In 2016, Michigan experienced the largest outbreak of shigellosis, a type of bacillary dysentery caused by Shigella spp., since 1988. Following this outbreak, we isolated 16 novel Shigella -infecting bacteriophages (viruses that infect bacteria) from environmental water sources. Most well-known bacteriophages infect the common laboratory species Escherichia coli and Salmonella enterica , and these phages have built the foundation of molecular and bacteriophage biology. Until now, comparatively few bacteriophages were known to infect Shigella spp., which are close relatives of E. coli . We present a comprehensive analysis of these phages' host ranges, genomes, and structures, revealing genome sizes and capsid properties that are shared by very few previously described phages. After sequencing, a majority of the Shigella phages were found to have genomes of an uncommon size, shared by only 2% of all reported phage genomes. To investigate the structural implications of this unusual genome size, we used cryo-electron microscopy to resolve their capsid structures. We determined that these bacteriophage capsids have similarly uncommon geometry. Only two other viruses with this capsid structure have been described. Since most well-known bacteriophages infect Escherichia or Salmonella , our understanding of bacteriophages has been limited to a subset of well-described systems. Continuing to isolate phages using nontraditional strains of bacteria can fill gaps that currently exist in bacteriophage biology. In addition, the prevalence of Shigella phages during a shigellosis outbreak may suggest a potential impact of human health epidemics on local microbial communities. IMPORTANCE Shigella spp. bacteria are causative agents of dysentery and affect more than 164 million people worldwide every year. Despite the need to combat antibiotic-resistant Shigella strains, relatively few Shigella -infecting bacteriophages have been described. By specifically looking for Shigella -infecting phages, this work has identified new isolates that (i) may be useful to combat Shigella infections and (ii) fill gaps in our knowledge of bacteriophage biology. The rare qualities of these new isolates emphasize the importance of isolating phages on “nontraditional” laboratory strains of bacteria to more fully understand both the basic biology and diversity of bacteriophages.

Viruses depend on host cells to propagate themselves. In mixed populations and communities of host cells, finding these susceptible host cells may have to be balanced with avoiding nonhost cells.

Shared host cells can serve as melting pots for viral genomes, giving many phylogenies a web-like appearance due to horizontal gene transfer. However, not all virus families exhibit web-like phylogenies. Microviruses form three distinct clades, represented by φX174, G4, and α3. Here, we investigate protein-based barriers to horizontal gene transfer between clades. We transferred gene G, which encodes a structural protein, between φX174 and G4, and monitored the evolutionary recovery of the resulting chimeras. In both cases, particle assembly was the major barrier after gene transfer. The G4φXG chimera displayed a temperature-sensitive assembly defect that could easily be corrected through single mutations that promote productive assembly. Gene transfer in the other direction was more problematic. The initial φXG4G chimera required an exogenous supply of both the φX174 major spike G and DNA pilot H proteins. Elevated DNA pilot protein levels may be required to compensate for off-pathway reactions that may have become thermodynamically and/or kinetically favored when the foreign spike protein was present. After three targeted genetic selections, the foreign spike protein was productively integrated into the φX174 background. The first adaption involved a global decrease in gene expression. This was followed by modifications affecting key protein–protein interactions that govern assembly. Finally, gene expression was re-elevated. Although the first selection suppresses nonproductive reactions, subsequent selections promote productive assembly and ultimately viability. However, viable chimeric strains exhibited reduced fitness compared with wild-type. This chimera’s path to recovery may partially explain how unusual recombinant viruses could persist long enough to naturally emerge.

Podophages are, by far, the least well studied of all the bacteriophages. Despite being classified together due to their short, noncontractile tails, there is a huge amount of diversity among members of this group. Of the podophages, the N4-like Schitoviridae family is the least well studied structurally and is quite divergent from well-characterized podophages such as T7 and P22. In this work, we isolate and fully characterize two members of the Schitoviridae family by cryo–electron microscopy, genetics, and biochemistry. We describe the capsid features of Moo19 and B2, including a decoration protein. In addition, we have fully modeled the tail machinery for both phages and identify proteins with esterase activity. Genetic knockouts of the host reveal factors specific for host attachment including key modifications to the O-antigen on the lipopolysaccharide. Moo19 and B2 are both Schitoviridae members, yet some distinct differences in the genome and structure place them into distinct clades.