| More and more works to solve the problem of environmental pollution have been recently carried out, among which most are mainly physical and chemical methods. However, the physical and chemical methods in environmental pollution usually include the new problems sucha as high cost and secondary pollutant generation. Compared with these conventional processes, biodegradation offered an alternative method and demonstrated the unique advantages of cost effectiveness, less secondary pollutant, high ecological safety, and human ecosystem stability.Mustard gas or Yperite is the most produced and stored chemical warfare agent in the world. It is also a markedly cytotoxic, carcinogenic and mutagenic alkylating agent. Furthermore, under alkaline condition, it is commonly hydrolyzed and transformed into another toxic compound thiodiglycol (TDG), which thus needs the further degradation. In this paper, the degradation performance of TDG by Burkholderia sp.01S was investigated. Then the factors that may have effects for biodegrading TDG, sucha as the TDG concentrations, pHs, dissolved oxygen, or some other substrates were identified. In order to illustrate the TDG catabolism pathway in Burkholderia sp.01S, the intermediates during TDG degradation were analyzed by HPLC and GC-MS, and two possible degradation routes were proposed. Finally, the proteomic analysis was employed to study the global protein expression profiles of Burkholderia sp.01S. The candidate genes from proteomic analysis would be disrupted to interpret the potential mechanisms underlying TDG biodegradation. Main results are obtained in our current investigation as follows:l.The factors that might have effects on the biodegradation of TDG were determined. In order to determine the optimal concentration of01S degradation TDG, a series of TDG concentration gradient were studied. Our experimental results show that the best growth concentration was3000ppm. Furthermore, the effects of tempretures, pHs, dissolved oxygens, or the other similar substrates were also investigated.2. The possible pathways to biodegradate TDG in the Burkholderia sp.01S was proposed.HPLC and GC-MS techniques were employed to analyzed the metabolic intermediate in the degradation of TDG. Based on the appraisal to the five compounds and in combination with related literature, proposed Burkholderia sp.01S degradation of TDG have two possible metabolic pathways, which involved the breakdown of carbon sulfur bond, the oxidation by adding oxygen or some other chemical reactions.3.By using the method of proteomics to explore the proteins that were potentially involved in the biodegradation of TDG in Burkholderia sp.01S. In this section, iTRAQ was applied to identify the differentially expressed proteins in the glycerol and TDG of Burkholderia sp.01S. A total of180proteins were detected with the ratio above1.5times and the P value less than0.05. Those differentially expressed proteins included124up-regulated proteins and56down-regulated proteins. After analyzing the data of differentially expressed proteins, it was found that three carboxylic acid and glyoxylate cycles should be the main metabolic pathways to supply the energy for degradating TDG. Furthermore, a variety of genes related to transporting and metabolism of amino acids, carbohydrates and energy were involved in this process.4.Since the genomic sequencing of Burkholderia sp.01S has been finished in our laboratory, we compared the up-regulated proteins with the genes of Burkholderia xenovorans LB400. The latter strain has the most closest relative to Burkholderia sp.01S, but it couldn’t degradate TDG Our analysis demonstrated that eight proteins in Burkholderia sp.01S are unique. According to protein function annotation and significant differences of expression, we select BurkGL001217, BurkGL001688, BurkGL002992, BurkGL006539four proteins for constructing the gene knock-out plasmids. Using the method of homologous homologous single-crossover, the four candidate genes were disrupted to verify their functions in TDG degradation.In summary, the innovative includes:1.This is the first time to report that the pathway about TDG biodegradation in Burkholderia genus.2. Two catalytic pathways of TDG degradation were firstly proposed in Burkholderia genus, and we further discussed the potential proteins involved in TDG degradation through proteomic analysis. |
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