Cloning and expression of the phosphotriesterase gene hocA from Pseudomonas monteilii C11.

The cloning of a gene encoding the novel phosphotriesterase from Pseudomonas monteilii C11, which enabled it to use the organophosphate (OP) coroxon as its sole phosphorus source, is described. The gene, called hocA (hydrolysis of coroxon) consists of 501 bp and encodes a protein of 19 kDa. This protein had no sequence similarity to any proteins in the SWISS-PROT/GenBank databases. When a spectinomycin-resistance cassette was placed in this gene, phosphotriesterase activity was abolished and P. monteilii C11 could no longer grow with coroxon as the sole phosphorus source. Overexpression and purification of HocA as a maltose-binding protein fusion produced a protein having a broad substrate specificity across oxon and thion OPs. Michaelis-Menten kinetics were observed with the oxon OPs, but not with the thion OPs. End-product inhibition was observed for coroxon-hydrolytic activity. Increased expression of hocA was observed from an integrative hocA-lacZ fusion when cultures were grown in the absence of phosphate, suggesting that it might be part of the Pho regulon, but the phosphate-regulated promoter was not cloned in this study. This is believed to be the first study in which a gene required for an organism to grow with OP pesticides as a phosphorus source has been isolated.

Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate.

We isolated a bacterial strain, Agrobacterium radiobacter P230, which can hydrolyze a wide range of organophosphate (OP) insecticides. A gene encoding a protein involved in OP hydrolysis was cloned from A. radiobacter P230 and sequenced. This gene (called opdA) had sequence similarity to opd, a gene previously shown to encode an OP-hydrolyzing enzyme in Flavobacterium sp. strain ATCC 27551 and Brevundimonas diminuta MG. Insertional mutation of the opdA gene produced a strain lacking the ability to hydrolyze OPs, suggesting that this is the only gene encoding an OP-hydrolyzing enzyme in A. radiobacter P230. The OPH and OpdA proteins, encoded by opd and opdA, respectively, were overexpressed and purified as maltose-binding proteins, and the maltose-binding protein moiety was cleaved and removed. Neither protein was able to hydrolyze the aliphatic OP malathion. The kinetics of the two proteins for diethyl OPs were comparable. For dimethyl OPs, OpdA had a higher k(cat) than OPH. It was also capable of hydrolyzing the dimethyl OPs phosmet and fenthion, which were not hydrolyzed at detectable levels by OPH.

Isolation of a Pseudomonas monteilli strain with a novel phosphotriesterase.

A Pseudomonas monteilli strain (designated C11) that uses the phosphotriester coroxon as its sole phosphorus source has been isolated. Native PAGE and activity staining identified a single isozyme with significant phosphotriesterase activity in the soluble fraction of the cell. This phosphotriesterase could hydrolyse both coumaphos and coroxon. The hydrolysis product of coroxon, diethylphosphate, and the thion analogue, coumaphos, could not serve as phosphorus sources when added to the growth medium. The majority of the phosphotriesterase and phosphatase activity was contained in the soluble fraction of the cell. Phosphatase activity was inhibited by vanadate as well as by dialysis against the metal chelator, EDTA. Phosphotriesterase activity was not affected by either vanadate or dialysis with EDTA or 1,10-phenanthroline. Phosphotriesterase activity was regulated by the amounts of both phosphate and coroxon in the medium, whereas total phosphatase activity was regulated by phosphate but not coroxon. A lack of hybridisation using a probe against the opd (organophosphate degradation) gene encoding a phosphotriesterase from Flavobacterium sp. ATCC27551 against bulk DNA from P. monteilli C11 suggested that this strain does not contain opd. The work presented here indicates the presence of a novel phosphotriesterase in P. monteilli C11.

Synthesis of (Diethyl-d(10)) coumaphos and related compounds.

