Structure and size determination of bacteriophage P2 and P4 procapsids: Function of size responsiveness mutations
Introduction
Bacteriophage P2 is a temperate, double-stranded (ds) DNA phage of the Myoviridae family that infects Escherichia coli and other enterobacteria (Bertani and Bertani, 1970, Bertani and Six, 1988, Nilsson and Haggård-Ljungquist, 2006). P2-like prophages are common in the environment (Breitbart et al., 2002), are present in about 30% of strains in the E. coli reference collection (Nilsson et al., 2004), and likely play an important role in horizontal gene transfer in bacterial evolution.
The P2 virion consists of a 60 nm diameter isometric capsid, or head, attached to a 135 nm long contractile tail tipped by a base plate with six tail fibers attached. The capsids consist of 415 copies of capsid protein, gene product (gp) of the N gene (gpN) arranged with T = 7 icosahedral symmetry (Dokland et al., 1992). P2 capsids are assembled as roughly spherical precursors called procapsids from gpN (40.2 kDa), several copies of a gpO scaffolding protein and a dodecameric connector or portal (gpQ) that forms a unique vertex through which the DNA is subsequently packaged (Fig. 1A) (Lengyel et al., 1973, Chang et al., 2008). At some point during the assembly process, gpN is processed by the removal of 31 N-terminal residues, yielding a mature cleavage product called N∗ (36.7 kDa) (Lengyel et al., 1973, Rishovd and Lindqvist, 1992). Likewise, gpQ is processed to Q∗ by the removal of 26 N-terminal amino acids, while gpO is cleaved between residues 141 and 142, leaving an N-terminal fragment, O∗, that remains inside the mature capsid (Rishovd et al., 1994, Chang et al., 2008). These cleavage events are thought to be carried out by gpO, which contains a serine protease domain and possesses autoproteolytic activity (Wang et al., 2006, Chang et al., 2009). The proteolytic activity of gpO resides in the N-terminal half of the protein, while the C-terminal 90 amino acids comprise a scaffolding domain, which is required and sufficient for promoting capsid assembly (Wang et al., 2006, Chang et al., 2008, Chang et al., 2009).
Circular P2 genomic DNA substrates (33.6 kb) are packaged into these empty procapsids through the action of the terminase proteins gpM and gpP (Bowden and Modrich, 1985), upon which the capsid undergoes major structural transitions that lead to a more angular mature capsid (Lengyel et al., 1973, Marvik et al., 1994a, Chang et al., 2008) (Fig. 1B). The tails, which are assembled via a separate pathway, are attached to the gpQ connector at the unique vertex (Lengyel et al., 1974). A head completion protein, gpL, is added to the final assembly product (Chang et al., 2008).
P4 is an 11.6 kb replicon that can exist either as a plasmid or integrated into the host genome like a prophage (Briani et al., 2001, Deho and Ghisotti, 2006). It is genetically unrelated to P2, except in the cohesive (cos) ends. P4 does not normally get mobilized at high frequency and lacks genes encoding major structural proteins. In the presence of P2, however, P4 gets packaged into phage particles using structural proteins supplied by the P2 helper (Six, 1975). The capsid produced in the presence of P4 is smaller (45 nm) than the normal P2 capsid and contains 235 copies of gpN arranged with T = 4 symmetry (Dokland et al., 1992, Lindqvist et al., 1993). This size difference is effected by the P4-encoded Sid (Size determination) protein (Barrett et al., 1976), which forms an external scaffold surrounding the P4 procapsids (Marvik et al., 1995) (Fig. 1C). P4 sid mutants fail to form small capsids; however, P4 DNA can still get packaged as dimers or trimers into large capsids at low efficiency (Shore et al., 1978). Mutants in gpN, called sir (sid responsiveness) render the capsid protein unable to form small capsids even in the presence of functional Sid (Six et al., 1991). Furthermore, certain mutations in Sid, named super-sid or nms (N mutation sensitive), can act as second-site suppressors of the sir mutations and enable the formation of small capsids even on a P2 Nsir background (Kim et al., 2001). Capsid assembly is completed by removal of Sid, cleavage of gpN, gpO and gpQ, and addition of the Psu decoration protein (Dokland et al., 1992, Rishovd and Lindqvist, 1992, Dokland et al., 1993, Chang et al., 2008) (Fig. 1D–F).
We showed previously that P4-size procapsids can be produced by co-expression of gpN and Sid alone (Dokland et al., 2002). Likewise, P2 procapsids can be produced by co-expression of gpN with a protease–defective version of gpO generated either by truncating the protein at the N-terminus or by mutating one of the three active site residues (Wang et al., 2006, Chang et al., 2009). In this paper, we present three-dimensional reconstructions of P2 and P4 procapsids produced by co-expression of gpN with gpO and Sid, respectively. The gpN capsid protein has the HK97 fold seen in all other tailed dsDNA phages studied to date (Bamford et al., 2005, Johnson and Chiu, 2007). Unusually, the P2 procapsid has a T = 7 dextro lattice, unlike the T = 7 laevo lattices of all other bacteriophage capsids with the HK97 fold observed to date. gpO is not ordered with icosahedral symmetry and does not appear in the reconstruction. In the P4 procapsids, Sid forms a dodecahedral cage that surrounds the procapsid and interacts with the gpN hexamers. Sid–gpN interactions occur at a loop in the apical domain of gpN where the sir mutations are located.
Section snippets
Cryo-EM of P2 and P4 procapsids
P4 procapsids were produced by the co-expression of gpN and Sid, while P2 procapsids were produced by co-expression of gpN and protease-defective forms of gpO, either with or without the gpQ portal protein (Dokland et al., 2002, Wang et al., 2006, Chang et al., 2008) (Table 1). The procapsids were purified on sucrose gradients and prepared for cryo-EM as previously described (Dokland et al., 2002, Huiskonen et al., 2004, Dokland and Ng, 2006). The gpQ portals were not incorporated into capsids,
Structure and assembly of gpN
Over the past decade it has become clear that all tailed dsDNA bacteriophages (order Caudovirales) are structurally related and share a common capsid protein fold (Bamford et al., 2005, Johnson and Chiu, 2007), first described at high resolution in the E. coli bacteriophage HK97 (Wikoff et al., 2000). Apart from the HK97 gp5 capsid protein, examples of this fold are found in the structures of T4, T7, φ29, P22 and ε15 as well as herpesviruses and the archaeal virus-like particle PfV (Baker et
Cloning and expression of co-expression constructs
The plasmids used are listed in Table 1. pLucky7, which expresses gpN and Sid under control of separate T7 promoters from a chimeric vector, was previously described (Dokland et al., 2002). pET16b-derived plasmids pSW101 and pJRC49 express protease-deficient truncated and mutated versions of gpO, respectively, in tandem with gpN under control of the same T7 promoter (Wang et al., 2006, Chang et al., 2009). The Q gene was inserted after OΔ25 and N in pSW101 to generate plasmid pCMRP1 that
Acknowledgments
Funding for this work at UAB was partly provided by The National Institutes of Health grants R21 AI071982 and R01 AI083255 to T.D. This work was initiated while T.D. was at the Institute for Molecular Agrobiology, Singapore, and was funded by Singapore’s Agency for Science, Technology and Research (A*STAR). We thank the Biocenter Finland National Cryo Electron Microscopy Unit, Institute of Biotechnology, Helsinki University, for providing services.
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