Study Of The Arsenite Oxidation Regulatory Mechanism In Achromobacter Arsenitoxydans SY8

Arsenic (As) is a widespread toxic metalloid element, which exists maily in arsenite [As(Ⅲ)] and arsenate [As(Ⅴ)] in the natural environment. Some microorganisms could oxidize the more toxic As(Ⅲ) to the less toxic As(Ⅴ) through arsenite oxidase (AioA/AioB) resulting in detoxification for itself. The oxidation process is confirmed to be regulated by the two-component signal transduction system AioS/AioR and arsenic-binding protein AioX, both upstream from aioA/aioB genes in Agrobacterium tumefaciens5A, however it is not yet clear how the As(Ⅲ) signal is transducted to AioS/AioR from AioX, and eventually initiates the expression of aioA/aioB. Thus, this research mainly studies the function of aioX gene in arsenite oxidation process, the As(Ⅲ) signal transduction, as well as the feasibility of quorum sensing involved in this process in the arsenite-oxidizing bacterium Achromobacter arsenitoxydans SY8.This project studies the arsenite oxidation regulatory mechanism in A. arsenitoxydans SY8via multiple approaches, including gene knockout/complementation and protein-protein interaction. Gene knockout experiment indicated that aioX disruption (refer to SY8△aioX) could not lead to the loss of arsenite oxidation capability for A. arsenitoxydans SY8, nevertheless, the mutant SY8△aioX spent approximately40h more to completely oxidize the600μM As(Ⅲ) in the same condition, compared to the wide type strain. After the mutant was complemented with aioX, its capability of arsenite oxidation was completely restored. These above findings showed that aioX played a critical role in regulating the arsenite oxidation process. The yeast two-hybrid protein-protein interaction system was used to explore the potential signal trasduction roles of these proteins in this process. The results showed that there was a weak interaction present between the AioX and the periplasmic domain of the transmembrane histine kinase AioS, but a strong interaction present between the cytoplasm domain of AioS and the response regulator AioR. Taken together, we could infer that A. arsenitoxydans SY8may employ the AioX to bind the periplasmic As(Ⅲ) signal, then transmits the signal to inside by interacting with AioS, and finally initiats the expression of aioA/aioB using AioS/AioR through the typical TCS pathway. However no apparent evidence was found that quorum sensing mechanism participating in regulating the process of arsenite oxidation in A. arsenitoxydans SY8.This thesis elaborated the arsenite oxidation regulatory pathway in A. arsenitoxydans SY8through molecular biology methods and identified the essential role of AioX in regulation of arsenite oxidation process. We firstly proposed that the three-component system AioX/AioS/AioR regulates the arsenite oxidation by protein-protein interaction, meanwhile the robust results suggested that except the AioS/AioR pathway, there may be other regulatory pathway involved in regulation of arsenite oxidation in A. arsenitoxydans SY8.