RESISTANCE TO LYSIS FROM WITHOUT IN BACTERIA INFECTED WITH T2 BACTERIOPHAGE'

RESISTANCE TO LYSIS FROM WITHOUT IN BACTERIA INFECTED WITH T2 BACTERIOPHAGE' N. VISCONTI Department of Genetics, Carnegie Institution of Washington, Cold Spring Harbor, New York Received for publication February 18, 1953 When a large number of phage particles is adsorbed to a bacterium, lysis, as measured by decrease in bacterial turbidity, is induced very rapidly. No new phage is liberated; on the contrary the adsorbed phage is lost. Delbrtick (1940) first described this phenomenon and called it lysis from wihout to distinguish it from normal lysis which occurs at the end of the latent period with liberation of new phage. In the same paper Delbruck described some microscopic observations of lysis from without. Bacteria infected with a multiplicity of 200 phage particles per bacterium start swelling to an oval or spherical shape about 18 minutes after infection. As a result of the swelling the bacterial turbidity decreases. These morphological observations were repeated by Watson (1950) who found, furthermore, that X-ray-inactivated phage could induce lysis from without. Watson described another method of measuring lysis from without. Bacteria were infected with active T2 phage at a multiplicity of 0.02 per bacterium, and X-rayinactivated T2 phage at multiplicities ranging from 1 to 90 was added. The frequency of suppression of active phage was considered a measure of lysis from without. Watson found that as the multiplicity of the X-ray-inactivated phage was raised some bacteria underwent lysis from without. It is possible also to induce lysis from without with only a few phage particles if any one of a variety of metabolic inhibitors such as iodoacetate or 2,4-dinitrophenol is present in addition to the phage (Cohen, 1949; Heagy, 1950). During experiments performed to test for the maximum multiplicity of phage infection attainable without encountering lysis from without, it was noticed that bacteria become resistant to lysis from without after infection at a low multi- 1 This investigation was supported in part by a research grant from the National Microbiological Institute of the National Institutes of Health, Public Health Service. plicity. This fact can be demonstrated in a simple manner by comparing a turbid growing bacterial culture infected with phage with a noninfected control culture. Five or six minutes after the first culture has been infected, a large amount of phage is added to both cultures to give a multiplicity of over 500 phage particles per bacterium. The control culture clears within a few minutes while the turbidity of the superinfected culture remains unchanged. This paper will describe some quantitative experiments on this acquired resistance to lysis from without and its relation to other changes also induced in the bacterial cell by infection at low multiplicity. MATERIALS AND METHODS Coli-phage T2 and its mutants T2h and T2r (Hershey and Rotman, 1949) were used in these experiments. Mutant h is a host-range mutant that lyses certain bacteria (B/2) that are resistant to T2h+. T2h can be detected in the presence of T2h+ by plating on a mixture of B and B/2; h gives clear plaques, h+ turbid plaques. The r mutant can be distinguished from the r+ by plaque morphology. The r used in these experiments and the h marker are genetically unlinked; this r locus is referred to as r1 in the work of Hershey and Rotman. The methods for preparing and titrating the phage have been described by Adams (1950). "UV phage" means phage irradiated with a germicidal ultraviolet lamp at a dose which reduces the titer to 10-6. The ability of the phage to kill bacteria is unimpaired after irradiation (Dulbecco, 1952). The same preparation of ultraviolet-irradiated phage was used throughout these experiments to obtain lysis from without. The preparation was maintained under refrigeration for one month; no appreciable difference in its lysis from without inducing power was found after this time. The phage-adsorption medium contained per litermgso4, 1 mm; CaC12, 0.1 mm; gelatin, 0.01 g; 247

248 N. VISCONTI [VOL. 66 Na2HPO4, 3.0 g; KH2PO4, 1.5 g; KCI, 5 g; and NaCl, 4.0 g at ph 7.0. Adsorption was measured by mixing known concentrations of bacteria and phage in this medium and, after a given interval of time, centrifuging down the bacteria and titrating the supernatant for unadsorbed phage. In this medium no growth of the phage occurs after adsorption (B3enzer, 1952). In order to start phage growth, broth was added to the infected bacteria. The time at which the broth was added will be referred to as time zero. For analyzing the phage yield from single bacteria ("single bursts"), samples of the culture, diluted to contain about one infected bacterium in 3 or 4 samples, were distributed in a series of tubes. After allowing 2 hours at 37 C for lysis of the bacteria, each tube was plated separately and the phage yield analyzed. TABLE 1 