bialaphos and phosphinothricin

Topics: Adsorption; Charcoal; Environmental Monitoring; Glyphosate; Herbicides; Hydrogen-Ion Concentration; Kinetics; Solutions

Stable transformation of common bean (Phaseolus vulgaris L.) has been successful, to date, only using biolistic-mediated transformation and shoot regeneration from meristem-containing embryo axes. In this study, using precultured embryo axes, and optimal co-cultivation conditions resulted in a successful transformation of the common bean cultivar Olathe using Agrobacterium tumefaciens strain EHA105. Plant regeneration through somatic embryogenesis was attained through the preculture of embryo axes for 12 weeks using induced competent cells for A. tumefaciens-mediated gene delivery. Using A. tumefaciens at a low optical density (OD) of 0.1 at a wavelength of 600 nm for infection and 4-day co-cultivation, compared to OD600 of 0.5, increased the survival rate of the inoculated explants from 23% to 45%. Selection using 0.5 mg L-1 glufosinate (GS) was effective to identify transformed cells when the bialaphos resistance (bar) gene under the constitutive 35S promoter was used as a selectable marker. After an 18-week selection period, 1.5% -2.5% inoculated explants, in three experiments with a total of 600 explants, produced GS-resistant plants through somatic embryogenesis. The expression of bar was confirmed in first- and second-generation seedlings of the two lines through reverse polymerase chain reaction. Presence of the bar gene was verified through genome sequencing of two selected transgenic lines. The induction of regenerable, competent cells is key for the successful transformation, and the protocols described may be useful for future transformation of additional Phaseolus germplasm.

Topics: Agrobacterium tumefaciens; Aminobutyrates; DNA, Plant; Drug Resistance, Bacterial; Genetic Vectors; Herbicides; Organophosphorus Compounds; Phaseolus; Phenotype; Plant Breeding; Plants, Genetically Modified; RNA, Plant; Transformation, Genetic

Natural products containing phosphonic or phosphinic acid functionalities often display potent biological activities with applications in medicine and agriculture. The herbicide phosphinothricin-tripeptide (PTT) was the first phosphinate natural product discovered, yet despite numerous studies, questions remain surrounding key transformations required for its biosynthesis. In particular, the enzymology required to convert phosphonoformate to carboxyphosphonoenolpyruvate and the mechanisms underlying phosphorus methylation remain poorly understood. In addition, the model for non-ribosomal peptide synthetase assembly of the intact tripeptide product has undergone numerous revisions that have yet to be experimentally tested. To further investigate the biosynthesis of this unusual natural product, we completely sequenced the PTT biosynthetic locus from Streptomyces hygroscopicus and compared it with the orthologous cluster from Streptomyces viridochromogenes. We also sequenced and analyzed the closely related phosalacine (PAL) biosynthetic locus from Kitasatospora phosalacinea. Using data drawn from the comparative analysis of the PTT and PAL pathways, we also evaluate three related recently discovered phosphonate biosynthetic loci from Streptomyces sviceus, Streptomyces sp. WM6386 and Frankia alni. Our observations address long-standing biosynthetic questions related to PTT and PAL production and suggest that additional members of this pharmacologically important class await discovery.

Topics: Actinobacteria; Amino Acid Sequence; Aminobutyrates; Bacterial Proteins; Dipeptides; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Molecular Structure; Organophosphorus Compounds; Streptomyces; Thiolester Hydrolases

We developed a simple and rapid method for the simultaneous determination of phosphorus-containing amino acid herbicides (glyphosate, glufosinate, bialaphos) and their major metabolites, aminomethylphosphonic acid (AMPA) and 3-methylphosphinicopropionic acid (MPPA), in human serum. Serum samples were filtrated through an ultrafiltration membrane to remove proteins. The filtrate was then washed with chloroform, and injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. Chromatographic separation was achieved on a hydrophilic interaction chromatography (HILIC) column. Determination of the target herbicides and metabolites was successfully carried out without derivatization or solid phase extraction (SPE) cartridge clean-up. The recoveries of these compounds, added to human serum at 0.2μg/mL, ranged from 94% to 108%, and the relative standard deviations (RSDs) were within 5.9%. The limits of detection (LODs) were 0.01μg/mL for MPPA, 0.02μg/mL for AMPA, 0.03μg/mL for both glyphosate and glufosinate, and 0.07μg/mL for bialaphos, respectively.

