Phage hijacks host phosphorothioate DNA modification machinery to circumvent bacterial Ssp defences.

The bacterial Ssp defence system discriminates self from non-self by introducing sequence-specific phosphorothioate (PT) modifications in host DNA (via SspABCD) and cleaving unmodified foreign DNA (via SspFGH or SspE). Here we report PptA, a phage-encoded [4Fe-4S] cluster-containing protein, which hijacks cognate host cysteine desulfurase IscS homologues to assemble a streamlined PT modification machinery. Integrated biochemical and structural data delineate a model for intermolecular sulfur transfer within the IscS-PptA complex. Upon infection, robust expression of PptA, not merely its presence, drives sufficient PT incorporation into the phage genome, enabling molecular mimicry of host PT patterns. By masquerading as 'self', the modified phage DNA evades recognition and cleavage by SspFGH/SspE. Notably, PptA can reprogramme the Ssp-sensitive λ phage into an immune-evasive variant. These results reveal a co-evolutionary strategy used by phages to overcome PT-based bacterial immunity and provide a foundation for engineering therapeutic phages that bypass this widespread defence system.