Bacterial resistance to lysis from without when bacteria first infected with a multiplicity between 10 and 20 phage particles per bacterium are superinfected after different intervals of time with a multiplicity of 500 phage particles per bacterium INTERVAL OF TIME IN INFECTED INFECTED BACTERIA MINUTES BETWEEN BACTERIA WHICH YIELD PHAGE IRNFECTION PE RML (MOTTLED PLAQUES) 0 10' 108 1 10' 106 2 10' 2.107 3 108 5.107 RESULTS Resistance to lysis from without in relation to the interval of time between first and second infection. The most sensitive measure of lysis from without is the suppression of phage production in infected bacteria after adsorption of new phage at a very high multiplicity. In order to measure acquired resistance to lysis from without, the survival of bacterial infective centers (i.e., plaques originating from infected bacteria) was determined at different intervals of time between the initiation of phage growth and the addition of new phage. The following two methods were utilized for these measurements: Method 1. An aerated growing broth culture with a concentration of 2 X 107 bacteria per ml is centrifuged and resuspended in broth to give a concentration of 2 X 10' per ml. To 1 ml of this suspension of bacteria, 1 ml of phage in buffer at a concentration of 3 X 109 per ml is added. Half the added phage is r and half wild type r+ so that each bacterium will be infected with both types. A plaque formed by a bacterium mixedly infected with r and r+ will give rise to a mottled plaque, which can be distinguished readily from pure r or r+ plaques (Hershey, 1946; Dulbecco, 1949, 1952). Five minutes at 37 C are allowed for adsorption (80 per cent or more) of the phage. At the end of the adsorption period 2 ml of broth are added, and the culture is divided into three samples. At 1, 2, and 3 minute intervals thereafter phage is added to one of the samples with a multiplicity of 500 phage particles per bacterium. The added phage is half r and half r+, as in the first infection. After 5 minutes at 37 C T2 antiserum is added, and 4 minutes later the culture is diluted and plated. Only infected bacteria and free phage particles that survive the antiserum treatment will give plaques. Free phage will give either r or r+ plaques; mixedly infected bacteria will give mottled plaques. This method is important in these experiments since it is difficult to eliminate all the free phage by using antiserum when the multiplicity is very high. The results of this experiment, as shown in table 1, indicate that the survival of bacterial infective centers increases with the time interval between the two infections. Method B. In method 1 the first infection with r and r+ phage must be multiple to obtain mottled plaques from the infected bacteria. In order to measure resistance to lysis from without in single infected cells, the following procedure is used. A bacterial culture growing in aerated broth at a concentration of 2 X 107 bacteria per ml is washed in adsorption medium and resuspended to give a concentration of 2 X 10' per ml. This suspension is aerated for 30 minutes to bring the bacteria into a resting condition (Benzer, 1952). The bacteria are infected then with hr phage at a rate of one phage particle per ten bacteria. The suspension is diluted and centrifuged, and the supernatant is discarded in order to eliminate the unadsorbed phage. The bacterial pellet is resuspended in adsorption medium at the original concentration of 2 X 108 per ml. Two different experiments now may be performed with this preparation of adsorbed phage

1953] LYSIS FROM WITHOUT IN T2 BACTERIOPHAGE 249 to show that killing of the bacterial infective centers by lysis from without depends on the multiplicity of the second infection and on the interval of time between the first and second infections. In the first experiment the time of the second infection is kept constant and its multiplicity is varied by adding at time zero different concentrations of h+r+ ultraviolet-irradiated is left for another 5 minutes at 37 C and then diluted and plated, and the survival of hr infective centers, which measures the resistance to lysis from without, is determined. The results, plotted in figure 2, show that lysis from without destroys most of the bacterial infective centers at time zero, whereas at 8 minutes the infected.9 MULTIPLICITY OF SECOND INFECTION Figure 1. Bacterial resistance to lysis from without when singly infected bacteria are superinfected at different multiplicities of phage particles per bacterium. The first infection takes place in buffer, the second one at the moment the bacteria are transferred to broth. Ordinate, the proportion of surviving bacterial infective centers from singly infected bacteria; abscissa, the multiplicity per bacterium of superinfecting ultraviolet-irradiated phage. Under these conditions half the bacteria undergo lysis from without at a multiplicity of 100 phage particles per bacterium. phage to samples