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

ABSTRACT: Bacteriophages represent a largely untapped reservoir of regulatory elements that can expand the genetic toolbox for engineering non‑model hosts. 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. Our results position phage transcriptomics as a scalable strategy for mining regulatory elements tailored to non‑model bacterial hosts and accelerate the development of new synthetic biology platforms.

ORGANISM(S): Pseudomonas phage pphageB21 Pseudomonas savastanoi pv. phaseolicola Pseudomonas fluorescens Pseudomonas phage KNP Pseudomonas phage vB_PsyM_KIL5 Pseudomonas phage vB_PsyM_KIL3b Pseudomonas phage phi2 Pseudomonas coronafaciens pv. porri

PROVIDER: GSE328282 | GEO | 2026/07/13

REPOSITORIES: GEO

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Project description:Cyanobacteria are highly abundant in the oceans where they are constantly exposed to lytic viruses. Some viruses are restricted to a narrow host range while others infect a broad range of hosts. It is currently unknown whether broad-host range phages employ the same infection program, or regulate their program in a host-specific manner to accommodate for the different genetic makeup and defense systems of each host. Here we used a combination of microarray and RNA-seq analyses to investigate the interaction of three phylogentically distinct Synechococcus strains, WH7803, WH8102, and WH8109, with the broad-host range T4-like myovirus, Syn9, during infection. Strikingly, we found that the phage led a nearly identical expression program in the three hosts despite considerable differences in host gene content. On the other hand, host responses to infection involved mainly host-specific genes, suggesting variable attempts at defense against infection. A large number of responsive host genes were located in hypervariable genomic islands, substantiating genomic islands as a major axis of phage-bacteria interactions in cyanobacteria. Furthermore, transcriptome analyses and experimental determination of the complete phage promoter map revealed three temporally regulated modules and not two as previously thought for cyanophages. In contrast to T4, an extensive, previously unknown regulatory motif drives expression of early genes and host-like promoters drive middle-gene expression. These promoters are highly conserved among cyanophages and host-like middle promoters extend to other T4-like phages, indicating that the well-known mode of regulation in T4 is not the rule among the broad family of T4-like phages. We investigated the infection process and transcriptional program of the P-TIM40 cyanophage during infection of a Prochlorococcus NATL2A host. The results are discussed in conjunction with results obtained from the infection process for the Syn9 cyanophage in three different Synechococcus hosts: WH7803 (Dufresne et al. 2008), WH8102 (Palenik et al. 2003) and WH8109 (sequenced as part of this study).

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

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