| Synthetic organophosphorus pesticides (OPs), kinds of insecticides with broad spectrum, are widely used to control major insect pests in agriculture since its invention. OPs are frequently applied in modern agricultural systems to ensure good harvests. However, the extensive use of OPs may easily lead to widespread environmental pollution. So the remediation of OPs has caused growing concern among environmental scientists. Microorganisms play key roles in the detoxification of xenobiotics, and the use of microorganisms for bioremediation of OPs-contaminated sites has received increasing attention as an efficient and cost effective biotechnological approach compared to physical and chemical one.The study on the microbial degradation of OPs in our lab has a history of 20 years, and several high-efficient OPs-degrading bacterial strains, such as Pseudomonas sp. DLL-1, Sphingomonas sp. Dsp-2, Ochrobacter sp. DDV-1 and Paracoccus sp. M-1, have been isolated. However, there are some OPs such as isocarbophos and dimethoate, that can not be degraded by those isolated strains. The aim of this study is to isolate some high-efficient strains capable of degrading isocarbophos and dimethoate that are not degraded by the previously isolated strains. With the goal of elucidating a possible application in OPs-contaminated environmental remediation, degrading characteristics of OPs by isolated strains are also to be studied. To make the isolated strain more potential in the bioremediation of multiple OPs contaminates sites, the cloning of the organophosphorus hydrolase gene and construction of genetically engineered microogansisms (GEMs) with high efficiency and broader rang substrate specificity will also be studied.In this paper, a highly effective isocarbophos-degrading strain of scl-2 was isolated from isocarbophos-polluted soil. The strain scl-2 was preliminarily identified as Arthrobacter sp. based on its morphological, physiological and biochemical properties, as well as 16S rRNA analysis. The strain scl-2 could utilize isocarbophos as its sole source of carbon and phosphorus for growth.100 mg·l-1 isocarbophos could be degraded to a non-detectable level in 21 hours by scl-2 in cell culture, and isofenphos-methyl, profenofos and phosemet could also be degraded. During the degradation of isocarbophos, the metabolites isopropyl salicylate, salicylate and gentisate were detected and identified based on MS/MS analysis and their retention times in HPLC. Tansformation of gentisate to pyruvate and fumarate via maleylpyruvate and fumarylpyruvate was detected by assaying for the activities of gentisate 1,2-dioxygenase (GDO) and maleylpyruvate isomerase. Therefore, we have identified the degradation pathway of isocarbophos in Arthrobacter sp. scl-2 for the first time. When used strain scl-2 to degrade the isocarbophos residue in Nanfeng Orange, we found that the degradation rate reached 75%, showed that strain scl-2 has a strong prospect.A highly effective dimethoate-degrading Paracoccus sp. strain lgjj-3 was isolated from treatment wastewater. Strain lgjj-3 can utilize dimethoate as its sole carbon source for growth and degrade an initial concentration of 100 mg·1-1 dimethoate to non-detectable levels within 6 hours in liquid culture. Strain lgjj-3 could efficiently degrade dimethoate at temperatures ranging from 25℃to 40℃and pH values ranging from 6 to 9. The optimal temperature and initial pH value for dimethoate biodegradation were 35℃and 7.0, respectively. High concentrations of dimethoate (up to 500 mg l-1) could also be degraded by Lgjj-3 During the degradation of dimethoate, seven metabolites, including dimethoate carboxylic acid,2-(hydroxy(methoxy)phosphorylthio)acetic acid, O,O,S-trimethyl thiophosphorothioate, O-methyl O,S-dihydrogen phosphorothioate, phosphorothioic O,O,S-acid,O,O,S-trimethylphosphorothiate and O,O,O-trimethyl phosphoric ester, were successfully detected and identified based on MS/MS and GC-MS analysis. A biochemical degradation pathway of dimethoate by Paracoccus sp. lgjj-3 is proposed for the first time.To construct GEMs with broad range substrate specificity and without bringing exogenous antibiotic resistance gene, a set of random transposon vectors pUTTns that facilitates the markerless integration of new functions into the chromosome of gram-negative bacteria has been developed. The vectors, which are derived from mini-Tn5 transposons, are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis, but they are external to the mobile element. The vectors’ conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor. Internal to the mini-Tn5 element is a cassette that contains a selectable antibiotic resistance marker (kanamycin, chloramphenicol, or tetracycline resistance gene), a counter-selectable marker (sacB), a 430-bp repeat of the sacB gene 3’end acted as the directly-repeated (DR) sequence, and modified multiple cloning sites (MCS). After two total rounds of transposon integration and recombination between the two DRs, only the exogenous DNA inserted into the MCS (passenger genes) and a single 430-bp scar sacBDR fragment remained in the chromosome after excision. The utility of these vectors was demonstrated by integrating the organophosphorus insecticide hydrolase gene (mpd) into the chromosome of Escherichia, Pseudomonas, Sphingomonas, and Paracoccus species. Sequential integration of another organophosphorus insecticide hydrolase gene (oph) into the previously engineered bacteria, without bringing any selectable markers, was also successful. These engineered bacteria were relatively stable. Cell viability and original degrading characteristics were not affected compared with the original recipients. This shows that the developed system is very useful for the markerless integration of exogenous genes into the chromosome of gram-negative eubacteria. |
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