Ferric Uptake Regulator(AcFur) Participates In The Extreme Acid-resistance Of Acidithiobacillus Caldus

Acidithiobacillus caldus(A.caldus)shows high-efficiency sulfur oxidation ability and extreme environmental(low pH,high concentration of heavy metals)adaptabilities It has attracted wide attention,because A.caldus has wide application prospects and great industrial application value for bioleaching and bioremediation.According to related research,one of the main challenge for A.caldus industrial application is the extremely low pH environment in the late bioleaching process.It will strongly inhibit its physiological activity and seriously affects its growth and reproduction.Therefore,studying the interaction between A.caldus and extremely acidic environment and clarifying the mechanism and acid-resistance model of A.caldus are important for understanding the environmental adaptability and ecological behavior of A.caldusCompared with the acid-resistance model of neutralophilic bacteria,the acid-resistance model of acidophilic bacteria has both the same acid-resistance systems and its unique acid-resistance mechanisms.In our lab,earlier study found that the transcription level of ferric uptake regulator(Fur)increased about 3 times,under acid stimulation(pH=0.5),suggested that Fur plays a key role in the acid tolerance response of A.caldus.Based on the lab research and previous papers,this paper performed research on Fur of A.caldus in three aspectsFirst,the firefly luciferase reporter system was first developed and in situ studied the expression level of Fur under acid shock.The results showed that the fluorescence intensity increased sharply during acid shock;the growth curve showed that the deletion fur gene caused A.caldus to exhibit acid-sensitive phenotypic characteristics.Moreever,the growth curve of complementation and overexpression strains also showed corresponding phenotypic characteristics.The above experiments revealed that the fur gene of A.caldus responds to acid shock and participates in the acid-resistance of A.caldus.Next,based on the RN A-seq analysis of wild-type(WT)and mutant(?fur)strains under acid shock.Compared with the WT strain,a total of 302 genes were differentially expressed in ?fur,including 214 up-regulated genes and 88 down-regulated genes KEGG enrichment of differentially expressed genes(DEGs)indicates that some physiological metabolic pathways are affected by the deletion fur genes in A.caldus,including bacterial chemotaxis,flagellar assembly,sulfur metabolism,nitrogen metabolism,and two-component system,etc..RNA-seq suggested that Fur could affect the physiological behavior of cells by regulating the expression level of some genes under acid shock.Then,the DNA pull-down sequence was designed and tested for the first time-the direct binding site of Fur in A.caldus was studied in vitro.Based on RNA-seq and DNA pull-down seq,the Fur-based acid-resistance network in A.caldus was reconstructed.This work verified the feasibility of the DNA pull-down seq experiment scheme.It would be helpful in the later experiments.Finally,the related genes were discovered from the transcriptome data,and related functions and phenotypes were verified by experiments such as EMSA,RT-qPCR and crystal violet biofilm staining.Moreover,we found that Fur directly regulated iron transport and indirectly regulated Pel system in A.caldus.Ultimately,universality and conservation of Fur in acidophilus bacteria were analyzed by bioinformatics.The analysis showed that Fur was widely distributed in acidophilus bacteria and the amino acid sequence was highly conserved.At the same time,Fur of A.caldus was predicted and identified the key regulatory sites.Expression of Fur protein,EMSA,mutant gene knockin,RT-qPCR,and growth phenotype analysis were used to determine the relationship between iron transport and acid resistance,and further explore the possible involvement regulatory pathways of Fur in vitro and in vivo.In summary,the importance of Fur in A.caldus response and adaptation to acidic environments was identified through fluorescence intensity and phenotypic analysis under acid shock conditions.Bioinformatics analysis found that Fur is ubiquitous in acidophilus bacteria and the amino acid sequence is highly conserved.The close correlation between key regulatory sites of Fur and acid tolerance were identified through multiple sequence alignment,site-directed mutagenesis,and biochemical analysis.RNA-seq and DNA pull-down seq data indicate that Fur participates in the extreme acid resistance of A.caldus by regulating the expression of some key genes in cell physiological activities,such as iron transport,biofilm formation,sulfur metabolism,bacterial chemotaxis and biosynthesis of flagella genes.In this work,Fur-based acid-resitence regulation strategy in A.caldus and the ecological behavior of A caldus under low pH conditions was explained.This study provides new insights into the adaptation of acidophilic bacteria to acidic ecosystems.The study of Fur protein function would promote the design and development of environmentally adapted engineering biological systems of acidophilic bacteria.