Topics: Aminobutyrates; Chromatography, Liquid; Female; Forensic Medicine; Glycine; Glyphosate; Herbicides; Humans; Hydrophobic and Hydrophilic Interactions; Isoxazoles; Male; Middle Aged; Organophosphonates; Organophosphorus Compounds; Propionates; Reproducibility of Results; Sensitivity and Specificity; Solid Phase Extraction; Tandem Mass Spectrometry; Tetrazoles

We describe a new practical capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) method for the forensic analysis of phosphorus-containing amino acid-type herbicides, glyphosate (GLYP), glufosinate (GLUF) and bialaphos (BIAL). A new sheathless interface, a high sensitivity porous sprayer (HSPS), was used in this study. The limits of detections of GLYP, GLUF and BIAL were 7.6, 0.61 and 0.57 pg, respectively. These values were 4-36 times lower than these obtained by conventional CE/ESI-MS using a sheath liquid. The developed method was successfully applied to the analysis of beverages spiked with the herbicides.

Topics: Amino Acids; Aminobutyrates; Electrophoresis, Capillary; Glycine; Glyphosate; Herbicides; Molecular Structure; Organophosphorus Compounds; Phosphorus; Porosity; Spectrometry, Mass, Electrospray Ionization; Surface Properties

The symptoms of acute poisoning caused by ingestion of bialaphos (BIAL), an ingredient of herbicide, are supposed to be due to the L-glufosinate (L-GLUF), which is formed by the degradation of bialaphos. To elucidate the pharmacokinetics of BIAL and L-GLUF, we attempted a simultaneous analysis of BIAL and L-GLUF in biological samples by exploiting a reversed phase HPLC method. The derivatization reaction of BIAL and L-GLUF using (+) -1- (9-fluorenyl) ethyl chloroformate was completed in 30 min at 40 degrees C and both derivatives were stable for 48 hr at 25 degrees C. A fluorescence detector were used for HPLC; the exicitation wavelength was set at 265 nm and the emission wavelength at 315 nm. Respective calibration curves prepared by adding BIAL and L-GLUF to serum were linear within ranges of 0.01-10.0 and 0.005-10.0 microg/mL in derivatived liquid samples for introducing into HPLC. The lower limits of detection for BIAL and L-GLUF were 0.005 and 0.001 microg/mL, respectively. An 83-year old male who ingested approximately 350 mL of Herby Liquid, a herbicide containing 18% BIAL and 82% surfactant, in an attempt to commit suicide developed delayed respiratory depression and seizures. L-GLUF was detected in the serum of the patient 2.7 hr after ingestion, but BIAL was not. The change in serum L-GLUF concentration measured over time was consistent with a 2-compartment model, with a distribution half-life of 1.70 hr and an elimination half-life of 6.03 hr.

Topics: Aged, 80 and over; Aminobutyrates; Chromatography, High Pressure Liquid; Herbicides; Humans; Male; Organophosphate Poisoning; Organophosphorus Compounds; Specimen Handling; Suicide, Attempted; Time Factors

A novel absolute determination method using chirality without any calibration curves or comparison standards has been proposed for phosphorus-containing amino acid-type herbicides, glufosinate (D,L-GLUF) and bialaphos (BIAL). This method is based on a change in the enantiomeric ratio after the spiking of a known amount of the enantiomers with different enantiomeric ratios to a sample. D,L-GLUF was determined by adding a known amount of L-GLUF to the sample, derivatizing them with dansyl chloride, and measuring the ratio of the peak area of the D-isomer to that of the L-isomer by means of gamma-cyclodextrin modified capillary zone electrophoresis. The accuracy and precision of the method were evaluated using a synthetic sample. The mean values obtained for D- and L-GLUF agreed with the values taken within 1.6%; also the reproducibility was as good as less than 2.8%. The determination of BIAL was achieved by determining GLUF quantitatively produced by the acid hydrolysis of BIAL. The proposed methods were applied to the analysis of commercial herbicides and the validity and usefulness were evaluated.

