Exploration Of Synthesis Method Of Pseudomonas Aeruginosa Phage With Headful Packaging Mechanism

The world is on the cusp of the "post-antibiotic era",multidrug-resistant,extensively drug resistant and pandrug-resistant pathogens pose a huge challenge to global public health.P.aeruginosa is one of the members of the common multidrug-resistant pathogen "ESKAPE".WHO listed the carbapenem-resistant P.aeruginosa as one of the superbug for which new antibiotics are urgently needed for control.Phages,bacterial virus,have become a promising strategy in the treatment of drug resistant P.aeruginosa infection due to its strong specificity to the host.However,due to the phage resistance and natural phages may encode virulence factors or toxins during phage therapy,engineered phages and de novo synthetic phages could increase the safety of the phage therapy and reduce the complexity of safety assessment.Headful packaging mechanism is a main model for cleavage and packaging of concatemeric phage ds DNA,it accounts for nearly 47% of the packaging mechanism of Pseudomonas phages that we known.Due to the genomes of phage with headful packaging mechanism were heterogeneous population of terminally redundant and circularly permuted DNA molecules,it had no reports on the synthesis of phages with headful packaging mechanism by yeast TAR(transformation-associated recombination)cloning.This study aims to explore the synthesis strategy of P.aeruginosa headful packaging phage based on yeast TAR cloning.Firstly,we tested the reboot of phage genomes from two P.aeruginosa headful packaging phage S1 and S4 in P.aeruginosa,E.coli,S.typhimurium and cell free systems based on E.coli.We found that phage S1 genome could only be rebooted in P.aeruginosa PAO1.As for phage S4,its genome could not be rebooted in S.typhimurium ATCC 14028 and cell free systems,but could be efficiently rebooted in P.aeruginosa ATCC 9027 and E.coli NEB 10-beta.Then,we tried to synthesize phage S1 and S4 based on yeast TAR cloning.After completing the assembly of phage genome fragments in vivo by yeast TAR cloning,we transformed the yeast plasmid containing phage S1 genome into its host P.aeruginosa PAO1 for rebooting,but we did not observe any plaque in plaque assay.Then we tried to transform the yeast plasmids p YEP-?-S1-60 and p YEP-?-S1-160 containing the phage S1 genome into E.coli cells,there were few transformants but no positive transformants.For phage S4,we transformed the yeast plasmid containing its genome into E.coli NEB 10-beta and its host P.aeruginosa ATCC 9027 for rebooting,plaques were observed in plaque assay.Based on this synthesis strategy,we successfully synthesized the synthetic phage S4 containing RFP label using two different vectors,and proved that the circularly permuted terminal redundancy in this synthesis strategy is critical for the reboot of the synthetic phage genome.This study explored the synthetic strategy of P.aeruginosa phages with headful packaging mechanism based on yeast TAR cloning.We failed to reboot the synthetic genome of phage S1,but we firstly report the systhesis of a P.aeruginosa headful packaging phage S4 based on yeast TAR cloning.We hope the work of this study will be helpful for the synthesis,application and research of phages with headful packaging mechanism.