Bacteriophage T4 self-assembly: localization of gp3 and its role in determining tail length

Abstract

Gene 3 of bacteriophage T4 participates at a late stage in the T4 tail assembly pathway, but the hypothetical protein product, gp3, has never been identified in extracts of infected cells or in any tail assembly intermediate. In order to overcome this difficulty, we expressed gp3 in a high-efficiency plasmid expression vector and subsequently purified it for further analysis. The N-terminal sequence of the purified protein showed that the initial methionine had been removed. Variant C-terminal amino acid sequences were resolved by determining the cysteine content of the protein. The molecular mass of 20.6 kDa for the pure protein was confirmed by Western blotting, using a specific anti-gp3 serum for which the purified protein was the immunogen. We also demonstrated, for the first time, the physical presence of gp3 in the mature T4 phage particle and localized it to the tail tube. By finding a nonleaky, nonpermissive host for a gene 3 mutant, we could clearly demonstrate a new phenotype: the slow, aberrant elongation of the tail tube in the absence of gp3.

FIG. 1

Construction of pAVgp3 (pAV9 · 3 · 1). See Materials and Methods for details.

FIG. 2

Induction kinetics of gp3. Proteins from E. coli BL21(DE3)/pAVgp3 cell lysates were separated by SDS-PAGE. (A) Coomassie blue-stained gel of samples taken at 30-min intervals after addition of IPTG to the culture. gp3 as a percentage of total protein is given below each lane. (B) Western blot of the same samples as shown in panel A except that fivefold-less protein was used.

FIG. 3

Purification of gp3. SDS-polyacrylamide gels of gp3 at various stages of purification are shown. Lane A, supernatant of the cell lysate after sedimentation; lane B, ammonium sulfate precipitate; lane C, purified gp3 after DEAE–Sephadex A-50 and Superdex-75 column chromatographies; lane D, molecular mass markers. The arrow denotes the 55-kDa band of gp3.

FIG. 4

Comparison of two sequences of T4 phage gene 3. Amino acids are indicated by boldface capital letters at top and bottom; variant nucleotides are indicated by boldface and underlining.

FIG. 5

Sulfhydryl content of gp3. Open circles, standard curve for the reaction of cysteine with Ellman's reagent. Solid circles, the same but in the presence of 6 M guanidinium chloride. +, reactivity of a 0.38 mM gp3 sample in Ellman's reagent with and without 6 M guanidinium chloride.

FIG. 6

Kinetics of gp3 production of T4-infected cells. Infected cells were sampled at 15-min intervals after infection and lysed. They were run on SDS-polyacrylamide gels, and a Western blot probed with anti-gp3 rabbit serum was prepared. Lanes a through g correspond to 0, 15, 30, 45, 60, 75, and 90 min after infection, respectively. Lane h is purified gp3.

FIG. 7

Content of gp3 in various phage structures. Shown are a Coomassie blue-stained polyacrylamide gel (A) and a Western blot of an identical gel probed with anti-gp3 antiserum (B). Quantities of the samples were adjusted to show a clear band on the Western blot. Lanes 1, molecular mass markers; lanes 2, purified gp3; lanes 3, T4D⁺ phage particles; lanes 4, tails; lanes 5, tube baseplates; and lanes 6, tubes.

FIG. 8

T4 gp3 prevents elongation of tails beyond the correct length. (A and B) Electron micrographs of negatively stained lysates of cells infected with tail-producing mutants that have either inactive gp3 (A) or active gp3 (B). Notice the long tails in panel A (the tails are considered long whether or not elongated tubes are completely covered with sheath). (C) Electron micrograph of the extra-long tubes found in a preparation of purified 3⁻/15⁻ tails after storage for a week at 4°C. The extra-long tubes apparently form in vitro when subunits depolymerize from a fraction of the tubes and repolymerize onto tubes which are not properly terminated at the correct length by gp3. The mutants T43⁻/15⁻/23⁻/34⁻ (A and C) and T43⁺/15⁻/23⁻/34⁻ (B) are described in Table 1.

FIG. 9

Length distribution of tails made with or without gp3. The lengths of tails present in electron micrographs made from lysates of mutant infected cells (Fig. 8) were measured in arbitrary units as described previously (12). The top panel is from a 3⁺/15⁻/23⁻/34⁻ lysate (as in Fig. 8B), and the bottom panel is from a 3⁻/15⁻/23⁻/34⁻ lysate (as in Fig. 8A). The means and standard deviations of the measurements and the numbers of particles counted (N) are shown in each panel. The values along the x axis are tail lengths in millimeters, measured directly on electron micrograph negatives at ×24,000 magnification.