Cryo-EM analysis of Pseudomonas phage Pa193 structural components.

The World Health Organization has designated Pseudomonas aeruginosa as a critical pathogen for the development of new antimicrobials. Bacterial viruses, or bacteriophages, have been used in various clinical settings, commonly called phage therapy, to address this growing public health crisis. Here, we describe a high-resolution structural atlas of a therapeutic, contractile-tailed Pseudomonas phage, Pa193. We used bioinformatics, proteomics, and cryogenic electron microscopy single particle analysis to identify, annotate, and build atomic models for 21 distinct structural polypeptide chains forming the icosahedral capsid, neck, contractile tail, and baseplate. We identified a putative scaffolding protein stabilizing the interior of the capsid 5-fold vertex. We also visualized a large portion of Pa193 ~ 500 Å long tail fibers and resolved the interface between the baseplate and tail fibers. The work presented here provides a framework to support a better understanding of phages as biomedicines for phage therapy and inform engineering opportunities.

Integrative structural analysis of Pseudomonas phage DEV reveals a genome ejection motor.

DEV is an obligatory lytic Pseudomonas phage of the N4-like genus, recently reclassified as Schitoviridae. The DEV genome encodes 91 ORFs, including a 3,398 amino acid virion-associated RNA polymerase. Here, we describe the complete architecture of DEV, determined using a combination of cryo-electron microscopy localized reconstruction, biochemical methods, and genetic knockouts. We built de novo structures of all capsid factors and tail components involved in host attachment. We demonstrate that DEV long tail fibers are essential for infection of Pseudomonas aeruginosa and dispensable for infecting mutants with a truncated lipopolysaccharide devoid of the O-antigen. We identified DEV ejection proteins and, unexpectedly, found that the giant DEV RNA polymerase, the hallmark of the Schitoviridae family, is an ejection protein. We propose that DEV ejection proteins form a genome ejection motor across the host cell envelope and that these structural principles are conserved in all Schitoviridae.

Use of Localized Reconstruction to Visualize the Shigella Phage Sf6 Tail Apparatus.

Cryogenic electron microscopy (cryo-EM) single-particle analysis has revolutionized the structural analysis of icosahedral viruses, including tailed bacteriophages. In recent years, localized (or focused) reconstruction has emerged as a powerful data analysis method to capture symmetry mismatches and resolve asymmetric features in icosahedral viruses. Here, we describe the methods used to reconstruct the 2.65-MDa tail apparatus of the Shigella phage Sf6, a representative member of the Podoviridae superfamily.

Molecular Architecture of Salmonella Typhimurium Virus P22 Genome Ejection Machinery.

Bacteriophage P22 is a prototypical member of the Podoviridae superfamily. Since its discovery in 1952, P22 has become a paradigm for phage transduction and a model for icosahedral viral capsid assembly. Here, we describe the complete architecture of the P22 tail apparatus (gp1, gp4, gp10, gp9, and gp26) and the potential location and organization of P22 ejection proteins (gp7, gp20, and gp16), determined using cryo-EM localized reconstruction, genetic knockouts, and biochemical analysis. We found that the tail apparatus exists in two equivalent conformations, rotated by ∼6° relative to the capsid. Portal protomers make unique contacts with coat subunits in both conformations, explaining the 12:5 symmetry mismatch. The tail assembles around the hexameric tail hub (gp10), which folds into an interrupted ß-propeller characterized by an apical insertion domain. The tail hub connects proximally to the dodecameric portal protein and head-to-tail adapter (gp4), distally to the trimeric tail needle (gp26), and laterally to six trimeric tailspikes (gp9) that attach asymmetrically to gp10 insertion domain. Cryo-EM analysis of P22 mutants lacking the ejection proteins gp7 or gp20 and biochemical analysis of purified recombinant proteins suggest that gp7 and gp20 form a molecular complex associated with the tail apparatus via the portal protein barrel. We identified a putative signal transduction pathway from the tailspike to the tail needle, mediated by three flexible loops in the tail hub, that explains how lipopolysaccharide (LPS) is sufficient to trigger the ejection of the P22 DNA in vitro.