Summary
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 64 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 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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
In the revised manuscript we have looked closely at the significance of the tailed F-plasmid dependent phage FtMidnight, and found it to be related to phages that depend on the type 4 pilus (T4P) as a receptor, an orthogonal contractile structure that is thought to be unrelated to conjugal pili. We believe this finding has interesting implications for the evolution of plasmid-dependent phages, as both other phage groups that F-plasmid dependent phages belong to (the ssDNA inoviruses and the ssRNA fiersviruses) also include T4P targeting phages. Secondly, we have conducted further experiments to understand the genetic basis of plasmid-dependent tectivirus host range, by assessing the presence of defense systems in the host genomes, and by creating recombinant phages with expanded host ranges. Our results show that variation in the phage holin protein is associated with host range differences, and imply that these phages have to adapt their lysis machinery to accommodate different host cell physiologies. Along with these large additions, we have discovered one additional ssRNA-phage that targets IncP plasmids which is closely related to our F-plasmid targeting phages. Finally, we have made improvements to the language and data interpretation throughout the manuscript, and changed use of viruses to phages in the title and throughtout.