Topics: Aminobutyrates; Electrophoresis, Capillary; gamma-Cyclodextrins; Herbicides; Methods; Organophosphorus Compounds; Reproducibility of Results; Stereoisomerism

Manganese peroxidase (MnP) is a major extracellular component of the lignin-degrading system of the white-rot fungus, Phanerochaete chrysosporium. Homologous expression of recombinant MnP isozyme 1 (rMnP1) in P. chrysosporium was achieved using a novel transformation system for this fungus, which utilizes the Streptomyces hygroscopicus bialaphos-resistant gene, bar, as the selectable marker. The transformation frequency for this system is approximately 100 bialaphos-resistant transformants per microgram of plasmid DNA. Transformed strains all contain plasmid DNA, ectopically integrated into the fungal genome. Using this transformation system, the promoter region of the P. chrysosporium translation elongation factor gene was used to drive expression of mnp1, encoding MnP1, in primary metabolic cultures of P. chrysosporium, where endogenous MnP was not expressed. Approximately 2-3 mg of active recombinant MnP1 per liter of extracellular medium was produced in agitated cultures of transformants.

Topics: Aminobutyrates; Antifungal Agents; Base Sequence; Drug Resistance; Gene Expression Regulation; Genes, Dominant; Genetic Markers; Genetic Vectors; Molecular Sequence Data; Organophosphorus Compounds; Peroxidases; Phanerochaete; Plasmids; Promoter Regions, Genetic; Recombinant Proteins; Transformation, Genetic

A rapid and practical method for direct detection of the herbicides (glufosinate, bialaphos and glyphosate) in anion-exchange chromatography has been developed with integrated pulsed amperometric detection (IPAD). The electrochemical behavior of these herbicides showed catalytic currents based on the oxidation of amines in their structures. Waveform in IPAD was similar to that for amino acids, which exhibited adsorption/desorption catalytic features at gold electrode surface in alkaline solution. Under optimized conditions, detection limits (signal-to-noise ratio of 3) for glufosinate, bialaphos and glyphosate were 20, 65 and 50 ng ml(-1), respectively, with correlation coefficients of 0.995, 0.997 and 0.996 over concentration ranges of 0.1-45, 0.3-32 and 0.1-50 microg ml(-1), respectively. The relative standard deviations (n=5) were 1.7-3.0%. The present method was successfully applied to the determination of glyphosate in urine and serum.

Topics: Aminobutyrates; Chromatography, Ion Exchange; Electrochemistry; Electrodes; Glycine; Glyphosate; Gold; Herbicides; Organophosphorus Compounds

Metarhizium anisopliae var. acridum (syn. M. flavoviride) is recognized as a highly specific and virulent mycopathogen of locusts and grasshoppers and is currently being developed as a biological control agent for this group of insects in Brazil. Intact conidia of M. anisopliae var. acridum strain CG423 were transformed using microparticle bombardment. Plasmids used were: (1) pBARKS1 carrying the bar gene of Streptomyces hygroscopicus fused to the Aspergillus nidulans trpC promoter, encoding resistance to glufosinate ammonium (or phosphinothricin) and modified by addition of the telomeric repeat (TTAGGG)(18) of Fusarium oxysporum and 2.pEGFP/gpd/tel carrying a red-shifted variant gene for Aequorea victoria green fluorescent protein (EGFP) which we have fused to the A. nidulans gpd promoter and trpC terminator. Highly fluorescent co-transformants were selected on solid minimal medium containing 100 microg ml(-1) glufosinate ammonium using an inverted microscope with 450-490 nm excitation/510 nm emission filter set. Southern blot analysis of co-transformants revealed varying multiple chromosomal integrations of both bar and egfp genes at both telomeric and non-telomeric loci. Transformants retained pathogenicity in bioassays against Rhammatocerus schistocercoides and showed unaltered lack of pathogenicity against larvae of the non-target insect Anticarsia gemmatalis. One co-transformant from four tested, however, showed a significant, but non-dose-dependent, elevation in virulence against Tenebrio molitor.

