Transcriptomics-Driven Characterization of LUZ100, a T7-like Pseudomonas Phage with Temperate Features

Abstract

Bacteriophage T7, considered the “model phage” of the Autographiviridae family, is marked by a strictly lytic life cycle and conserved genome organization. Recently, novel phages within this clade have emerged which display characteristics associated with a temperate life cycle.

Sourcing Phage-Encoded Terminators Using ONT-cappable-seq for SynBio Applications in Pseudomonas

“…The ONT-cappable-seq method enables full-length sequencing of primary prokaryotic transcripts and can be readily leveraged to mine regulatory features from densely coded phage genomes, including transcriptional terminators (Figure ). Indeed, the transcriptional landscapes ofPseudomonasphages LUZ7 and LUZ100 obtained by ONT-cappable-seq were leveraged to identify 61 and 22 transcription termination sites, respectively. , These phage-encoded terminator sequences are short and highly diverse and display varying levels of transcription read-through, making them interesting candidate terminator parts for SynBio applications. About 20% of the terminator sequences from LUZ7 and LUZ100 are predicted to be intrinsic, factor-independent terminators, which are hallmarked by a canonical GC-rich hairpin and an uridine-rich terminal segment to destabilize the RNA polymerase (RNAP) elongation complex .…”

“…The transcriptional landscapes of phage LUZ7 and phage LUZ100 were previously identified using ONT-cappable-seq. , In short, RNA was extracted at different time points during phage infection and subjected to library preparation. For LUZ7, the different time points were sequenced individually to obtain a temporal transcriptional overview.…”

“…This approach has been established usingPseudomonas viruses LUZ7 and LUZ100. 18,19 In this work, we mine bacterial virus genomes using ONTcappable-seq to identify short, highly potent, and sequencediverse terminators for nonmodel hosts. Bacteriophages, or phages in short, have a compact genome which, from an evolutionary perspective, is fully adapted to their host in terms of GC content and codon usage.…”

“…In contrast to Illumina-based, short-read RNA-seq methods, , long-read transcriptomic approaches allow sequencing of full-length transcripts and hence provide a comprehensive manner to delineate the 5′ and 3′ transcript boundaries. − For example, ONT-cappable-seq, a recently established nanopore-based cDNA sequencing method, enables end-to-end sequencing of primary prokaryotic transcripts, yielding a genome-wide map of key regulatory features encoded by viruses and their bacterial hosts, including transcription start sites (TSSs) and transcription termination sites (TTSs) . By specifically capturing intact, full-length primary RNA molecules by their triphosphate 5′ end during the ONT-cappable-seq library preparation, this method is ideally suited to accurately identify RNA 5′ and 3′ ends. , In addition to mapping transcription termination events, ONT-cappable-seq provides insights in their apparent termination strengths and dynamics. This approach has been established usingPseudomonas viruses LUZ7 and LUZ100. , …”

“…By specifically capturing intact, full-length primary RNA molecules by their triphosphate 5′ end during the ONT-cappable-seq library preparation, this method is ideally suited to accurately identify RNA 5′ and 3′ ends. , In addition to mapping transcription termination events, ONT-cappable-seq provides insights in their apparent termination strengths and dynamics. This approach has been established usingPseudomonas viruses LUZ7 and LUZ100. , …”