Studies of phage growth in irradiated cells

ARCHIVES

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BIOCHEMISTRY

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Studies

BIOPHYSICS

of Phage

86, 286293

Growth

(1960)

in Irradiated

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H. T. EPSTEIN From Brandeis

University, Received

Waltham, Massachusetts May 26, 1959

The reduction in number of targets resulting from irradiation of cells after phage infection is equal to the reduction resulting from irradiation of cells before infection. The results of radiological and genetic experiments on cells irradiated before infection are entirely compatible with the interpretation of this number as the number of phage-synthesizing centers. These centers contain primarily nucleic acid, as can be concluded from the results of a rough action spectrum for inactivation of phage production by infected cells. INTRODUCTION

The effects of irradiation on bacterial ability to support phage growth can be studied by irradiating the bacteria either before or after infection. In the case of irradiation before infection, the data give what has been termed the capacity of the bacterium. The capacity has been studied for a few bacterium-phage systems by Benzer and Jacob (1)) Tessman (2)) Bertani (3), and Pollard et ~2. (4). Luria and Latarjet (5) initiated the study of infected cells by irradiating them \;rith ultraviolet light (uv) at various times after infection. Since then, similar studies have been made by several investigators (1, 6-8). In a previous note (9), published survival curves for phage-infected bacteria were interpreted as revealing the existence within the bacteria of a small number of entities which are not phages but which are necessarily associated with the synthesis of mature phages. Since then Dulbecco (10) has found a similar situation in studies of polioinfected monkey kidney cells; he has termed these entities cellular centers of virus reproduction. In both phage and animal virus infections the pattern of radiosensitivity changes may be interpreted as follows. The first third of 1 Supported b.y grant Institutes of Health.

E-1274 from the National 286

the latent period is taken up with alterations of the cell and the virus with the first cellular center being activated at about one-third of the latent period. The other centers are then activated until all are functioning by about one-half of the latent period. The number of centers remains constant throughout the remainder of the latent period. For each system the number of targets revealed by capacity curves turns out to be similar to the number of cellular centers revealed in the Luria-Latarjet type of experiment. In this paper we present experiments designed to clarify the nature of these centers of virus reproduction. MATERIALS

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METHODS

Escherichia coli B and phages T2r+ were obtained initially from Dr. Mark Adams. T2r was prepared from a single plaque isolate. Pseudomonas pyocyanea (both lysogenic and sensitive strains) were obtained from the University of Manchester type culture collection, with the help of Dr. J. 0. Tobin who had obtained the cultures from Dr. F. Jacob and had deposited the strains in the collection. Both systems were propagated on 0.8% Difco nutrient broth plus 0.5% NaCl. For some experiments 1.6% broth was used. This is called 2X broth. The non-uv absorbing buffer was 0.1 M phosphate with 0.01 M MgC12 added; the pH was about 6.7.

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Most uv irradiations were carried out using a sterilamp as source of 2537-A. radiation. Buffer suspensions (3 ml.) in Petri dishes were exposed about 18 in. from the lamp. The dose rate is then about 3 ergs/sq. mm./sec. For some experiments, the source was a medium pressure AH4 lamp from which the wavelengths below 2800 A. had been filtered by use of a Corning 9710 filter. This lamp contains about 1.6 times as much 2800-A. radiation as 2600-A. radiation. After passing through the filter, only 1.2% of the 2600-A. radiation remains and about 21% of the 2800-A. radiation, as measured with a Cary spectrophotometer. All wavelengths greater than 2800 A. remain undiminished in intensity. In all instances when exposures were longer than a few seconds, the buffers were chilled in ice-water baths, and the Petri dishes sat in icewater baths during the irradiations. The anti-T2 serum was made by Dr. T. E. Cartwright of the University of Pittsburgh by injecting rabbits with wild-type T2 which had been extensively purified by cycles of high- and low-speed centrifugation. Its k value was about 140/min. The source of x-rays was a Phillips 100.kv. constant potential machine. The machine was operated with a 2.7-mm. Al filter and with the cups used for the solutions in contact with the filter. The dose rate was then about 10,000 r./min. The medium was broth at 40 g./l., a concentration which previous work has shown to be adequate for preventing indirect effects of radiation. The temperature for growth experiments was 37°C. EXPERIMENTAL