Topics: Aminobutyrates; Animals; Biolistics; Drug Resistance, Microbial; Fungi; Grasshoppers; Green Fluorescent Proteins; Herbicides; Luminescent Proteins; Organophosphorus Compounds; Pest Control, Biological; Transformation, Genetic; Virulence

Phosphinothricyl-alanyl-alanine (Pt tripeptide (Ptt), bialaphos) is a metabolite produced by Streptomyces viridochromogenes and Streptomyces hygroscopicus. It contains the unique phosphinoamino acid phosphinothricin (Pt), which after cleavage from Ptt is active as an inhibitor of glutamine synthetase. We have isolated three enzymes that assemble the building block of the Ptt peptide backbone in a nonribosomal mechanism. The first enzyme, named Ptt-synthetase I (PTTS I), activates N-acetyldemethylphosphinothricin (AcDMPt) as adenylate and thioester. Pt is not activated. PTTS I can also activate N-acetylphosphinothricin (AcPt) or N-acetylglutamate as structural analogues of AcDMPT. Native PTTS I has an estimated size of 62 kDa whereas the denatured form displays a size of 76 kDa. Immunoblot analysis and determination of its N-terminal protein sequence revealed that PTTS I is identical with the gene product of phsA. The phsA gene was previously identified near the Pt-resistance gene pat in the Ptt biosynthesis gene cluster in S. viridochromogenes. Besides PTTS I, two alanine-activating enzymes (PTTS II/III) were partially purified from S. viridochromogenes with estimated native sizes of ca. 120 kDa (enzyme 1) and ca. 140 kDa (enzyme 2). Both enzymes bind alanine as a thioester via the corresponding adenylate. Level of PTTS II/III and product formation were correlated with each other in several different strains of S. viridochromogenes. These results indicate that Ptt is synthesized by three peptide synthetases, each activating one single amino acid. The data also confirm previous genetic data, which suggest that AcDMPt-Ala-Ala is the precursor of Ptt.

Topics: Alanine; Amidohydrolases; Aminobutyrates; Enzyme Activation; Herbicides; Organophosphorus Compounds; Peptide Synthases; Streptomyces; Substrate Specificity

Enhanced expression of the bialaphos resistance (bar) from Streptomyces hygroscopicus, which confers resistance to the herbicides bialaphos and phosphinothricin (PPT), has been obtained in Escherichia coli using a vector system based on translational coupling. The gene product, PPT acetyltransferase, was purified to homogeneity and its enzymatic properties were analyzed. Hybrid gene constructs with gene fragments fused to the 3'-terminus of bar yield fusion proteins having acetyltransferase activity, with a Michaelis constant for the PPT substrate comparable to the unmodified enzyme. The bar gene represents a selectable and assayable reporter gene especially suitable for 3'-terminal gene fusions.

Topics: Acetyltransferases; Aminobutyrates; Blotting, Western; Escherichia coli; Gene Expression; Hydrogen-Ion Concentration; Kinetics; Organophosphorus Compounds; Recombinant Fusion Proteins; Streptomyces; Temperature

Two oligopeptides containing phosphinothricin were accumulated in the culture of bialaphos (BA) producer Streptomyces hygroscopicus SF1293 when large amount of BA was added to the culture at idio phase. One of the oligopeptides was a new substance, BA dimer (phosphinothricyl-alanyl-alanyl-phosphinothricyl-alanyl-alan ine), and the other was a known substance called If (phosphinothricyl-alanyl-alanyl-phosphinothricin). Though BA none producing mutants which were blocked at the steps 1, 10 and 13, respectively in BA biosynthesis also converted BA to BA dimer and If, the mutants blocked at the step 11 (alanylation) could not curry out the conversion. Antibiotic activities of BA dimer and If were lower than 1/100 of BA.

Topics: Aminobutyrates; Anti-Bacterial Agents; Magnetic Resonance Spectroscopy; Microbial Sensitivity Tests; Oligopeptides; Organophosphorus Compounds; Streptomyces

Topics: Aminobutyrates; Chromatography, Paper; Gastric Juice; Glycine; Glyphosate; Herbicides; Humans; Organophosphorus Compounds