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

## TL;DR

This study reveals how the JSS1_004 protein from a phage neutralizes a bacterial defense system and how it can be modified for safer phage therapy.

## Contribution

The study identifies the dual functional domains of JSS1_004 and engineers less toxic variants for biotechnological use.

## Key findings

- Both the N-terminal kinase and C-terminal shutoff domains of JSS1_004 are essential for antagonizing the DndBCDE-FGH system.
- JSS1_004 represses the dndBCDE-FGH operon and regulates host genes involved in protein synthesis.
- Engineered JSS1_004 variants maintain antagonistic activity while reducing host toxicity for phage therapy applications.

## Abstract

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 dndBCDE-FGH 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.

Our 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.