Excavate And Study On Elemental Sulfur Metabolism Genes In Acidithiobacillus

Acidithiobacillus is an acidophilic,autotrophic,gram-negative bacterium of Proteus.Strains in this genus can efficiently oxidize various reduced inorganic sulfur compounds（RISCs）and elemental sulfur,some populations also have the ability to oxidize ferrous.Acidithiobacillus has broad application prospects in fields such as biometallurgy and heavy metal pollution treatment due to its efficient sulfur oxidation ability and adaptability to extreme environments.Among them,the research and application of Acidithiobacillus ferrooxidans,Acidithiobacillus thiooxidans,and Acidithiobacillus caldus are the most extensive and in-depth.As an important energy substance of Acidithiobacillus,elemental sulfur plays an important role in the growth and reproduction of microorganisms.However,due to the complexity of its elemental sulfur oxidation process,the specific intracellular metabolism process and genes involved in elemental sulfur metabolism have not been defined.Therefore,exploring the elemental sulfur metabolism genes in Acidithiobacillus is of great significance for exploring the mechanism of sulfur metabolism and completing the sulfur metabolism network of Acidithiobacillus,as well as for the transformation of engineering strains in industrial applications.A.ferrooxidans contain two sets of energy systems,sulfur oxidation and ferrous oxidation,which are important for the study of sulphur oxidation.This study was conducted by starting from A.ferrooxidans,a systematic comparison and identification of the elemental sulfur metabolism process in the Acidithiobacillus genus were conducted from the following four aspects:First,the transcriptomics and proteomics studies of A.ferrooxidans using elemental sulfur or ferrous as the sole energy source were carried out,analyzing from RNA level and protein level respectively.A.ferrooxidans were cultured in elemental sulfur or ferrous medium until the logarithmic metaphase,and the cells were collected for multi-group comparative study.RNA-seq transcriptome study showed that there were 1409 differentially expressed genes in cells cultured with elemental sulfur compared with those cultured with ferrous iron,including 689 up-regulated genes and 720 down-regulated genes.The enrichment of differentially expressed gene KEGG involves oxidative phosphorylation of ribosomes and biofilm formation.The up-regulated genes and their functions in sulfur culture were emphasized.It was found that the genes related to sulfur metabolism,such as hdr gene cluster of sulfur metabolism electron transfer chain,were obviously up-regulated Proteomic analysis based on liquid chromatography tandem mass spectrometry（LC-MS/MS）showed that there were 1267 differentially expressed proteins including 788 up-regulated proteins and 479 down-regulated proteins in sulfur culture compared with ferrous culture;KEGG is involved in oxidative phosphorylation system and phosphate hypophosphite metabolism Through the transcriptome and proteomic techniques of A.ferrooxidans in different energy sources of sulfur and iron,the genes related to sulfur metabolism in A.ferrooxidans were preliminarily excavated,which provided favorable conditions and basis for the study of sulfur metabolism in Acidithiobacillus.Second,comparative genomics studies were conducted on typical populations of Acidithiobacillus（A.ferrooxidans、A.thioxidans and A.caldus）,and the genes related to elemental sulfur metabolism in the genus of Acidithiobacillus were discovered in combination with the multi-combination comparison system of A.ferrooxidans under different energy sources.All groups of Acidithiobacillus have the oxidation characteristics of elemental sulfur.And current research suggests that they share the same mechanism of elemental sulfur metabolism.By comparative genomics,it is concluded that there are 1568 common genes in all groups of Acidithiobacillus.The combined transcriptome and proteome of A.ferrooxidans showed that 428 genes expressed differently in both groups of data,and 132 genes were up-regulated in elemental sulfur culture.Protein functional annotation of 132 genes showed 33 annotations as hypothetical proteins including 17 genes ubiquitous in various groups of Acidithiobacillus.The discovery of these