Bioinformatics Analysis And Mutation Of Leptospirillum Ferriphilum Arsenic Resistance System (ARS) Cluster

Recently, the consumption rate of mineral resources was accelerating, and the environmental pollution caused by conventional physical and chemical mining method was very serious, which runs counter to the social requirements of sustainable development today. Under such circumstances, people pay more and more attention to biometallurgy.Leptospirillum ferriphilum is obligate autotrophy iron oxidizing bacterium, spirilla-shaped, motile, flagellate, Gram-negative, and grows best in pH 2.0 and at 40℃. L. ferriphilum is one of the main bacterium used in microbial leaching, and plays an important role in bioleaching because it bears low pH and high oxidation-reduction potential (ORP) and high temperature.However, the drawbacks of slow growth rate, low cell yield, and being sensitive to heavy metals have limited its further applications and hindered the development of bioleaching. So the genetic transformation has become an effective method. Analyzing the genome sequences to identify resistant genes to clarify gene expression and regulation mechanism is the primary problem.Our laboratory conducted the whole genome sequence on L. ferriphilum, using methods of bioinformatics for functional annotation, analysis the relationship between gene and gene product. We find that the L. ferriphilum contain two different arsenic resistant gene clusters. Our research focuses primarily on the large gene cluster which contains functional genes, regulatory genes and a phosphotransferase system. And we conduct the sequence similarity analysis in different bacteria contained resistant genes.Based on the vector pSDU1 and pSDU1-remob which knocked mob gene that is related to conjugation transformation, with the chloramphenicol gene as the reporter gene, pSDU 1-tac-Ars1,pSDU1-remob-tac-Ars1,pSDU1-tac-Ars2和pSDU1-ArsⅠ-S which carrying arsenic resistant gene from plasmid R773 and Leptospirillum ferriphilum was constructed by us. The arsenic resistant plasmids were transferred into E. coli, and the MIC value of E.coli harboring pSDU1-remob-tac-Ars1 was 20mmol/L, which has the higher arsenic resistance capacity, also arsenic resistant gene driven by tac promoter can be highly expressed.