Basic Study On The Electron Transport Chain Of Ferrous Oxidation In Acidithiobacillus Ferrooxidans

Acidithiobacillusspp.are chemoautotrophic bacteria.These bacteria can oxidize iron or sulfur in crustal ores to obtain the energy required for growth and metabolism in the presence of trace amounts of O2.The generated Fe3+can be used as an oxidant to precipitate metals in ores.Based on this characteristic,Acidithiobacillus spp.are regarded as the dominant strains in bioleachingAcidithiobacillus ferrooxidans,which belongs to the genus Acidithiobacillus,has the ability to metabolize sulfur and ferrous iron.It is a typical representative of ferrous oxidizing bacteria（FeOB）.The ferrous oxidation model predicted in A.ferrooxidans ATCC 23270 is a bifurcated electron transport chain divided into"uphill" and "downhill" branches involved in the ferrous oxidation process.The uphill pathway encoded by the rus operon accepts electrons from the oxidation of Fe2+to Fe3+in the external environment.The cytochrome c protein Cyc2,as the first acceptor,is crucial for the oxidation process.The electrons are transferred to O2 through a series of electron transport proteins and ATP are finally generated as a result.The downhill pathway is encoded by the petl operon.Small amount of electrons obtained by Cyc2 are transferred along this electron transport chain at the Rus branch point,and finally generate the reducing force required for cell growth and metabolism.The model provides a reference for studying the ferrous oxidation mechanism.However,the model is still lacking experimental verification due to the difficulty of genetic manipulation in this bacterium.The efficiency of biological metallurgy depends largely on the concentration of Fe3+and the low oxidizing ability of ferrous iron is currently an important rate-limiting step in bioleaching.Therefore,further study on the ferrous oxidation metabolism mechanism of A.ferrooxidans to clarify the key proteins in this metabolism can lay a theoretical foundation for the construction of engineering bacteria with higher ferrous oxidation efficiency.In order to study the ferrous oxidation mechanism of A.ferrooxidans,the comparative genomics was firstly carried out between A.ferrooxidans ATCC 23270,which can oxidize both reduced inorganic sulfur compounds（RISCs）and ferrous iron,and A.thiooxidans ATCC 19377,which can only oxidize RISCs,to explore the functional genes related to ferrous oxidation and to improve the understanding of the ferrous oxidation mechanism.Then,the two operons of rus and petⅠ were studied by constructing recombinant plasmids pJRD215-ΔSm-Orus and pMSD1-Opetl respectively based on broad host range plasmids pJRD215-ΔSm and pMSD1,and transferring into A.ferrooxidans ATCC 23270 and A.thiooxidans ATCC 19377 by conjugation;respectively.The results showed that the overexpression of rus operon or petⅠ operon in A.ferrooxidans could enhance the ferrous oxidative capacity and growth status of A.ferrooxidans,indicating that the two operons play important roles in the ferrous metabolism of the bacterium.It was also detected that the heterologous expression of rus operon or petⅠ operon in A.thiooxidans could not endow the strain with the ability to grow with ferrous iron as the sole energy source,but could promote the growth of A.thiooxidans with sulfur powder as the energy source.And the promotion on growth was more apparent when Fe2+ was added.What’a more,the decreased concentration of Fe2+could also be detected by the heterologous expression of rus operon in A.thiooxidans when sulfur powder and Fe2+were used as energy sources,indicating that the rus operon could enable A.thiooxidans have the ability to oxidize Fe2+.Although the petⅠ operon could not enable the strain to directly obtain the ability to oxidize Fe2+,it was likely helpful for the growth of A.thiooxidans on sulfur powder.Therefore,the role of rus operon in ferrous oxidation is more important,and it is necessary to construct A.thiooxidans engineering bacteria containing both rus and petⅠ operons,and to test their ferrous oxidation ability in the future.In the previous work,the gene cyc2 encoding the first receptor protein Cyc2 in the electron transport chain was knocked out in A.ferrooxidans ATCC 23270.But the A.ferrooxidans Acyc2 mutant still had the ability to oxidize ferrous iron,and the expression of AFE₁428 was found significantly up-regulated.Since the extracellular membrane protein Cyc2 is the only first electron acceptor discovered in the bacterium and is responsible for transferring electrons to the Rus protein in the periplasm,AFE₁428 protein was hypothesized to have the same function as Cyc2,but the evidence is lacking.In this paper,the protein structures of AFE 1428 and Cyc2 were firstly compared,and a significant high similarity was found in the two proteins.Both have a conserved heme A site,and can form a tertiary structure with a β-barrel structure similar to the porin domain.Then,the proteins of AFE₁428,Cyc2 and Rus were heterologous expressed in E.coli and the purified proteins from E.coli were characterized by in vitro Pull-down experiments.The results showed that both Cyc2 and AFE₁428 could interact specifically with Rus in vitro,respectively.Provides support that AFE₁428 protein could probably replace Cyc2 as the first electron acceptor in A.ferrooxidans Δcyc2 knockout mutant.Firefly luciferase（Luc）is widely used as a reporter gene in the study of gene expression and regulation.but no application of Luc has been reported in A.ferrooxidans.In this paper,the P1428 and PIPII promoters from AFE₁428 and cyc2,respectively,were selected to construct promoter-luc cassettes on a promoter-detecting plasmid and the luciferase activity expressed on the plasmids in E.coli and A.ferrooxidans were determined,respectively.The experimental results showed that the luciferase could be used as a reporter gene in A.ferrooxidans cultured by using sulfur as energy source.Due to the inhibition of luciferase activity by Fe2+,the application of luc in A.ferrooxidans cultured by using Fe2+as energy source was limited.Moreover,P1428 and PIPII promoters were detected to have high activity in A.ferrooxidans,so they can be used as candidate promoters for the efficient transcription and expression of exogenous genes in this bacterium in the future.In conclusion,the functions of the rus operon and petⅠ operon were characterized in this paper,by their overexpression in A.ferrooxidans and heterologous expression in A.thiooxidans,respectively.It was verified that AFE₁428 protein could interact with Rus protein like Cyc2 by the in vitro Pull-down experiment.The firefly luciferase reporter gene can be applied in A.ferrooxidans cultured by using sulfur powder as energy source.PIPII and P1428 can be used as candidate promoters for high expression of genes in A.ferrooxidans.