Functional Analysis Of Sulfur Metabolism-related Genes In Acidithiobacillus Caldus

Acidithiobacillus caldus(A.caldus)is a moderately acidophilic,thermophilic,chemolithoautotrophic sulfur-oxidizing bacterium.It has the capability of growing on reducing sulfur energy in acid mine drainage and has been widely used in bioleaching industry for recovering heavy metals and environmental protection for biological sulfur removal.A.caldus has several drawbacks during its application,such as the slow growth and bad environmental adaptability,thus the study of sulfur oxidation mechanism for A.caldus is not only conducive to the genetic engineering of this strain,but also has important implications for its industrial applications.However,in the published sulfur oxidation models of A.caldus,the metabolism of intracellular sulfite and sulfurous acid in A.caldus has not been reported yet.It is of great theoretical and practical significance to study these metabolisms.In this study,the sulfite oxidase SIO(sulfite oxidase)and the sulfite reductase Srx(sulfiredoxin)in A.caldus were discovered,thus the studies about the two enzymes using the knowledge and techniques from bioinformatics,molecular genetics and enzymology were performed to determine the role of SIO in intracellular sulfite metabolism and the role of Srx in the reduction of cysteine sulfinic acid and the conversion of inorganic sulfides to organic sulfides.The study mainly contains the following aspects:1.Analysis of sio and srx in A.caldus MTH-04 using bioinformatics:Through the analysis of the orf1689 and orf1690 protein sequences and domains in A.caldus MTH-04,it was predicted that they encode the sulfite oxidase SIO and the sulfite reductase Srx,respectively.the sio,srx gene clusters and phylogenetic trees were constructed to observe the arrangement of genes and analyze evolutionary relationships.The results showed that the clusters were basically the same in A.caldus,but no obvious alignment rules were found in the same genus,ie,Thiobacillus acidophilus and other strains.The protein sequences from A.caldus strains also showed high homologies.2.Construction of the mutants of sio genes and srx genes in A.caldus MTH-04:According to the markerless gene knockout technique of the A.caldus,the sio and srx knockouted strains of A.caldus MTH-04 were successfully constructed.F urtherly,overexpression strains of WT(p JRD215-PtetH-sio)and WT(p JRD215-PtetH-srx),and the corresponding gene complement strain ?sio(pJRD215)-PtetH-sio)and ?srx(pJRD215-PtetH-srx)were generated to study the gene function.3.Physiological analysis of the mutants in A.caldus MTH-04:For the mutants ?sio and ?srx,as well as the corresponding overexpression engineering strains and replenishing strains,the growth characteristics and gene transcription levels of these strains were investigated.The results showed that the SIO played a role in the high concentration of sulfur powder(1.2 gS0).When the elemental sulfur energy is obviously insufficient,?srx appears to be in the stable stage earlier than the wild strain in the absence of sulfur powder(0.4 gS0),and the growth in the stable stage is only 2/3 of the wild strain.The overexpression strain of srx had higher maximum bacterial concentration then that of wild type.In addition,under the condition that the tetrathionate was the sole energy source,the deletion and high expression of sio and high expression of srx caused the increase of the delay period.However,these strains had the almost the same maximum bacterial concentration at the stationary stage compared to that of wildtype.It was speculated that the loss of SIO led to the disruption of the electronic production of this pathway,affecting the growth of the bacterial cells.The RT-qPCR results showed that knockout of the sio,resulting in significant changes in the expression of major genes in sulfur metabolism,such as the up-regulation of tetH,soxX-?,hdrC,sqr,and sdo.It was speculated that the lack of SIO led to the accumulation of intracellular SO32-and electron reduction,so that accelerated other metabolic pathways.SIO as a metabolic terminal enzyme,its overexpression had little effect on the growth and metabolism of the bacteria.The deletion of the srx resulted in the up-regulation of sulfur-oxidizing genes in the periplasmic space and the downregulation of sulfur-oxidizing genes in cytoplasmic space when the sulfur powder was lack,indicating that Srx is associated with the metabolic pathway of tetrathionate or thiosulfate,and SIO may have a certain link with Srx in metabolic function.4.Expression sio and srx genes in E.coli and purification of the proteins:Based on the A.caldus MTH-04 genome,the sio and srx gene expression vectors were constructed.The heterologous expression of the target genes was completed in Escherichia coli BL21(DE3).The two proteins were successfully expressed in E.coli and the Srx protein was successfully purified using His tag.These results provided the experimental foundation for the studies of their enzyme activities.In this study,the functions of SIO and Srx were studied,their role in the sulfur metabolism network was explored,and the sulfur metabolic system was further analyzed and improved.