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RESULTS

There is a discrepancy between the number of cellular centers deduced from the results of Luria and Latarjet (5) and those of later workers (6, 7) studying the same E. coli T2 system. It seemed possible that the explanation lay in the use by the later workers of the radiation-resistant strain, B/r, whereas the original work was done with strain B. With the latter, the target number was about 3; with B/r the target number was 5 or 6. The capacity curves for T2 were determined for both these strains. Logarithmic phase cells were centrifuged, resuspended at about 4 X 108/ml. in buffer, and given various doses of uv. The cells were then diluted twofold into 2X concentrated broth, and lo6 T2/ml. was added. After 1 min. of adsorption, the suspension was diluted for assay, and anti-T2 serum was added to

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FIG. 1. The fraction of T2r phages producing plaques after adsorption to E. coli cells irradiated with ultraviolet light before infection.

eliminate free phages. By this time more than 90 % of the phages were adsorbed. Both suspensions were assayed before the end of the latent period. The results of these experiments are shown in Fig. 1 where it is seen that the capacity curves do, indeed, give results paralleling those deduced from experiments on infected cells. In this figure the curves are theoretical 3-target and 5-target curves chosen because they were judged to give best fit to the data with the following fact in mind. At reasonably great doses (more than 20 min. of uv), the plaque counts on B/r were almost twice those on B. From this one can conclude that the curves become parallel and that the capacity of B/r is almost twice that of B. A discrepancy also exists between the number of centers deduced from uv capacity (8) of E. coli B and the x-ray capacity (4). Because there were several points in the experiments in Ref. (4) which made us unwilling to accept the results, we redid the x-ray capacity curve for E. coli B-T2. These results, in excellent agreement with our uv results, are shown in Fig. 2. We next determined some relevant aspects of phage growth in irradiated cells. One-step growth curves showed that as the uv dose to the cells before infection increases from zero to about 100 bacterium-lethal hits to about 200 bacterium-lethal hits, the latent period increases from about 21 min. to about 22

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times after infection; this resulted in the loss of all infective centers for more than half the latent period. An average of one phage per cell was reached only after about 22 min. in an experiment in which the latent period was about 25 min. Two experiments with 400 bacteriumlethal doses of uv resulted in a burst size of 2 and a latent period of about 30 min. With these data in mind, it is possible to do experiments on cells irradiated so that an average of only two or only one center remains per cell; this would be 10 and 20 min. I of uv, respectively, on the scale of Fig. 1. 200 100 A very few experiments with “two-center MINUTES OF X-RAYS cells” showed that the Luria-Latarjet curves FIG. 2. The fraction of T2r phages producing indeed seemed restricted to developing to plaques after adsorption to E. coli cells irradiated about the 2-center stage (normal 9-min. with 100-kv. x-rays before infection. Dose rate curve), but that it would be difficult to make was about 10,000 r./min. a case based on small differences in curvature such as exist between the normal 9- and 11-min. curves. Accordingly, attention was focussed on “single-center cells.” Logarithmic phase cells were centrifuged, suspended in buffer at a concentration of 4-8 X lO*/ml., and given 20 min. of uv. These were diluted twofold into 2X broth and infected with about lo6 T2/ml. Over 90% of the phages were absorbed within 2 min., after which free phages were eliminated with antiserum. The infected cells were sampled at various times by diluting an aliquot 1:50 into chilled buffer. These cells were irradiated with uv to obtain the survival curve for the infective centers. Figure 3 contains the results of a typical experiment. In the figure the relevant curves for phage development in unirradiated cells are given as dashed lines. Development in 20 40 60 6D 100 W DOSE IN SECONDS infected cells is normal for about 5 min., but FIG. 3. The fractional survival of plaque-prothe normal 7-min. radiosensitivity is reached duction by T2r-infected preirradiated E. coli after only after about 10 min. in irradiated cells. varying doses of ultraviolet light. The number on After this time the radiosensitivity does not each curve is the time in minutes after cells were change. Thus, the target number in the infected. The dashed curve is the fractional surLuria-Latarjet experiment has been reduced vival in cells not irradiated before infection. by the same factor as the capacity target number. min., then to about 25-28 min. CorrespondThe existence of a limited number of ceningly, the burst size decreases from about 100 ters of virus reproduction could be the source to 10-15, then to about 2-5. At the greatest of a limitation on the number of phages that dose of uv mentioned, about 30 % of the cells still yield at least one phage. Cells were lysed can replicate within the same cell. Dulbecco (11) had measured this number by infecting by the addition of chloroform at various

