The Gene Regulatory Networks Of Magnetosome Synthesis In Acidithiobacillus Ferrooxidans BYM

Magnetosome is a kind of mineral crystal with magnetism,nanoscale,homogeneity,dispersion and biocompatibility,which has a broad application prospect in medicine and bioengineering.Magnetotactic bacteria(MTB)are the main microorganisms that can produce magnetosome.However,the production conditions of magnetosome in MTB are very strict,requiring tight control of iron and oxygen concentration.It is difficult to achieve large-scale production of magnetosome.Acidithiobacillus ferrooxidans(A.ferrooxidans),as one of the important microorganisms driving iron and sulfur circulation in the environment,can also produce magnetosome under suitable environmental conditions,and its separation,culture and magnetosome synthesis conditions are simpler than MTB.Therefore,it has become a potential strain to achieve large-scale production of magnetosome.At present,the magnetosome synthesis mechanism of A.ferrooxidans is not clear.So,the industrial production of magnetosome has been greatly restricted.In order to fully explore the genes related to magnetosome synthesis and construct the magnetosome synthesis gene regulatory network in A.ferrooxidans.In this paper,transmission electron microscope and transcriptome sequencing were performed on A.ferrooxidans BYM treated with different Fe(II)concentration,growth time and static magnetic field intensity.Bioinformatics analysis was used to analyze the function of differentially expressed gene(DEGs)and gene expression trend in samples.The key genes related to magnetosome synthesis were screened by weighted gene co-expression network analysis(WGCNA).The relative expression levels of genes were obtained by RT-q PCR,and the correlation between genes and environmental conditions and genes and traits was analyzed by polynomial fitting and redundancy analysis(RDA).Finally,the transcription factors in the genome and their interaction patterns with DEGs were predicted to construct a gene regulatory network of magnetosome synthesis in A.ferrooxidans BYM.The results of transmission electron microscopy and the analysis of intracellular total iron content showed that the number of magnetosome and intracellular total iron content reached the maximum(15.67±2.08 per cell and 2.82±0.22?g/mg)at 40 g/L Fe(II).The maximum size of magnetosome was 44.00±4.58 nm at 80 g/L Fe(II).When the growth time was 36 h,the number and size of magnetosome were the minimum(7.00±1.00 per cell,20.00±2.00 nm),and the intracellular total iron content was the maximum(5.93±1.03?g/mg).When the magnetic field intensity was 3.5 m T,the number of magnetosome and the intracellular total iron content reached the maximum(19.33±2.52 per cell,4.69±0.66?g/mg),and the size of magnetosome was about 50.00 nm,which was similar to that of the magnetic field intensity was 15 m T.Fe(II)concentration,growth time and static magnetic field intensity were significantly correlated with the number and size of magnetosome and intracellular total iron content in A.ferrooxidans BYM(R~2>0.683,P<0.05).The results of DEGs and gene expression trend analysis showed that treatment conditions mainly affected the biological processes such as ion absorption and transport,redox reaction,plasma membrane structure,signal transduction and quorum sensing.Based on WGCNA,the results showed that there were 2 gene profile of positive correlation with the number and size of magnetosome(R~2>0.35,P<0.09)and 2 gene profile of negative correlation with the number and size of magnetosome(R~2<-0.42,P<0.04),and 24 key genes related to magnetosome synthesis were screened from them.Through NCBI database annotation and sequence alignment analysis,10 genes related to magnetosome synthesis were selected as the objects of RT-q PCR,including chr?2549,chr?1390,chr?1157,chr?251,chr?974,chr?2620,chr?1779,chr?2224,chr?1243 and chr?1062.The functions of these genes are mainly related to cell redox,ion transport,ferrous chelation,membrane structure and cell chemotaxis.The relative expression levels of 10 genes were analyzed and it was found that the gene expression were significantly correlated with conditions(P<0.01).Based on RDA,the results showed that the expression of10 genes was significantly correlated with the number and size of magnetosome.The predicted constructed magnetosome synthesis gene regulation network shows that the biological processes constituting the network mainly include transcription regulation,energy generation and transformation,redox,inorganic ion transport and metabolism,cell wall/membrane/envelope biogenesis,which correspond to the magnetosome synthesis process.In conclusion,appropriate Fe(II)concentration and magnetic field intensity can promote the formation of magnetosome,and the synthesis ability of magnetosome is the strongest at the end of logarithmic growth or stable stage.Environmental conditions mainly affect the magnetosome synthesis by changing the biological processes such as ion transport,redox reaction and membrane structure.The predicted constructed magnetosome synthesis gene regulatory network has biological process corresponding to MTB magnetosome synthesis,indicating that it has reference value.This study provides a basis for genetic manipulation of A.ferrooxidans based on magnetosome synthesis genes and revealing the magnetosome synthesis mechanism.