A fruit fly bioassay with phosphotriesterase for detection of certain organophosphorus insecticide residues

Chiang, Tom; Dean, Mary C.; McDaniel, C S

doi:10.1007/bf01609811

Cited by 7 publications

(5 citation statements)

References 6 publications

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“…This phenomenon is not limited to organophosphorus neurotoxins, since plasmid-borne genes for degradative enzymes of herbicides have been well documented (9,27). In the case of the opd genes, a wide range of pesticides sharing a common chemical structure are degraded (6), providing the potential for rapid evolution of genes to degrade a variety of pesticides and challenging the agrochemical rationale of substituting pesticides of similar chemical structure or increasing application rates for extended pest control. Rapid mutational adaptation in an enriched soil bacterial population could render ineffective any subsequent applications of a similar chemical.…”

Section: Methodsmentioning

confidence: 99%

“…Pseudomonas putida MG and Flavobacterium spp. have been shown to possess the ability to degrade an extremely broad spectrum of organophosphorus phosphotriesters as well as thiol esters (4,6). Recently, certain mammalian neurotoxins, such as diisopropyl phosphonofluoridate (1) and Soman (1,2,2-trimethylpropyl-methylphosphonofluoridate; J. DeFrank, personal communication), have been shown to be hydrolyzed by selected bacteria.…”

mentioning

confidence: 99%

“…Recently, certain mammalian neurotoxins, such as diisopropyl phosphonofluoridate (1) and Soman (1,2,2-trimethylpropyl-methylphosphonofluoridate; J. DeFrank, personal communication), have been shown to be hydrolyzed by selected bacteria. Several of the bacterial strains possess constitutively expressed phosphotriesterases with broad substrate ranges including many commonly used organophosphorus pesticides (4,6). None of these strains has shown the ability to utilize these neurotoxins as sole nutrient or energy sources, thus making mutant selection difficult (C. S. McDaniel and J. R. Wild, Arch.…”

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Cloning and sequencing of a plasmid-borne gene (opd) encoding a phosphotriesterase

1988

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Plasmid pCMS1 was isolated from Pseudomonas diminuta MG, a strain which constitutively hydrolyzes a broad spectrum of organophosphorus compounds. The native plasmid was restricted with PstI, and individual DNA fragments were subcloned into pBR322. A recombinant plasmid transformed into Escherichia coli possessed weak hydrolytic activity, and Southern blotting with the native plasmid DNA verified that the DNA sequence originated from pCMS1. When the cloned 1.3-kilobase fragment was placed behind the lacZ' promoter of M13mplO and retransformed into E. coli, clear-plaque isolates with correctly sized inserts exhibited isopropyl-,-D-thiogalactopyranoside-inducible whole-cell activity. Sequence determination of the M13 constructions identified an open reading frame of 975 bases preceded by a putative ribosome-binding site appropriately positioned upstream of the first ATG codon in the open reading frame. An intragenic fusion of the opd gene with the lacZ gene produced a hybrid polypeptide which was purified by ,-galactosidase immunoaffinity chromatography and used to confirm the open reading frame of opd. The gene product, an organophosphorus phosphotriesterase, would have a molecular weight of 35,418 if the presumed start site is correct. Eighty to ninety percent of the enzymatic activity was associated with the pseudomonad membrane fractions. When dissociated by treatment with 0.1% Triton and 1 M NaCl, the enzymatic activity was associated with a molecular weight of approximately 65,000, suggesting that the active enzyme was dimeric.Synthetic organophosphorus neurotoxins are used extensively as agricultural and domestic pesticides including insecticides, fungicides, and herbicides. Naturally occurring bacterial isolates capable of metabolizing this class of compounds have received considerable attention (20, 25) since they provide the possibility of both environmental and in situ detoxification (reviewed in reference 18). Pseudomonas putida MG and Flavobacterium spp. have been shown to possess the ability to degrade an extremely broad spectrum of organophosphorus phosphotriesters as well as thiol esters (4, 6). Recently, certain mammalian neurotoxins, such as diisopropyl phosphonofluoridate (1) and Soman (1,2,2-trimethylpropyl-methylphosphonofluoridate; J. DeFrank, personal communication), have been shown to be hydrolyzed by selected bacteria. Several of the bacterial strains possess constitutively expressed phosphotriesterases with broad substrate ranges including many commonly used organophosphorus pesticides (4, 6). None of these strains has shown the ability to utilize these neurotoxins as sole nutrient or energy sources, thus making mutant selection difficult (C. S. McDaniel and J. R. Wild, Arch. Environ. Contam. Toxicol., in press). The hydrolysis of organophosphorus compounds by the pseudomonad phosphotriesterase has been shown to proceed via nucleophilic addition of a molecule of water across the acid anhydride bond

