Study On The Function Of Quorum Sensing System In Sulfur Oxidation Population Of Acidithiobacillus Spp.

Biometallurgy generally refers to the biological oxidation of ore by using microorganisms in nature,and under the metabolic action of the microorganisms themselves,the conversion of metal minerals contained in the ore into metal oxides is accelerated,which is also called biological leaching Acidithiobacillus spp.is the dominant bacterial group in the biometallurgical process.According to the sulfur and iron metabolism capacity of this type of microorganisms,it can be divided into sulfur-oxidizing populations that can only oxidize sulfur and those that can both oxidize sulfur and iron.Among them,Acidithiobacillus caldus and Acidithiobacillus thiooxidans are the most widely studied and used sulfur oxidizing species,and Acidithiobacillus ferrooxidans are typical iron sulfide oxidation population.Quorum Sensing(QS)is a communication process between cells that allows bacteria to share information about cell density and regulate their gene expression accordingly.The AfeI/R type QS system encoded by the afeI-orf3-afeR operon is widespread in A.ferrooxidans,where afel encodes signal molecule synthetase and afeR encodes transcriptional regulatory protein.AfeI/R type QS system also exists in A.thiooxidans,but some strains such as A.thiooxidans ATCC19377 do not contain this system.In A.caldus,there is no AfeI/R system.Therefore,the distribution of Afel/R type QS system inAcidithiobacillus spp.has significant differences in species and strains.In this study,the analysis of the role of QS systems from different populations in different types of Acidithiobacillus spp.was carried out to reveal the function of QS system in different types of populations,laying the foundation for the establishment of QS system-based control strategies for different populations of Acidithiobacilus spp.The main research content is carried out from the following aspects:(1)The protein structure analysis and comparison of the AfeI/R system of different population types were carried out:the AfeI and AfeR proteins from the QS system in A.ferrooxidans ATCC23270 and A.thiooxidans A01 were analyzed by bioinformatics.The homology is 82%and 85%,respectively,and the homology is high.The analysis of physical and chemical properties showed that AfeI protein is a hydrophilic unstable protein,AfeR protein is an unstable protein,but the AfeR protein derived from A.thiooxidans A01 is a hydrophobic protein,and the AfeR protein derived from A.ferrooxidans ATCC23270 is hydrophilic protein.And to predict its structure,it is found that there are differences in the secondary and tertiary structure predictions.The analysis of AfeI and AfeR in different types of Acidithiobacillus spp.has laid a theoretical foundation for the functional research and application of AfeI/R.(2)Carried out a study on the regulatory effects of AfeI/R systems from different sources in different populations:The artificial synthesis of the AfeI/R system in A.thiooxidans A01 was carried out,and the function of the AfeI/R system in A.thiooxidans ATCC19377 without QS was realized by constructing an overexpression plasmid.It was found that the presence of the AfeI/R system can promote the cell concentration of A.thiooxidans ATCC 19377 in the stationary phase reveals the regulatory effect of the AfeI/R system in A.thiooxidans,indicating that the differences in AfeI/R between different strains of A.thiooxidans can be artificially introduced into the QS system to achieve functional replenishment.A functional study of the AfeI/R system in A.thiooxidans A01 in A.caldus MTH-04 was carried out.It is found that the introduction of AfeI/R into A.caldus without QS system can promote strain growth and EPS synthesis.The results show that AfeI/R from A.thiooxidans can perform biological functions in A.caldus,revealing for the first time that AfeI/R The regulatory effect of the type Q S system on A.caldus indicates that the distribution of AfeI/R among different populations of Acidithiobacillus spp.can be artificially introduced into the QS system to achieve functional empowerment and performance improvement.A functional study of the AfeI/R system derived from A.thiooxidans in A.ferrooxidans was carried out.The AfeI/R system of A.thiooxidans A01 was introduced into A.ferrooxidans ATCC23270,and it was found that it had no significant effect on the growth and metabolism of the strain,indicating that the QS system derived from A.thiooxidans may not perform biological functions in A.ferrooxidans.The regulation function of the AfeI/R system derived from A.ferrooxidans in sulfur oxidized populations has been studied.The AfeI/R of A.ferrooxidans ATCC23270 was introduced into the sulfur oxidizing bacteria A.thiooxidans and A.caldus,and it was found that there was no significant effect on cell growth and flora density.It indicates that the QS system derived from A.ferrooxidans may not perform biological functions in the sulfur-oxidized population of Acidithiobacillus spp.Based on the above results,it shows that the QS system of the sulfur-oxidizing population of Acidithiobacillus spp.and the iron-sulfur-oxidizing population have different mechanisms of action and cannot achieve functional complementarity.(3)Analysis and research of promoters in A.thiooxidans:In order to obtain strong promoters in A.thiooxidans,which can be used in the future synthetic biology modification of AfeI/R system in sulfur-oxidized populations,we carried out the promoters in A.thiooxidans ATCC19377 analysis and study of its expression activity:using bioinformatics technology,many potential promoters were obtained on the chromosome of A.thiooxidans ATCC19377,involving the promoters of related genes in cell sulfur metabolism,carbon metabolism,nitrogen metabolism and other major metabolic processes;a series of different types of promoter detection plasmids were constructed using the luciferase reporter gene;probing the promoter strength of the plasmid in E.coli,found that P2255,P3076,P2290,P0351,P0375,P3203 have strong transcriptional activity;research in A.thiooxidans found that P3203,P0375 and P3076 have strong initiation of transcription activity,among which P3203 is better than tac promoter.The results show that the promoters in A.thiooxidans and E.coli have significant differences in transcriptional activity,and the strong promoters obtained in A.thiooxidans can be used in the future transformation of the QS system.To sum up,this study is based on the research work on the AfeI/R type QS system among different populations of Acidithiobacillus spp.The versatility of AfeI/R in sulfur oxidizing bacteria A.thiooxidans and A.caldus has been discovered.The AfeI/R system derived from A.ferrooxidans has functional compatibility problems with sulfur oxidizing bacteria;the artificial construction of the AfeI/R system can make up for the difference in the distribution of the QS system between strains and populations.Some strong promoters discovered in A.thiooxidans in this study provide important genetic elements for the future QS system synthetic biology modification and the construction of high-efficiency strains.The above research results are of great significance for guiding the construction of a high-efficiency thiobacillus acidophilus artificial flora system based on the QS system,improving the growth and metabolism of the flora and its industrial application value.