Analysis Of Transcriptional Initiation Element And Deletion Of Reverse Gyrase Gene In Sulfolobus Islandicus

Reverse Gyrase (RG) was first identified in hyperthermophiles—Sulfolobus. RG is wide spread in hyperthermophiles and it is the only protein that is conserved in all hyperthermophilic archaea and bacteria. Reverse gyrase probably functions in the stabilization of the genome structure under high temperature. Due to lack of the genetic system, the research of reverse gyrase is limited to the biochemical characterization and physiological study of the hyperthermophilic organisms. In vivo functional analysis of RG was not conducted in crenarchaea due to the lack of a proper genetic system. This research is based on a spontaneous pyrEF mutant of Sulfolobus islandicus E233 isolated in our lab. The pyrEF genes were then used as the selection marker for transformtion. Genetic system was used to study whether the RG gene is essential in Sulfolobus islandicus. In order to do that, the gene knockout plasmid pRG is constructed. After transformed into S. islandicus E233S, the plasmid was integrated into the host chromosome through double crossover, yielding uracil-proficient transformants that formed colonies on the selective plates.A novel gene deletion scheme, designated marker integration and unmarked target gene deletion or MID, was developed for S. islandicus. Moreover, MID involves specific selection at each step of the mutant construction, select double crossover mutants since the insert of M-arm and L-arm and in the counter selection at the second step which specifically kills transformants and only allows mutant cells to grow. which is in strict contrast to other gene knockout methods that infer gene essentiality from mutant colonization capability. To our knowledge, MID represents the first knockout method that allows a direct study of mutant cell propagation.We obtained the reverse gyrase gene deletion mutant through the novel gene deletion scheme at 65℃which was lower than the optimal temperature. S. islandicus can propagate even lack of the RG gene and the RG gene is not essential for S. islandicus’ survival at the 65℃condition at least, but undeniable the RG gene is very important for the stabilization of the genome structure of S. islandicus’survival under high temperature through introduces positive supercoils into covalently closed DNA or other ways independent on the enzyme activity.we testified the great role RG gene played under high temperature condition and provide groundwork for further research.Microorganisms can utilize different kinds of sugars as carbon source and energy source and the genes involved in sugar metabolism are usually highly regulated. The Sso 1300、Sso3117、Sso3118 and Sso3124 genes from Sulfolobus solfataricus P2 can be induced by D-arabinose. Study on the cis-acting DNA element of these genes are very important to know the transcription initiation mechanism of archaea.In order to characterize the c/s-acting DNA elements on these promoters, S. islandicus reporter gene system was employed in this study. In S. islandicus reporter gene system, lacS gene is used as the reporter gene, pZC1 as E. coli-Sulfolobus shuttle vector and S. islandicus E233SΔpyrEF/ΔlacS as host. The promoters contain IE sequences were cloned to the upstream of lacS gene on the shuttle vector and controlled the expression of lacS gene. After transformantion of reporter plasmids into S. islandicus E233S, transformants were obtained and cultured in defined media. The function of IE was identified according to changes of lacS activity from different transformants.The promoter containing IE sequence of Sso1300、Sso3117 and Sso3124 down to 0.94、0.66 and 0.71 times of the former promoter respectively, but the Sso3118 up to 8.3 times of the former promoter due to the high A/T ratio of IE sequence. The lacS mRNA level of S-3118IE was 6.2 higher than S-3118, which indicated the IE was very important for transcription initiation in Sso3118 promoter.