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Cloning and sequencing of a plasmid-borne gene (opd) encoding a phosphotriesterase

1988

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“…Purification of Phosphotriesterase. The phosphotriesterase from P. diminuta is a membrane-associated enzyme capable of hydrolyzing organophosphotriesters in intact whole cells (Chiang et al, 1985). Sonic disruption or explosive decombustion of cells in a French pressure cell resulted in 95% of the enzymatic activity being located in the particulate fraction of the cell (removed by lOOOOg centrifugation).…”

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confidence: 99%

Mechanism and stereochemical course at phosphorus of the reaction catalyzed by a bacterial phosphotriesterase

Lewis¹,

Donarski²,

Wild³

et al. 1988

Biochemistry

185

140

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The reaction mechanism for the phosphotriesterase from Pseudomonas diminuta has been examined. When paraoxon (diethyl 4-nitrophenyl phosphate) is hydrolyzed by this enzyme in oxygen-18-labeled water, the oxygen-18 label is found exclusively in the diethyl phosphate product. The absolute configurations for the (+) and (-) enantiomers of O-ethyl phenylphosphonothioic acid have been determined by X-ray diffraction structural determination of the individual crystalline 1-phenylethylamine salts. The (+) enantiomer of the free acid corresponds to the RP configuration. The RP enantiomer of O-ethyl phenylphosphonothioic acid has been converted to the SP enantiomer of EPN [O-ethyl O-(4-nitrophenyl) phenylphosphonothioate]. (SP)-EPN is hydrolyzed by the phosphotriesterase to the SP enantiomer of O-ethyl phenylphosphonothioic acid. The enzymatic reaction therefore proceeds with inversion of configuration. These results have been interpreted as an indication of a single in-line displacement by an activated water molecule directly at the phosphorus center of the phosphotriester substrate. (RP)-EPN is not hydrolyzed by the enzyme at an appreciable rate.

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“…Pseudomonas diminuta MG and a Flavobacterium sp. (ATCC 27551) have the ability to degrade a broad spectrum of organophosphorous triesters (2,4) by virtue of a constitutively expressed organophosphorous acid anhydrase (11,14). This enzyme stereospecifically hydrolyzes the triester bond common to organophosphorous pesticide molecules without a phosphorylated enzyme intermediate (7).…”

mentioning

confidence: 99%

Dissimilar plasmids isolated from Pseudomonas diminuta MG and a Flavobacterium sp. (ATCC 27551) contain identical opd genes

Harper

McDaniel

Miller

et al. 1988

Appl Environ Microbiol

123

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The opd (organophosphate-degrading) gene derived from a 43-kilobase-pair plasmid (pSM55) of a Flavobacterium sp. (ATCC 27551) has a sequence identical to that of the plasmid-borne gene of Pseudomonas diminuta. Hybridization studies with DNA fragments obtained by restriction endonuclease digestion of plasmid DNAs demonstrated that the identical opd sequences were encoded on dissimilar plasmids from the two sources.

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A fruit fly bioassay with phosphotriesterase for detection of certain organophosphorus insecticide residues

Cited by 7 publications

References 6 publications

Cloning and sequencing of a plasmid-borne gene (opd) encoding a phosphotriesterase

Cloning and sequencing of a plasmid-borne gene (opd) encoding a phosphotriesterase

Mechanism and stereochemical course at phosphorus of the reaction catalyzed by a bacterial phosphotriesterase

Dissimilar plasmids isolated from Pseudomonas diminuta MG and a Flavobacterium sp. (ATCC 27551) contain identical opd genes

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