Fig. 1: A method for systematic discovery of plasmid-dependent phages by fluorescence assisted co-culture (Phage DisCo).

a Comparison between monocultured lawns and a co-cultured lawn. All images show merged GFP and mScarlet fluorescence channels (GFP shown in blue for visualization purposes). In monocultured lawns with exclusively S. enterica ^RP4 (red) or P. putida ^RP4 (blue), only plasmid-dependent phage PRD1 and the appropriate species-specific phages (S. enterica phage 9NA or P. putida phage SVOϕ44) generate plaques. In the co-culture lawn (magenta, showing the overlap of both bacterial hosts), the species-specific phages form plaques on one of the species while plasmid-dependent phage PRD1 forms plaques on both species. b Schematic of the Phage DisCo method and screening strategy. Environmental samples were collected from around Boston, USA, and processed into a co-culture lawn with two plasmid-carrying bacterial hosts labeled with different fluorescent markers. After incubation, the plates were imaged in both fluorescence channels. The merged image was then used to distinguish species-specific phages (forming red or blue plaques) from plasmid-dependent phages (forming dark plaques). c Imaging of co-cultured lawn with white light or fluorescent light channels, with approximately equimolar concentrations of phages shown in (a) to simulate a screening plate from an environmental sample containing plasmid-dependent and species-specific phages. Individual plaques are clearly discernible as 9NA (blue plaques), SVOϕ44 (red plaques), and PRD1 (dark plaques). d Sankey diagram summarizing the properties of the 65 novel plasmid-dependent phages isolated in this study, broken down by plasmid-dependency, phage genus, and genome type. In total, 63 phages were obtained from the broad-host range phage DisCo screen (light gray horizontal blocks), and an additional two phages were isolated from a narrow-host range screen (dark gray horizontal blocks).