Phage-encoded small RNA hijacks host replication machinery to support the phage lytic cycle.

Bacteriophages (phages) are major drivers of bacterial population dynamics, yet the significance of post-transcriptional regulation during infection remains largely unexplored. Central to this regulatory layer are small RNAs (sRNAs), which regulate target mRNAs via base-pairing, typically facilitated by RNA chaperones such as Hfq. Here, we applied RNA interaction by ligation and sequencing (RIL-seq) to comprehensively map the in vivo RNA-RNA interaction network in Escherichia coli during phage lambda infection. This analysis revealed extensive reprogramming of E. coli-E. coli interactions, phage-specific lambda-lambda interactions, and interkingdom interactions between phage and host RNAs. Among these, we identified a phage-encoded sRNA, phage replication enhancer sRNA (PreS), embedded within the early left operon. PreS regulates essential host genes, including dnaN, which encodes the DNA polymerase β sliding clamp. This regulation enhances DNA replication and fine-tunes the phage lytic cycle. These findings uncover an RNA-level regulatory layer in phage-host interactions and demonstrate how a phage-encoded sRNA can hijack host replication machinery to optimize infection.