expressed differential hypothetical proteins suggests that these genes may be involved in the sulfur metabolism of Acidithiobacillus,which provides an important research basis for further functional verification and revealing the sulfur metabolism of Acidithiobacillus.Third,using IPTG-inducible promoter and constitutive promoter to analyze the effect of elemental sulfur metabolism related genes on the growth and metabolism of A.ferrooxidans by overexpression of elemental sulfur metabolism related genes.Firstly,the applicability of Ptac-modified inducible promoter Ptac（Ⅰ&Ⅱ）in A.ferrooxidans was investigated.The results showed that the promoter activity was increased 12-fold and 25-fold by 1.0 mM IPTG on the fourth and ninth day,respectively.Then,inducible promoter Ptac（Ⅰ&Ⅱ）was used to study the effect of elemental sulfur metabolism gene on different growth stages of A.ferrooxidans:the overexpression strains induced by IPTG were constructed and induced by IPTG on the fourth and ninth days respectively,and the concentrations of AFE₁991 and AFE₂009 were found after induction on the ninth day;the delayed period of AFE 2680 induced expression was shortened;the growth curves of other mutants did not change significantly after induction.In addition,the effect of elemental sulfur metabolism potential genes on the growth and metabolism of A.ferrooxidans was studied by using constitutive promoter Ptac.Five elemental sulfur metabolism overexpression strains were constructed.Compared with control strain A.f（pJRD215）,the final concentration of A.f（pJRD215-Ptac-AFE₁389）was 16.5%higher than that of control strain at 28d OD600;The growth trends of A.f（pJRD215-Ptac-AFE₂587）and A.f（pJRD215-Ptac-AFE₂560）were significantly higher than that of the control strain OD600,which increased by 27%and 25%respectively;A.f（pJRD215-Ptac-AFE 2676）was weaker than the control;A.f（pJRD215-Ptac-AFE₂680）was weaker than that of the control strain in the early logarithmic growth period.The key genes which can significantly affect the growth of elemental sulfur in A.ferrooxidans were successfully identified by the expression of several genes.Fourth,by constructing gene overexpression and knockout strains,the functional verification of elemental sulfur metabolism-related genes shared by Acidithiobacillus species was carried out in A.caldus.Utilizing the excavated 8 common genes related to elemental sulfur metabolism in Acidithiobacillus,8 overexpression strains induced by IPTG were constructed in A.caldus,and the study found that the cell concentration of A.c（pJRD215-Ptac（Ⅰ&Ⅱ）-RS11075）in the post-logarithmic and stationary phases was higher than that of the control strain after induction on the fourth day and the ninth day;the cell concentration of A.c（pJRD215-Ptac（Ⅰ&Ⅱ）-RS01515）in the late and stable stages of logarithmic growth after induction on the ninth day was higher than that of the control strain;the growth curves of the other mutants had no significant changes,and there was no significant difference in the final concentration in the stationary phase.Then,obtained separately ΔA5904_RS11475 and ΔA5904_RS1147 knockout strainusing gene traceless knockout technology,and carride out the growth characteristics of A.caldus wild-type and knockout strains under elemental sulfur culture.It was found that compared with WT,ΔA5904_RS11475 showed decrease in stable growth phase,ΔA5904_RS1147 had no significant difference in the whole growth cycle.The results showed that A5904_RS11075,A5904_RS01515 and A5904_RS11475 had certain effects on A.caldus elemental sulfur metabolism.In summary,through the transcriptomics and proteomics of A.ferrooxidans with sulfur or ferrous as the sole energy source,the sulfur metabolism gene of A.ferrooxidans was preliminarily discovered.By comparing and analyzing its genome with other Acidithiobacillus,we identified genes related to elemental sulfur metabolism in Acidithiobacillus.Finally,through the overexpression and knockout strains of related genes were constructed in A.ferrooxidans and A.caldus,respectively,to study their effects on cell growth and elemental sulfur utilization.Key genes related to elemental sulfur metabolism were identified,and the research results provided new ideas and directions for the study of elemental sulfur metabolism in Acidithiobacillus.