Functional Studies On The Roles Of Cyc1 And Cyc2 Gene In Fe²⁺ Oxidation Of Acidophilic Acidithiobacillus Ferrooxidans

Acidithiobacillus ferrooxidans as an extremely acidophilic,chemolithoautotrophic bacterium plays a significant role in bioleaching and environmental protection.The bacteria can oxidize both ferrous iron(Fe2+)and also inorganic sulfur compounds.Biomass of A.ferrooxidans is a crucial parameter in both the fundamental research and the applications.However,the common cell counting approaches were unstable and inaccurate due to its unique characteristics of cultivation.In this study,two standard curves between cell numbers and total cell proteins of A.ferrooxidans grown on ferrous iron and elemental sulfur were developed,which were tested by real-time qPCR and modified Lowry protein assay respectively.Based on the standard curves,it is more rapid and practical to quantify the biomass of A.ferrooxidans by measuring cell protein.In addition,the specific ferrous iron oxidation rate was investigated and subsequently applied to evaluate the ferrous iron oxidation capacity of two A.ferrooxidans strains on the cellular level.In sulfide minerals,the bacteria depend on the mechanism of biological oxidation of ferrous ions to catalyze mineral dissolution.But there are only a few speculative models of iron metabolism-related electron transport system,mainly dependent on techniques such as bioinformatics analysis,proteomics analysis,quantitative PCR analysis and membrane separation and lack of direct experimental data to support,for example,direct gene knockout in the model.Due to the lack of direct experimental evidence,many enzymatic reactions in the model and enzymes in the models remain to be further experimental verification.This paper relied on the remarkless gene knockout technique on A.ferrooxidans and acquired the two mutants lack of iron metabolism-related gene cyc1,cyc2 and their gene regulation of wild-type and mutant strains were studied.In addition,we constructed engineering bacteria containing plasmids with overexpressed cyc2 and cyc1 genes respectively and studied their metabolic characteristics and gene expression regulation in comparision to the wild strain A.ferrooxidans ATCC23270.The roles of genes cycl as well as cyc2 in A.ferrooxidans ATCC 23270 ferrous iron metabolism were also explored,to further study A ferrooxidans ATCC 23270 ferrous iron metabolic pathways as a reference...