Genome-wide Transcriptional Characteristics During Magnetosome Formation And Functional Analysis Of Two Ferric Uptake Regulators In Magnetospirillum Gryphiswaldense MSR-1

Magnetotactic bacteria(MTB)can synthesize magnetosomes composed of Fe304 particles,and the iron content of MTB is more than 3%of cell dry weight which is over 100-fold higher than the content in Escherichia coli.But the high intracellular iron does not induce the Fenton reaction and oxidative stress which suggests that the role of ferric uptake regulator(Fur)is significant.Because of the importance of iron and oxygen for magnetosome formation,we focus on the function of two ferric uptake regulators(Fur3137 and Fur3149)in Magnetospirillum gryphiswaldense MSR-1,and investigate the metabolism and regulation mechanisms of iron and oxygen in the cell growth and process of magnetosome synthesis by transcriptome analysis and molecular biological methods,which not only illustrates the mechanism of biomineralization but also provides the information for artificial modification or cultivation of MTB.In order to further obtain the information of key genes participated in magnetosome formation,the RNA-seq was firstly introduced in the study of MTB.Firstly,flask culturing MSR-1 cells in the presence vs.absence of 20 ?M ferric citrate,magnetosomes formation began at 6 h and crystal maturation occurred from 10 to 18 h,which only occurred in the high-iron cells.RT-qPCR analysis showed that 13 iron and oxygen related genes have similar expression patterns which reflected the coordination relationship in the cell growth and magnetosome formation.Secondly,RNA-seq analysis showed that there were 1619 differentially expressed genes(DE genes)in the early of magnetosome formation(8 h),which included 568 up-regulated and 1051 down-regulated genes under the high iron condition respectively;80 differentially expressed genes in the mature period of magnetosome formation(18 h),which included 53 up-regulated and 27 down-regulated genes under the high iron condition respectively.GO enrichment and KEGG analysis found that DE genes in the early stage mainly participated in the oxidative phosphorylation,ribosome,nitrogen metabolism and flagellar assembly,and DE genes in the mature stage were related to oxidation-reduction process,ion transport,sulfur metabolism and amino acid metabolism.Notably,genes in magnetosome island(MAI)had high transcriptional level only in the early stage,which took part in the magnetosome synthesis.Additionally,bioinformatics analysis for the promoter of 80 DE genes,it indicated that they could be regulated by Fur3137,Crp,CytR,NarL,SigH and GerE and most of them were under the regulation of 2-4 regulators,which established the coupling regulatory network jointly and the foundation for revealing the iron and oxygen metabolism of MTB.According to the RNA-seq information in the mature stage of magnetosome formation(18h),only one fur gene(fur3149)belonged to the down-regulated DE genes under the high iron condition.Amino acid analysis by BlastP showed that Fur3149 was an iron response regulator(Irr)protein which had HHH motif that can bind heme.By comparing the wild type(WT)of MSR-1,fur3149 deletion mutant(Afur3149)and complement strain(CF3149),it was found that the growth of Afur3149 was obvious lower than WT under the condition with 30 ?M 2,2-Dipyridyl(DIPy).When cultivated in the 60 ?M ferric citrate,Afur3149 can produce smaller and less magnetosomes than WT,which suggested that Fur3149 play a supplementary role in the magnetosome formation.Furthermore,the iron uptake and tolerance of H2O2 of Afur3149 were both decreased than WT,and it indicated that Fur3149 participated in the iron metabolism and resistance to oxidative stress.However,EMSA results showed that Fur3149 can not directly regulate the expression of hemB,bhuA,hemE,hemA,nifU,nifS,sufA,nsrR,which suggested that its regulatory strategy was different from non-magnetotactic bacteria.To detect the role of other furs in the ?fur3149,the results proved that the complementary function between Furs in MSR-1,and they consisted of the regulation network for controlling the balance of iron and oxygen metabolism.It made sense of coexisting Furs for MSR-1.Previously,the results of ChIP proved that Fur3137 could directly regulate two iron metabolism related genes(feoABl and feoAB2)and two oxygen metabolism related genes(katG and sodB).Now using DNase I footprinting,it was further determined the sequence of Fur box in the four genes'promoter.Based on the crystal structure of Fur3137,the biological function of several amino acids was tested using conjugation,RT-qPCR and physiological parameters measurement.The results showed that Cys9,Met 16 and Met 19 were sensitive for oxygen concentration.Two key amino acids in DNA binding domain Arg57 and Lys15,His33 and His90 in the metal site 1,Glu108 and His125 in the metal site 2 were all crucial for the regulatory function of Fur3137.These results filled the blank of the research in the relationship between the structure and functions of Fur3137.