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cells with a fixed multiplicity of, e.g., T2r+ and varying the multiplicity of a mutant phage (e.g., T2r). If a multiplicity of one T2r+ was used, the number of cells yielding both phages began to drop off when the multiplicity of T2r was about 5 or 6. Thus, assuming a random selection of phages for replication, only about 5 or 6 phages can replicate within a single cell, for then, by chance, the phages which replicate might all be of the same type. If the centers are real, the limitation could result from having all the centers utilized by one type of phage. On this hypothesis, the reduction (by uv) of the number of centers before infection should be paralleled by a reduction in the number of phages that can replicate in single cells. Logarithmic phase cells were centrifuged, resuspended in buffer at 4-8 X 108/ml., given various doses of uv, and diluted twofold into 2X broth. A multiplicity of 1 T2r+ and 4.5 T2r was added to the cells and, after allowing enough time for absorption to take place (almost all phages were absorbed within 1 minute), free phages were eliminated with antiserum and infected cells were assayed well before the end of the latent period. In some experiments the multiplicities of the phages were interchanged. As in Dulbecco’s original experiments, the mixed-yielding cells were scored primarily by counting mottled plaques. In six instances all the plaques on one plate were picked and replated to check the identification of the plaques. The resultant uncertainty in the number of mottled plaques on any one plate was never as much as 20 %. Figure 4 contains the results of several experiments. The decrease in fraction of mottled plaques is evident. In the two previous experiments there is a major source of difficulty in that the burst size in such preirradiated cells is so low (about 2) that one cannot be certain that the reduction in target number and in limitation number is not due to the very small total yield of new phages. Accordingly, a search was made for a more suitable system. The Pseudomonas pyocyanea phage p8 system studied by Jacob and his collaborators [see, e.g., Benzer and Jacob (l)] is, to our knowledge, the only system in which the phage is more radioresistant than the center

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FIG. 4. The ordinate is the fraction of all plaques which are mottled relative to the fraction mottled when T2 infects unirradiated cells. The indicated spread of the results represents the actual range of the data from nine experiments.

and in which the center is also so sensitive to radiation that the capacity number can be reduced to unity (single-center cells) by low uv doses. Indeed, single-center status can be achieved by only 5 or 6 bacteriumlethal hits, and the burst size is thereby reduced only from about 300 to somewhat more than 200. Logarithmic phage cells were concentrated to 4-8 X 108/ml. in buffer. One aliquot was given a 60-sec. uv dose (sufficient to reduce the capacity to 1 %), and then diluted twofold into 2X broth. The other aliquot was diluted similarly without being irradiated. The cells were infected with about 5 X lo6 p8 phages/ml. More than 90% of the phages were adsorbed within 1 min., as checked by centrifuging aliquots. At various times after infection, the cells were diluted IOO-fold into chilled buffer for uv irradiation, and then assayed well before the end of the latent period. The results of a typical experiment are contained in Fig. 5. The radiosensitivity curves for phage growth in unirradiated cells are entirely similar to those obtained by Benzer and Jacob (1). The curves for growth in irradiated cells show a reduction of target number from 3 to 1, and the slope of the

