Study On The Metabolic Function And Regulation Mechanism Of Sox Sulfur Oxidization System In Acidithiobacillus Caldus MTH-04

Acidithiobacillus caldus（A.caldus）is a Gram-negative chemolithototrophic acidophilic sulfur-oxidizing bacterium,which can grow with various reduced inorganic sulfur compounds（RISCs）as the energy resource such as tetrathionate（S4O62-）,thiosulfate（S2O32-）,sulfite（SO32-）,sulfide（S2-）and elemental sulfur（S0）.A.caldus,as one kind of moderately thermophilic bacteria,the optimum growth temperature is 45℃ and the optimum pH is 2～2.5.It has been widely utilized in bioleaching industry,the biological desulfuration of coal and the treatment of sulfur-containing wastewater.A.caldus has evolved a complicated sulfur-metabolizing network to oxidize a variety of RISCs,and the metabolism of thiosulfate is the key part of sulfur oxidization,which is primarily metabolized through the Sox sulfur oxidization system.There are two sets of the truncated Sox system,lacking Sox（CD）2,in the genome of A.caldus,which are encoded by two gene clusters sox-Ⅰ and sox-Ⅱ.SoxXA,a c-type cytochrome,can medicate the oxidation of SoxYZ by binding the sulfur substrate at the cysteine residue of SoxY protein,thereby reducing the SoxXA.SoxB is a sulfate thiol hydrolase which hydrolyzes the SoxYZ bound sulfur substrate to form sulfate.The regeneration pathway of SoxYZ,the working way and the regulation mechanism of the truncated Sox system has not been reported.Therefore,the study of the sulfur oxidization related gene transcription pattern,growth changes and metabolite changes in the gene knockout strains,on the basis of the gene knockout technology and other molecular technique,is important for the understanding of the mechanism of Sox system function and regulation in A.caldus.Meanwhile,we can know about the sulfur oxidizing and regulatory network,reveal the molecular mechanism of sulfur metabolism,support for the genetic engineering of A.caldus.The study is conducted mainly through the tollowing aspects:1.Bioinformatics and transcription analyses of the Sox system genes in A.caldus MTH-04:The gene order and components of sox-Ⅰ and sox-Ⅱ in A.caldus were analyzed.They all contained the three proteins SoxXA,SoxYZ and SoxB,which are essential for the sulfur oxidation in vitro,and Sox（CD）2 was not found.The same protein in the two sox clusters shared high identities and the same evolution pathway was obtained through the phylogenetic analysis.A σ54-dependent two component system TspR/TspS（A5904₂484 and A5904₂485）was first discovered right upstream of the sox-Ⅰ cluster through bioinformatic analysis,which may regulate the transcription initiation of sox-Ⅰ.The protein TspS was a histidine kinase,the protein TspR was a regulator,and Tsp was short for the two component system（TCS）of sox pathway.The tspSR-soxXAYZB like gene cluster was also found in other sulfur oxidizing bacteria.The transcription level analysis of sox genes in the elemental sulfur and tetrathionate media were performed by RT-qPCR.The sox gene had enhanced expression levels in the tetrathionate media,and the almost opposite trend of sox-Ⅰ and sox-Ⅱ across the growth cycle may indicate the different regulation mechanism and different function of the two Sox system.2.Construction of the mutants of sox genes in A.caldus MTH-04:The method used in the gene knockout was based on homologous recombination.The recombination plasmid pSDUDI::soxB-Ⅱ（UHA+DHA）was constructed firstly,which contained the homologous arm of soxB-Ⅱ and the enzymatic sites of I-Scel.The transformation of the plasmid pSDUDI::soxB-Ⅱ（UHA+DHA）into wild type A.caldus can promote the first homologous recombination to get the single crossover strain,and the transformation of the plasmid pSDU-I-Scel lead to another recombination to obtain the gene knockout strain The deletion of △sox-Ⅱ was on the basis of △soxAX-Ⅱ,and the plasmid pSDUDI::soxYZB-Ⅱ（UHA+DHA）was constructed.The successful construction of the mutant △soxB-Ⅱ and △sox-Ⅱ made the analysis of the gene function possible.A lot of efforts were made to get the mutants of sox-Ⅰ,however,no one succeed,which may indicate the importance of Sox-Ⅰ to the growth of A.caldus.3.Physiological analysis of the mutants in A.caldus MTH-04:The research on the mutant △soxYZ-Ⅱ,△soxXA-Ⅱ,△soxB-Ⅱ and △sox-Ⅱ was conducted through the following aspects:growth properties,transcription analysis and detection of the metabolites by HPLC.The growth curves with the substrate elemental sulfur,tetrathionate and thiosulfate as the sole energy resources were measured,the mutant △soxB-Ⅱ and△soxYZ-Ⅱ had the pretty similar character with the wild strain,while the growth amount of△soxXA-Ⅱ and was significantly reduced to less than a half of the wild strain.RT-qPCR was performed to analyze the transcription levels of sulfur oxidizing genes in the mutant △soxB-Ⅱ,△sox-Ⅱ,△soxYZ-Ⅱ and △soxXA-Ⅱ compared with the wild type.The deletion of and soxYZ-Ⅱ can be made up by the up-regulated of another sox cluster,which was consistent with the same growth curve with the control strain.However,the up-regulated of another sox cluster genes did not compensate for the deletion of soxXA-Ⅱ and sox-Ⅱ,hence the expression of tetH and doxD was up-regulated,which resulted in the transfer of sulfur oxidization from