| Object: In order to solve the cause and mechanism of this phenomenon that difference in the ability of bacteriophage to lyse host,their differences are explored based on the genome,transcriptome and proteome to provide a basis for the development of efficient and broad-spectrum bacteriophage preparations.Methods:(1)Using the clinical strains of Escherichia coli isolated from chicken as host bacteria,the bacteriophage was separated and purified from sewage with double-layer agar culture method;its morphology was observed by transmission electron microscopy;bacteriophage nucleic acid type was analyzed by enzyme digestion and gel electrophoresis.(2)The biological characteristics of one-step growth curve,thermal stability and acid-base stability of phage were determined by double-layer agar culture.(3)The genome of phages were sequenced and assembled by Illumina platform and software;using online tools to completed prediction of open reading frames(ORFs)and coding sequences(CDSs),search of t RNA and r RNA genes;combining with mauve and BLAST software for the homology analysis;using MAGE and other software for genetic evolution analysis;CGView and Snap Gene 1.1.3 were used to construct bacteriophage whole genome map.(4)Samples at 3 time points after IME540 infection E.coli 01,IME537 infection E.coli 01 and E.coli 02 were sequenced by Illumina high-throughput sequencing platform,the differentially expressed genes identified were analyzed for GO and KEGG functional annotation.(5)On the basis of transcriptome analysis,the differentially expressed proteins were identified by quantitative analysis of label free proteomics,and subjected to GO and KEGG functional annotation analysis.Results:(1)Two strains of lysing Escherichia coli phage v B?Eco M?IME540(IME540)and v B?Eco M?IME537(IME537)were successfully isolated.Combining electron microscopy and nucleic acid identification,both bacteriophage belong to the ds DNA group with Myoviridae phage;(2)The phage IME540 has a lysis rate of 6.67%(3/45),which can be kept active at 70? for 40 min,the titer is stable in the range of p H 4.0 ? 13.0,the optimal MOI=0.000001,the incubation period is about 20 min,the burst time is about 130 min,the lysis amount is about 23 PFU/cell;the phage IME537 has a lysis rate of 55.56%(25/45),which inactivated at 70? for 20 min,the titer is stable in the range of p H5.0?9.0,the optimal MOI=0.0001,the incubation period is about 30 min,the burst time is about 160 min,the lysis amount is about 2 PFU/cell.(3)It was found that the genome size of the phage IME540 is 170 237 bp,G + C: 39.46%,2 t RNA genes,and 271 CDSs,IME540 is highly homologous to Enterobacter bacteriophage Bp7,and belongs to the Dhakavirus of Tevenvirinae;the whole genome size of IME537 is 168 642 bp in size,G + C: 35.42%,containing 8 t RNAs and 274 CDSs,homology analysis showed that IME537 belongs to T4 virus and highly homologous to Enterobacter phage IME340.(4)In 5 560 genes signal expression data,both IME540 and IME537 caused significant changes in bacteria transcriptome in the early stage of infection,the expression of differential genes in the phages-infected group was significantly different from that in the control group,and the differential genes increased gradually with the increase of infection time,the differential genes between IME540 infection group and IME537 infection group were also different,and changed significantly with the infection time in the late stage of infection.(5)The proteomic differences of both IME540-infected bacteria and IME537-infected bacteria were compared and analyzed,among 2 567 proteins detected in the bacterioprotein,66 proteins were up-regulated and 14 down-regulated by IME540-E1 infection from that in the control group,97 proteins were up-regulated and 40 down-regulated by IME537-E1 infection from that in the control group,117 proteins were up-regulated and 107 down-regulated by IME537-E2 infection from that in the control group;12 proteins were significant expressed after IME537-E1 infection and IME537-E2 infection compared with IME540-E1 infection group at 5 min;2 proteins were significant expressed after IME537-E1 infection and IME537-E2 infection compared with IME540-E1 infection group at 20 min;2 proteins were significant expressed after IME537-E1 infection and IME537-E2 infection compared with IME540-E1 infection group at 30 min;IME540 and IME537 infection both can cause significant changes in host bateria levels.(6)totally 74 DEPs were identified in the transcription-protein combination analysis with consistent changes trend in transcriptional and protein levels by IME540-E1 infection,DEPs were enriched in RNA degradation,sulfur metabolism and purine metabolism;totally 95 DEPs were identified in the transcription-protein combination analysis with consistent changes trend in transcriptional and protein levels by IME537-E1 infection,DEPs were enriched in pyrimidine metabolism,sulfur metabolism and so on;totally 167 DEPs were identified in the transcription-protein combination analysis with consistent changes trend in transcriptional and protein levels by IME537-E2 infection,DEPs were enriched in purine metabolism,ABC transporters and sulfur metabolism and so on.Conclusion: In this study,two lysing Myoviridae phages v B?Eco M?IME540 and v B?Eco M?IME537 were isolated and its biological characters and genomic was analyzed.The phage-host mutual infection was used as the research object,based on high-throughput sequencing and label free quantitative proteomics techniques,explored thedifferences of phage-infected host-specific transcriptome and proteome.Moreover,this study enriched the phage transcriptome and proteome databases. |
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