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FIG. 5. The fractional survival of plaque-production by P. pyocyanea cells infected with phage P8. a, free phage survival X, p8 infection of P 13 t = 10 min. 0, p8 infection of P 13 t = 25 min. A, p8 infection of 60-sec. uv P 13 t = 10 min. Cl, p8 infection of 60-sec. uv P 13 t = 25 min. n , IO-sec. induction of P 13(p8) 1 = 50 min. 0, 60.sec. induction of P 13(p8) t = 50 min.

single-target curve is the same as that of the asymptote of the S-target curve. The experiments just described may also be carried out with lysogenic cells. In these experiments the log-phase cells were concentrated in buffer as before and given the optimal lo-sec. uv induction dose or a 60-sec. dose. The cells were then diluted twofold into 2~ broth. Aliquots taken at various times were diluted loo-fold into chilled buffer, irradiated with uv, and assayed well before the end of the latent period. The results of these experiments are accurately superposable upon the curves obtained with externally infected cells. For example, the data for two of these curves have been included in Fig. 5. One additional experiment was carried out with the Pseudomonas system. The number of prophages in the lysogenic Pseudomonas bacteria has been estimated (12) by infecting uv-induced cells with various multiplicities of mutant phages and examining the progeny to discover the multiplicity of mutants giving equal outputs of carried and mutant phages. This occurred when the mutant

EPSTEIN

multiplicity was three; the result was interpreted as showing that there are three prophages per cell. We repeated this experiment with both unirradiated and preirradiated cells and can say that, when using preirradiated single-center cells, the carried and mutant phage outputs are equal at onehalf to one-third the multiplicity required for equal outputs using unirradiated cells. Because of the leakiness of the host-range mutant utilized, the result is only qualitative, but we were unable to isolate a better mutant phage. Finally, a set of experiments was done to study the nature of the centers. This can be done by doing an action spectrum for the Luria-Latarjet experiment. We have done part of this by studying the relative effectiveness of 260- and 280-rnp radiation in killing the infected cells. Logarithmic phase E. coli B at a concentration of 5 X 107/ml. were infected in broth with T2r at a multiplicity of about 1: 10, and after 1 min. for adsorption, anti-T2 serum was added to reduce free phage titer to less than 1OP’ in 4 min. This protocol resulted in an effective multiplicity of about 1: 50. Samples taken at various times were diluted 1: 50 into chilled buffer, and aliquots were irradiated with the two lamp setups already described . The inactivation curves were obtained after infections lasting 5, 7, 9, 11, and 15 min. In general the curves obtained with the filtered AH4 lamp radiation were quite similar to those obtained with 2537A radiation; the latter results were in good agreement with the previously published results of Benzer (7). The curves could then be superposed by a simple change of time scale. The relative effectiveness of the two lamps was defined as this time-scale factor. The lamps were set so that the factor was about 2 for the killing of free phages. The results were that the factor remained two throughout the experiment. The results of a typical single experiment are shown in Fig. 6. It can be seen that the uncertainty in time-scale factor is surprisingly small and is surely less than 50%. Thus there is little or no change in the light absorption properties of the materials controlling phage synthesis from

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what they were initially. The Hershey-Chase (13) experiment tells us that the starting materials are primarily nucleic acid. Further, the action spectrum for inactivation of T2 has been found by Zelle and Hollaender (14) to be like the absorption spectrum of nucleic acid. Thus the centers must also be nucleic acid-like in their light absorption. DISCUSSION

