the role oflysozyme in the biological activity of phage ghosts. We used a phage which has a nonsense muta- tion in its lysozyme gene rendering it incapable of.
JOURNAL OF VIROLOGY, Jan. 1969, p. 92-94 Copyright © 1969 American Society for Microbiology
Vol. 3, No. 1 Printed in U.S.A.
Role of Lysozyme in the Biological Activity of Bacteriophage Ghosts DONNA HARDY DUCKWORTH Department of Microbiology, School of Medicine, University of Virginia, Charlottesville, Virginia 22901
Received for publication 10 October 1968
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 92
NOTES
VOL. 3, 1969
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MULTIPLICITY (PHAGE-EQUIVALENTS) FIG. 1. Inihibition of induced ,3-galactosidase by "normal" (am E957) and lysozyme-less (am H26) ghosts. E. coli B was grown to a concentration of 4 X 108 cells/ml in broth, and 10-ml samples were transferred to 50-nrd Erlenmeyerflasks warmed to 37 C. The indicated number ofphage-equivalents (titer before osmotic shock) were added, and after 5 miti 0.1 ml oJ freshly prepared 0.0.5 M isopropyl-4-D thiogalactopyranloside was added. The flasks were shiakenz for 8 miii at 37 C, then chilled, centrifuged at 6,000 X g for 5 min, and resuspended in 5 ml of 0.02 M sodium phosphate buffer, pH 7.5. The cells were sonically treated, and the resulting suspension was assayed for f3-galactosidase. Symbols: 0, "normal" glhosts; A, lysozyme-less ghosts.
titer before osmotic shock, as measured by the ability of the phage to inhibit induced enzyme synthesis. The titer of phage measured in this way was either equal to or slightly greater than the titer obtained by measuring plaque-forming ability (4). Ghosts of both the lysozyme-less and lysozyme-producing phages caused inhibition of induced enzyme synthesis with the same efficiency. The inhibition obtained with ghosts was, however, only approximately one-half that produced by either phage before osmotic shock. At 37% f3-galactosidase induction, where there should have been a multiplicity of one ghost per bacterium, there were actually two phage-equivalents per bacterium. This does not mean that two ghosts are needed to produce the inhibition. Rather, approximately 50%0 of both the normal and the
93
lysozyme-less particles were inactivated. The inhibition seen in Fig. 1 could not be produced by residual live phage in the ghost suspensions, as their concentration was too low to have any detectable effect. These results were readily reproducible in experiments with ghosts of am H26, am E957, and wild-type T4. Although cellkilling is not routinely measured for reasons previously discussed (4), one experiment with the lysozyme-less ghosts showed that they acted to inhibit colony formation with approximately the same efficiency as ghosts of wild-type T4. In summary, we found no difference in the biological activity of ghosts made from phage which could not produce lysozyme and ghosts from phage which could produce lysozyme. These data do not rule out leakage of cellular substances as the primary cause of inhibition of macromolecular synthesis and cell death after ghost infection. It seems clear, however, that the product of the lysozyme gene is not causing this leakage. Since this work was undertaken, Emrich and Streisinger have found that lysozyme plays no detectable role in infection or in lysis from without (G. Streisinger, personal communication). I am indebted to Robert Kretsinger for providing the lysozymeless amber mutant and for performing the lysozyme assays. I also thank George Streisinger for sending me reports of his work before publication.
LITERATURE CITED 1. Barrington, L. F., and L. M. Kozloff. 1956. Action of bacteriophage on isolated host cell walls. J. Biol. Chem. 223: 615-627. 2. Champe, S. P. 1963. Bacteriophage reproduction. Ann. Rev. Microbiol. 17:87-114. 3. Doerman, A. H. 1952. Liberation of intracellular bacteriophage T4 by premature lysis with another phage or with cyanide. J. Gen. Physiol. 35:645-656. 4. Duckworth, D. H., and M. J. Bessman. 1965. Assay for the killing properties of T2 bacteriophage and their "ghosts." J. Bacteriol. 90:724-728. 5. Duckworth, D. H., and M. J. Bessman. 1967. A biochemicalgenetic study of the deoxynucleotide kinase induced by wild-type and amber mutants of phage T4. J. Biol. Chem. 242:2877-2885. 6. Herriott, R. M., and J. L. Barlow. 1957. The protein coats or "ghosts" of coli phage T2. I. Preparation, assay, and some chemical properties. J. Gen. Physiol. 40:809-825. 7. Herriott, R. M., and J. L. Barlow. 1957. The protein coats or "ghosts" of coli phage T2. II. The biological functions. J. Gen. Physiol. 41:307-331. 8. Koch, G., and W. J. Dreyer. 1958. Characterization of an enzyme of phage T2 as a lysozyme. Virology 6:291-293. 9. Koch, G., and E. M. Jordan. 1957. Killing of E. coli B by phage-free T2 lysates. Biochim. Biophys. Acta 25:437. 10. Kozloff, L. M., and M. Lute. 1957. Viral invasion. II. The role of zinc in bacteriophage invasion. J. Biol. Chem. 228:529-536. 11. Luria, S. E., and J. E. Darnell, Jr. 1967. General Virology. John Wiley and Sons, Inc., New York. 12. Mukai, F., G. Streisinger, and B. Miller. 1967. The mechanism of lysis in phage T4-infected cells. Virology 33b: 398-402. 13. Muller-Jensen, K., and P. H. Hofschneider. 1964. The nature
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of infectious urea-T2 particles. Biochim. Biophys. Acta 80:422-430. 14. Puck, T. T., and H. H. Lee. 1956. Demonstration of cyclic permeability change accompanying virus infection of E. coli B cells. J. Exptl. Med. 101:151-175.
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15. Stolp, H., and M. P. Starr. 1965. Bacteriolysis. Ann. Rev. Microbio 1. 19:79-104. 16. Weidel, W., and J. Primosigh. 1957. Die gemeinsame Wurzel der Lyse von E. coli B durch Penicillin oder durch Phagen. Z. Naturforsch. 12:421.
