Presentation Information
[P03-325]Translation–Enhanced Anti–Plasmid Defense by the RDP System of Prokaryotic PIWI–RE
○Hao Sun1 (1. Shanghai Jiao Tong University (China))
Keywords:
Bacterial defense,Argonaute,PIWI family,Multi–enzyme complex,Translational regulation
[Purpose] Prokaryotic PIWI–RE proteins, together with their co–encoded restriction endonuclease (REase) and SFII family helicase (DinG), form the RDP system. However, whether the RDP system possesses defense functions and the underlying mechanisms remain largely unknown. This study investigates the defense mechanism of the RDP system derived from Pseudomonas species, reveals the molecular basis for triggering its defense function, and elucidates the enhancing effect and regulatory role of exogenous plasmid translation on RDP–mediated defense.
[Method] Plasmid interference assays, colony–forming unit (CFU) determination, flow cytometry, construction of GFP reporter plasmids containing various expression elements (promoters, ribosome binding sites), construction of GFP reporter plasmids with RBS mutants exhibiting gradually increasing translation initiation rates, quantitative real–time PCR (qRT–PCR), and co–immunoprecipitation (Co–IP) assays.
[Results] We first validated the defense capability of the RDP system against exogenous plasmids. The results showed that the defense function of the RDP system requires the simultaneous presence of REase, DinG, and PIWI–RE; mutations at the binding interface of the RDP complex significantly impaired defense capability. Further investigation into the molecular mechanism revealed that translation of exogenous plasmids markedly enhanced RDP–mediated defense: transcription alone increased plasmid clearance efficiency, whereas concurrent transcription and translation led to a further increase. Using RBS mutants with gradually increasing translation initiation rates, plasmid clearance efficiency correlated positively with the strength of protein translation. This mechanism enables the RDP system to preferentially recognize and eliminate exogenous plasmids with high expression levels. Co–IP results demonstrated that PIWI–RE protein associates with multiple transcription– and translation–related proteins.
[Consideration] These findings provide the first evidence that translation of exogenous plasmids enhances the defense activity of a prokaryotic Argonaute system. Unlike previously characterized pAgo–based immune modules that rely primarily on guide nucleic acid recognition or abortive infection, the RDP system integrates translational cues as a regulatory signal to potentiate its anti–plasmid function. This discovery expands our understanding of the regulatory landscape of prokaryotic Argonautes, revealing that the host’s protein synthesis machinery can directly modulate the activity of a DNA–targeting immune complex. The positive correlation between translation strength and plasmid clearance efficiency further suggests that the RDP system has evolved to sense metabolic burden or gene expression levels, thereby prioritizing the elimination of highly active foreign genetic elements while minimizing fitness costs to the host.
[Conclusion] PIWI–RE binds these transcription– and translation–associated proteins; enhanced transcription and translation of exogenous genes potentiate the recognition of foreign genetic elements by PIWI–RE, leading to the recruitment of more REase and DinG proteins and thereby achieving more efficient clearance of exogenous genetic material.
[Method] Plasmid interference assays, colony–forming unit (CFU) determination, flow cytometry, construction of GFP reporter plasmids containing various expression elements (promoters, ribosome binding sites), construction of GFP reporter plasmids with RBS mutants exhibiting gradually increasing translation initiation rates, quantitative real–time PCR (qRT–PCR), and co–immunoprecipitation (Co–IP) assays.
[Results] We first validated the defense capability of the RDP system against exogenous plasmids. The results showed that the defense function of the RDP system requires the simultaneous presence of REase, DinG, and PIWI–RE; mutations at the binding interface of the RDP complex significantly impaired defense capability. Further investigation into the molecular mechanism revealed that translation of exogenous plasmids markedly enhanced RDP–mediated defense: transcription alone increased plasmid clearance efficiency, whereas concurrent transcription and translation led to a further increase. Using RBS mutants with gradually increasing translation initiation rates, plasmid clearance efficiency correlated positively with the strength of protein translation. This mechanism enables the RDP system to preferentially recognize and eliminate exogenous plasmids with high expression levels. Co–IP results demonstrated that PIWI–RE protein associates with multiple transcription– and translation–related proteins.
[Consideration] These findings provide the first evidence that translation of exogenous plasmids enhances the defense activity of a prokaryotic Argonaute system. Unlike previously characterized pAgo–based immune modules that rely primarily on guide nucleic acid recognition or abortive infection, the RDP system integrates translational cues as a regulatory signal to potentiate its anti–plasmid function. This discovery expands our understanding of the regulatory landscape of prokaryotic Argonautes, revealing that the host’s protein synthesis machinery can directly modulate the activity of a DNA–targeting immune complex. The positive correlation between translation strength and plasmid clearance efficiency further suggests that the RDP system has evolved to sense metabolic burden or gene expression levels, thereby prioritizing the elimination of highly active foreign genetic elements while minimizing fitness costs to the host.
[Conclusion] PIWI–RE binds these transcription– and translation–associated proteins; enhanced transcription and translation of exogenous genes potentiate the recognition of foreign genetic elements by PIWI–RE, leading to the recruitment of more REase and DinG proteins and thereby achieving more efficient clearance of exogenous genetic material.
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