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. 2002 May;76(10):4866-72.
doi: 10.1128/jvi.76.10.4866-4872.2002.

The small viral membrane-associated protein P32 is involved in bacteriophage PRD1 DNA entry

A Marika Grahn  1 , Rimantas Daugelavicius, Dennis H Bamford
Affiliations

The small viral membrane-associated protein P32 is involved in bacteriophage PRD1 DNA entry

A Marika Grahn et al. J Virol. 2002 May.

Abstract

The lipid-containing bacteriophage PRD1 infects a variety of gram-negative cells by injecting its linear double-stranded DNA genome into the host cell cytoplasm, while the protein capsid is left outside. The virus membrane and several structural proteins are involved in phage DNA entry. In this work we identified a new infectivity protein of PRD1. Disruption of gene XXXII resulted in a mutant phenotype defective in phage reproduction. The absence of the protein P32 did not compromise the particle assembly but led to a defect in phage DNA injection. In P32-deficient particles the phage membrane is unable to undergo a structural transformation from a spherical to a tubular form. Since P32(-) particles are able to increase the permeability of the host cell envelope to a degree comparable to that found with wild-type particles, we suggest that the tail-tube formation is needed to eject the DNA from the phage particle rather than to reach the host cell interior.

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Figures

FIG. 1.
FIG. 1.
(A) An electron micrograph of thin-sectioned DS88 cells infected with PRD1 [lacZα]-9. The image, obtained 55 min postinfection, shows a large number of peripheral filled phage particles (bar, 500 nm). (B) Protein composition of PRD1 wt and P32-deficient mutant particles is shown in a Coomassie blue-stained Tricine SDS-polyacrylamide gel. In the mutant phage preparation the only protein missing is the 5.4-kDa P32 (marked by an arrow). Molecular weight markers and some PRD1 proteins are indicated on the left and right, respectively.
FIG. 2.
FIG. 2.
Electron micrographs of negatively stained wt and P32-deficient particles showing extended or short tail-tube structures, respectively. The arrow points to the tail appendage. (A) Phage particles treated with 20 mM Tris-HCl, pH 7.4, for 48 h at 22°C. (B) Disruption of the wt or P32− particles with 2 M GuHCl treatment reveals membrane vesicles with or without tail-tubes, respectively (bar, 200 nm).
FIG. 3.
FIG. 3.
(A) An electron micrograph of thin-sectioned DS88 cells infected with P32-deficient mutant particles. The image, obtained 50 min postinfection, shows several phage particles bound on the cell surface (bar, 500 nm). (B) DNA synthesis, determined by [3H]thymidine incorporation into replicating DNA, in DS88 cells treated with nalidixic acid (open circles) and cells infected with PRD1 wt (filled circles) or P32− (squares) particles. The DNA gyrase inhibitor does not affect PRD1 DNA replication, unlike the case with host replication.
FIG. 4.
FIG. 4.
Electrochemical measurements of PRD1-infected cells. Accumulation of TPP+ by EDTA-treated (A) or intact (B) DS88 cells, and efflux of K+ (from intact cells) followed by phage infection (C). The experiments were performed at 37°C in 50 mM Tris-HCl, pH 7.5. Cells, phages, GD, and PMB were added to the final concentration of 3 × 109 CFU/ml, 24 μg of protein/ml (that is, MOI of ∼100), 5 μg/ml, and 100 μg/ml, respectively. Ion-selective electrodes were used to monitor the concentration of the indicator ions in the external medium. For interpretation of the experiment, see the text.
FIG. 5.
FIG. 5.
Organization of the late operon OL3 of phage PRD1 (nucleotides 8464 to 13886) (8, 22). Roman numerals and lowercase letters identify genes and open reading frames, respectively. The inset shows the alignment of proteins P32 and P34, sharing an overall similarity of 66%. The regions predicted (26) to form a membrane-spanning α-helix are underlined.
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References

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