Cleavage and Structural Transitions during Maturation of Staphylococcus aureus Bacteriophage 80α and SaPI1 Capsids

Abstract

In the tailed bacteriophages, DNA is packaged into spherical procapsids, leading to expansion into angular, thin-walled mature capsids. In many cases, this maturation is accompanied by cleavage of the major capsid protein (CP) and other capsid-associated proteins, including the scaffolding protein (SP) that serves as a chaperone for the assembly process. Staphylococcus aureus bacteriophage 80α is capable of high frequency mobilization of mobile genetic elements called S. aureus pathogenicity islands (SaPIs), such as SaPI1. SaPI1 redirects the assembly pathway of 80α to form capsids that are smaller than those normally made by the phage alone. Both CP and SP of 80α are N-terminally processed by a host-encoded protease, Prp. We have analyzed phage mutants that express pre-cleaved or uncleavable versions of CP or SP, and show that the N-terminal sequence in SP is absolutely required for assembly, but does not need to be cleaved in order to produce viable capsids. Mutants with pre-cleaved or uncleavable CP display normal viability. We have used cryo-EM to solve the structures of mature capsids from an 80α mutant expressing uncleavable CP, and from wildtype SaPI1. Comparisons with structures of 80α and SaPI1 procapsids show that capsid maturation involves major conformational changes in CP, consistent with a release of the CP N-arm by SP. The hexamers reorganize during maturation to accommodate the different environments in the 80α and SaPI1 capsids.

Structure of the host cell recognition and penetration machinery of a Staphylococcus aureus bacteriophage

“…This process most likely requires conformational changes in the baseplate in order to expose the hydrolase domains of Tal and Hyd ( Fig 6B). We previously observed that treatment of 80α with heat or low pH led to clustering of baseplates, presumably due to exposure of hydrophobic sequences resulting from a conformational change [16]. The observed conformational differences in the stem between the 80α and ϕ11 RBPs and the phage P68 tail fiber ( Fig 5B and 5C) suggest that the hinge might constitute a pivot point that allows the RBPs to rotate.…”

“…80α tails were produced using an 80α lysogen (strain ST247) with a deletion of the first 13 amino acids of the scaffolding protein (gp46), which results in failure to form capsids and accumulation of tails in the lysate ( Fig 1C) [16]. Initial cryo-EM datasets of up to �7,000 baseplate particle images were collected with a Philips CM20 microscope on SO-163 film or using an FEI Titan Krios microscope equipped with a Direct Electron DE-20 detector, and reconstructed with sixfold (C6) symmetry to 9.3 Å resolution (FSC 0.5 ) using EMAN and EMAN2 [17].…”

“…80α tails were produced by mitomycin C induction of S. aureus strain ST247, which is an 80α lysogen with a deletion of residues 1-13 from the scaffolding protein, gp46 [16]. The tails were purified from the lysate by PEG precipitation and CsCl gradient purification, as previously described.…”

“…Phage 80α is a typical staphylococcal siphovirus with a 43.8 kbp genome and a 63 nm icosahedral capsid, attached to a 190 nm long flexuous tail that is capped by an ornate baseplate [14][15][16]. 80α is one of the best-described S. aureus phages, and is closely related to ϕETA2 and other phages involved in specifying bacterial pathogenicity [14].…”

Structure of the host cell recognition and penetration machinery of a Staphylococcus aureus bacteriophage

“…Phage 80α is a typical staphylococcal siphovirus with a 43.8 kbp genome and a 63 nm icosahedral capsid, attached to a 190 nm long flexuous tail that is capped by an ornate baseplate (14)(15)(16). 80α is one of the best-described S. aureus phages, and is closely related to φETA2 and other phages involved in specifying bacterial pathogenicity (14).…”