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1.  Assembly of bacteriophage 80α capsids in a Staphylococcus aureus expression system 
Virology  2012;434(2):242-250.
80α is a temperate, double-stranded DNA bacteriophage of Staphylococcus aureus that can act as a “helper” for the mobilization of S. aureus pathogenicity islands (SaPIs), including SaPI1. When SaPI1 is mobilized by 80α, the SaPI genomes are packaged into capsids that are composed of phage proteins, but that are smaller than those normally formed by the phage. This size determination is dependent on SaPI1 proteins CpmA and CpmB. Here, we show that co-expression of the 80α capsid and scaffolding proteins in S. aureus, but not in E. coli, leads to the formation of procapsid-related structures, suggesting that a host co-factor is required for assembly. The capsid and scaffolding proteins also undergo normal N-terminal processing upon expression in S. aureus, implicating a host protease. We also find that SaPI1 proteins CpmA and CpmB promote the formation of small capsids upon co-expression with 80α capsid and scaffolding proteins in S. aureus.
doi:10.1016/j.virol.2012.08.031
PMCID: PMC3518739  PMID: 22980502
molecular piracy; virus assembly; size determination; procapsid; electron microscopy; scaffolding protein; ribosomal protein L27; co-expression; Staphylococcus aureus pathogenicity island 1
2.  The roles of SaPI1 proteins gp7 (CpmA) and gp6 (CpmB) in capsid size determination and helper phage interference 
Virology  2012;432(2):277-282.
SaPIs are molecular pirates that exploit helper bacteriophages for their own high frequency mobilization. One striking feature of helper exploitation by SaPIs is redirection of the phage capsid assembly pathway to produce smaller phage-like particles with T=4 icosahedral symmetry rather than T=7 bacteriophage capsids. Small capsids can accommodate the SaPI genome but not that of the helper phage, leading to interference with helper propagation. Previous studies identified two proteins encoded by the prototype element SaPI1, gp6 and gp7, in SaPI1 procapsids but not in mature SaPI1 particles. Dimers of gp6 form an internal scaffold, aiding fidelity of small capsid assembly. Here we show that both SaPI1 gp6 (CpmB) and gp7 (CpmA) are necessary and sufficient to direct small capsid formation. Surprisingly, failure to form small capsids did not restore wild-type levels of helper phage growth, suggesting an additional role for these SaPI1 proteins in phage interference.
doi:10.1016/j.virol.2012.05.026
PMCID: PMC3423473  PMID: 22709958
Staphylococcus aureus pathogenicity island; procapsid; bacteriophage assembly; cryo-electron microscopy; molecular piracy
3.  Structure and size determination of bacteriophage P2 and P4 procapsids: function of size responsiveness mutations 
Journal of Structural Biology  2012;178(3):215-224.
Bacteriophage P4 is dependent on structural proteins supplied by a helper phage, P2, to assemble infectious virions. Bacteriophage P2 normally forms an icosahedral capsid with T=7 symmetry from the gpN capsid protein, the gpO scaffolding protein and the gpQ portal protein. In the presence of P4, however, the same structural proteins are assembled into a smaller capsid with T=4 symmetry. This size determination is effected by the P4-encoded protein Sid, which forms an external scaffold around the small P4 procapsids. Size responsiveness (sir) mutants in gpN fail to assemble small capsids even in the presence of Sid. We have produced large and small procapsids by co-expression of gpN with gpO and Sid, respectively, and applied cryo-electron microscopy and three-dimensional reconstruction methods to visualize these procapsids. gpN has an HK97-like fold and interacts with Sid in an exposed loop where the sir mutations are clustered. The T=7 lattice of P2 has dextro handedness, unlike the laevo lattices of other phages with this fold observed so far.
doi:10.1016/j.jsb.2012.04.002
PMCID: PMC3361666  PMID: 22508104
molecular piracy; capsid assembly; morphogenesis; cryo-electron microscopy; three-dimensional reconstruction; HK97 fold
4.  The Staphylococcus aureus pathogenicity island 1 protein gp6 functions as an internal scaffold during capsid size determination 
Journal of molecular biology  2011;412(4):710-722.
