Study On The Genes Involved In The Ferrous Oxidation And Sulfur Oxidation In Acidithiobacillus Ferrooxidans

The rational exploitation and utilization of mineral resources is of great significance to our country's economic development.Considering the increasing decrease of non renewable mineral resources and the serious environmental pollution in the process of resource exploitation,the bioleaching technology has become an effective way for the high efficiency and green development of natural resources.Acidithiobacillus ferrooxidans is a kind of extremely acidophilic chemolithotrophic bacterium with two sets of energy metabolism systems.It can live on ferrous ion as the sole energy source,as well as elemental sulfur and a variety of reduced inorganic sulfur compounds.It plays an important role in the biometallurgy and environment protection.However,the low efficiency of energy metabolism limits its wide industrial application.The study of the Fe2+and sulfur oxidation systems of A ferrooxidans is not only theoretically important for its physiology and metabolism,but also the basis for the efficiently industrial application.Therefore,the study on the Fe2+and sulfur oxidation mechanism of A.ferrooxidans to improve its oxidation capacity is undoubtedly theoretical and practical signifcant.A widely accepted electron transfer modle of ferrous oxidation has been available in A.ferrooxidans.The Cyc2 encoded by cyc2 gene was considered as the first electron acceptor,and it is presumed to be the key protein to determine the oxidation efficiency of A.ferrooxidans.However,the cyc2 could be knocked out and the mutant did not lose its capacity of Fe2+ oxidation,indicating that there were other genes to function as cyc2.A candidate AFE₁428 gene was then screened out by bioinformatics and transcriptional analysis,and further studies are needed to prove the function of AFE₁428 gene in the Fe2+ oxidation in A.ferrooxidans.In this paper,the AFE₁428 gene was successfully knocked out from A.ferrooxidans ATCC 23270 wild type,as well as from cyc2 gene knockout mutant,and two AFE₁428 knockout mutants ?AF1428 and ?cyc2-?AF1428 were constructed.The growth capacity and Fe2+oxidizing activity of the knockout mutants were detected and compared with that of the wild type.By using Fe2+as the energy source,the growth capacity of wide type>?AF1428>? cyc2>? cyc2-?AF1428 and the Fe2+ oxidizing activity of wide type>? F1428>?cyc2>? cyc2-?AF1428 were obtained.The results indicated that AFE₁428 was certainly involved in the Fe2+oxidation of A.ferrooxidans as cyc2,but the role of Cyc2 is more important.The transcriptional level of related genes from different strains was analyzed by qRT-PCR.The up-regulation of genes in the downhill electron pathway and the down-regrlation of genes in the uphill electron pathway were detected in the mutant strains,indicating that the knockout of the AFE₁428 or cyc2 gene could increase the production of ATP and reduce the production of reducing force,resulting in the weaken of growth and Fe2+oxidation.The double knockout mutant ?cyc2-?AF1428 did not lose its Fe2+oxidation ability,indicating that other electron transfer pathways or other genes that have similar functions to cyc2 might exist,which need further study.The mechanism of sulfur oxidation in A.ferrooxidans has not been perfectly disolved,and the metabolism of elemental sulfur?S0?is not clear.It was predicted that the heterodisulfide reductase complex?HdrABC?and dioxygenase?SDO?might be involved in the S0 oxidation.In our previous study,the six genes?AFE₂550-2555?encoding HdrABC were detected in hdr operon,which is composed of ten genes.In the upstream of hdrABC cluster in hdr operon,three genes,which were rhd?AFE₂558?,tusA?AFE₂557?,dsrE?AFE₂556?,respectively,were predicted to act as transferase in the S0 oxidation.However,direct evidence is needed to support this conjecture.In this paper,the AFE₂556-2558 genes were successfully knocked out from A.ferrooxidans ATCC 23270 wild type and the knockout mutants ?AF2556-2558 was constructed.The growth property of A.ferrooxidans ATCC 23270 wild type and ?AF2556-2558 were detected and compared by using the sulfur powder and thiosulfate as energy source,respectively.The knockout of AFE₂556-2558 resulted in the obviously decreased growth in the mutant only by metabolising sulfur powder,indicating that the three genes are involving in the S0 oxidation.The transcriptional levels of related genes were also determined by qRT-PCR when sulfur powder was used as energy source.And the transcriptional profiles from ?AF2556-2558 was similar to that from ?hdrABC when compared with that of the wild type.Therefore,AFE₂556-AFE₂558 genes were speculated to be part of HdrABC system and function in the S0 oxidation together with other genes in hdr operon.The function of sulfur transferase encoded by AFE₂556-AFE₂558 remains to be studied further.In summary,the function of AFE₁428 in the Fe2+ oxidation and the function of AFE₂556-AFE₂558 in the S0 oxidation were characterized respectively by the employment of the markerless gene knockout,studying of the properties of the mutants,as well as analysing of the transcriptional levels of related genes.These results would provide evidence for improving the models of Fe2+ oxidation and S0 oxidation in A.ferrooxidans and benefit the genetic modification in this bacterium in future.