Biology subjects

Putzeys, L.

Publications and source records attributed to Putzeys, L..

3 recordsLinked to original sources

bioRxiv2022

Tail-tape-fused virion and non-virion RNA polymerases of a thermophilic virus with an extremely long tail

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP)1 that defines the length of its extraordinarily long 800 nm tail2,3. 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.

molecular biology↗

bioRxiv2022

Transcriptomics-driven characterisation of novel T7-like temperate Pseudomonas phage LUZ100

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 birds-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. ImportanceBacteriophage T7, considered the model phage of the Autographiviridae family, is marked by a strictly lytic lifecycle and conserved genome organisation. Recently, novel phages of this clade are emerging and showing characteristics associated to a lysogenic lifecycle. Screening for temperate behaviour is of outmost importance in fields like phage therapy, where strictly lytic phages are generally required for therapeutic applications. In this study, weve used an omics-driven approach to characterise the T7-like Pseudomonas aeruginosa phage LUZ100. These results led to the identification of actively transcribed lysogeny-associated genes in the phage genome, pointing out that temperate T7-like phages are emerging more frequent than initially thought. In short, the combination of genomics and transcriptomics allowed us to obtain a better understanding of the biology of non-model Autographiviridae phages, which can be used to optimize the implementation of phages and their regulatory elements in phage therapy and biotechnological applications, respectively.

microbiology↗

bioRxiv2022

Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages

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.

genomics↗