Role of lysozyme in the biological activity of bacteriophage ghosts.

Although it is generally accepted that phage lysozyme is necessary for the release of mature phage from infected cells (3, 12), its role in the initiation of infection is less clear. It has been considered for some time that lysozyme is incorporated into the protein coat of some phages (1, 9, 16), thereby assisting in the injection of the phage deoxyribonucleic acid into the cell by hydrolyzing the cell wall at the site of phage attachment (2, 13, 15). Lysozyme has also been considered as the cell-killing factor of the empty protein coat, or ghost, of the T-even phages (7, 11). This suggestion is supported by reports of leakage of cellular substances from ghost-infected cells that is greater than that observed from phageinfected cells and by the fact that cells infected with ghosts lyse sooner than cells infected with an equal number of intact phage (6, 14; C. D. Prater, Ph.D. Thesis, Univ. of Pennsylvania, Philadelphia, 1951). Conversely, however, isolated lysozyme does not have the same killing ability as do intact phage or ghosts (8), and cell death can be observed under conditions in which lysozyme activity is inhibited (10). These contradications and the report that no lysozyme can be detected in a suspension of T4 phage particles which have been purified by sucrose density gradient centrifugation and then osmotically shocked (R. Kretsinger, personal communication) prompted an investigation into the role of lysozyme in the biological activity of phage ghosts. We used a phage which has a nonsense mutation in its lysozyme gene rendering it incapable of producing any lysozyme when it infects the nonpermissive host Escherichia coli B. Whole phage and ghosts made from this lysozyme-less phage were tested for their ability to inhibit the induction of f-galactosidase synthesis. This inhibition is one of the known biological functions of ghosts that can be correlated with their killing ability (4). The amber mutant of T4 which is defective in the lysozyme gene (am H26) was kindly supplied by R. Kretsinger of the University of Virginia. The phage were grown in E. coli B and released after 40 min of infection by grinding the cells with glass beads (0.11 mm in diameter) in a Waring Blendor. Although no lysozyme could be detected in the suspension of disrupted cells or in the partially purified phage under conditions that could measure 0.1 jig of egg white lysozyme and readily detect lysozyme in a lysate of wild-type phage, the phage were judged to be lysozyme-less primarily by the behavior of the nonpermissive host when infected with the mutant. Virtually no phage were released from the infected cell spontaneously, or by the addition of chloroform, or by any other procedure short of mechanical disruption of the cell. This is in marked contrast to the behavior of the permissive host E. coli CR63, which spontaneously lyses and releases phage when infected with the same mutant. Ghosts were prepared by incubating the phage in 2 M sodium acetate for 15 min at 0 C and then rapidly diluting into 100 volumes of cold distilled water. The ghosts are stable for several weeks when purified and concentrated (6) and stored in a neutral phosphate buffer containing 10-3 M Mg++ and Ca++. The phage titer (plaque-forming units) after osmotic shock usually dropped by a factor of 100 to 500. The assay for inhibition of 3-galactosidase synthesis was carried out as previously described (4), except that the cells were grown in a nutrient broth, 5 min were allowed for adsorption, and 8 min were allowed for enzyme induction. The cells were broken by ultrasonic treatment for 60 sec in a Bronwill Biosonik operating at 90% intensity and were assayed for ,B-galactosidase as previously described (4). "Normal" ghosts were prepared either from wild-type T4 or from a stock of T4 am E957 (5) grown in the permissive host. The latter was selected for routine use to prevent any multiplication of residual phage in experiments of longer duration. Both the normal and the lysozyme-less phage were tested for their ability to inhibit the synthesis of f3-galactosidase before and after osmotic shock. Figure 1 shows the inhibition produced by the two kinds of phage ghosts. The multiplicity (phage-equivalents) was determined by the phage