Refining the transcriptional landscapes for distinct clades of virulent phages infectingpseudomonas aeruginosa

Abstract

The introduction of high-throughput sequencing has resulted in a surge of available bacteriophage genomes, unveiling their tremendous genomic diversity. However, our current understanding of the complex transcriptional mechanisms that dictate their gene expression during infection is limited to a handful of model phages. Here, we applied ONT-cappable-seq to reveal the transcriptional architecture of six different clades of virulent phages infecting Pseudomonas aeruginosa. This long-read microbial transcriptomics approach is tailored to globally map transcription start and termination sites, transcription units and putative RNA-based regulators on dense phage genomes. Specifically, the full-length transcriptomes of LUZ19, LUZ24, 14-1, YuA, PAK_P3 and giant phage phiKZ during early, middle and late infection were collectively charted. Beyond pinpointing traditional promoter and terminator elements and transcription units, these transcriptional profiles provide insights in transcriptional attenuation and splicing events and allow straightforward validation of Group I intron activity. In addition, ONT-cappable-seq data can guide genome-wide discovery of novel regulatory element candidates, including non-coding RNAs and riboswitches. This work substantially expands the number of annotated phage-encoded transcriptional elements identified to date, shedding light on the intricate and diverse gene expression regulation mechanisms in Pseudomonas phages, which can ultimately be sourced as tools for biotechnological applications in phage and bacterial engineering.

Conserved jumbo phage factors required for protein import into a phage nucleus

“…Additionally, EcoRI-Imp3 fusions were inactive (data not shown) and thus whether it is proximal to phage DNA could not be ascertained. Finally, we suspect that co-encoded genes in a putative operon 14 with imp3 are required for proper expression or function as these genes ( p18-imp3-orf60-orf61 ) were required to achieve partial complementation EcoRI-Imp1 ΦPA3 and EcoRI-TopA escape (Extended Data Fig. 4c, 5b).…”