Isolation And Identification Of Pesticides-Degrading Bacterium Pseudomonas Stutzeri YC-YH1 And Study Of Degradation Mechanism

Organophosphorus pesticides(OPPs) are widely used to wipe out pests and protect crops, however, pesticide residues in environment harmed to human health. Great concerns have been raised about the rapidly and efficient strategy to eliminate pesticides residues of OPPs in environment. Bacteria was isolated from contaminated environment and was used to degrade pesticides residues, which was one of the important means of bioremediation of pesticide contaminated environment.In the study, a bacterial strain YC-YH1 was isolated from sludge and could utilize chlorpyrifos as the sole phosphorus source for growth. According to 16 S r RNA gene analysis incorporation with physiological, Biolog test and biochemical tests, YC-YH1 was identified as a Pseudomonas stutzeri strain. Response surface methodology was used to optimize the parameters that significantly affected chlorpyrifos biodegradation by strain YC-YH1 and their interactions. Under optimum conditions(p H 7.3, 32 ℃), P. stutzeri YC-YH1 degraded 100% of chlorpyrifos(100 mg/L) initially added within 4 days. Simulated chlorpyrifos-containing wastewater was prepared for the application of YC-YH1 in bioreactor and YC-YH1 showed a good tolerance to high concentration of chlorpyrifos. P. stutzeri YC-YH1 also showed efficient degrading capacity to broad range substrates, which has great degradation efficiency for organophosphorus pesticides, pyrethroid pesticides, and some PAHs. Metabolites from chlorpyrifos degradation and components of biosurfactant were identified by liquid chromatography coupled with mass spectrometry, and a possible degradation pathway was deduced.This study sequenced the genome of the strain YC-YH1 using Illumina Hiseq2000 sequencing platform, and carried out bioinformatics analysis for genomic data. Genome analysis showed that strain YC-YH1 has organophosphate degradation enzyme genes mpd and ophc2 and contains genes coding for flagellum, bacterial chemotaxis, the enzymes involved in naphthalene and phenanthrene degradation. These results were consistent with the identification of degradation characteristics of strain YC-YH1 which was observed in our previous study. Strain YC-YH1 contains genes which code for the protein secretion system and biological membrane protein and possesses special potential to metabolize pollutants and for bioremediation applications. The study provided an insight into the metabolic pathway and regulation network of exogenous compounds degradation in strain YC-YH1.Two-dimensional fluorescence gel electrophoresis analyzed the differential expressed proteins of Pseudomonas stutzeri YC-YH1 in chlorpyrifos stress and normal conditions. 377 differential expressed proteins were observed according to the abundance comparison and statistical analysis, in which 113 were increased and 264 were decreased. MPH and OPHC2 were induced in strain YC-YH1 under the condition of chlorpyrifos stress. At the same time, 20 of genes which involved in pesticide response were validated by real-time fluorescence quantitative PCR at the transcriptional level. The results of validation showed that the expression trends of 18 genes were consistent with proteomic results, however, the expression trends of other two genes were opposite.The 42 genes encoding Lys R type regulatory proteins were cloned from the genome of strain YC-YH1, and these proteins were expressed and purified. Biacore T200 biological macromolecules interactions system was used to filter regulatory proteins for mpd gene, and regulatory protein Lys R1661 which interacted with promoter sequence of mpd gene was elected. Electrophoretic mobility shift assayby was used to verify the specially binding between regulatory protein Lys R1661 and promoter sequence of mpd gene.