X-radiation studies of a lysogenic bacteriophage

X-Radiation

Studies of a Lysogenic Bacteriophage*’ H. T. Epstein2 and S. W. Englander

From

the Biophysics

Department,

University

of Pittsburgh,

Pittsburgh, Pennsylvania

Received January 21, 1954

Weigle and Delbriick (1) have reported that the lysogenic phage lambda, obtained from E. CO& strain K12, is inactivated by ultraviolet light at a rate about 1/13th that of the coliphage T5 which is.about the same size as lambda. They pointed out the consequent desirability of biochemical study of lambda to see if there could be found a biochemical abnormality which would correlate with the radiological abnormality. In taking up this question it seemed desirable first to study the sensitivity of lambda to ionizing radiations because ultraviolet-inactivation data do not permit an interpretation in terms of a radiosensitive volume. A second purpose of this work was to obtain an apparently severe test for the proposition of equality of target volume and nucleic acid volume which had been observed (2) to hold for most viruses. Since the ultraviolet radiosensitivy of lambda is so small relative to that of T5 and T2, the nucleic acid contents should differ by an easily detectable amount. First, however, it was necessary to discover the target size difference between lambda and T5 or T2, and this difference, as was explained above, cannot be obtained from ultraviolet-irradiation data, so that it is necessary to study the sensitivity of lambda to ionizing radiation. A third impetus for starting the present work arose from the question as to whether the ability to lysogenize might be knocked out at a different rate from the ability to lyse. Subsequent studies will deal with this question. * These studies were aided Department’ of the Navy, and No. Nonr-624(03). 1 Publication No. 27 of the 2 Present address: Brandeis

by a contract between the Office of Naval Research, the University of Pittsburgh, NR 135-110, Contract Department University, 394

of Biophysics. Waltham, Mass.

LYSOGENIC

BACTERIOPHAGE

This paper reports the results of x-ray inactivations and virulent forms of lambda. METHODS

AND

39.5 of the temperate

MhTERl.4LS

,4 200.kv. x-ray unit was used in this work. The dose rate of 1600 roentgens (r.)/ min. was obtained both by means of a Victoreen r-meter and by a biological calibration with coliphage T2 whose inactivation dose (dose to obtain 37% survival) was taken to be 40,000 r. (3). One-half-milliliter samples were placed in 2-cm. diameter glass cups covered with a single sheet of household aluminum foil which was taped to the outside of the cups to prevent concentration changes due to evaporation and condensation. The cups sat in an ice-water bath throughout the irradiation. Unirradiated samples were kept in a refrigerator during the irradiat,ion and assayed in parallel with the irradiated samples. The medium in which the viruses were suspended for the irradiation was nutrient broth containing 40 g. solids/l., which is five times the usual broth concentration. This medium has been tested for protect.ive action by lengthy irradiation of T2 and, for times about twice as long as those shown in Fig. 1, a linear survival curve was obtained. A few experiments were carried out on lambda in which the irradiation lasted as long as 6 hr., and in these experiments there was a slight indication of some so-called indirect effect. Accordingly, the data presented include only data from irradiations lasting up to 3 hr., although the straight line given in the figure continues straight for at least one, and probably two more hours of irradiation. Further consider:tCions deriving from the method of carrying out these experiments will be presented in the discussion. The K12 bacteria were obtained from Dr. 8. S. Cohen; the sensitive bacteria Kl2S (also known as W1485) were obtained from Dr. E. Lederberg. The stock of temperate lambda was obtained from ultraviolet-induced K12; and the virulent lambda were obtained from Dr. M. Lieb. RESULTS

Figure I shows the inactivation data for lambda, with the data for the virulent form being plotted separately from the data for the temperat,e form. There was no a priori reason for making this separation, but the appearance of an apparent difference in inactivation rates in the early esperimtbnts led to subsequent experiments designed to determine whether the difference was real. In these later experiments, the two forms of the phages were inactivated simultaneously (though in separate cups) to avoid the experiment-to-experiment and intraexperimental dose rate fluctuations which might occur. Tn order to utilize the fact that the two forms were inactivated simul‘csneously, the following analysis was carried out. If it is assumed that t,he two forms of lambda have different inactivation doses (D, and D, for t’he virulcntS and t,emperate forms, respectively), the following equations

396

H.

