Takeo Uchiyama

Biodegradation of organophosphorus insecticides by bacteria isolated from turf green soil

Several organophosphate-degrading bacteria were isolated from test turf green soil using clear zones formed around their colonies on plates supplemented with organophosphate isoxathion. The degrading activity of the isolates for isoxathion was tested by incubation in liquid cultures and evaluated by gas chromatography. Strain B-5 exhibited the highest isoxathion degrading ability in the isolates and it was identified as an Arthrobacter sp. A high concentration of nutrients in the media affected the isoxathion degrading activity of strain B-5. The bacterium could not utilize isoxathion as a sole source of carbon and phosphorus. The degradation products of isoxathion by B-5 washed cells were identified as its hydrolysis preducts, 3-hydroxy-5-phenylisoxazole and diethylthiophosphoric acid, suggesting that strain hydrolyzes the heterocycle ester bond in isoxathion. Arthrobacter sp. strain B-5 also hydrolyzed diazinon, parathion, EPN, fenitrothion, isofenphos, chlorpyrifos, and ethoprophos at rates dependent on the substrate. Of the organophosphorus insecticides examined, isofenphos was affected most by the hydrolytic activity of the bacterium, which completely removed the compound (10 mg/l) from cultures within 1-h incubation.

Participation of Oryza sativa leaf wax in appressorium formation by Pyricularia oryzae.

Abstract Appressorium formation of Pyricularia oryzae P2 on cover-glass coated with each of the components of rice leaf wax was examined. Wax esters, aldehydes and alcohols, having polar groups and low contact angles, promoted appressorium formation, but alkanes, non-polar molecules having high contact angles, had no effects. Germination of conidia, however, was not aftected with those constituents.

Molecular cloning and nucleotide sequencing of organophosphorus insecticide hydrolase gene from Arthrobacter sp. strain B-5.

The organophosphorus insecticide hydrolase (OPH) gene of Arthrobacter sp. strain B-5, isolated from turf green soil was cloned into Escherichia coli JM109. Three clones, termed EpB511, EpB521 and EpB531, exhibiting OPH activity were obtained. However, these three clones showed lower OP-degrading ability than strain B-5. A 7.7-kb inserted fragment of the plasmid pB521 harbored by EpB521 was subcloned, resulting in construction of a plasmid, pB526, carrying the 2.6-kb inserted fragment with OP-degrading ability. In this sequence, an open reading frame (ORF) that encodes a 43,607 Da polypeptide composed of 415 amino acids was identified. The N-terminal amino acid sequence deduced from the nucleotide sequence was identical to that of purified OPHs. The deduced amino acid sequence was compared with the sequences in the data bank and a 58.1% amino acid identity was found with the aryldialkylphosphatase from Nocardia sp. strain B-1, an enzyme that possesses catalytic functions similar to OPH.

O-acetylated xyloglucan in extracellular polysaccharides from cell-suspension cultures of Mentha

Extracellular polysaccharide produced by suspension-cultured Mentha cells consisted of 50% neutral sugars, 32% uronic acid and 10% of protein. The ammonium oxalate-soluble fraction of this polysaccharide contained 30% hemicellulose and 70% pectic substances. The purified hemicellulose contained xylose, glucose, arabinose, galactose, mannose and fucose residues in a molar ratio of 41.6:31.3:13.1:11.1:1.3:1.6. It was identified as a xyloglucan from its neutral sugar composition and by methylation analysis and cellulase treatment: the principal neutral sugar was arabinose. The presence of O-acetyl residues was confirmed with 1H NMR, 13C NMR and GC-mass spectrometry. The total acetyl content in the polysaccharide was 4%. The point of attachment of the O-acetyl residue was shown to be at position 6 of the galactosyl residue.

Early biosynthetic pathway to abscisic acid in Cercospora cruenta.

A new biosynthetic intermediate of ABA, (2Z,4E)-γ-ionylideneacetaldehyde, was isolated from young mycelia of Cercospora cruenta. Under an 18O2 atmosphere, an oxygen atom of this endogenous aldehyde was exclusively labeled. Similarly, three 18O atoms were incorporated into the ABA molecule recovered after prolonged incubation; selectively labeled were one of the carboxyl oxygen atoms and the two on the ring portion of ABA. A feeding experiment with [1-13C]glucose proved the exclusive operation of the mevalonate pathway for the formation of both ABA and β-carotene. These results suggest that (2Z,4E)-γ-ionylideneacetaldehyde can be a key ABA biosynthetic intermediate formed by the oxidative cleavage of a carotenoid precursor.

