Study On Ferrous Oxidation Related Genes And Their Functions In Acidithiobacillus Ferrooxidans.

Acidithiobacillus ferrooxidans is a strictly obligate aerobic autotrophic Gram negative bacterium with an optimal growth temperature of 30? and an optimal growth pH range of 2.0-3.0.The bacterium obtains energy from the oxidation of Fe2+and the reduced inorganic sulfide,to fix carbon dioxide and nitrogen,and synthesize sugar,protein,nucleic acid and other substances needed for its own growth.A.ferrooxidans is a kind of acidophilic inorganic autotrophic bacterium.It is a dominant strain in the biometallurgical industry.It has been widely used in the fields of biometallurgy,biological desulfurization and sulfur-containing wastewater treatment.As a model organism for biological leaching,the whole genome sequencing of A.ferrooxidans ATCC 23270 has been completed,and its genome information has been published and available on the TIGR website and NCBI website.Therefore,the biological research data of this bacterium are the most detailed among that of all leaching microorganisms.Among various biometallurgical microorganisms,A.ferrooxidans plays an important role in bioleaching due to its unique energy metabolism system,which uses ferrous and sulfur compounds as energy sources,especially its ferrous oxidizing ability.In-depth study of the ferrous oxidation-related genes and their functions of the bacterium will help to improve its ferrous oxidation ability of and make it better used in application.Therefore,the study has the dual significance of theoretical research and application.The ferrous oxidation ability is mainly controlled by its ferrous oxidation electron transport chain.Through the overexpression of some genes of the rus operon of the bacterium,the key functional genes affecting the ferrous oxidation electron transport chain were analyzed.The results showed that cyc2 and rus genes play a key role in the electron transport pathway of A.ferrooxidans.The results also showed that AFE 1428 and AFE₁429 genes were closely related to the overexpression of cup gene.Overexpression of the cyc2 and rus genes in the rus operon could significantly increase the ferrous oxidation capacity,indicating that both cyc2 and rus are probably the key rate-limiting sites of the ferrous oxidation electron transport chain of the strain.The bioinformatical analysis of the genome and transcriptome of A.ferrooxidans ATCC 23270 was further carried out,focusing on the study of the first receptor protein coding gene of the electron transport chain,namely the first key gene cyc2 of the rus operon.Cyc2 may play an important regulatory role in biological pathways such as hydrogen sulfide metabolism,sulfate assimilation,and protein synthesis in A.ferrooxidans ATCC 23270.Among them,based on DEG analysis,reaction characteristics,sequence alignment and evolutionary relationship analysis,AFE₁428 was selected as the candidate gene of the first electron acceptor of the electron transport chain of Fe2+ oxidation.It could be used as an important target for studying the ferrous oxidation,as well as for subsequent functional research.By comparing the ferrous oxidation curves,growth curves,and qPCR data of related genes of cyc2 and AFE 1428 overexpression strains under different conditions,the results showed that the growth of cyc2 and AFE 1428 overexpression strains using ferrous iron as energy source were similar.The AFE₁428 gene was supposed to have a similar ferrous oxidation effect in the electron transport pathway in A.ferrooxidans,though its ferrous oxidation efficiency was slightly lower than that of cyc2.Subsequently,the cyc2 and AFE 1428 were expressed in E.coli and the ferrous oxidase enzyme activities were successfully detected on the purified proteins by improving the determination method of o-phenanthroline iron measurement method.The results provided evidence that both the cyc2 and AFE₁428 are the coding genes for the first electron acceptor protein involved in the ferrous iron oxidation of A.ferrooxidans ATCC 23270.The results obtained in this paper could theoretically supplement and improve the important metabolic pathway of the ferrous oxidation electron transport chain of A.ferrooxidans ATCC 23270,and further clarify the key coding genes and their functions involved in this process.It could also provide methodological guidance for the construction of excellent engineering bacteria with improved ferrous oxidation metabolism ability for industrial application.