic interactions.     

Expression of antimicrobial peptides thanatin(S) in transgenic Arabidopsis enhanced resistance to phytopathogenic fungi and bacteria

Thanatin(S) is an analog of thanatin, an insect antimicrobial peptide possessing strong and broad spectrum of antimicrobial activity. In order to investigate if the thanatin could be used in engineering transgenic plants for increased resistance against phytopathogens, the synthetic thanatin(S) was introduced into Arabidopsis thaliana plants. To increase the expression level of thanatin(S) in plants, the coding sequence was optimized by plant-preference codon. To avoid cellular protease degradation, signal peptide of rice Cht1 was fused to N terminal of thanatin(S) for secreting the expressed thanatin(S) into intercellular spaces. To evaluate the application value of thanatin(S) in plant disease control, the synthesized coding sequence of Cht1 signal peptide (Cht1SP)-thanatin(S) was ligated to plant gateway destination binary vectors pGWB11 (with FLAG tag). Meanwhile, in order to observe the subcellular localization of Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP, the sequences of Cht1SP-thanatin(S) and thanatin(S) were respectively linked to pGWB5 (with GFP tag). The constructs were transformed into Arabidopsis ecotype Col-0 and mutant pad4-1 via Agrobacterium-mediated transformation. The transformants with Cht1SP-thanatin(S)-FLAG fusion gene were analyzed by genomic PCR, real-time PCR, and western blots and the transgenic Arabidopsis plants introduced respectively Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP were observed by confocal microscopy. Transgenic plants expressing Cht1SP-thanatin(S)-FLAG fusion protein showed antifungal activity against Botrytis cinerea and powdery mildew, as well as antibacterial activity against Pseudomonas syringae pv. tomato. And the results from confocal observation showed that the GFP signal from Cht1SP-thanatin(S)-GFP transgenic Arabidopsis plants occurred mainly in intercellular space, while that from thanatin(S)-GFP transgenic plants was mainly detected in the cytoplasm and that from empty vector transgenic plants was distributed uniformly throughout the cell, demonstrating that Cht1 signal peptide functioned. In addition, thanatin(S) and thanatin(S)-FLAG chemically synthesized have both in vitro antimicrobial activities against P. syringae pv. tomato and B. cinerea. So, thanatin(S) is an ideal candidate AMPs for the construction of transgenic crops endowed with a broad-spectrum resistance to phytopathogens and the strategy is feasible to link a signal peptide to the target gene.     

Pocket analysis of the full-length cholix toxin. An assessment of the structure–dynamics of the apo catalytic domain

Cholix toxin from Vibrio cholerae is the third member of the diphtheria toxin (DT) group of mono-ADP-ribosyltransferase (mART) bacterial toxins. It shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae DT. Cholix toxin is an important model for the development of antivirulence approaches and therapeutics against these toxins from pathogenic bacteria. Herein, we have used the high-resolution X-ray structure of full-length cholix complexed with NAD⁺ to describe the properties of the NAD⁺-binding pocket at the residue level, including the role of crystallographic water molecules in the NAD⁺ substrate interaction. The full-length apo cholix structure is used to describe the putative NAD⁺-binding site(s) and to correlate biochemical with crystallographic data to study the stoichiometry and orientation of bound NAD⁺ molecules. We quantitatively describe the NAD⁺ substrate interactions on a residue basis for the main 22 pocket residues in cholix_f, a glycerol and 5 contact water molecules as part of the recognition surface by the substrate according to the conditions of crystallization. In addition, the dynamic properties of an in silico version of the catalytic domain were investigated in order to understand the lack of electronic density for one of the main flexible loops (R-loop) in the pocket of X-ray complexes. Implications for a rational drug design approach for mART toxins are derived.