Elucidating patterns of prophage-mediated viral defense and viral counter defense was greatly facilitated by access to a large collection of completely sequenced bacteriophages of a common bacterial host. This collection emerged from the Science Education Alliance phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program that provides authentic research experiences to early career (primarily first year) undergraduate students. SEA-PHAGES students isolate their own phages and annotate sequenced genomes, and give their phages interesting and unusual names, with Charlie, Phrann, Tweety, and Butters, highlighted in this report (see phagesdb.org and seaphages.org for further information).
The largest group of phages emerging from the SEA-PHAGES program are those that infect a single strain of Mycobacterium smegmatis. The >1200 individual phages with sequenced genomes are amazingly diverse genetically and represent a truly fantastic resource, especially given the slow progress of phage genomics relative to other genomic projects. Eleven closely-related phages are grouped in Cluster N, all of which were isolated and genomically characterized by SEA-PHAGES students, followed by detailed bioinformatic analyses in a workshop for SEA-PHAGES faculty.All are temperate phages and form stable lysogens in M. smegmatis. The patterns of viral defense provided by Cluster N prophages were elucidated by testing 80 genomically diverse phages for the plating efficiencies on ten of these lysogens.
The prophage-encoded defense genes were elucidated by transcriptomic analyses of the lysogens, by cloning and expressing the genes in M. smegmatis, and by constructing lysogens in which specific genes were deleted. The specificity with which the defense systems target attacking phages is remarkable; for example, Charlie gene 32 targets only a single phage (Che9c) of those we tested. If we can sample the phage collection more broadly then it seems probable there are more defense systems to be discovered, even among Cluster N phages.Moreover, the majority of the mycobacteriophages are temperate, and we predict that many of them – spanning the full diversity of the collection – code for their own defense systems. The prospects for continued discovery using simple microbiology and straightforward molecular genetics is an especially pleasing thought.
We ought not to be surprised that phages have evolved systems to counter these defenses. In one intriguing instance, phage Tweety codes for a counter-defense system that appears to be tunable against a specific defense system. This helps to remind us that the complex patterns of phage infectivity of lysogens results from both the action of the defense systems and the counter-defenses of the attacking phages. The counter-defense systems as well as the genetic basis for targeting by the defenses can be addressed by isolating and characterizing defense escape mutants, but much of that work lies ahead.
Finally, the apparent abundance of prophage-encoded viral defense systems has notable implications for the roles of temperate phages in microbial ecology. Temperate phages play prominent roles in some microbial communities, and lysogeny can offer a special advantage: success relative to non-lysogenic competitors in the face of vial attack. As such, temperate phages may not just piggyback-the-winner, but make-the-winner, serving as the microbial kingmakers.
Video at: https://youtu.be/KpLA6RkVRnc
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