The Regulatory Mechanism Of The Tetrathionate Intermediate Pathway ?S₄I?for Thiosulfate Oxidation Pathway In Acidithiobacillus Caldus MTH-04

Acidithiobacillus caldus(A.caldus)is a kind of autotrophic bacterium,which can oxidize the elemental sulfur and various reduced inorganic sulfur compounds(RISCs),and derive energy and reducing power from the course,to complete its metabolism and growth.A.caldus is a common applied bacterium in bioleaching industry,with the optimal 40-50? growth temperature and the optimal 1.5-2.5 pH for its growth.A.caldus possesses a sophisticated and highly efficient inorganic sulfur compound metabolism network,and thiosulfate is an important intermediate metabolite in the sulfur metabolism system,which could be metabolized via the tetrathionate intermediate(S4I)pathway.Therefore,it is very important to reveal the role and regulation mechanism of S4I pathway for understanding the whole sulfur metabolism system and relative regulatory mechanisms in A.caldus.The S4I pathway includes two enzymes,thiosulfate:quinol oxidoreductase(Tqo or DoxDA)and tetrathionate hydrolase(TetH),whose encoding genes are both located in tetH gene cluster.Based on bioinformatics analysis,it was found that there were two genes,rsrR and rsrS,located upstream of tetH gene in the cluster,encoding a two-component regulatory system.Therefore,based on the transcriptional changes of genes related to sulfur metabolism and regulation when A.caldus was cultured with element sulfur,it was induced that the two-component regulatory system RsrS-RsrR might regulate the S4I pathway.To solve the above problems,research was carried out according to the following parts in the work.1.Gene expression profiling for A.caldus in time-course.There would be various sulfur compounds when A.caldus is in the cultivation process with elemental sulfur as energy,along with the activation of related metabolic pathways.To illustrate the situation for transcriptional changes of genes related to sulfur metabolism and regulation,gene expression profiling on the cultivation with elemental sulfur(2,4,6,8 and 10 days)for A.caldus was conducted.The results showed that the genes related to sulfur metabolism and regulation of A.caldus had obvious transcriptional changes,which would provide whole insights and new clues for the overall understanding of sulfur metabolism.Based on the gene expression profiling analysis,it was found that there were two two-component systems in A.caldus,which might be associated with sulfur metabolism regulation.And the two-component system,RsrS-RsrR,might regulate the S4I way in A.caldus.2.To construct the mutant strains for deletion of genes encoding RsrS-RsrR by using non-trace knockout technology.The rsrR and rsrS genes knockout mutant strains were constructed based on the principle of homologous recombination as follow.Suicide plasmids pSDUDI::rsrR(UHA + DHA)and pSDUDI::rsrS(UHA + DHA)were constructed firstly,and transferred into A.caldus MTH-04.Secondly,?rsrR and ArsrS strains were obtained by screening using the primers located in&out homologous arms and in genes,respectively.The obtainment for mutant strains is the most basic and most important step for study on the two-component systems,RsrS-RsrR.3.Growth curve analysis for ArsrR and ArsrS strains.The growth situations for ArsrR and ?rsrS strains were detected in Starkey-S0 and Starkey-K2S4O6 medium,respectively,and the differences compared to the wild type were analyzed.Results showed that there was little difference between the mutants and wild type when cultured in SLarkey-S0 medium.However,?rsrR and ?rsrS had a two-day and four-day growth delay in lag phase,respectively when compared to the wild type when cultured in Starkey-K2S4O6 medium.In addition,the covering strains for the two genes were constructed,and their growth curves were consistent with the wild type.The growth curve analysis for ?rsrR and ArsrS strains is important for deducing the regulatory mechanism for RsrS-RsrR regulating the S4I way.4.Transcriptional expression changes for genes included in tetH gene cluster of A.caldus with the addition of K2S4O6.The S4I way is currently the only clear pathway which could metabolize tetrathionate in the microbes containing sulfur oxidization.And the two-component system,RsrS-RsrR,is likely to regulate the pathway.Therefore,when cultured in Starkey-S0 mediun,K2S4O6 was added as a stimulus.The transcriptional changes for genes included in tetH gene cluster were detected using RT-qPCR.Results showed that the relative RNA transcript levels of these genes in the wild type increased by many folds.However,the data in the two mutants did not show any obvious increase.This result supported the notion that there is a K2S4O6-dependent positive regulation of RsrS-RsrR on the tetH gene cluster.5.Analysis of tetH clusters in A.caldus.In A.caldus MTH-04,the S4I pathway is an important part of sulfur metabolism system.The thiosulfate:quinol oxidoreductase(Tqo or DoxDA)and tetrathionate hydrolase(TetH)included in this pathway exist in a variety of microbes containing sulfur oxidation.Comparison of tetH clusters between A.caldus and other sulfur oxidizers which contains these two enzymes was conducted.And it showed that an unique two-component system,RsrS-RsrR,was located upstream of tetH and doxDA genes in the genera Acidithiobacillus,which is necessary for regulating the complex sulfur oxidation system more orderly and efficient.On the other hand,the potential promoters in the tetH cluster were predicted using bioinformatics analysis.They were verified by detecting the enzyme activity of GusA(expressed using the constructed exogenous verification plasmids),and transcription