of bacteria already infected with hr. After 5 minutes each sample is diluted and plated. The results of this experiment (figure 1) show that as the multiplicity of infection is increased the percentage of surviving bacterial infective centers drops to a very low level. In the second experiment the multiplicity of the second infection is kept at a constant value of 1,000 whereas the time of superinfection is varied. After the second phage is added, the suspension 7.6 A 3.2.1 a I I I 0 1% -2 I a s --- 4 5 6 7 8 MINUTES Figure B. Bacterial resistance to lysis from without when singly infected bacteria are superinfected at a high multiplicity of phage particles per bacterium at different intervals after the first infection. Ordinate, the proportion of surviving bacterial infective centers; abscissa, the interval of time, measured in minutes, between the two infections. Singly infected bacteria are superinfected with ultraviolet-irradiated phage at a multiplicity of 1,000 phage particles per bacterium. Bacterial resistance to lysis from without is complete, under these conditions, by about 8 minutes. bacteria have acquired complete resistance to lysis from without. The exclusion effect in relation to the multiplicity of the second and first infecion. Dulbecco (1952) has shown that when bacteria infected with phage are superinfected with a related phage, the first phage excludes the second from the yield. Exclusion becomes more and more complete as the interval of time between the two infections increases. This phenomenon has been

250 N. VISCONTI [vol. 66 called mutual exclusion between related phages, to distinguish it from mutual exclusion between unrelated phages which is an entirely different phenomenon (Delbriick and Luria, 1942). Dulbecco correlates this effect with the stimulation to breakdown discovered by French et al. (1951), and with the destruction of chromatinic bodies of infected bacteria as described by Luria and Human (1950). In Dulbecco's expermnents, the MINUTES Figure 3. Proportion of bursts yielding both first and superinfecting phage when superinfection is either at a low or high multiplicity. Ordinate, the proportion of mottled plaques among the total number of surviving bacterial infective centers; abscissa, the interval of time between first and second infections. The multiplicity of the first infection is one phage particle per bacterium, the multiplicity of superinfection is 500 for the upper curve (0) and 2 for the curve below (0). multiplicity was approximately 1 for both the first and the second infection. Bacteria were plated before lysis, and since r and r+ strains were used for both infections, mixed bursts gave mottled plaques. Delaying the second infection greatly reduced the frequency of mottled plaques. This type of exclusion can be explained in two different ways: (1) as the interval of time between the two infections increases, more and more bacteria develop an all-or-none exclusion ability towards any number of superinfecting phage particles or (2) as the interval of time between the two infections increases, the probability that a superinfecting phage particle will be excluded also increases. The discovery of acquired resistance of infected bacteria to lysis from without now makes it possible to superinfect with large multiplicities of phage and therefore to distinguish between these two explanations of exclusion. If the second explanation is correct, the frequency of mixed bursts should increase with the multiplicity of the second infection..6.5.a.3.2.1 I I I I. 4,1 125 2.5 5 10 20 MULTIPLICITY OF FIRST INFECTION Figure 4. Proportion of mixed bursts yielding both first and superinfecting phage when the first infection occurs at different multiplicities of phage per bacterium. Ordinate, the proportion of mottled plaques among the total number of surviving infective centers; abscissa, the multiplicity of the first infection. All bacteria were superinfected at a multiplicity of 1,000 phage particles per bacterium 6 minutes after the first infection. Figure 3 shows the results of such an experiment using a multiplicity of 1 for the first infection with r and multiplicities of both 2 and 500 for the superinfection with r+. The frequency of mottled plaques is plotted against the time interval between first infection and superinfection. These results show that the superinfecting phage has a much greater probability of growing when the multiplicity of the superinfection is high. It should be mentioned that with a multiplicity of 500 the observations are limited to those bacteria which are resistant to lysis from without at the moment of superinfection. The effect of the multiplicity of the first infection on the mutual exclusion phenomenon also