The experiments described in this paper all support the idea that there are cellular centers necessarily associated with phage replication and that the number of these centers is related to the number of some structures existing in the cell before infection and to the number of prophages in lysogenic cells. The nucleic acid-like uv absorption of the centers might be expected because the first center is activated at about one-third of the latent period, which is the time of appearance of the first phage nucleic acid (15, 16). In addition, the suicide experiments have shown the replication of phagesynthetic ability at about the same time: one-third of the latent period. In the normal E. coli B cells, there is an average of about 3 Feulgen-staining nuclear bodies per cell; see, e.g., Luria and Human (17) or Murray et al. (18). Thus, there is a ready interpretation of the centers as being nuclear bodies. It is reasonable for phage nucleic acid to be replicated at the site where the cell has already established its nucleic acid replication mechanism. After T2 infection the nuclear bodies are disrupted in the sense that the Feulgen - staining material disperses throughout the cell; but, beginning at about one-third of the latent period, there is a regranulation into a few Feulgen-staining bodies. After this time the number of Feulgen-staining centers remains constant although the amount of Feulgen-staining material increases, presumably due to the appearance of mature phages within the cells. One can interpret this cytological picture as revealing the stripping of cellular nucleic acid from the nuclear bodies and the eventual establishment of phage DNA on the nuclear bodies. The appearance of the new Feulgenpositive bodies should signal the completion of the first new phage-synthesizing center,

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FIG. 6. The fractional survival of plaque production by E. coli B cells infected with phage T2r. The number on each curve is the time in minutes after cells were infected. Irradiation with two different ultraviolet light sources is indicated by the two symbols for each curve. The time scales on the abscissa are chosen to show that there is essentially a constant dose equivalence factor for all times after infection.

and it is interesting that this occurs cytologically at the expected time: one-third of the latent period. One of the difficulties with identifying the cellular centers with nuclear bodies is that for Tl and T3 the number of centers revealed both by Luria-Latarjet and by capacity experiments is unity, so that the number of centers in a given host depends upon the infecting phage. The cytological studies provide a surprising resolution of this apparent failure of the cellular center idea. When Tl is used to infect E. coli B cells, it has been observed (17) that the three nuclear bodies coalesce into one large one, or one of the bodies enlarges and darkens while the other two fade away. Thus we see that the number of cellular centers deduced from both the capacity and Luria-Latarjet experiments is equal to the number deduced from cytological observations. The difference in center number between B and B/r would make it desirable to do cytological studies on infections of these two strains of coli. It should be noted that the difference in number of centers between B and B/r has also been observed in our lab-

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oratory by Tessman and Epstein (19) who found a capacity number of unity for T3 infection of B and two for T3 infection of B/r. The experiments on relative effectiveness of 2600- and 2800-A. radiation were initially done to test one of the interpretations of the suicide experiments. Stent (20) had pointed out that one could understand the stabilization of P32-containing cells infected with P32containing phages in Ps2-containing medium if the control of phage synthesis passed to some non-phosphorus-containing materials. Our experiments rule out this possibility because if this happened, the relative effectiveness of wavelengths other than 2600 A. should be enhanced. For example, if protein alone became the controlling substance, the relative effectiveness of 2800-A. radiation would be several times greater than that of 2600-A. If the nucleic acid controlled synthesis initially, the relative effectiveness of 2800-A. radiation would be about 35 that of 2600-A. radiation. Thus, if there were a shift, the relative effectiveness would change by at least a factor of ten. The experimental uncertainty of constancy of the number two given by our experiments is no more than 50%. Furthermore, all the wavelengths greater than 2800 A. are present in filtered output of the lamp so that the results of the experiment permit an interpretation that no materials other than those which are nucleic acid-like in their absorption become markedly predominant in controlling phage synthesis. This experiment says nothing about the state and type of the nucleic acid. Thus, the stabilization might be due to some stabilization of the infecting deoxyribonucleic acid (DXA) by combination with other materials or it could be associated with ribonucleic acid (RNA) or some stabilized RNA. The results of our experiments on the limitation number for phages replicating within a single cell conflict with those of Edgar and Steinberg (21) who took advantage of the rI1 system to follow the r+ phenotype as increasing multiplicities of T4r were adsorbed to the same cells. They found that of the order of 30 phages had to infect before

