Efficient DNA packaging of bacteriophage PRD1 requires the unique vertex protein P6

Abstract

The assembly of bacteriophage PRD1 proceeds via formation of empty procapsids containing an internal lipid membrane, into which the linear double-stranded DNA genome is subsequently packaged. The packaging ATPase P9 and other putative packaging proteins have been shown to be located at a unique vertex of the PRD1 capsid. Here, we describe the isolation and characterization of a suppressor-sensitive PRD1 mutant deficient in the unique vertex protein P6. Protein P6 was found to be an essential part of the PRD1 packaging machinery; its absence leads to greatly reduced packaging efficiency. Lack of P6 was not found to affect particle assembly, because in the P6-deficient mutant infection, wild-type (wt) amounts of particles were produced, although most were empty. P6 was determined not to be a specificity factor, as the few filled particles seen in the P6-deficient infection contained only PRD1-specific DNA. The presence of P6 was not necessary for retention of DNA in the capsid once packaging had occurred, and P6-deficient DNA-containing particles were found to be stable and infectious, albeit not as infectious as wt PRD1 virions. A packaging model for bacteriophage PRD1, based on previous results and those obtained in this study, is presented.

FIG. 1.

Analysis of the protein content of 2× purified virus particles by SDS-PAGE and Coomassie brilliant blue staining. Lane 1, empty Sus621; lane 2, DNA-filled Sus621; lane 3, empty wt; and lane 4. DNA-filled wt particles. Since both 1× and 2× purified preparations produced similar patterns, only 2× particles are shown here.

FIG. 2.

Effect of P6 deficiency on particle assembly. (A and B) Sections of S. enterica DS88 cells infected with (A) wt PRD1 (50 min p.i.) and (B) mutant sus621 (60 min p.i.). Examples of filled particles are depicted with arrows. (C to H) Negatively stained purified virus particles. (C) Empty Sus621 particles, (D) filled Sus621 particles, (E) empty Sus1 particles, (F) filled wt particles, (G) SDS-treated empty Sus621 particles, and (H) SDS-treated empty Sus1 particles.

FIG. 3.

Restriction enzyme analysis of DNA extracted from filled Sus621 and wt PRD1 particles. Lane 1, DraI-digested sus621 DNA; lane 2, DraI-digested wt PRD1 DNA; and lane 3, DNA standard, PstI-digested bacteriophage λ DNA.

FIG. 4.

In vitro packaging of Sus621 particles. Columns indicate the total amount of PFU/ml of packaging reaction obtained in host DS88. This is shown on a logarithmic scale (depicted by the left y axis), and the mean values for each reaction are shown above each column. The gray portion of each column represents the relative frequency of blue plaques (linear scale). In vitro packaging of Sus621 particles was achieved only with the addition of P9 (columns “Sus621+P6+P9” and “Sus621+P9”) but not with the addition of P6 alone (column “Sus621+P6”). The standard in vitro packaging assay with P9-deficient Sus1 particles and P9 extract (Sus1+P9) is shown as a positive control. Negative control reactions for both Sus621 and Sus1 particles, containing all other components except the lacZα-containing DNA, are marked “no DNA.”