| Bioleaching refers to a technology that uses the microorganisms or their metabolic reaction products to chemical oxidation or biological oxidation of finger ore to immerse insoluble metal compounds in the ore into solution in the form of ions.Acidithiobacillus ferrooxidans(A.ferrooxidans)is a widely studied model strain in the bioleaching process.It is a chemolithoautrophic bacteria.It can grow by oxidizing Fe2+ or reducing sulfur to obtain energy.It is widely used in acid mines and environments containing iron or sulfur,and is widely used in the fields of biological leaching,industrial metal recovery,heavy metal pollution biological treatment,environmental purification,and biomedicine.Quorum sensing(QS)is a communication process.When the cell density and species around the microbial community change,bacteria can change their behavior collectively,bacteria can change gene express at low cell density(LCD)and high cell density(HCD).Studies have found that quorum sensing systems are widely distributed in Gram-negative bacteria and play an important role in the regulation of many biological processes,such as:biofilm formation,virulence factor secretion,bioluminescence,antibiotic production,spore formation,and DNA uptake.Studies have shown that there are two density sensing systems in A.ferrooxidans.One is a typical LuxI/R type,including the functional genes afel,afeR,and unknown functional genes orf3.afel encodes AHL synthase to synthesize AHL signal molecules.afeR encodes the AHL signal molecule receptor protein,which is a transcriptional regulator of the LuxR protein family;the second type is an atypical QS system,and the glyQ,glyS,gph and act genes are located in the same operon,which respectively encode glycyl-tRNA synthetase ?,? subunits,phosphatases,acyltransferace.However,the mechanism of action and the regulation mechanism are not clear.Therefore,to further study the regulation mechanism of QS in A.ferrooxidans,in order to improve its metabolic regulation network,understand the mechanism of QS system in this type of extreme bacteria,clarify Its mechanism for adapting to extreme metallurgical environments is of great significance..In this paper,we research two genes afe R and gph in A.ferrooxidans QS,study their functions in the QS system and their role in environmental adaptation to A.ferrooxidans,and their potential for industrial applications.The main research contents include:TTT(1)Study on the transcription level of A.ferrooxidans QS gene and promoter under different energy conditions.The promoter of each gene in QS was fused to the gusA reporter gene,and the activity of the promoter of the QS gene in different environments was detected by detecting GusA enzyme activity and RT-qPCR.Studies have shown that under different energy conditions,the transcription level of afel gene is at the top of each gene.In addition,compared with Fe2+ energy,afeI has a higher transcription level under S0 energy.(2)Functional study of afeR gene in A.ferrooxidans QS.A.ferrooxidans AQS I knockout strain established in our laboratory was used to construct afeR knockout strain(?afeR).At the same time,afeR gene overexpression engineered strain WT(pJRD215-Ptac-afeR)was constructed.The characteristics of afeR knockout,overexpression strains under different energy conditions,growth curves,energy metabolism,EPS synthesis,and biofilm formation were determined and RT-qPCR verified the transcriptional changes of genes in related pathways,and found that overexpressed afeR gene can positively regulate cell growth and energy metabolisI under So energy conditions,promote EPS synthesis and biofilm formation,and inhibit iron oxidation ability under Fe2+ energy conditions.(3)Exploration of gph gene function in A.ferrooxidans QS system,Using the gene-free knockout method constructed in the laboratory,a knockout strain(Agph)and an overexpression strain(WT(pJRD215-Ptac-gph))of the gph gene were constructed.Measurement and analysis of the growth curve,energy metabolism,heavy metal resistance and other characteristics of gph mutant strains under different energy conditions,and the transcriptomics method to study Agph and(WT(pJRD215-Ptac-gph))when S0 is used as an energy source.The study found that gph gene express can promote the growth of A.ferrooxidaUs cells under SO energy conditions,and can enhance the resistance of Cu2+ heavy metals.When stimulated by a specific copper ion concentration,it can promote the expression of copper resistance genes to resist heavy metal stress.Analysis of transcriptome data found that gph gene has an important role on A.ferrooxidans.and it is associated with bacterial secretion system,two-component system,and ABC protein transport,the biosynthesis of lipopolysaccharide,and the pathway of oxidative phosphorylation are closely linked.(4)In summary,this paper has comprehensively studied the biological functions of A.ferrooxidans QS system receptor protein gene afeR and phosphatases gene gph,opening up new ways for further elucidating QS regulatory mechanism and regulatory network and providing important Data support. |
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