The Function Analysis Of OxyR Homologs In Magnetospirillum Gryphiswaldense MSR-1

Magnetotactic bacteria is a specialized bacteria,which synthesizes intracellular organelles named magnetosmes and aligns along the earth's magnetic field.Magnetosomes,whose size are distributed from 30 nm to 120 nn,are composed of bio-membrane enveloped magnetite?Fe3O4?or greigite?Fe3S4?.Because of the simple cellular structure and genome composition of these bacteria,it has been selected as the model organism for the research of biomineralization.For decades,most of the work on magnetotactic bacteria has been concentrated on the formation mechanism of magnetosomes,especially on the magnetosome island?MAI?genes.But the study on genes located outside the MAI is limited.It has been stated a lot that the formation of magnetosme is closely related to redox control in magnetotactic bacteria.In this study,the regulatory mechanism of two homolog genes of oxyR is studied in Magnetospirillum gryphiswaldense MSR-1.In the previous study,oxyR-Like deleted mutant and complementary strains were construced.By the measurement of the phenotype of these strains,it was stated that the formation process of magnetosomes in the mutant strain was greatly impaired.Further more,the expression level of the genes in the whole genome was measured by expression profile.However,the regulatory mechanism of OxyR-Like still remained unrevealed.In this work,the regulatory mechanism was studied.The results indicated that OxyR-Like bind to the promoter regions of MGMSRv-2₀006,pdhA and mcpA.The DNA binding sites of these genes were confirmed by DNase I foot printing assay,while the consensus binding sequence?5'-ATN{3}AN{5}TTATCA-3'?was found by multiple sequence alignment.Thereafter,the qRT-PCR results indicated the down regulation of all of the genes related to tricarboxylic acid cycle,suggesting the greatly impairment of energy metabolism in the mutant strain.oxyR-4250 is another oxyR homolog in MSR-1 genome.The oxyR-4250 deletion mutant and complementary strains were constructed.The disruption of oxyR-4250 resulted in weak growth and the lost of magnetic response.Interestingly,transmission electron microscopy analysis revealed that the number and size of magnetosomes in the mutant strain decreased dramatically,and the magnetosomes did not align along the long axis of the bacteria any more.EMSA assay indicated OxyR-4250 regulated ahpC and sodB,which proved the relationship between OxyR-4250 and redox control regulatory mechanism.However,the katE and katG gene,which were regulated by OxyR in E.coli,could not be regulated by OxyR-4250,indicating a novel antioxidant mechanism existed in MSR-1.Further more,when DTT was added to the binding system for EMSA assay,OxyR-4250 can no longer bind to the promoter region of these genes,indicated the OxyR-4250 can respond to the redox signals in the environment.The binding affinity between OxyR-4250 and the promoter region of mam clusters can not be detected,hinted a indirect relationship between OxyR-4250 and the formation of magnetosomes.Additionally,DNase I foot printing assay showed that OxyR-4250 bind to the conserved 5'-TCGATTTNTNTNANNANNANAACTCNT-3' region within the oxyR-4250 and ahpC-0756 promoters.It was confirmed that the consensus amino acid,212Cys,in the OxyR-4250 plays a key role in binding target DNA sequence by site-directed mutation and EMSA assay.In conclusion,this work concentrated on the regulatory mechanism of two oxyR homolog genes in M.gryphiswaldense MSR-1.The study on the regulatory mechanism of OxyR-Like again proved the close relationship between energy metabolism and magnetosome formation process.While,the study on OxyR-4250 firstly revealed the anti-oxidant mechanism in magnetotactic bacteria,and also indicated the direct or indirect link between redox control and magnetosome formation.