Chemical Composition fo the Glue From Appressoria of Magnaporthe grisea

The chemical composition of the glue substance that attaches the appressoria of Magnaporthe grisea bar artificial leaf wax was investigated. As a percentage of fresh weights, the glue was crude lipids 29.0%, proteins 12.2%, sugars 7.6%, water 26.5% and other substances 24.7%. The major fatty acid components of the crude lipids were hexadecanoic and octadecanoic acids. Unsaturated fatty acids and branched fatty acids were also detected as minor components. The protein was abundant in glycine, glutamic acid, and serine. Glucose, xylose, and mannose were the major monosaccharides.

Production and composition of extracellular polysaccharide from cell suspension cultures of Mentha

A suspension culture of Mentha was established from callus which formed on the tips of young shoots of a Mentha hybrid (M. arvenis × M. spicata). Changes in growth parameters during a culture cycle were recorded. The general appearance of cells during division and growth, including the changes in cell form, was also represented.Suspension-cultured cells of Mentha hybrid released a large amount of extracellular polysaccharides (ECP) mainly at the logarithmic phase of the growth cycle. The ECP contained galacturonic acid as major components and arabinose, galactose, glucose, xylose, rhamnose and mannose as minor components. The ratio of the uronic acid content to total sugar content in the ECP was below 40% at day 7, but increased up to 90% at day 21. The relative contents of xylose and glucose in the ECP decreased during the culture period, while the arabinose content increased and those of rhamnose, mannose and galactose remained constant.The IR spectrum suggested that the ECP were low-methoxylated pectic polysaccharides. The presence of lignin and related compounds in the ECP was not detected. The protein content of the ECP was about 10% and the main amino acids were alanine, proline, hydroxyproline, valine, asparticacid and serine, in that order.

Influences of Metabolic Inhibitors and Hydrolytic Enzymes on the Adhesion of Appressoria of Pyricularia oryzae to Wax-coated Cover-glasses.

For discovering the components that contributed to the bonding strength of the glue substances produced by appressoria of Pyricularia oryzae on wax-coated cover-glasses, the influences of metabolic inhibitors and hydrolytic enzymes were investigated. The bonding strength of appressoria was assessed by the ratio of the remaining appressoria after sonication to the appressoria formed before sonication. Remaining appressoria decreased with increasing concentrations of cerulenin, an inhibitor of lipid synthesis, but isoprothiolane and compactin showed no influence on bonding strength. Tunicamycin, an inhibitor of glycoprotein synthesis, weakened the bonding strength of appressoria, but castanospermin had no effect. Of the hydrolytic enzymes tested, protease particularly weakened the bonding strength of appressoria. On the surfaces of substrata, the appressorias bonding strength was higher on the hydrophobic surfaces than on the hydrophilic. These results suggest that lipid components and glycoprotein were closely associated with appressoria bonding strength to the surface of wax-coated cover-glass.

Hydroxycinnamic Acids and Their Dimers Involved in the Cessation of Cell Elongation in Mentha Suspension Culture

The contents of wall-bound hydroxycinnamic acids and their dimers were compared between elongated and non-elongated cells of suspension-cultured Mentha. Wall-bound peroxidase activity was also investigated. The main hydroxycinnamic acids esterified to these two kinds of cell walls were ferulic and caffeic acids. Eleven dehydrodicaffeic acid isomers and six dehydrodiferulic acid isomers formed through C-C and C-O-C coupling processes, were detected by GC-MS from the extracts released from the walls of non-elongated cells. On the other hand, only four dehydrodicaffeic acid isomers and three dehydrodiferulic acid isomers were found in the walls of elongated cells. Amounts of monomers of ferulic and caffeic acids and their 5,5′-dehydrodimers in non-elongated cell walls were about ten and twenty times higher, respectively, than those in the elongated cell walls. There was a close correlation between the amount of 5,5′-dehydrodimers and activity of wall-bound peroxidase in non-elongated and elongated cells. The level of 5,5′-dehydrodimers accumulated at a higher rate than monomers in non-elongated cell walls. These results suggest that the dimerization of ester-linked ferulic and caffeic acids by peroxidase and the increase in amounts of their 5,5′-dehydrodimers are important factors in the cessation of cell elongation in Mentha suspension culture.