Research On Phages Of Shewanella Oneidensis MR-1 And Its Influence On MR-1 Diagenesis Process

Shewanella oneidensis strains are facultative anaerobic bacteria that belong to?-Proteobacteria and can use alternative ter minal electron acceptors under anaerobic conditions.S.oneidensis MR-1 can promote the reaction of smectite-to-illite by reducing structural Fe?III?of ferric oxide minerals under anaerobic conditions.Without microbial activity,this reaction usually needs condition of 300 to 350?,100 megapascals and 4 to 5months.However,MR-1 can finish this reaction at room temperature and 1 atmosphere within14 days.This ability of MR-1 has been regarded as a proof of microbes playing roles in geological evolution process and made MR-1 a model organism in diagenesis research.Interestingly,recent study revealed that bacteria is not the only microbials that participated in diagenesis processes,phages also affect diagenesis process.It was found that mineral precipitation can occur directly on free phage particles and cell debris generated after phage infection.Phage particles were per mineralized by amorphous magnesium silicates and then were transferred into magnesium carbonate nanospheres of⁸0-200 nm in diameter during diagenesis.In the meantime,bacteriophages also manipulate bacterial mortality.It was estimated that total phage particles in biosphere were in the order of 10³¹,which outnumber total microbes as a factor of ten.Analysis of bacterial genome sequences also revealed that prophages are ubiquitous in bacterial chromosomes,with 2.6 prophages in every bacterial genome on average and more than twenty prophages at maximum in some bacteria.Once infected,phages can either transferred into lysogenic pathway or start lytic pathway.Phages affect co mmunity composition and impact organic matter and nutrients supply through lysing their host cells.In lysogeny,the lysogenic conversion can alter host phenotypes and provide homo-i mmunity to protect lysogen from infection by closely related phages.These features together suggest that phages have a potential in regulating diagenesis process.No research on lytic phage of MR-1 was reported and systematic investigations on prophages of MR-1 are still needed.Previous studies on MR-1 prophages demonstrated that MR-1 has four prophages,LambdaSo,MuSo1,MuSo2 and Cp4So.A large number of MuSo1,MuSo2 and LambdaSo genes were induced after ultra-violet C exposure.Phage particles with similar morphology to Siphoviridae were observed,which were presumed to be phage particles of LambdaSo.During biofilm formation process,DNA replication and structural protein synthesis of LambdaSo and MuSo2 were detected.Besides,supernatants of strain??1??2 and????1 can form plaques on????MR-1 strain deletion of prophage LambdaSo,MuSo1 and MuSo2?,indicating that infectious LambdaSo and MuSo2 were synthesized.Analysis of MuSo1 genome indicated that MuSo1 is deficient in function genes and lots of insertion were detected in MuSo1 genome,indicating that MuSo1 is unable to form intact offspring.The forth prophage CP4So,a P4-like cryptic phage,can only be induced by temperature downshift.At low temperature CP4So excised itself from host chromosome,resulting in the generation of a subpopulation?0.1-3%?of prophage-free cells.However,this prophage was unable to form intact and infectious phage particles during induction.Hence,prophage CP4So was not involved in this research.To investigate the potential roles of phages in diagenesis process,we investigated the basic characteristics of lytic and lysogenic phages of MR-1.We isolated five individual lytic phages of MR-1.Through transmission electronic microscopy?TEM?,we confirmed all five phages are consisted of head and tail subunits.Among them,MSO-1,MSO-4 and MSO-5 have contractile tails.Therefore,they belong to phage family Myoviridae.Tails of MSO-2 and MSO-3 were unable to shrink.Therefore,they belong to phage family Caudovirales.Analysis of phage genomes proved that all five phage genomes were constituted of double strand DNA.genomes of MSO-1 and MSO-5 were constituted of linear DNA and genomes of MSO-2,MSO-3 and MSO-4 were constituted of circular DNA.By proceeding one-step growth of phages,we found differences were existed between replication process of these three phages.MSO-1 and MSO-5 have low lethality to host cells and generated fewer offspring per infected cell than MSO-4.Besides,difference on release time was also existed between those phages.These diversities of phages provide abundant materials for researches in phages manipulating MR-1 diagenesis process.During the research of investing lytic phages,we found that phage MSO-1 was different with other phages.First of all,MSO-1 was resisted to a lot of restriction endonucleases,which included enzymes that can digest methylated DNA of Dam,Dcm and SssI.Secondly,lipid is composition of MSO-1,which is not a co mmon composition of Myoviridae.But the chemical component and function of lipid is unclear.In this study,we constructed MR-1 prophage deletion mutants.By infecting MR-1 and its prophage deletion mutants,we detected infectivity of supernatants of MR-1 and its prophage deletion mutants.We found that supernatants of strain only containing prophage LambdaSo can form plaques on MR-1 LambdaSo deletion mutants and supernatants of strain only containing prophage MuSo2 can form plaques on MR-1 MuSo2 deletion mutants.Hence,we obtained indicator strains of LambdaSo and MuSo2.With indicator strains,we investigated basic characteristics,release curves and stability under different conditions of LambdaSo and MuSo2.We proved that LambdaSo preceded over MuSo2 in release time,maximum burst size,acid-base and NaCl tolerance,indicating that adaptivity of LambdaSo is superior to MuSo2 in enviro nment.By TEM,we observed phage particles share similar morphology with phage family Siphoviridae and Myovirida,indicating they were LambdaSo and MuSo2phage particles respectively.Competition between MR-1 prophage was also observed.Comparing to wild type MR-1,replication of MuSo2 in MR-1 LambdaSo deletion strains had earlier release time,shorter replication cycle and higher burst size.This result indicated that the existence of LambdaSo repressed MuSo2 reproduction.Further investigation proved that cluster R of LambdaSo completed this repression by inhibition on host cell and MuSo2 DNA replication.We investigated phage influence on MR-1 diagenesis process by adding MSO-1 to smectite to illite reaction and found that MSO-1 affected diagenesis process by regulating number of host cells.We also investigated difference between MR-1 and MR-1 prophage deletion mutants.So far,no evidence of prophage affecting MR-1 diagenesis process was obtained.By investigating MR-1 phages,we gathered basic information of lytic phages and prophages of MR-1,which is fundamental to research in phage affecting MR-1 diagenesis process.Research on LambdaSo repressing MuSo2 reproductivity make us understand means of competition between MR-1 prophages.This interaction between phages belong to different phage families indicating interactions between phages in enviro nment might be severely underestimated.Although,lytic phage affected MR-1 diagenesis process by regulating host cell number,the abundant existence of phage in enviro nment indicating phages have the potential in diagenesis process.Hence,roles of phages in diagenesis should be seriously considered.