Two deuterated insecticides were prepared for use as internal standards for gas-liquid chromatographic-mass spectrometric analyses. Diethyl chlorothiophosphate-d(10) was prepared by reaction of ethanol-d(6) with P(2)S(5) to give labeled diethyldithiophosphoric acid, followed by chlorination. Treatment of the acid chloride with 3-chloro-4-methyl-7-hydroxycoumarin and potassium carbonate in acetone at reflux gave labeled coumaphos. An analogous reaction with 4-methyl-7-hydroxycoumarin gave labeled potasan, and the technique should be usable for synthesis of labeled forms of other dialkyl thiophosphate insecticides.

Multiple forms of sheep serum A-esterase activity associated with the high-density lipoprotein.

Five lipoproteins of sheep serum expressing A-esterase activity, but with differing activities towards four organophosphate substrates, were separated by a combination of gel filtration and ion-exchange chromatography. Each had an Mr of approx. 360,000 and contained a major peptide of Mr 28,000-30,000 that appeared to be present as several isoforms on urea/agarose isoelectric focusing. In every case this peptide split into a number of bands on urea/agarose isoelectric focusing. The bands appear to represent isoforms of the peptide, and four lipoproteins yielded characteristic patterns of bands. This peptide resembles the apolipoprotein A-I of human serum, and available evidence suggests that this is the protein that expresses A-esterase activity. Evidence is presented for the existence of different species of high-density lipoprotein HDL2 particles containing different complements of peptide isoforms and expressing contrasting substrate specificities towards organophosphates.

Biochemical genetics of resistance to organophosphorus acaricides in three strains of the cattle tick, Boophilus microplus.

Three aspects of the biochemical genetics of resistance to organophosphorus compounds in the Biarra (B), Mackay (M) and Ridgelands (R) strains of the cattle tick B. microplus were studied. These were: decreased acetylcholinesterase (AChE) activity in adult brains of strains B and M; decreased AChE sensitivity to inhibitors in adult brains and in larvae of strains B, M and R; and increased detoxication in larvae and adult females of strain M. Comparisons were made with a susceptible reference strain (S). Microspectrophotometric estimations of AChE activity in histochemical preparations of whole brains showed that hybrids had levels of activity approximately intermediate between those of the parental strains. Homogenates of brains from hybrids assayed biochemically gave similar but more precise results which indicated that decreased brain AChE activity was neither recessive nor dominant (degree of dominance, D = +0-02) in strain B and incompletely recessive (D = -0-26) in strain M. The proportions of brains showing decreased AChE activity in testcross and F2 progenies indicated that decreased AChE activity in strains B and M is controlled by single autosomal genes. Inhibition of AChE at diagnostic concentrations of coroxon in brains of B, B x S hybrid and S types suggested that decreased sensitivity of AChE in strain B is incompletely dominant (D = +0-10). Kinetic studies on coroxon inhibition of AChE in brain homogenates of B, B x S hybrid and S types revealed the presence of each parental AChE component in hybrids in equal amounts and the absence of a hybrid enzyme. Dimethoxon inhibition of AChE in brains, their homogenates and larval homogenates of B, M and R types showed that decreased AChE sensitivity was a major mechanism of resistance to dimethoate strongly expressed in B x S and M x S hybrids. The proportion of brains showing decreased AChE sensitivity to coroxon in testocross and F2 progenies indicated that decreased AChE sensitivity in strain B is controlled by a single autosomal gene. The degree of dominance of increased degradative metabolism of coumaphos in strain M was variable; the hydrolytic rate in all M x S hybrids was similar to that of M but the overall detoxication rate in hybrids was lower. Genetic control of detoxication is discussed.

Determination of coumaphos and its oxygen analog in eggs by in situ fluorometry.

A method is reported for the simultaneous determination of coumaphos (o,o-diethyl o(3-chloro-4-methyl-2-oxo-2H-1-benzopyran-7-yl)phosphorothioate) and its oxygen analog, Coroxon, in eggs in the ppb range. The residues are extracted with acetone and chloroform. The extract is purified by liquid-liquid partition followed by column chromatography and then by 2-dimensional thin layer chromatography. The fluorescence is measured directly on the chromatogram.