Screening Of Efficient Promoters And Transcriptomic Analysis Of Ferrous Oxidation Related Gene Knockout Mutants Of Acidithiobacillus Ferrooxidans.

Biometallurgical technology can be used to bio-oxidize ores and accelerate the transformation of metal minerals into metal oxides by natural micro-organisms under their own metabolism.It is also called bio-leaching technology.Acidithiobacillus ferrooxidans?A.ferrooxidans?,as a dominant strain in bio-metallurgy,is an important metallurgical industrial micro-organism.It can use ferrous ions as sole energy source for growth.It also can use elemental sulfur or reducing inorganic sulfide to obtain energy and reducing power for its own metabolism.It's energy metabolism pathways are diverse and complex,and metabolic mechanisms are still quite unclear.Further exploration of the genetic background and energy metabolic system of this bacteria can not only clarify the relationship between ferrous metabolism and sulfur metabolism theoretically,but also promote the efficient application of the bacteria in bio-metallurgical industry.The mechanism of ferrous oxidation in A.ferrooxidans is still quite unclear.Now,a model of ferrous oxidation electron transport chain,proposed by Bonnefoy et al.in A.ferrooxidans ATCC 23270,has been widely accepted.According to the model,Cyc2 is the sole first electron transfer acceptor.In our early study,the knockout of cyc2 and its replacement gene AFE 1428 did not break the ability of ferrous oxidation in mutants.In order to improve the efficiency of ferrous oxidation of A.ferrooxidans,the screening of efficient promoters and transcriptomic analysis of ferrous oxidation related gene knockout mutants of A.ferrooxidans were carried out in this study.Based on the RNA-seq and bio-informatics analysis,the possible efficient promoter sequences were searched in the whole genome,especially focusing on the transcriptional regulatory elements and promoter activities of genes involved in ferrous oxidizing metabolism to provide alternative promoters for efficient gene expression in A.ferrooxidans.And the promoter of 11 genes predicted by bio-informatics was ligated to the reporter gene gusA,respectively.The promoter activity was first detected in E.coli.Among the tested promoters from A.ferrooxidans,six promoters were stronger than Ptac,and two promoters were weaker than Ptac in E.coli.They were P0781>P2727>P1658>P2254>P2026>P2278>Ptac>P0422>P2998.However,the activities of the remaining three promoters,P0041,P0696 and P1636,were very low,which need to be confirmed.The three promoters,as well as two active promoters,P2026 and P2998,were further detected in A.ferrooxidans.The results showed that one was stronger than Ptac,and two were weaker than Ptac.They were P2026>Ptac>P2998>P0041.Confirmed promoter activity was detected on P0041,which was not active in E.coli.But P0696 and P1696 were also almost inactive in A.ferrooxidans.The results are consistent with in E.coli.The different results on promoter activity of genes with high transcriptional activity may be due to the different regulation on promoters and accuracy of the promoter prediction software.Anyway,the availability of efficient promoters is conducive to the construction of bioleaching strains with improved metabolism on ferrous and sulfur.In order to further study the function of the key genes cyc2 and AFE 1428,as well as other functional replacement genes involved in ferrous oxidation in A.ferrooxidans,the transcriptome analysis was performed on A.ferrooxidans ATCC 23270 wild type strain and its three gene knockout mutants,?cyc2,AAFE 1428 and cyc2-?AFE 1428.The sample was prepared and collected according to the growth and ferrous-oxidizing curves of each strain when cultivated on ferrous iron as the only energy source.By the analysis and comparison of the transcriptome data,the significant differentially expressed genes were found out and verified by RT-qPCR.Finally,the functions of cyc2,AFE₁428 and several significantly differentially expressed genes in ferrous oxidation metabolism were analyzed according to the transcriptome data,RT-qPCR results and bioinformatics,which could be verified by further experiments.In summary,the screening of efficent promoters and transcriptomic analysis of ferrous oxidation related gene knockout mutants of A.ferrooxidans were carried out in this paper.The results provide clues and data for further study on the electron transfer chain model of ferrous oxidation and on other key electron transport proteins which have not been involved in the model.