situation of endogenous gene gusA using RT-qPCR.Otherwise,the promoter regions and transcription start site of gene doxDA were determined by using the cDNA end rapid amplification technology.All these work raised an important significance for understanding the transcription of tetH gene cluster and revealing the regulatory mechanism.6.Study on RsrS-RsrR regulating the S4I pathway.The interaction between the RsrR protein and the promoter sequence of tetH gene was verified using the optimized electrophoretic mobility shift assay(EMSA).On the other hand,a series of detecting plasmids with report gene,pJRD-P360IRS-gusA,pJRD-P148IRS-gusA and pJRD-P90-gusA,were constructed,to verify the interaction between the RsrR protein and the promoter sequence of tetH gene in vivo.It was preliminarily determined that there was a 19 bp reverse repetitive sequence(IRS,AACACCTGTTACACCTGTT)located upstream of tetH gene based on the experiment results combined with bioinformatics analysis,which might be the regulatory sequence for RsrR protein.However,the regulatory sequence was 22 bp,using the DNase I footprint analysis.There are advantages and disadvantages when comparing these two kinds of experimental methods,and it is necessary to determine the accurate regulatory sequence for RsrR by mutating the key sites in the sequence.7.Study on the structures of RsrS and RsrR,and the cross-talk.Based on the evolutionary tree adjacency method(Neighbor Joining...),the sources and classification for RsrS and RsrR were analyzed.In the mean,mathematical modeling for structure and superposition for the two proteins were conducted.It was found that RsrS-RsrR has a highly similarity with another two-component regulatory system,EnvZ-OmpR,which is involved the regulation of osmotic pressure.This result is the important theory basis for studying the co-regulation,or the cross-talk between the two regulatory systems.Combined with the result of the growth delays of mutants cultured in Starkey-K2S4O6 medium,it was deduced that EnvZ-OmpR played the role of RsrS-RsrR when they were absent to ensure the two mutant strains could survive in the medium.Meanwhile,EMSA experiment between several relevant two-component proteins(RRs)and tetH gene promoter sequence was conducted,and Orf2590(OmpR)could also bind to regulatory sequence.On the other hand,some key amino acids included in the HTH domain for the combination with DNA fragments of RsrR protein were analyzed.The results of the analysis and experiments has the vital significance for explaining the growth delays of mutants cultured in Starkey-K2S4O6 medium and perfecting the regulatory mechanism of S4I pathway.8.Transcriptional analysis of genes related to sulfur metabolism and regulation in?rsrR??rsrSThe S4I pathway is involved in the transformation between tetrathionate and thiosulfate,and thiosulfate is the important intermediate metabolite in the sulfur metabolism system,which indicates that there is close relationship between the S4I pathway and other sulfur oxidation pathways.It has been suggested that he two-component system,RsrS-RsrR,could regulate the S4I pathway.Therefore,it is of great significance for detecting transcriptional changes of genes related to sulfur metabolism using RT-qPCR to reveal the relationship between the S4I pathway and other sulfur oxidation pathways.On the other hand,there were growth delays for mutants when cultured in Starkey-K2S4O6 medium.The research results show that it is likely to be with the co-regulation or cross-talk between RsrS-RsrR and other regulators.Therefore,transcriptional changes of genes included in other two-component regulatory system and one-component regulatory system were also detected.The results of RT-qPCR analysis showed that there were influence in transcription of genes related sulfur metabolism and regulation both in ?rsrR and?rsrS.However,ArsrR had significantly greater influence than ArsrS referring both the number of influenced genes and the influenced extent of changes in gene expression.9.The signal transduction and transcriptional regulation model of RsrS-RsrR on the S4I pathway in A.caldus.Based on the experimental results,combining with the bioinformatics analysis,the signal transduction and transcriptional regulation model of RsrS-RsrR on the S4I pathway in A.caldus was proposed.In the S4I pathway,the histidine kinase,RsrS,senses the stimulus coming from tetrathionate,and activates its DHp domain by transferring a phosphoryl group to the conservative His.After the catalytic action of the CA domain,the phosphoryl group is transferred to the conservative Asp located in the REC domain of RsrR.Then the actived RsrR forms homologous dimer,binds to the regulatory sequence located upstream of tetH gene,which activates the RNA polymerase to regulate transcription of the tetH cluster.Then the expressed proteins,TetH and doxDA,enter the periplasmic space to conduct the transformation between tetrathionate and thiosulfate,which referring to the metabolism of sulfur oxide system.In this work,gene expression profiling on A.caldus MTH-04 in time-course was conducted,which helped to learn the situation for transcriptional changes of genes related to sulfur metabolism and regulation.?rsrR and ArsrS strains were constructed using non-trace knockout technology.The positive regulation RsrS-RsrR plays on the S4I pathway was verified both in vivo and in vitro.And the basic regulation model of RsrS-RsrR on the S4I pathway in A.caldus was proposed.The whole work is an innovation for study the regulatory mechanisms included in sulfur oxidization system of A.caldus MTH-04.This helps to construct research strategies and raise new problems and challenges to be solved.