19531 LYSIS FROM WITHOUT IN T2 BACTERIOPHAGE 251 has been investigated. For this experiment the bacteria were superinfected with a constant multiplicity of 1,000 at an interval of 6 minutes after the first infection. Figure 4 shows the results of this experiment, which demonstrate clearly that the exclusion as tested by the frequency of mottled plaques depends on the multiplicity of the first infection. TABLE 2 Single bursts obtainedfrom a culture infected at time O with rh phage at a multiplicity of I phage particle per bacterium and superinfected at 4 minutes with h+r+ phage at a multiplioity of 600. Of 90 tubes seeded with infected bacteria before burst, 20 yielded phage. The value p is calculated from the formula rh + r+h/2 + rh+/2 - rh + r+h + rh+ + r+h BUIRST Is 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 23 131 6 2 4 1 178 223 1 161 306 351 182 719 506 223 103 76 248 326 175 120 182 271 147 224 147 r+k 81 29 20 112 91 172 23 76 3 88 122 8 9 rhk 44 94 16 32 141 71 16 43 22 74 105 23 6 TOTALI 309 442 388 182 869 831 249 106 76 287 376 207 124 522 271 543 147 256 162 p.28.17.b0.09.25.06.01.07.07.09.02.50.62.06.05 Experiments on the exclusion effect using the single burst technique. Some single burst experiments were made by infecting with hr phage at multiplicity 1 and then, either at 4 minutes or at 7 minutes, adding h+r+ phage at multiplicity 500. All bacteria that escape infection by the first phage undergo lysis from without on the second infection and therefore do not contribute to the bursts. Large differences were found among the separate bursts, both in size and in the frequency of appearance in the yield of the markers carried by the two parental phage types. When a single burst yields both parental types (mixed yield), recombinants are formed in approximately the ratio expected for unlinked markers (Visconti and Delbriick, 1953). Table 2 shows the data of a representative single burst experiment. Figure 5 illustrates the way in which the markers carried by the first infecting phage are distributed in the bursts. The frequency of the first-phage markers Figure 5. Analysis of single bursts from bacteria infected with hr phage at a multiplicity of one phage particle per bacterium, and superinfected after 4 minutes and 7 minutes with h+r+ phage at a multiplicity of 500. Ordinate, the number of single bursts. Abscissa, the parameter p defined in table 2. appearing in the yield was calculated in the following way: + rh+/2+ r+h/2 freueny ( ) -rh frequcy (r + h) =rh + rh+ + r+h + r+h+ The frequency of appearance of the first-phage markers increased markedly when the time between first and second infection was raised from 4 minutes to 7 minutes. Another finding was that in some cases the h+r+ superinfecting phage completely suppressed the growth of the phage that had been adsorbed first. When the interval of time between the two infections was 4 minutes, complete suppression of the first phage occurred in 6 out of 33 bursts; when the interval was increased to 7 minutes, suppression still occurred in 4 out of 31 bursts. These bursts with complete suppression of the first phage represented the extreme end of the distribution shown in figure 5.

252 N. VISCONTI [VOL. 66 DISCUSSION When bacteria are infected with phage at sufficiently high ratios of phage per cell, lysis from without occurs. This can be recognized both from the rapid visible lysis and from the failure of the cells to yield phage. The lysed cells remain visible as swollen intact membranes (Delbrulck, 1940). The experiments reported here show that bacteria become resistant to both effects of lysis from without shortly after infection with phage at low multiplicity. The greater the interval of time between the first and second infections, the higher is the proportion of bacteria that are resistant to lysis from without. Resistance to lysis from without is not due to lack of adsorption of the superinfecting phage since infected bacteria adsorb phage up to the moment of lysis, even when lysis is greatly delayed (Levinthal and Visconti, 1953). The experiments on lysis from without described here are pertinent to previous studies on mutual exclusion between related phages (Dulbecco, 1952). In Dulbecco's experiments superinfection was with a low multiplicity, and the effect measured was exclusion of the superinfecting phage from growth, whereas in our experiments superinfection was with a high multiplicity and the effect measured was resistance to lysis from without. The discovery that infection with a high multiplicity does not necessarily cause lysis from without made it possible to extend Dulbecco's experiments on exclusion to include superinfection at a higher multiplicity. Thus, the results of varying the time interval between first and second infection, and the multiplicities of both first and second infections, could be studied independently. In the experiments described here, the extent of the exclusion is indicated by the number of mottled plaques obtained after infecting first with r and then superinfecting with r+. The results of these experiments show that the probability of obtaining mottled plaques is related directly to the multiplicity of the second