Mechanistic insights into JSS1_004-mediated antagonism of the DndBCDE-FGH restriction system and engineering applications.

The bacterial DNA phosphorothioate (PT) modification system is orchestrated by the DndCDE enzymatic complex. This system collaborates with restriction components DndF, DndG, and DndH to establish a widespread prokaryotic anti-phage defense network. In the evolutionary arms race, phages such as JSS1 have evolved counter strategies, including the expression of the JSS1_004 protein, to subvert PT-mediated host immunity. Although the N-terminal kinase domain of JSS1_004 is known to inhibit DndFGH through phosphorylation-dependent inactivation, the functional role of its C-terminal shutoff domain remained enigmatic. Here, we demonstrate that both domains are indispensable for full antagonistic activity against the DndBCDE-FGH system. Genetic dissection revealed that the absence of either domain substantially compromises suppression efficacy. Notably, JSS1_004 exhibits pleiotropic effects on host physiology, including repressing the operon confirmed by transcriptome. ChIP-seq analysis further revealed that JSS1_004 binding to promoter regions and coding sequences of genes associated with protein synthesis machinery, including tRNA, rRNA, and ribosomal subunit genes (SSU/LSU), thereby hijacking the host's translational apparatus. Although the potent inhibitory efficacy and broad-spectrum antagonistic properties make JSS1_004 a promising functional element for engineered phage applications, the severe cytotoxicity of JSS1_004 constrains its biotechnological applicability. To overcome this limitation, we engineered attenuated JSS1_004 variants. These modified constructs maintained robust antagonism against the DndBCDE-FGH system while significantly reducing host toxicity, enabling cross-protection of heterologous phages against PT-based restriction and expanding the potential of phage therapy. Our findings advance the understanding of phage-host interactions and provide a framework for optimizing phage therapeutics to circumvent bacterial defense barriers.IMPORTANCEOur recent investigation elucidated the molecular mechanism by which bacteriophage JSS1 counteracted the bacterial DndBCDE-FGH anti-phage defense system. Upon host invasion, the early gene transcriptional fragment of JSS1 facilitated the rapid expression of JSS1_004, with a N-terminal kinase domain that mediated phosphorylation of serine, threonine, and tyrosine residues within the DndFGH defense complex. This post-translational modification induced conformational changes in the complex, effectively neutralizing its restriction activity against phage propagation. However, the role of the C-terminal shutoff domain remains to be elucidated. In this study, we revealed that both the kinase and shutoff domains were crucial for the antagonistic function of JSS1_004. Moreover, transcriptomic and ChIP-seq analyses revealed JSS1_004's broad expressional regulation of host gene expression, thus establishing a cellular environment conducive to bacteriophage replication. Furthermore, we successfully developed and integrated attenuated-cytotoxicity variants of JSS1_004 into the genome of M13 phages, conferring robust resistance against the DndBCDE-FGH defense system. These findings provide critical insights into the molecular arms race between phages and prokaryotic hosts while expanding the toolkit for developing phage-based biotechnological applications.