Assessing the transcriptional landscape of <i>Pseudomonas</i> phage 201ϕ2‐1: Uncovering the small regulatory details of a giant phage

The transcriptional architecture of phages can deepen our understanding of the phage-host infection process and can be of key importance for phage engineering and biotechnological applications. Here, we applied ONT-cappable-sequencing, a long-read RNA-sequencing technique, to study the regulatory mechanisms of Pseudomonas infecting giant phage 201ϕ2-1. We identified 67 promoters and 132 terminators that together represent 92 transcriptional units. A full comparison of these data to the transcriptome of model Pseudomonas phage ϕKZ confirmed that the transcriptional programs of these prototypes of the Serwervirus and Phikzvirus genera are largely conserved, despite some subtle regulatory differences. Evidence supporting these shared mechanisms include the identification of highly similar sequence motifs for regulatory elements in both phages and the conservation of regulatory elements loci relative to homologous genes in each phage. Moreover, we discovered a sRNA in 201ϕ2-1 that is highly conserved among prototype members of different giant phage genera. Sequencing of the 201ϕ2-1 host genome resulted in its reclassification as Pseudomonas atacamensis, a close relative of the important agricultural biocontrol agent Pseudomonas chlororaphis. Finally, we conducted in vivo assays of eight 201ϕ2-1 terminators and found them to strongly terminate transcription in P. chlororaphis. Control elements from phage transcriptional programs have a rich history for applications in biotechnology. In these studies, we demonstrate new insight into the transcriptional program of 201ϕ2-1 and demonstrate the potential of its regulatory elements for novel and useful tools for synthetic biology circuitry.