ABSTRACT 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. Importance Bacteriophage 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, we’ve 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.

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