Regulation of the phage phi 29 prohead shape and size by the portal vertex

Abstract

Bacteriophage phi 29 of Bacillus subtilis packages its double-stranded DNA into a preformed prohead during morphogenesis. The prohead is composed of the scaffold protein gp7, the capsid protein pg8, the portal protein gp10, and the dispensable head fiber protein gp8.5. Our objective was to elucidate the phi 29 prohead assembly pathway and to define the factors that determine prohead shape and size. The structural genes of the phi 29 prohead were cloned and expressed in Escherichia coli individually or in combination to study form determination. The scaffold protein was purified from E. coli as a soluble monomer. In vivo and in vitro studies showed that the scaffolding protein interacted with both the portal vertex and capsid proteins. When the scaffold protein interacted only with the capsid protein in vivo, particles were formed with variable size and shape. However, in the presence of the portal vertex protein, particles with uniform size and shape were produced in vivo. SDS-PAGE analysis showed that the latter particles contained the proteins of the scaffold, capsid, head fiber, and portal vertex. These results suggest that the scaffolding protein serves as the linkage between the portal vertex and the capsid proteins, and that the portal vertex plays a crucial role in regulating the size and shape of the prohead.

Fig. 1

Outline of construction of plasmids pARgp7, pARgp7-8-8.5, and pARgp7-8-8.5-10 expressing or coexpressing the scaffolding protein gp7, the capsid protein gp8, the fiber protein gp8.5, and the portal protein gp10. Individual DNA fragments were cloned into the Ndel site of the plasmid pET-3c. P_T7, bacteriophage T7 promotor; Hairpin, a dyad structure that produces a hairpin loop in RNA which protects the RNA transcripts from degradation by exonuclease; gp, gene product; Term, φ10 transcription termination sequence.

Fig. 2

SDS–PAGE of the scaffolding protein demonstrating solubility and purity. (Lane A) Purified portal protein gp10; (lane B) purified scaffolding protein gp7; (lane C) the four structural proteins of purified proheads; (lane D) lysate of E. coli HMS174(DE3) as a control; (lanes E and F) pellets and supernatant, respectively, of induced HMS174(DE3)pARgp7 lysate after 16,000 g sedimentation. The gp7 expressed in E. coli was present in the supernatant, indicating it is in a soluble form.

Fig. 3

Native 15% polyacrylamide gel of purified gp7, showing the monomeric form of gp7 in solution. (Lane A) Structural proteins of purified proheads denatured in 1% SDS and 0.1% β-mercaptoethanol; (lane B) denatured gp7, prepared as above; (lane C) gp7 prepared in nondenaturing buffer. No difference in the migration rate between the denatured and nondenatured gp7 was detected, indicating that gp7 is present in a monomeric form in solution.

Fig. 4

Size determination of purified gp7 by sucrose gradient sedimentation. Purified gp7 was sedimented in a 5–20% linear sucrose gradient with molecular weight markers. Samples from every other fraction were run on SDS–PAGE, which was first stained with Coomassie blue and then with silver stain after decoloration. Sedimentation was from right to left. (Lane A) Purified proheads; (lane B–S) fractions of the sucrose gradient; (lanes O and P) gp7 migrated with a rate close to the 12-kDa marker. gp7 was clearly identified by its typical color reaction, staining with Coomassie blue but not with silver stain. The 12-kDa molecular marker was only stained by silver stain.

Fig. 5

Interaction of the scaffolding protein gp7 with the portal vertex gp10. Varying concentrations of gp7 and a fixed concentration of portal vertex were mixed and dialyzed together against TBE. After 30 min at ambient temperature, the mixture was transferred to TMS and allowed to dialyze another 30 min. The mixture was then run on a 1.0% native agarose gel. Lane A contained only the portal vertex and lane H only gp7. The final concentration of portal vertex in lanes A–G was 1 mg/ml and gp7 in Lanes B to H was 0.08, 0.16, 0.32, 0.48, 0.64, 0.80, and 1.6 mg/ml, respectively. The migration rate of the products increased with the increasing concentration of gp7, indicating the interaction of gp7 with the portal vertex.

Fig. 6

Coexpression of gp7 and gp8 after IPTQ induction as demonstrated by SDS–PAGE and Coomassie blue staining. (Lanes A and C) Crude lysate of E. coli HMS174(DE3)pARgp7-8-8.5 overexpressing both gp7 and gp8; (lane B) lysate of E. coli HMS174(DE3) as a control.

Fig. 7

Identification of the components of purified particles by SDS–PAGE and Coomassie blue staining. (Lane A) Proheads from infected B. subtilis cells; (lane B) particles from pARgp7-8-8.5-10. Comigration of gp7, gp8, gp8.5 with gp10 at the prohead position in the gradient indicates incorporation of the portal vertex into particles.

Fig. 8

Micrographs of frozen-hydrated φ29 particles. (A) Scaffold–capsid particles from E. coli that coexpressed gp7 and gp8. The particles vary greatly in size and shape. (B) Uniform scaffold–capsid–portal vertex particles from E. coli that expressed gp7, gp8, and gp10. (C) Purified proheads from φ29-infected B. subtilis cells. In (B) and (C), some particles are oriented with their long axes perpendicular to the plane of view and observed as circles (arrows). Bar = 200 nm.