Plasmid-mediated degradation of organophosphate pesticides

Abstract

A plasmid (pCMS1) of approximately 51 Kbp was isolated from a Pseudomonas diminuta strain (PD3) known to constitutively degrade a variety of organophosphate pesticides, including parathion. The plasmid was sized and partially mapped by restriction endonuclease digestion and a PstI digest was used to subclone the entire degradative plasmid into pBR322 and transformed into E.coli. One transformant containing pBR322 with an insert of 1270 bp was capable of degrading parathion on a whole cell basis. CsCl-purification and retransforming the recombinant plasmid into E. coli HB101-4442 showed that the plasmid did encode a phosphotriesterase. Southern blotting of this labelled insert against the native plasmid DNA verified that the phosphotriesterase activity originated from pCMSl. Analysis of whole cell and cellular subfractions of both the native strain and cloned derivatives by high resolution SDS-PAGE demonstrated that the gene product had a molecular weight of approximately 30,000 and was predominately associated with the membrane fraction of the cell. Using this recombinant plasmid, a fine structure restriction map was constructed of the cloned phosphotriesterase gene (opd). The 1270 bp fragment was transferred into coliphage M13mpl0 and retransformed into E. coli strain JM103. Half of the clear-plaque isolates with the correct insert exhibited a whole-cell phosphotriesterase activity under the control of M13's lacZ' promoter. Several phage with 1270 bp inserts but lacking the degradative capability had opposite orientations to those exhibiting activity. Subcloning of the 1270 bp PstI fragment was accomplished for the purpose of initiating the dideoxy sequencing of the gene along both strands. Initial sequencing in the 5 '-flanking region identified potential translational and transcriptional control sequences, an initiation codon and a downstream open reading frame of approximately 300 bp (the sequenced 5' portion). In addition to the core research accomplished above, a useful plate assay for the detection of Opd^+ colonies was developed and subsequently used to determine the background rate of plasmid loss in the Pseudomonas strain to be greater than 1% per generation. A bioassay using Drosophila and a crude extract of phosphotriesterase was developed to distinguish certain organophosphates from other insecticides at residue levels.