there was an appreciable falling off of cells yielding the r+ phenotype which could be sensitively scored by plating the mixedly infected E. coli B on strain K on which only the wild-type phage can make plaques. In this experiment one cannot distinguish between wild-type phages which replicate and those which are excluded from replication but which sit around inside the cells and are eventually repackaged into protein-coated mature phages without having taken part in replication. In other words, these workers measured marker survival, not marker replication. They found an exclusion number of about 30, which is to be expected since the pool size is about 30 (22, 23). When the number of infecting mutant phages is of the order of the pool size, the wild-type phage may be randomly lost as the sampling mechanism takes out phages for maturation. The Dulbecco (11) experiment nicely selects out those cells in which the wild-type phage has been able to replicate, because otherwise no mottled plaque results. However, the existence of a very few r+ phages in a plaque could almost surely not be noticed. Therefore, it must underestimate the limitation number somewhat because of the unobserved mottling. It is not necessary to postulate that a center is irreversibly activated by one type of phage. If this were true then, for example, it would be hard to explain the existence of clones of recombinant phages. However, the phages which first utilize a center would make more of themselves so that a phage which did not get to a center in the first round of replications would then become even less likely to get to a center because it forms a smaller fraction of all the phages and because the newly formed phages presumably have the additional advantage of being located close to these centers of reproduction. If the statistical factor outweighs the localization factor, the occurrence of an early mutation could result in a reasonably large clone, whereas later mutations would have a less-than-average chance of being replicated. Thus a clone size frequency distribution would tend to be peaked in favor of having many more clones with a very small number of mutants.

PHAGE

ACKNOWLEDGMENTS The author is help with some Dr. M. Fox for action spectrum periment .

indebted to Mrs. H. Z. Grover for of the experimental work and to pointing out implications of the study of the Luria-Latarjet exREFERENCES

1. BENZER, S., AND JACOB, F., Ann. inst. Pasteur 84, 186 (1953). 2. TESSMAN, E. S., ViroZogy 2, 679 (1956). 3. BERTANI, L. E., Virology 7, 92 (1959). 4. POLLARD, E., SETLOW, J., AND WATTS, E., Radiation Research 8, 77 (1958). 5. LURIA, S. E., AND LATARJET, R., J. Bacterial. 63, 149 (1947). 6. LATARJET, R., J. Gen. Physiol. 31, 529 (1948). 7. BENZER, S., J. Bacterial. 63, 59 (1952). 8. SYMONDS, N., Virology 3, 485 (1957). 9. EPSTEIN, H. T., Bull. Math. Biophys. 18, 265 (1956). 10. DULBECCO, R., in “The Nature of Viruses.” J. & A. Churchill Ltd., London, 1957.

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11. DULBECCO, R., Genetics 34, 126 (1949). 12. JACOB, F.,‘AN; WOLLMAN, E. L., Cold Spring Harbor Symposia &ant. Biol. 18, 101 (1953). 13. HERSHEY, A. D., AND CHASE, M., J. Gen. Physiol. 36, 39 (1952). 14. ZELLE, M. R., AND HOLLAENDER, A., J. Bacterioz. 68,210 (1954). 15. COHEN, S. S., AND ARBOGAST, R., J. Exptl. Med. 91, 637 (1950). 16. VIDAVER, G. A., AND KOZLOFF, L. M., J. BioZ. Chem. 226, 335 (1957). 17. LURIA, S. E., AND HUMAN, M. L., J. BacterioZ. 69, 551 (1950). 18. MURRAY, R. G. E., GILLEN, D. H., AR’D HEAGY, F. C., J. Bacterial. 69, 603 (1950). 19. TESSMAN, E. S., AND EPSTEIN, H. T., unpublished experiments. 20. STENT, G. S., J. Gen. Physiol. 38, 853 (1955). 21. EDGAR, R. S., AND STEINBERG, C. M., Virology 6, 115 (1958). 22. LEVINTHAL, C., AND VISCONTI, N.? Genetics 38, 599 (1953). 23. HERSHEY, A. D., J. Gen. Physiol. 37, 1 (1953).