Staphylococcus aureus pathogenicity island 1 (SaPI1) is a mobile genetic element that carries genes for several superantigen toxins. SaPI1 is normally stably integrated into the host genome, but can become mobilized by “helper” bacteriophage 80α, leading to the packaging of SaPI1 genomes into phage-like transducing particles that are composed of structural proteins supplied by the helper phage, but having smaller capsids. We show that the SaPI1-encoded protein gp6 is necessary for efficient formation of small capsids. The NMR structure of gp6 reveals a dimeric protein with a helix-loop-helix motif similar to that of bacteriophage scaffolding proteins. The gp6 dimer matches internal densities that bridge capsid subunits in cryo-EM reconstructions of SaPI1 procapsids, suggesting that gp6 acts as an internal scaffolding protein in capsid size determination.
doi:10.1016/j.jmb.2011.07.036
PMCID: PMC3175317  PMID: 21821042
bacteriophage; mobilization; virus assembly; NMR spectroscopy; cryo-electron microscopy
5.  Mobilization of pathogenicity islands by Staphylococcus aureus strain Newman bacteriophages 
Bacteriophage  2012;2(2):70-78.
Staphylococcus aureus pathogenicity islands (SaPIs) are mobile genetic elements that encode virulence factors and depend on helper phages for their mobilization. Such mobilization is specific and depends on the ability of a phage protein to inactivate the SaPI repressor Stl. Phage 80α can mobilize several SaPIs, including SaPI1 and SaPIbov1, via its Sri and Dut proteins, respectively. In many cases, the capsids formed in the presence of the SaPI are smaller than those normally produced by the phage. Two SaPI-encoded proteins, CpmA and CpmB, are involved in this size determination process. S. aureus strain Newman contains four prophages, named φNM1 through φNM4. Phages φNM1 and φNM2 are very similar to phage 80α in the structural genes, and encode almost identical Sri proteins, while their Dut proteins are highly divergent. We show that φNM1 and φNM2 are able to mobilize both SaPI1 and SaPIbov1 and yield infectious transducing particles. The majority of the capsids formed in all cases are small, showing that both SaPIs can redirect the capsid size of both φNM1 and φNM2.
PMCID: PMC3442828  PMID: 23050217
mobile genetic elements; SaPI1; SaPIbov1; bacteriophage assembly; capsid size determination
6.  A conformational switch involved in maturation of Staphylococcus aureus bacteriophage 80α capsids 
Journal of molecular biology  2010;405(3):863-876.
Bacteriophages are involved in many aspects of the spread and establishment of virulence factors in Staphylococcus aureus, including the mobilization of genetic elements known as pathogenicity islands (SaPIs), which carry genes for superantigen toxins and other virulence factors. SaPIs are packaged into phage-like transducing particles using proteins supplied by the helper phage. We have used cryo-electron microscopy and icosahedral reconstruction to determine the structure of the procapsid and the mature capsid of 80α, a bacteriophage that can mobilize several different SaPIs. The 80α capsid has T = 7 icosahedral symmetry with the capsid protein organized into pentameric and hexameric clusters that interact via prominent trimeric densities. The 80α capsid protein was modeled based on the capsid protein fold of bacteriophage HK97, and fitted into the 80α reconstructions. The models show that the trivalent interactions are mediated primarily by a 22-residue β hairpin structure called the P loop that is not found in HK97. Capsid expansion is associated with a conformational switch in the spine helix that is propagated throughout the subunit, unlike the domain rotation mechanism in phages HK97 or P22.
doi:10.1016/j.jmb.2010.11.047
PMCID: PMC3017672  PMID: 21129380
procapsid; structure; assembly; three-dimensional reconstruction; pathogenicity island

Results 1-6 (6)