Characterization Of A Mp-Degrading Strain Sphingopyxis Sp.DLP-2, Cloning And Functional Verification Of Tnmpd

Organophosphorus pesticides (OPs),a kind of insecticide with broad spectrum and high efficiency, are widely used in agriculture to ensure good harvests. However, the extensive use of OPs also caused environmental pollution. Microorganisms play a key role in the detoxification of xenobiotics, and the use of microorganisms for bioremediation of OPs-contaminated sites has received increasing attention, because it is an efficient and cost effective biotechnological approach.This research aimed at isolating the bacteria that can use methyl-parathion (MP) as the sole carbon source, investigating their biological characteristics as well as degrading characteristics of MP. At the same time, the degrading related gene and gene clusters were cloned, analyzed, the occurrence of horizontal gene transfer was functionally validated, which provided the foundation for the bioremediation of environments contaminated by OPs.MP-degrading strain DLP-2 was isolated from the activated sludge from the wastewater treating system of an OPs producer. Strain DLP-2 was preliminarily identified as Sphingopyxis sp. (GenBank Accession NO. JF833116) according to its physiological & biochemical characteristics and 16S rRNA gene sequence analysis.The optimal pH and temperature for the growth of strain DLP-2 was 7.0 and 30℃respectively. The optimal pH and temperature for the degradation of strain DLP-2 was 7.0 and 30℃respectively which is in agreement with the optimal growth condition. The degradation rate of strain DLP-2 against 50 mg·L-1 methyl parathion was 99% within 12h.But it could only hydrolyze MP to PNP under the concentration of 100 mg·L-1, indicating that the accumulated PNP was toxic to strain DLP-2 and inhibited the further degradation. The degradation rate against MP showed positive correlation with the inoculums size. The degradation rate of strain DLP-2 against 50 mg·L-1 ethyl-parathion and phoxim was above 80% within 24 h, exceed 50% against chlorpyrifos and fenitrothion, but only less 20% against isofenphos-methyl, methidathion and triazophos.The organophosphorus hydrolase gene mpd (996bp in length) of strain DLP-2 was cloned by PCR, the sequence was analyzed it had 99% similarity to mpd gene(AF338729.1)(DQ173274.1) and mph gene (AY251554.2) respectively. The mpd gene of strain DLP-2 was cloned into the expression plasmid vector pET29a and functional expressed in E.coli BL21(DE3). SDS-PAGE analysis of revealed the production of the protein with the right molecular size (35KD) as the predicted.The Tnmpd cluster of strain DLP-2 was cloned by PCR, the sequence was analyzed, (4552bp in length). It had 99% similarity with Tnmph gene (GenBank accession No.EF463103). The structure of Tnmpd cluster showed that the upstream and downstream of the mpd was associated with a typical mobile element IS6100 respectively. We deduced that mpd gene was likely transferable among the methyl parathion-degrading bacteria.To verified the ability of horizontal gene transfer of mpd, the Tnmpd was ligated to the suicide plasmid pJQ200SK, and transferred to the chromosome of Pseudomonas putida KT2440 by the method of triparental mating, and Pseudomonas putida KT2440 could degrade methyl-parathion, which proved the occurrence of horizontal gene transfer of Tnmpd, and provided the foundation for the gene enhancement bioremediation of environment polluted with OPs.