Mining phage-genomes for regulatory elements to develop a SynBio toolbox for non-model Pseudomonads

Using ONT‑cappable‑sequencing, we mapped full‑length primary transcripts across five Pseudomonas phages and identified 232 transcription start sites (TSSs) and 176 transcription termination sites (TTSs), including 38 intrinsic factor‑independent terminators. Motif discovery revealed 59 s70-like promoters in KIL5, 37 in KIL3b, and unique phage‑encoded RNA polymerase (RNAP) promoter motifs for KNP, ϕ2, and pphageB21, enabling discrimination between host‑ and phage‑dependent transcriptional control.

To verify these predictions, a subset of ten promoters and five terminators was quantitatively characterized in vivo in P. fluorescens strain GL-S-306, demonstrating promoter activities spanning a large dynamic range and terminator efficiencies allowing complete transcriptional termination. These data establish the first experimentally validated, phage‑derived library of promoters and terminators for P. fluorescens and P. syringae, providing chassis‑specific regulatory parts that enable more finetuned gene‑expression than current cross‑species SynBio tools.