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Phages have demonstrated significant potential as therapeutics in bacterial disease control and as diagnostics due to their targeted bacterial host range. Host range has typically been defined by plaque assays; an important technique for therapeutic development that relies on the ability of a phage to form a plaque upon a lawn of monoculture bacteria. Plaque assays cannot be used to evaluate a phage’s ability to recognize and adsorb to a bacterial strain of interest if the infection process is thwarted post-adsorption or is temporally delayed, and it cannot highlight which phages have the strongest adsorption characteristics. Other techniques, such as classic adsorption assays, are required to define a phage’s “adsorptive host range.” The issue shared amongst all adsorption assays, however, is that they rely on the use of a complete bacteriophage and thus inherently describe when all adsorption-specific machinery is working together to facilitate bacterial surface adsorption. These techniques cannot be used to examine individual interactions between a singular set of a phage’s adsorptive machinery (like long tail fibers, short tail fibers, tail spikes, etc.) and that protein’s targeted bacterial surface receptor. To address this gap in knowledge we have developed a high-throughput, filtration-based, bacterial binding assay that can evaluate the adsorptive capability of an individual set of a phage’s adsorption machinery. In this manuscript, we used a fusion protein comprised of an N-terminal bioluminescent tag translationally fused to T4’s long tail fiber binding tip (gp37) to evaluate and quantify gp37’s relative adsorptive strength against the Escherichia coli reference collection (ECOR) panel of 72 Escherichia coli isolates. Gp37 could adsorb to 61 of the 72 ECOR strains (85%) but coliphage T4 only formed plaques on 8 of the 72 strains (11%). Overlaying these two datasets, we were able to identify ECOR strains incompatible with T4 due to failed adsorption, and strains T4 can adsorb to but is thwarted in replication at a step post-adsorption. While this manuscript only demonstrates our assay’s ability to characterize adsorptive capabilities of phage tail fibers, our assay could feasibly be modified to evaluate other adsorption-specific phage proteins.
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This content will become publicly available on June 17, 2026
Phage DisCo: targeted discovery of bacteriophages by co-culture
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.
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- Award ID(s):
- 2331228
- PAR ID:
- 10653758
- Editor(s):
- Reyes_Munoz, Alejandro
- Publisher / Repository:
- American Society for Microbiology
- Date Published:
- Journal Name:
- mSystems
- Volume:
- 10
- Issue:
- 6
- ISSN:
- 2379-5077
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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