The Role Of Prohage Phi6S In Phage Infection Of Pseudomonas Fluorescens W-6

Temperate phages have a dual life cycle,lytic and lysogenic.Once integrated into the host,the temperate phage and host strike a balance.Phage integration increases the ecological fitness of bacteria.For example,integration prevent further phage invasion,increase antibiotic resistance,and help bacteria to escape host infection by toxin or adhesion invasion.For phages,they benefit from the passage of bacterial offspring during chromosome replication and leave when the environment is unfavorable.It shows that prophages play an important role in the evolution of phages and hosts.However,there are few reports on study of prophages in Pseudomonas fluorescens.To study the function of the prophage phi6 s in W-6 genome,some genes of phi6 s were knocked out,and the function was verified.Combined with transcriptomic sequencing,the role of prophages phi6 s in interaction between phage VW6 S and host W-6 was explored.Based on the principle of homologous recombination,five genes W-6＿1403,W-6＿1404,W-6＿1405,W-6＿1406 and W-6＿1442 of prophage phi6 s were knocked out,and the function was verified.The kanamycin gene fragment containing promoter was amplified by using p ET28 a vector as template,and the target gene fragment to be knocked out was replaced by kanamycin fragment.Knockout homology arms replaced by antibiotics were obtained by overlapping PCR and cloned into suicide plasmid p EX18 Tc.The recombinant plasmid was transformed into SM10 λpir strain by conjugation,and then transferred into W-6 using SM10 λpir as a donor.Knockout strains were obtained by sac B counter selection marker selection.Mutant strains △1405,△1406 and double knockout strain△1405-1406 were successfully screened.Knockout verification by q PCR showed that W-6＿1405and W-6＿1406 genes had been deleted in the corresponding strains.It showed that single and double knockout strains did not affect the growth of W-6.The change in infectivity of phage VW6 S infection against the knockout strain was determined by double-plate method,and showed that the phage titer infecting △1406 was lower than that of WT within 72 h,and the difference appeared before 12 h.The infection rate of phage-infected △1406 at 4 time points within 100 min was determined.The phage titer of the knockout strain △1406 was consistently lower than that of WT.Thus,gene 1406 encodes a host-specific protein,but the specific receptor was not unique.It showed that prophage phi6 s was a defective phage based on the dynamic transcriptome analysis of 1406 and W-6,most of its genes were silenced in host,and only a few were expressed stably to maintain lysogenicity and self-replication.During the adsorption phase and lysis of phage infection,many genes involved in DNA replication,RNA and protein synthesis were differentiall y expressed.GO annotation and KEGG enrichment analysis were also significantly enriched for binding to RNA,macromolecular complexes,and ribosome synthesis,transcription,and so on.It showed that phages first hijack ed the host’s DNA,RNA and protein synthesis systems when infecting W-6,and then controlled the host’s metabolism to complete their own synthesis requirements.In this study,we further proved that the specific protein encoded by1406 gene on the prophage phi6 s was a receptor that can be recognized by phage VW6 S.To some extent,the host acquires immunity to phage VW6 S by mutating the receptor encoded by W-6＿1406 gene.The regulation mechanism of phage VW6 S on W-6 was initially revealed at the transcriptional level,which was of great significance for understanding the interaction mechanism between phage and host.