Studies On Stress Response And Biodegradation Of Burkholderia Capecia WZ1 Upon Quinclorac

An approach combining traditional biological methods and modern molecular biological techniques was used for investigating the stress response of strain Burkholderia cepacia WZ1 upon the quinclorac challenge. The effects of quinclorac were tested by the growth curves and response of superoxide dismutase (SOD) of strain WZ1 under different culture conditions . A proteomic technology-two dimensional electrophoresis (2-DE) was used to separate whole cell proteins of the strain and to identify the differential expressed proteins which were induced by quinclorac. The degrading pathway of quinclorac by strain WZ1 was analyzed based on the results of 2-DE, the GC/MS and enzymes activity assay. What's more, the enzymes which play important role in the process of quinclorac degradation were confirmed by using the Northern Blot and RT-PCR techniques. The results obtained from this study may give the reference on the application of remedaration technology remedying the pesticide pollution. The main results of this study are as follows:1. Based on the growth of strain WZ1 on different culture media and the various concentrations of quinclorac in the culture medium, it was found that strain WZ1 needed about 6 hours to adapt itself to the stress from quinlcorac, and then it could continue growing slowly. The concentration changes of quinclorac in the culture medium accorded with the growth of the treated strain WZ1, indicating that strain WZ1 indeed could degrade quinclorac for its growing.2. The superoxide dismutase (SOD) of strain WZ1 in response to quinclorac were analyzed because the antioxidant system can be changed according with the stress from the pollutants. The results showed that the SOD activities of strain WZ1 were different when treated with different concentrations of quinclorac and when cells at the different culture phases treated with a certain concentration of quinclorac. The Native PAGE analysis suggested that the strain could express more plentiful Fe-SOD than the normal level and exclusively express Mn-SOD to form a protection wall for itself away from the redox injury.3. An effective and comprehensive method, 2-DE combining with MALDI-TOF/TOF, was used to study the differential proteins induced by quinclorac. About 500 proteins were separated by 2-DE. Exposure to quinclorac altered the global protein expression pattern of B. cepacia WZ1. A total of 69 protein spots were up-regulated (exclusive appearance or at least two-fold expression), while 32 protein spots were down-regulated (disappearance or at least two-fold decrease). 34 protein spots of which were excised from the gels to identify by MALDI-TOF/TOF, 26 spots of which were from treated group and 8 spots from the reference, respectively. Taking into account their main biological functions, these proteins were classified into six different functional groups as amino acid biosynthesis and metabolism, energy synthesis, protein synthesis, detoxification and protection, transport relative, all of proteins may paly roles in the pathway of quinclorac degradation and some other unknowns.Such as 50S ribosomal protein L9, molecular chaperone DnaK and DnaK-related protein were obviously down-regulated or even disappeared, indicating that quinclorac not only influences the normal physiological activities and growth, but also show adverse impact on biosynthesis processes of strain WZ1. The strain protected itself by over-expressing some detoxification and protection proteins, like Mn-SOD, heat shock protein, DNA repair protein, CutAl divalent ion tolerance protein and so on.4. Two differential porteins-phthalate dioxygenase reductase (PDR) and chlorocatechol 1, 2-dioxygenase (1, 2-CCD) were considered as key enzymes in the degrading pathway of quinclorac. The changes of PDR and 1, 2-CCD activities at different culturing time accorded with the growth of B. cepacia WZ1 and the biodegradation rate of quinclorac. It illustrated that the quinclorac degradation and these enzyme activities were closely related. The key enzyme-1, 2-CCD was comfired by Northern Blot and RT-PCR. In addition, there are three products identified by GC-MS, and combining with the key enzymes in the degrading pathway of quinclorac, the degrading pathway of quinclorac may be presumed as follows: quinclorac is transformed into 3, 7-bichlorid quinoline firstly; 3, 7-bichlorid quinoline is catalyzed by dioxygenase, and then shifts to chlorophthalate via aminotransferase reaction. Secondly, chlorophthalate is transformed into 3-chlorocatechol through PDR, phthalate dihydrofiol dehydrogenase and 4, 5-dihydroxyphthalate decarboxylase. Finally, 1, 2-CCD catalyze 3-chlorocatechol into 2-chloro-cis, cis-muconate via an ortho-pathway, the subsequent metabolite is funneled into the TCA cycle.