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Defining the essential genome of diverse phages with Phage Tn-seq

ABSTRACT: Phages are important drivers of bacterial evolution with therapeutic potential as antimicrobials. However, gaps in our understanding of phages and ability to rapidly engineer them with new genetic cargo hinders progress towards phage-based therapies. To address the lack of unbiased, genome-wide mutational tools for phages, we developed transposon mutagenesis employing CRISPR-anti-CRISPR (Acr)-based selection and deep-sequencing (Phage Tn-seq). Transposon mutagenesis was effective for phages with unmodified or hypermodified genomes and a jumbo phage that protects its DNA within a nucleus. Phage Tn-seq enabled phage gene essentiality assignment consistent with structural proteomics and core gene conservation. Insertion biases allowed prediction of transcriptional direction and early injected phage DNA regions. We exploited the method to rapidly deliver new cargo to phage genomes in just a few days and used an AI-designed Acr to expand the phage transposon toolbox. Phage Tn-seq is versatile tool to advance our understanding and applications of phages.

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ORGANISM(S): Prodigiosinella Confusarubida

SUBMITTER: Torsten Kleffmann

LAB HEAD: Peter Fineran

PROVIDER: PXD072185 | Pride | 2026-07-30

REPOSITORIES: Pride

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Project description:Phages are viruses that infect prokaryotes and can shape microbial communitiesby lysis, thus offering applications in various fields. However, challengesexist in sampling, isolation and accurate prediction of the host specificity ofphages as well as in the identification of newly replicated virions in response toenvironmental challenges. A new workflow using biorthogonal non-canonicalamino acid tagging (BONCAT) and click chemistry (CC) allowed combinedanalysis of phages and their hosts, the identification of newly replicated virions,and the specific tagging of phages with biotin for affinity chromatography.Replication of phage λ in Escherichia coli was selected as a model for workflowdevelopment. Specific labeling of phage λ proteins with the non-canonicalamino acid 4-azido-L-homoalanine (AHA) during phage development in E. coliwas confirmed by LC–MS/MS. Subsequent tagging of AHA with fluorescentdyes via CC allowed the visualization of phages adsorbed to the cell surfaceby fluorescence microscopy. Flow cytometry enabled the automated detectionof these fluorescent phage-host complexes. Alternatively, AHA-labeled phageswere tagged with biotin for purification by affinity chromatography. Despitebiotinylation the tagged phages could be purified and were infectious afterpurification. Applying this approach to environmental samples would enablehost screening without cultivation. A flexible and powerful workflow for thedetection and enrichment of phages and their hosts in pure cultures has beenestablished. The developed method lays the groundwork for future workflowsthat could enable the isolation of phage-host complexes from diverse complexmicrobial communities using fluorescence-activated cell sorting or biotinpurification. The ability to expand and customize the workflow through thegrowing range of compounds for CC offers the potential to develop a versatiletoolbox in phage research. This work provides a starting point for these furtherstudies by providing a comprehensive standard operating procedure.

Project description:ICP1 is a predominant lytic phage of the Vibrio cholerae, the causative pathogen of diarrheal disease of cholera. A mobile genetic element called PLE (Phage inducible chromosomal island-like element) is often integrated into the V. cholerae chromosome, which is activated to excise only upon ICP1. PLE then redirects the ICP1 machinery to promote its own replication, packaging, and propagation while inhibiting ICP1 at multiple stages of infection. Thus, PLEs act as ‘phage satellites’ that parasitize ICP1 and provide anti-ICP1 phage defense to the V. cholerae population. The ICP1 and PLE virions have similar overall morphologies: an icosahedral capsid and a contractile tail. However, PLE does not encode a full suite of structural proteins. PLE remodels ICP1 capsid components to assemble a smaller capsid fitted for its shorter genomic DNA (Boyd C.M. et al., eLife 12, RP87611, 2024). This agrees with models of phage satellite assembly in the literature, which also propose that phage satellites hijack unmodified tails of the phages they parasitize. Surveillance of V. cholerae in cholera patient stool samples from 2019 to 2023 revealed PLE11, a novel PLE variant that potently inhibits ICP1 by the activity of a gene, Rta, which causes the production of tailless ICP1 particles. Genetic screening of escape mutations indicates the ICP1’s tape measure protein (TMP) as a probable target. However, PLE11 virions have functional contractile tails despite Rta-mediated inhibition of ICP1 tails. Further, bioinformatic analyses indicate PLE genomes encode a subset of tail protein, including a tail assembly chaperone and tape measure protein. Thus, we inquired whether PLE manipulates the ICP1 tail assembly to create hybrid PLE tails. In this proteomics study, we analyzed proteins constituting purified virions of ICP1 and PLE11 to decipher their structural compositions. The data identifies all predicted ICP1 structural proteins in ICP1 virions at expected abundances and demonstrates that PLE11 tails are hybrid, comprising PLE11-encoded TMP, baseplate hub, two proteins of unknown function, and the remaining tail proteins hijacked from ICP1. Together, the data substantiate the findings from our genetics and molecular biology approaches to uncover a novel phenomenon of tail assembly pathway manipulation by a phage satellite. Here, samples YM1 and YM4 are purified PLE11 and ICP1_2006_Dha_E virions (respectively), and the resulting peptides were searched against the translated genomes of ICP1 (Accession code: MH310934), V. cholerae (Accession code: N16961), and PLE11 (available on request). A combined database with fasta formatted sequences of the three proteomes is also provided with the data.

			Action	DRS
	17013_L2F.msf	Msf
	17013_L2F1_1.RAW	Raw
	17013_L2F1_2.RAW	Raw
	17013_L2F2_1.RAW	Raw
	17013_L2F2_2.RAW	Raw

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Defining the essential genome of diverse phages with Phage Tn-seq

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