Study On TetH And RpoS Genes In Acidithiobacillus Caldus

Acidilhiobacillus caldus is an acidophilic,thermophilic and chemoautotrophic bacterium in bioleaching and can utilize reduced inorganic sulfur compounds to generate energy,ATP and NADH.It plays a predominant role in bioleaching and its ability to biomine concerns with sulfur metablism and stress tolerance.We choose tetH gene which encodes tetrathionate hydrolase,an important enzyme in sulfur metablism,as research target to investigate its role and function in sulfur metablism.furtherly improving biomining ability.In the same time,we also choose rpoS gene which encodes RpoS.a global sigma regulator,as research target to investigate its role in stress tolerance.Our work contains 3 parts as followings:Firstly,a lot of transcriptomics are needed in research of tetH and rpoS,however.internal control gene is believed of vital improtance for data calibration and calculation.We have identified 6 potential internal control genes named rrs,era,map,alas,rpoc and gapdh by analysis of restored microarray information and articles reference.We cultured wild type in different conditions including various energy and stress and then got RNA to do qRT-PCR to have a Ct value respectively.With the help of 3 software commonly used in internal control genes analysis named geNorm.normfinder and bestkeeper,we successfully ranked 6 genes by stability value and got the best one or combination.Secondly,We do a series of research on tetH whose encode product is TetH,which can hydrolase tetrathionate into sulfate,thiosulfate and other sulfides.S41 pathway combinated by TetH and TQO is responsible for tetrathionate and thiosulfate metablism.We have got tetH knockout strain by markerless knockout technique,furtherly studying on colony characteristic,growth curve,transcriptome and metabolic products.The role of TetH is confirmed by all results above,declaring TetH is a core part of S4I and the only enzyme which can metablize tetrathionate.S4I pathway,a supplement way of thiosulfate metablism,is an important part of sulfur metablism system,but it dosen't play a core role in element sulfur metablism,the product of which is closedly related to sulfur metablism in cytoplasm.In addition,we also do research on RpoS,a alternative global regulator belonging to sigma factor family,which binds to RNA core polymerase to accomplish gene trascription by specific recognition.It is reported RpoS regulate many operons in various stress including stationary,hyperthermia,high omostic,oxidation,low pH conditions.A.caldus is faced up to serious stress including hyperthemia,high omostic,heavy metal,low pH and organic.We established rpoS knockout strain by markerless knockout technique,furtherly measuring growth curves under various stresses.A obvious difference was got under the stress of high omostic,low pH and organic,indicating RpoS regulate some genes in the condition.All these works lay a foundamental basis for furtherly investigating rpoS role in stress tolerance.