Research On The Mechanism Of Bacteriophage YopM Protein Activating Bacterial Toxin-antitoxin System MazEF

Quorum sensing is a signaling exchange mechanism ubiquitous in bacteria.In recent years,it has been found in Bacillus phage that there is a quorum-sensing system-like signal exchange that regulates the transition from lysis to lysogeny in the life cycle of temperate phages.Such quorum sensing systems are called arbitrium systems.The system mainly includes two components: signal short peptide Aim P and signal short peptide receptor Aim R.Through transcriptome analysis,our research group found that after SPbeta phage was infected with Bacillus subtilis,the signal peptide produced could up-regulate the expression level of yop M gene in the downstream region,and overexpression of yop M gene inhibited the formation of plaques.The possible interacting proteins of phage YopM were further screened by mass spectrometry,including the host bacterial antitoxin Maz E and toxin Maz F.How does the phage-encoded YopM protein interact with the host bacterial toxin-antitoxin system? My research work revolves around this scientific question and has achieved the following four results:(1)Obtained purified phage YopM protein(derived from SPbeta phage and phi3 T phage of SPbeta family,respectively),Bacillus subtilis toxin Maz F,antitoxin Maz E and toxin-antitoxin complex Maz EF,and proved by pull-down and co-expression experiments.The interaction between the phage YopM protein and the antitoxin Maz E was found.(2)Size-exclusion chromatography(SEC)assays proved that when the phage YopM protein was incubated with the Maz F-Maz E complex of the host bacteria,it could bind to the antitoxin Maz E,thereby releasing the toxin Maz F.(3)The reaction system of Maz F nuclease in vitro was established.Maz F protein can specifically cleave substrate RNA.When the Maz EF complex was incubated with YopM protein,with the increase of the amount of YopM protein,the phenomenon that the substrate was cleaved could be observed.It is proved that the phage YopM protein activates the nuclease activity of the toxin Maz F after binding to the host antitoxin protein Maz E.(4)The antitoxin Maz E was mutated by alanine scanning,and it was found that the amino acid 61-65 of the antitoxin Maz E could not be combined with YopM after the mutation,and this region is also the region between the antitoxin Maz E and the toxin Maz F,indicating that the phage YopM protein interacts with the bacterial antitoxin Maz E in a competitive binding manner,resulting in the release and activation of the toxin Maz F.In conclusion,this study explored the mode of action of bacteriophage YopM protein and host Bacillus toxin-antitoxin complex Maz F-Maz E,firstly demonstrated that bacteriophage protein can destroy bacterial toxin-antitoxin complex,and revealed the molecular mechanism of phage activation of host toxin,laying the foundation for understanding the phage infection process.