Characterization And Genome Analysis Of Phage VB_EfaP_IME195 And Phage GP4

Objectives:The biological and genomic characteristics of phages of Enterococcus faecalis and solanum Ralstonia solanacearum were studied,which can provide theoretical basis for the further study of phage therapy and phage genomics.Methods:Phage vB_EfaP_IME195 was isolated from hospital sewage by using Enterococcus faecalis as indicator bacteria,and phage GP4 was also isolated from ginger soil by using Ralstonia solanacearum as indicator bacteria.The biological characteristics of phages were analyzed by observing the plaques formed by phages cleavage of host bacteria on agar double-layer plates,the shape of phage under transmission electron microscope,the optimal infection number of phages,one-step growth curve,temperature sensitivity,chloroform sensitivity and lysis spectrum.Two phage were sequenced by second-generation high-throughput sequencing method.After sequencing,the whole genome sequence of the phage was assembled and spliced,and the genomic annotation,evolutionary tree and terminal analysis were performed on the obtained whole genome sequence.Results:?1?In this study,Enterococcus Faecalis?strain 2003?,which was isolated from the secretions of a female patient,was used as the indicator bacteria to isolate phages from the sewage of hospital,and a phage named as vB_EfaP_IME195?Abbreviation:IME195?was isolated.The phage was gradient diluted in PBS buffer,and the phages with clear edges and transparent plaques were obtained by two-layer medium plate method.Electron micrograph showed the phage contains a collar with protruding appendages which is the characteristic of phages classified within the subfamily Picovirinae of family Podoviridae,and it was very similar to the morphology of Enterococcus Faecalis phage vB_EfaP_IME199?Abbreviation:IME199?previously isolated from our laboratory.The optimal MOI of phage IME195was 0.01,and the one-step growth curve showed that the latent period was about 30min,and the outbreak was about 120 PFU/cell.The phage's lysis spectrum is relatively narrow and is sensitive to high temperatures,but is not sensitive to 5%chloroform.The whole genome sequence of phage IME195 was obtained by the next-generation sequencing technology,which contains a linear and double-stranded DNA molecule which had a genome size of only 18655bp?GenBank accession number:KT932700?and the GC content was only 33.2%.About 27 open reading frames?ORFs?were obtained by RAST annotation,and all the starting codons were ATG,in which 15 ORFs were hypothetical proteins and 12 ORFs were known functions.Run-off sequencing experiment showed that the end of the phage IME195 has an inverted repeat sequence of 59bp,which was very similar to the end of the aforementioned phage IME199.BLASTn analysis showed that IME195 was 92%similar to an unpublished phage and had low homology with other phages in NCBI library.?2?In this study,phage GP4 was isolated from the soil of ginger field using R.solanacearum JW20 as the indicator bacteria,and the phage was also belong to the family Podoviridae.The optimal MOI of phage GP4 was 10^-6,and the one-step growth curve showed that the latent period was about 2h,and the outbreak was about 106 PFU/cell.Phage GP4 has strong activity in40-70?.The lysis spectrum showed that 16 strains of R.solanacearum that could be lysed were all belonging to phylotype?,and the phylotype I bacteria could not be lysed by phage GP4.The genome of phage GP4 was a double-stranded DNA molecule with a total length of 61129bp?GenBank accession number:MH638294?determined by a next-generation sequencing,and the GC content was 64%.According to the online annotation with RAST,the phage has 83 open reading frames?ORFs?,of which 72codons starts with ATG and 11 ORF codons with GTG.Of these 83 ORFs,24 have known functions,12 were structural proteins,and the remaining 47 were hypothetical proteins.A cysteine tRNA?tRNA-cys?of a size of 90 bp was found by the on-line software tRNAscan-SE 2.0 between the 25202bp and 25291bp of the phage GP4genome.BLASTN analysis with the complete genome sequence indicates that GP4share very limited homology with four phages in Bcep22-like virus,and the coverage rates are Bcep22?13%?,BcepIL02?13%?,DC1?13%?,and BcepMigl?13%?,respectively.No fixed terminal sequences were found in the phage genome using the end analysis technique established by our laboratory,suggesting that the end of the phage genome may be random.Conclusions:?1?Phage vB_EfaP_IME195 was belong to subfamily Picovirinae of family Podoviridae.Its genome is small and has a special terminal,with a reverse repeating sequence of 59bp.So this simple,specialized phage can be used as a model for other phage genome.?2?Phage GP4 was belong to Bcep22-like virus of family Podoviridae.Biological characteristics experiments show that phage GP4 can lyse most of the R.solanacearum of phylotype?,so it can be used as antibacterial biological agent for R.solanacearum.