A P22 scaffold protein mutation increases the robustness of head assembly in the presence of excess portal protein

Abstract

Bacteriophage with linear, double-stranded DNA genomes package DNA into preassembled protein shells called procapsids. Located at one vertex in the procapsid is a portal complex composed of a ring of 12 subunits of portal protein. The portal complex serves as a docking site for the DNA packaging enzymes, a conduit for the passage of DNA, and a binding site for the phage tail. An excess of the P22 portal protein alters the assembly pathway of the procapsid, giving rise to defective procapsid-like particles and aberrant heads. In the present study, we report the isolation of escape mutant phage that are able to replicate more efficiently than wild-type phage in the presence of excess portal protein. The escape mutations all mapped to the same phage genome segment spanning the portal, scaffold, coat, and open reading frame 69 genes. The mutations present in five of the escape mutants were determined by DNA sequencing. Interestingly, each mutant contained the same mutation in the scaffold gene, which changes the glycine at position 287 to glutamate. This mutation alone conferred an escape phenotype, and the heads assembled by phage harboring only this mutation had reduced levels of portal protein and exhibited increased head assembly fidelity in the presence of excess portal protein. Because this mutation resides in a region of scaffold protein necessary for coat protein binding, these findings suggest that the P22 scaffold protein may define the portal vertices in an indirect manner, possibly by regulating the fidelity of coat protein polymerization.

FIG. 1.

Marker rescue strategy. To map escape mutations (E), a region of the mutant phage genome was cloned into a plasmid and introduced into Salmonella. The cells were then infected with a phage that contains a conditional selectable marker (x) under permissive conditions. During replication, some of the phage genomes undergo homologous recombination with the cloned escape mutant DNA and generate phage that no longer contain the marker. Subsequent plating under restrictive conditions allows the growth of only recombinant phage. If those recombinants exhibit the escape phenotype, then the cloned DNA fragment must have contained escape mutations.

FIG. 2.

Escape mutations. The amino acid alterations that result from mutations present in cloned DNAs isolated from escape mutant phage are indicated. The wild-type amino acid precedes the amino acid number, which is followed by the mutant amino acid. The cloned segments conferred the escape phenotype upon marker rescue, and the phage from which they were derived were independently isolated (except for P5′, which was derived from P5). Note that each clone contains the same Gly287Glu mutation in the scaffold gene. The clone names are indicated on the left. The gene names are shown in the rectangles that approximate their relative sizes. A prime symbol indicates a truncated gene.

FIG. 3.

Wild-type and Gly287Glu phage infections of the β-Gal host. β-Gal-expressing cells that had been infected with either wild-type or mutant Gly287Glu phage were resolved in sucrose gradients and analyzed for protein content by SDS-PAGE (top panels) and for particle content by native agarose gel electrophoresis (bottom panels). Load (L), samples of the total lysate; PC, procapsid peak position; φ, phage peak position. Dominant proteins (or the first letter thereof) are labeled to the left of each SDS gel, and the positions of the procapsid and phage particles are indicated to the left of each native agarose gel. The direction of sedimentation is indicated at the bottom. In the SDS gels, one-quarter of the bottommost fraction was loaded to reduce distortions caused by excessive CsCl present in the sample.

FIG. 4.

Wild-type and Gly287Glu phage infections of the portal protein host. P22 portal protein-overexpressing cells that had been infected with either wild-type or mutant Gly287Glu phage were resolved in sucrose gradients and analyzed for protein content by SDS-polyacrylamide gel electrophoresis (top panels) and for particle content by native agarose gel electrophoresis (bottom panels). Load (L), samples of the total lysate; PLP, procapsid-like particle peak position; φ, phage peak position. Dominant proteins (or the first letter thereof) are labeled to the left of each SDS gel, and the positions of the procapsid and phage particles are indicated to the left of each native agarose gel. The direction of sedimentation is indicated at the bottom. The ovals highlight regions in the native gels that contained aberrant, large, heterogeneous assemblies. Less sample was loaded from the bottommost fraction in the SDS gels to reduce CsCl distortion.

FIG. 5.

Multiple-tail procapsids. Shown are electron micrographs of negatively stained particles from the procapsid peak fraction of the wild-type phage-infected portal protein host. Tailspikes were detected on approximately 1% of the total procapsids. Because tailspikes attach only to portal vertices, they serve as a marker for the presence of portals within procapsid shells. No multiple-tail procapsids were detectable in the wild-type phage-infected β-Gal host, and no multiple-tail procapsids were present in any samples derived from Gly287Glu infections.

FIG. 6.

High-fidelity Gly287Glu mutation. Electron micrographs show negatively stained particles present in the phage peak fractions from the gradients shown in Fig. 4. The host in each example had overexpressed portal protein during the infection. Note that many of the particles in the sample derived from the wild-type phage infection are aberrant spirals and that practically all of the particles derived from the Gly287Glu infection have morphologies consistent with a T=7 capsid lattice.