infection and inversely to both the multiplicity of the first infection and the time interval between the two infections. We may summarize our present knowledge of the phenomenon of superinfection of the bacterial cell as follows. If the superinfecting phage is of the same type as that used in the first infection, three different effects are possible: lysis from without, participation in growth, or exclusion. When superinfecting phage is added at a given multiplicity at various times after the first infection, cells will react at time zero by lysing from without, at 3 minutes by admitting the superinfecting phage to growth, and at 6 minutes by excluding the superinfecting phage. Resistance to lysis from without may involve a different mechanism from that responsible for exclusion from growth. Both phenomena depend on the multiplicity of the second infection and on the interval of time between the two infections. It should be emphasized that when lysis from without is observed nothing can be said about whether or not the exclusion effect is operative. Finally, it has been shown that in some cases where the superinfecting phage participates in growth it can completely suppress the growth of the first infecting phage. Therefore, when ultraviolet irradiated phage is used for superinfection, the growth of the first phage can be suppressed as a result either of lysis from without or of the displacement of the first phage by nonviable ultraviolet-irradiated phage. This means that method 2 in which ultraviolet-irradiated phage is used to lyse from without may represent an underestimate of the number of cells resistanl to lysis from without. On the other hand, in method 1, which uses viable phage for superinfection, even if the first phage is suppressed by the second one the infected bacterium will still form a plaque on the plate. ACKNOWLEDGMENTS The author wishes to record his indebtedness to Dr. A. D. Hershey and Dr. A. Garen for invaluable advice concerning the execution of the experiments and the interpretation of the results, and to Mr. R. Coon for technical assistance. SUMMARY When bacteria are infected to give a multiplicity of the order of 200 T2 phage particles per cell, the ability of the bacteria to yield new phage is destroyed. At the same time the turbidity of the bacterial culture decreases rapidly, and for this reason the reaction to high multiplicity infection has been called lysis from without. When bacteria are first infected at low

19531 LYSIS FROM WITHOUT IN T2 BACTERIOPHAGE 253 multiplicity with T2 phage, they become resistant to lysis from without when superinfected at high multiplicity since they are able to yield new phage. By using strains of T2 with different genetic markers for the first infection and the superinfection, it has been found that the superinfecting phage can participate in growth within those bacteria which have become resistant to lysis from without. The frequency of genetically mixed bursts which is a measure of the growth of the second phage is related directly to the multiplicity of superinfecting phage and inversely to the interval of time between the two infections. REFERENCES ADAMS, M. 1950 Methods of study of bacterial viruses. Methods in Med. Research, 2, 1-62. BENZER, S. 1952 Resistance to ultraviolet light as an index to the reproduction of bacteriophage. J. Bact., 63, 59-72. COHEN, S. 1949 Growth requirements of bacterial viruses. Bact. Revs., 13, 1-24. DELBRtCK, M. 1940 The growth of bacteriophage and lysis of the host. J. Gen. Physiol., 23, 643-660. DELBRUCK, M., AND LURIA, S. E. 1942 Interference between bacterial viruses. Arch. Biochem., 1, 111-141. DULBECCO, R. 1949 The number of particles of bacteriophage T2 that can participate in intracellular growth. Genetics, 34, 126-132. DULBECCO, R. 1952 Mutual exclusion between related phages. J. Bact., 63, 209-217. FRENCH, R. C., LESLEY, S. M., GRAHAM, A. F., AND VAN ROOYEN, C. E. 1951 Studies on the relationship between virus and host cell. III. The breakdown of p32 labeled T2r+ bacteriophage adsorbed to E. coli previously infected by other coliphages of the T group. Can. J. Med. Sci., 29, 144. HEAGY, F. C. 1950 The effect of 2, 4-dinitrophenol and phage T2 on Escherichia coli B. J. Bact., 59, 367-373. HERSHEY, A. D. 1946 Mutation of bacteriophage with respect to type of plaque. Genetics, 31, 620-640. HERSHEY, A. D., AND ROTMAN, R. 1949 Genetic recombination between host range and plaque type mutants of bacteriophage in single bacterial cells. Genetics, 34, 44-71. LEVINTHAL, C., AND VISCONTI, N. 1953 Growth and recombination in bacterial viruses. Genetics. In pre88. LURIA, S. E., AND HUMAN, M. L. 1950 Chromatin staining of bacteria during bacteriophage infection. J. Bact., 59, 551-560. VISCONTI, N., AND DELBRtYCK, M. 1953 The mechanism of genetic recombination in phage. Genetics, 38, 5-33. WATSON, J. D. 1950 The properties of X-ray inactivated bacteriophage. I. Inactivation by direct effect. J. Bact., 60, 697-718.