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Phages interact with many components of bacterial physiology from the surface to the cytoplasm. Although there are methods to determine the receptors and intracellular systems a specified phage interacts with retroactively, finding a phage that interacts with a chosen piece of bacterial physiology a priori is very challenging. Variation in phage plaque morphology does not to reliably distinguish distinct phages, and therefore many potentially redundant phages may need to be isolated, purified, and individually characterized to find phages of interest. Here, we present a method in which multiple bacterial strains are co-cultured on the same screening plate to add an extra dimension to plaque morphology data. In this method,phage discovery by co-culture (Phage DisCo), strains are isogenic except for fluorescent tags and one perturbation expected to impact phage infection. Differential plaquing on the strains is easily detectable by fluorescent signal and implies that the perturbation made to the surviving strain in a plaque prevents phage infection. We validate the Phage DisCo method by showing that characterized phages have the expected plaque morphology on Phage DisCo plates and demonstrate the power of Phage DisCo for multiple targeted discovery applications, from receptors to phage defense systems.IMPORTANCEIn this work, we describe a targeted phage discovery method that allows immediate isolation of phages with specific traits. Currently, to find a phage with specific properties, huge libraries of phages must be collected and screened retroactively. This assay, Phage Discovery by Co-culture (Phage DisCo), works by co-culture of host strains that are identical except for one perturbation that may interfere with phage infection and a unique fluorescent marker. These strains are co-cultured with an environmental sample of interest in traditional plaque assay format, making phage characteristics easily identifiable by fluorescent signal after imaging of the screening plate. We validate that Phage DisCo can identify phages with specific properties, even when these phages are rare in samples. This approach allows rapid exploration of the diversity within phage samples with vastly streamlined processes, and we anticipate it will be widely adopted within the phage discovery field. 

Abstract Phages exert profound evolutionary pressure on bacteria by interacting with receptors on the cell surface to initiate infection. While the majority of phages use chromosomally encoded cell surface structures as receptors, plasmid-dependent phages exploit plasmid-encoded conjugation proteins, making their host range dependent on horizontal transfer of the plasmid. Despite their unique biology and biotechnological significance, only a small number of plasmid-dependent phages have been characterized. Here we systematically search for new plasmid-dependent phages targeting IncP and IncF plasmids using a targeted discovery platform, and find that they are common and abundant in wastewater, and largely unexplored in terms of their genetic diversity. Plasmid-dependent phages are enriched in non-canonical types of phages, and all but one of the 65 phages we isolated were non-tailed, and members of the lipid-containing tectiviruses, ssDNA filamentous phages or ssRNA phages. We show that plasmid-dependent tectiviruses exhibit profound differences in their host range which is associated with variation in the phage holin protein. Despite their relatively high abundance in wastewater, plasmid-dependent tectiviruses are missed by metaviromic analyses, underscoring the continued importance of culture-based phage discovery. Finally, we identify a tailed phage dependent on the IncF plasmid, and find related structural genes in phages that use the orthogonal type 4 pilus as a receptor, highlighting the evolutionarily promiscuous use of these distinct contractile structures by multiple groups of phages. Taken together, these results indicate plasmid-dependent phages play an under-appreciated evolutionary role in constraining horizontal gene transfer via conjugative plasmids.