Construction Of An Inducible Promoter And Mechanism Of Acid Resistance In Acidithiobacillus Caldus

Acidithiobacillus is an obligate aerobic,acidophilic,rod-shaped,chemoautotr-ophic Gram-negative bacteria widely distributed in acidic mine environments.Acidithiobacillus is an important model microorganism in geological microbiology and environmental microbiology,and it is also the core dominant functional flora in biometallurgy and other industries.Sulfuric acid is one of the important products produced by the metabolism of Acidithiobacillus,and the low pH caused by sulfuric acid is also one of the huge environmental pressures for its growth.Therefore,the adaptability of Acidithiobacillus to low pH directly determines its application potential.In Acidithiobacillus,the current research on the mechanism of acid resistance mainly focuses on the transcriptional regulation dominated by ferric uptake regulator(Fur),but ferrous iron transport protein B(FeoB)is directly regulated by Fur.The role of the FeoB protein in acid resistance is rarely studied.What role FeoB plays in acid resistance as a functional protein is of great significance for studying the response process of Thiobacillus acidophilus under acid stress,which is helpful for strain transformation and development Utilize,expand the scope and efficiency of its industrial application.Inducible promoters are important tools for microbial molecular biology and synthetic biology research,but the lack of inducible promoters in Acidithiobacillus currently hinders the development of research work in Acidithiobacillus.The inducible promoter is of great significance for the study of the molecular mechanism of this kind of microorganism and the transformation of synthetic biology.In conclusion,an inducible promoter suitable for Acidithiobacillus caldus was constructed in this paper,and the regulatory function of Fur was studied by using this promoter,and the functional study of FeoB under acid stress was carried out.The main research contents include the following aspects:First,an IPTG-inducible promoter was constructed in Acidithiobacillus caldus for the first time.The inducible promoter was constructed on the broad host plasmid pJRD215,containing the lacl gene encoding the repressor in the artificially optimized lactose operon and the modified Ptac promoter,and the firefly luciferase gene was used as the reporter gene.Different types of inducible promoters were constructed according to the positional difference of the repressor protein lacI binding sequence(lacO).The position of the lacO sequence in the Ptac(Ⅰ)type promoter between the-35 region and the-10 region of the tac promoter;the position of the lacO sequence in the Ptac(Ⅱ)type promoter after the transcription start site of the tac promoter;The lacO sequence in the Ptac(Ⅰ&Ⅱ)type promoter exists in the above two positions at the same time.The induction of different types of promoters under different IPTG concentrations was studied in E.coli,and it was found that Ptac(Ⅰ&Ⅱ)was the promoter with the lowest basal expression and 1.0 mM was the optimal induction concentration.The induction function of different types of promoters was studied in A.caldus,and it was found that 1.0 mM IPTG was used for induction on the 5th,9th,and 14th days of A.caldus growth.The 9th day was the best induction time and Ptac(Ⅰ&Ⅱ)had the best induction and expression effect,which was the most suitable inducible promoter in A.caldus.Second,the regulatory function of Fur protein in different A.caldus growth stages was studied using inducible promoters.The pJRD215-lacl-Ptac(Ⅰ&Ⅱ)-fur-luc plasmid was constructed by using an inducible promoter,and the plasmid was conjugated and transferred into Afur,which was induced with 1.0 mM IPTG on the 5th,9th,and 14th days of growth,respectively,and the fur gene were relatively increased by 154-fold,85-fold and 42-fold,respectively,indicating that the expression of fur could be significantly induced by adding IPTG at different periods.On the fifth day,fur-induced expression had little effect on gene transcription levels in iron metabolism,energy metabolism,cell chemotaxis,biofilm synthesis,and oxidative stress;Inducible expression of fur on day 9 caused down-regulation of genes in cell motility,biofilm synthesis and protection against oxidative stress;On the 14th day,furinduced expression caused cell chemotaxis-related genes cheY,membrane synthesis-related genes pelB,pelD,wbeA,bcsZ,etc.,and oxidative stress-related genes showed up-regulation.In conclusion,by using the inducible promoter in different growth stages of A.caldus,the regulation of Fur-induced expression on gene expression was studied,and it was proved that the use of IPTG inducible promoter can efficiently study the regulation of gene expression in A.caldus.Third,the AfeoB&fur double-knockout strain was obtained by using the traceless knockout technology,and the growth characteristics of different strains of WT,Δfur,AfeoB,ΔfeoB&fur under acid stimulation were studied.WT,Δfur,ΔfeoB,ΔfeoB&fur were cultured with elemental sulfur,and the pH of the medium was adjusted to about 0.5 on the second day.Growth analysis showed that the deletion of feoB gene had little effect on growth compared with wild-type strains under acid stimulation;ΔfeoB&fur strains had no effect in the early stage of growth,but showed a significant decrease in growth in the middle and late stages of growth,suggesting that the absence of fur and feoB at the same time would have adverse effects on the acid tolerance of the strain;Compared with Afur,the growth of ΔfeoB&fur was significantly better than that of Δfur in the middle and late stages of growth,The results showed that the deletion of feoB gene on the basis of fur deletion would improve the acid tolerance of the strain.Fourth,the transcriptomic studies of WT,Δfur,ΔfeoB,ΔfeoB&fur under acid stimulation were carried out,and the role of FeoB in acid tolerance was systematically analyzed.Compared with WT,other genes with the same function,such as tonB,tolQ,exbD,etc.,were up-regulated for functional compensation when feoB was deleted alone,indicating that iron ions play an important role in the process of acid tolerance.Compared with WT and ΔfeoB&fur,the genes related to iron transport were down-regulated in AfeoB&fur strains,resulting in downregulation of most proteins that use iron as a cofactor,including processes such as DNA replication and repair,pigment assembly,and sulfur metabolism,which is not conducive to bacterial tolerance to extremely low pH environments.Compared with Δfur,ΔfeoB&fur related genes encoding membrane components(glycosyltransferases,channel proteins,proton pumps,transporters)were up-regulated in transcription.When fur is absent,genes for flagellar assembly and sulfur metabolism are significantly up-regulated,indicating that when fur is absent,bacteria are more inclined to escape the acid stress environment through flagellar movement,and when fur and feoB are simultaneously absent,bacteria may be more inclined to strengthen Cells’ self-defense against damage caused by low pH.The lack of fur in Afur may lead to an increase in the intracellular iron content and the production of more hydroxyl radicals leading to intracellular oxidative damage,while in ΔfeoB&fur,the level of intracellular iron is reduced and the transcription of related genes in the glutathione system is up-regulated.It may make the intracellular oxidative damage smaller than that of Δfur,so the growth condition of AfeoB&fur is better than that of Afur.Iron is an important metal ion,and its reduction will affect some basic metabolic processes of cells,but excessive amounts can also adversely affect cells.Strict monitoring of iron transport is crucial for microorganisms.