Multifunctional roles of a bacteriophage phi 29 morphogenetic factor in assembly and infection

Abstract

Low copy number proteins within macromolecular complexes, such as viruses, can be critical to biological function while comprising a minimal mass fraction of the complex. The Bacillus subtilis double-stranded DNA bacteriophage phi 29 gene 13 product (gp13), previously undetected in the virion, was identified and localized to the distal tip of the tail knob. Western blots and immuno-electron microscopy detected a few copies of gp13 in phi 29, DNA-free particles, purified tails, and defective particles produced in suppressor-sensitive (sus) mutant sus13(330) infections. Particles assembled in the absence of intact gp13 (sus13(342) and sus13(330)) had the gross morphology of phi 29 but were not infectious. gp13 has predicted structural homology and sequence similarity to the M23 metalloprotease LytM. Poised at the tip of the phi 29 tail knob, gp13 may serve as a plug to help restrain the highly pressurized packaged genome. Also, in this position, gp13 may be the first virion protein to contact the cell wall in infection, acting as a pilot protein to depolymerize the cell wall. gp13 may facilitate juxtaposition of the tail knob onto the cytoplasmic membrane and the triggering of genome injection.

Figure 1

ϕ29 assembly pathway and cryo-EM 3D reconstruction. (a) Assembly begins with the formation of the head-tail connector (gp10) onto which the scaffolding (gp7) (hatched particle), major capsid (gp8) and head fiber (gp8.5) proteins and prohead RNA (pRNA) assemble to form the prohead. The DNA packaging motor is complete on assembly of the ATPase gp16 to pRNA, and on hydrolysis of ATP, drives translocation of DNA-gp3 into the prohead concurrent with scaffold extrusion (un-hatched particle). Lower collar and tail tube (gp11) assembly triggers release of gp16/pRNA after complete packaging (filled grey particle). The tail knob (gp9), pre-assembled with gp13 (this study), is fixed onto the tail tube. Lastly, the appendages (gp12*), which serve as adsorption organelles, are attached at the connector-lower collar junction to form the infectious virion. (b) ϕ29 structures revealed in a cryo-EM 3D reconstruction.

Figure 2

Western blot analysis of ϕ29 particles and tails using gp13-specific antiserum. gp13 is demonstrated in ϕ29 but not the prohead (a, top). Particle number is controlled and shown with connector-specific (anti-gp10) serum (a, bottom). gp13 is shown to be present in ϕ29 (b, top, lanes 2-4), ghosts (DNA-free particles) (lanes 7-8) and isolated tails (gp10, gp11, gp9, gp12*) (lanes 9-10) but not in proheads (lanes 5-6). gp9 does not react with anti-gp13 serum (b, top, lane 11), and gp13 does not react with anti-gp9 serum (b, bottom, lane 14). Anti-gp9 serum was used as a loading control, and ϕ29, ghosts and tails show equivalent signal (b, bottom, lanes 2-4 and 7-10).

Figure 3

Immuno-electron microscopy localization of gp13 in ϕ29 using anti-gp13 serum. Particles without appendages (12^-) form antibody-dependent tail-bound aggregates in the presence of anti-gp13 serum (a) while 12^- particles incubated with pre-immune serum (b) do not. Insets (a, i-iii) show magnified aggregates and isolated particles, demonstrating that tail knobs are obscured in the presence of anti-gp13 serum but not in the presence of non-specific IgG (b, i). Immuno-gold localization targets the tail in the presence of anti-gp13 IgG (c), as the only phage-associated gold is tail bound. Negative staining with phosphotungstate. Magnification bars = 100 nm.

Figure 4

Secondary structure prediction of the gp13 primary amino acid sequence (line 1), showing mutant glutamine (Q) residues, ochre mutations (arrows) and a His-x-His motif (circled green). Two secondary structure prediction models (psipred, line 2, and profsec, line 3) show predicted N-terminal α-helices (‘H’, red) and C-terminal β-sheets (‘E’, blue). Three putative Zn²⁺ ligands align with LytM-Zn²⁺ ligands (squares) following the canonical motif structure H-x(3,6)-D and H-x-H, and three residues provide putative anchoring for the fourth Zn²⁺ ligand as either a phosphate or water molecule in LytM (triangles). Zn²⁺-ligand residues in LytM are numbered below each residue. Amino acids N89 and I353 are italicized to denote differences compared to the published gp13 sequence.

Figure 5

Western blot analysis of gp13-defective particles produced in restrictive infection with the mutants sus13(330) and sus13(342). sus13(330) particles contain a long ochre fragment of gp13 (top panel, arrow with box down-shifted). sus13(342) particles contain no detectible gp13 (top panel). Reprobing of the blot with anti-connector (anti-gp10) serum serves as a loading control (bottom panel). Molecular weight standards are with visible light illumination.

Figure 6

Transmission electron microscopy of gp13-defective mutant particles. Particles produced in restrictive infection with the mutants sus13(342) (left panel) and sus13(330) (right panel) look grossly normal. However, on close inspection, sus13(330) particles have notched tail knobs (right panel, dashed arrows). For reference, ϕ29 is shown (insets). Negative staining with phosphotungstate. ϕ29 inset was stained with uranyl acetate. Magnification bar = 100 nm.

Figure 7

Immuno-electron microscopy detects the short sus13(342) ochre fragment of gp13 in DNA-filled but not empty particles. (A) Representative micrographs of DNA-containing and empty sus13(342) mutant particles incubated with anti-gp13 IgG. (B) ϕ29 aggregated with anti-gp13 IgG. Negative staining with phosphotungstate. Magnification bar = 100 nm.

Figure 8

The ϕ29 Accessory Tail Assembly Pathway. gp13 and gp9 pre-assemble to form the tail knob (step A), and this complex is then attached en bloc onto the lower collar and tail tube (gp11) of parallel assembling filled heads (step B) as the penultimate morphogenesis step (C) that precedes attachment of the adsorption organelle appendages (gp12*) (step D) to form the infectious virion.