Sox pathway to S4I pathway.Due to the reduction of the energy production,less energy was produced in the mutant △soxXA-Ⅱ and therefore the growth of the strain was affected.HPLC was utilized to detect the consumption of tetrathionate and the production of elemental sulfur.The consumption curve of tetrathionate was in accordance with the growth curve.The deletion oi sox genes caused the reduction of elemental sulfur contents,the order with the successively reduced sulfur content was△soxB-Ⅱ,△soxYZ-Ⅱ,△soxXA-Ⅱ and △sox-Ⅱ.When the strains were cultivated in the thiosulfate media,the wild strain can produce elemental sulfur and the weakened Sox system function could lead to the reduction of sulfur content.Therefore,we proposed that Sox YZ in the truncated Sox system could be regenerated through the accumulation of elemental sulfur by breaking down of the long chain sulfur at the end of SoxY to complete the metabolic cycle of thiosulfate.4.The relationship between sigma factors,sulfur metabolism and environmental stress in A.caldus MTH-04:Sigma factor is an important component for the gene transcription initiation in bacteria.The study focused on the transcription levels of sigma f:actors under different energy resources and different conditions in A.caldus,and the sigma54 overexpression strain was constructed to analyze the relation between Sox system and sigma54.We analyzed the transcription levels of sigma factors with elemental sulfur and tetrathionate as the substrate,the RT-qPCR results indicated that all the sigma factors were detected expression in the two energy resources,and the transcription levels changed at different growth periods,which may indicate that sigma factors were involved in the regulation of sulfur metabolism genes.The stimuli conditions pH,heat,osmotic pressure and copper were selected for RT-qPCR analysis,we found that the house keeping gene sigma factor may be involved in the adaption to the stimuli conditions,and the sigma factor belonging to the same family may participate in different gene regulation in different conditions.Through the analysis,we found that the expression of sigma54 kept relatively stable,and it may not participate in the regulation of stress related genes.To confirm whether the predicted sigma54 dependent two component system TspS and TspR has been involved in the regulation of Sox system,an rpoN overexpression strain was constructed.RT-qPCR analysis of the rpoN overexpression strain revealed that σ54 did function in the regulation of sox-Ⅰ by activating the transcription of sox-Ⅰgenes.5.The σ54-dependent Two-component Systel TspS/R Regulating Sox SysteI in A.caldus MTH-04:The σ54-dependent transcription initiation is characterized by recognition of the conserved-12/-24 region in the promoter and the participant of the activator.The activator binds the upstream activating sequence（UAS）,and it helps in the formation of the open complex between RNA polymerase and promoter DNA,TspR protein,upstream of sox-l,was the regulator of the two component system and meanwhile the activator protein of σ54.We discovered tspSR-sox like gene clusters in A.caldus and other sulfur oxidizing bacteria through analysis of the genome sequences.The overexpression of rpoN can activate the transcription of sox-Ⅰ was analyzed by RT-qPCR.A typical σ54-dependent promoter,P1,was identified upstream of soxX-Ⅰ in the sox-Ⅰ cluster of A.caldus MTH-04.The transcriptional start site（G）and the possible-12/-24 regions（GC/GG）of P1 were determined by rapid amplification of cDNA ends（5’RACE）.Firstly,the binding region of TspR on promoter P1 was confirmed by electrophoretic mobility shift assays（EMSAs）in vitro.Promoters of different sizes and mutations introduced into P1 were fused with gusA gene,and sets of promoter activity analysis plasmids were constructed.The-12/-24 regions（GC/GG）and the upstream activator sequences（UASs;TGTCCCAAATGGGACA）were determined by UAS-probe-plasmids assays in vivo.The high identity of TCS protein and the similar components of the presumed promoter may indicate the similar regulation mechanism in A.caldus and other sulfur oxidizing bacteria.On the basis of the results and the regulation mechanism of σ54-dependent TCSs,a signal transduction and transcriptional control model for the Sox system in A.caldus was proposed for the first time.6.Co-transcriptional analyses and promoter identification of sox-Ⅱ cluster in A.caldus MTH-04:Different from sox-Ⅰ,the genes of sox-Ⅱ were separated into two operon determined by eo-transcriptional analyses.No regulator protein was found near sox-Ⅱ.Hence,it is important to identify the promoters insox-Ⅱ.The selection of promoters was based on the results of co-transcriptional analyses and bioinformatic predictions.Four promoter-probe-plasmids pJRD215-P2515-gusA,PJRD215-P2519-gusA,pJRDZ15-P2523-gusA and pJRDZ15-P2525-gusA were constructed and promoter activity analyses indicated that four promoters all had the transcription activity.Rapid amplification of cDNA ends（5’RACE）was performed to determine the transcription initiation site（TSS）,and the TSS of A5904₂515 and A5904 2519 were determined successfully.We determined the regulatory element of sox-Ⅱ,which may lay foundation for further determination of the concrete transcription mechanism of sox-Ⅱ.