Fig. 1. SPP1 morphogenesis. Current knowledge of the sequence of assembly reactions during SPP1 capsid assembly (Dröge et al., 2000; Gual et al., 2000; Lurz et al., 2001; and references therein). Sizes are not scaled, to emphasize locations of minor capsid components. Gp1-gp2, terminase complex; gp6, portal protein; gp7, minor capsid protein; gp11, scaffolding protein; gp13, major capsid protein; gp15 and gp16, head completion proteins. The connector components are shown in the same colour code as in Figure 3. The assembly-naïve gp6 is shown in yellow, while the gp6 oligomer embedded in the capsid lattice is in blue to illustrate the different radial symmetry of the two gp6 forms (Lurz et al. 2001).

Fig. 3. Structure of the SPP1 connector and portal protein. The stereo view of the SPP1 connector structure at 10 Å resolution is shown in (A). The portal protein gp6 in the connector is shown in blue, gp15 in green and gp16 in orange. The structure of the SPP1 portal protein at 9 Å resolution is shown in (B) (stereo view). (C) Cut-away views of the connector and portal protein. The outlines show the location of the gp6 main domains: the crown of tentacles is in orange, the wing is outlined in green, the steam stalk is in magenta, the stem foot is in purple and the gp16 stopper is in red.

Fig. 5. Correlation between gp6 amino acid sequence and immunoreactive regions of the gp6 oligomer. The thick bar in (A) represents the gp6 amino acid sequence. The position of putative α-helices (>8 amino acids and with a reliability index of prediction ≥7) based on the secondary structure prediction PHD program (Rost, 1996) are shown by cylinders connected by a thin line above the thick bar. The thin lines below represent the peptides of gp6 used to define the regions of the protein that react with anti-gp6 polyclonal serum. Immunoreactive peptides in a western blot developed with anti-gp6 serum (peptides 1, 3 and 5) are shadowed in the thick bar. The relative strength of the western blot signal is indicated by vertical arrows on top of the bar. Affinity-purified antibodies specific for peptides 3 and 5 were used to identify the position of those peptides in gp6 present in tails with a connector by immunoelectron microscopy (B). More than 90% of the tail–connector complexes observed in the EM were immunolabelled, and the antibody–connector complexes revealed reproducibly the topology presented in (A). The topology found is indicated at the top of (A). Antibodies against the first 100 N-terminal amino acids, the most antigenic peptide of gp6 (A), could not be obtained due to lack of dissociation of the antibody–peptide 1 complexes. The bar on the micrograph represents 50 nm.

Fig. 2. Production of mature viral chromosomes during infection with SPP1 wild type and with SPP1 mutants defective in production of the connector proteins gp15 and gp16. (A) Position of the genes coding for the connector proteins in the SPP1 genome (black boxes) (nucleotide coordinates are according to Alonso et al., 1997). The enlarged region shows the position of mutations sus128 (codon Q₄→stop; Becker et al., 1997) and sus117 (Q₉₄→stop; this work) in genes 15 and 16, respectively. DNA packaging is from left to right, leading to encapsidation of a DNA molecule with ∼45.9 kbp (Tavares et al., 1996), which is 104% the size of the SPP1 genome (44 007 bp; Alonso et al., 1997). (B) Total DNA extracted from B.subtilis YB886 cells infected with SPP1 wild type (wt), SPP1sus128 (gp15^–) and SPP1sus117 (gp16^–) at the time points after infection indicated and resolved by PFGE. Samples treated (+) and untreated (–) with DNase were analysed. The position to where SPP1 unit-length chromosomes migrate is indicated by an arrow (45.9 kbp). The band observed at this position at 5 min post-infection is attributed to input phages used for infection that were not eliminated since SPP1 DNA packaging is only detected after 12 min of infection (our unpublished results). The gel was stained with ethidium bromide. A Southern blot hybridized with a probe specific for the SPP1 genome showed a pattern of SPP1 DNA distribution in the gel similar to that observed with total DNA staining (data not shown).

Fig. 4. Comparison between the structure of bacteriophage SPP1 portal protein, the SPP1 portal protein in the connector and bacteriophage φ29 portal protein. (A) Comparison of the gp6 12mer from the connector (in blue) with the model of the portal protein 12mer (in pink). Thin lines show alignment of structures. (B) Single subunits from gp6 in the SPP1 connector and isolated gp6. The upper row shows side views of subunits with fitted cylinders, corresponding to putative helices, superimposition of helices and the side view of the φ29 portal protein. In the bottom row are shown front views for each subunit from the two forms. The four cylinders are highlighted by colours matching the helices in the φ29 portal protein crystal structure (Simpson et al., 2000). The cylinders are labelled according to the nomenclature of Simpson et al. (2000). The most significant changes are observed in the area corresponding to helix a3.

Fig. 6. Fourier shell correlation curves of gp6 and of the connector in grey and black, respectively. The threshold curve is in thin black and corresponds to 10.6σ. The arrows are pointing to the frequency level, where the correlation twice exceeds the background level. The lower resolution for the connector as compared with the portal protein structure resulted from the smaller number of molecular images available for the analysis.