Bacteriophage T4 self-assembly: In vitro reconstitution of recombinant gp2into infectious phage

T4 gene 2 mutants have a pleiotropic phenotype: degradation of injected pha ge DNA by exonuclease V (ExoV) in the recBCD(+) host cell cytoplasm and a l ow burst size due, at least in part, to a decreased ability for head-to-tai l (H-T) joining, The more N terminal the mutation, the more pronounced is t he H-T joining defect. We have overexpressed and purified the recombinant g ene 2 product (rgp2) to homogeneity in order to test its role in H-T joinin g, during in vitro reconstitution. When we mix extracts of heads from a gp2 (+) phage infection (Ht) with tails from a gp2(+) or gp2(-) phage infection (T+ or T-), the H-T joining is fast and all of the reconstituted phage gro w equally well on cells with or without ExoV activity, When heads from gene 2 amber mutants (H-) are used, addition of rgp2 is required for H-T joinin g. In this case, PI-T joining is slow and only about 10% of the reconstitut ed phage can form plaques on ExoV(+) cells. When extracts of heads with dif ferent gene 2 amber mutations are mixed with extracts of tails (with a gene 2 amber mutation) in the presence of rgp2, we find that the size of the gp 2 amber peptide of the head extract is inversely related to the fraction of reconstituted phage with a 2(+) phenotype, We conclude that free rgp2 is b iologically active and has a direct role in H-T joining but that the proces s is different from H-T joining promoted by natural gp2 that is incorporate d into the head in vivo. Furthermore, it seems that gp2 has a domain which binds it to the head. Thus, the presence of the longer gp2am mutants (with this domain) inhibits their replacement by full-length rgp2.