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.
Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long 800 nm tail. We found that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular and viral two-barrel RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We determined the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab claw-like architecture similar to previously reported two-barrel RNAPs. The virion RNAP adopts a unique flat structure without a clamp, which likely reflects the requirement for its extrusion through the narrow channel in the phage tail for delivery into the cell. Structure and sequence comparisons of the P23-45 RNAPs with other phage and cellular RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate remarkable adaptability of two-barrel RNAPs that can be attained within a single virus species.
Efficient transcriptional
terminators are essential for
the performance
of genetic circuitry in microbial SynBio hosts. In recent years, several
libraries of characterized strong terminators have become available
for model organisms such as Escherichia coli. Conversely, terminator libraries for nonmodel species remain scarce,
and individual terminators are often ported over from model systems,
leading to unpredictable performance in their new hosts. In this work,
we mined the genomes of Pseudomonas infecting phages LUZ7 and LUZ100 for transcriptional terminators
utilizing the full-length RNA sequencing technique “ONT-cappable-seq”
and validated these terminators in three Gram-negative hosts using
a terminator trap assay. Based on these results, we present nine terminators
for E. coli, Pseudomonas
putida, and Pseudomonas aeruginosa, which outperform current reference terminators. Among these, terminator
LUZ7 T50 displays potent bidirectional activity. These data further
support that bacteriophages, as evolutionary-adapted natural predators
of the targeted bacteria, provide a valuable source of microbial SynBio
parts.
The Autographiviridae is a diverse yet distinct family of bacterial viruses marked by a strictly lytic lifestyle and a generally conserved genome organization. We here characterise Pseudomonas aeruginosa phage LUZ100, a distant relative of type phage T7. LUZ100 is a podovirus with a limited host range and identified LPS as the likely phage receptor. Interestingly, infection dynamics of LUZ100 indicated moderate adsorption rates and low virulence, hinting towards temperate behavior. This hypothesis was supported by genomic analysis, which showed that LUZ100 shares the conventional T7-like genome organization, yet encodes key genes associated with a temperate lifestyle. To unravel the peculiar characteristics of LUZ100, ONT-cappable-seq transcriptomics analysis was performed. This data generated a bird’s-eye view of the LUZ100 transcriptome and enabled the discovery of key regulatory elements, antisense RNA, and transcriptional unit structures. The transcriptional map of LUZ100 also allowed us to identify new RNAP-promoter pairs that can form the basis for biotechnological parts and tools for new synthetic transcription regulation circuitry. The ONT-cappable-seq data revealed that the LUZ100 integrase and a MarR-like regulator (proposed to be involved in the lytic/lysogeny decision), are actively co-transcribed in an operon. In addition, the presence of a phage-specific promoter transcribing the phage-encoded RNA polymerase, raises questions on the regulation of this polymerase, and suggests it is interwoven with the MarR-based regulation. This transcriptomics-driven characterisation of LUZ100 supports the increasing evidence that T7-like phages should not straightforwardly be marked as having a strictly lytic lifecycle.
RNA sequencing has become the method of choice to study the transcriptional landscape of phage-infected bacteria. However, short-read RNA sequencing approaches generally fail to capture the primary 5′ and 3′ boundaries of transcripts, confounding the discovery of key transcription initiation and termination events as well as operon architectures. Yet, the elucidation of these elements is crucial for the understanding of the strategy of transcription regulation during the infection process, which is currently lacking beyond a handful of model phages. To this end, we developed ONT-cappable-seq, a specialized long-read RNA sequencing technique that allows end-to-end sequencing of primary prokaryotic transcripts using the Nanopore sequencing platform. We applied ONT-cappable-seq to study transcription of Pseudomonas aeruginosa phage LUZ7, obtaining a comprehensive genome-wide map of viral transcription start sites, terminators, and complex operon structures that fine-regulate gene expression. Our work provides new insights in the RNA biology of a non-model phage, unveiling distinct promoter architectures, putative small non-coding viral RNAs, and the prominent regulatory role of terminators during infection. The robust workflow presented here offers a framework to obtain a global, yet fine-grained view of phage transcription and paves the way for standardized, in depth transcription studies for microbial viruses or bacteria in general.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.