T.

EPSTEIN

AND

S. W.

ENGLANDER

exist for the inactivations V

T

-D/D,

-=e vo

-D/Dt

-.-=e To

Here D is the total dose received at the time the number of virulent and temperate survivors per milliliter are, respectively, V and T; the initial concentrations of each are VO and TO. Elimination of D between the two equations yields T v =bIDt -= To (E > Thus, using the experimental survival ratios permits the ratio of the

‘2

I

DURATION FIG.

1. X-ray

inactivation

curves

OF

IRRADIATION

for temperate lambda.

‘3 IN

and

HOURS

virulent

forms

of phage

LTSOGEiXIC

TABLE

Inactivation of Temperate and Virulent Fractional Temperate

0.37 0.39 0.15 0.35 0.19 0.075 0.39 0.14 0.073 0.17 0.15 0.036 0.073

0.054 0.053

3!17

B.\(‘TERIOPH.A(;E

I Forms of the Lysogenic Phage Lambda

survival Virulent

Du,‘Dt

0.48 0.50 0.22 0.42 0.21 0.10 0.44 0.22 0.13 0.17 0.19 0.082 0.075 0.078 0.058

1.36 1.37 1.24 1.21 1.06 1.13 1.14 1.29 1.29 0.97 1.12 1.32 1.01 1.14 1.03

inactivation dosesto be calculated directly, while at the same time eliminating the possible dose rate fluctuations mentioned above. The data handled in this fashion are presented in Table 1. The mean ratio of inactivation doses was found to be 1.18, with the standard error of the mean being 0.03. The difference of the ratio from unity is 0.18 which is six times the standard error of the mean. To obtain the statistical significance of this result, the Student t test should be used. However, the number of observations is large enough, 15, so that, in this case, no serious error is made by using the standard error curve to estimate the chance of obtaining a result six standard deviat,ions from unity; thk turns out to be lessthan lO+. That is, if the true dose ratio were unity, then once in a million times would a set of 15 observations give the observed ratio. Consequently, it is likely that the observed ratio reflects a real difference in inactivation rates for the two forms. The inactivation doses for t’he virulent and temperate forms are, respectively, 1’20,000r. and 102,000 r. Discussion

Since the inactivation doses are both about 110,000 r., the target volumes for lambda are both about one-third those of T5 and T2 which are similar in size to lambda, but have inactivation doses (in the same medium) of about 40,000 r. (excluding, for the moment, complications

398

H.

T.

EPSTEIN

AND

S.

W.

ENGLAiVDER

introduced by the finding of hyperprotection by agents such as cysteine and very low temperatures). Consequently, the difference in inactivation rates for ultraviolet irradiation is much greater than for ionizing radiation of lambda and T5 or T2. It may be that the large difference in response to ultraviolet light reflects a qualitative difference in the target as well as a quantitative difference. It is a little disquieting that the inactivation rate difference between virulent and temperate lambdas is so small that it has been necessary to look to statistics to support the point. Furthermore, it is conceivable that some of the disappearance of temperate viruses derives from a radiationinduced increase of the tendency to lyse rather than lysogenize. If, however, one takes the data at face value, an interesting correlation appears. The biological difference between the temperate and virulent forms is that the latter has lost the ability to lysogenize. The physical difference is that the virulent virus has lost some radiosensitive volume. Thus, the present work correlates the loss of ability to lysogenize with loss of physical volume and suggests a direct structure-function relationship which should make even more desirable the biochemical analysis suggested by Weigle and Delbriick. This radiosensitivity difference between temperate and virulent forms may be a general property of lysogenic viruses, for two preliminary experiments with the P2 phage active on Shigella (both kindly supplied by Dr. G. Bertani) showed a difference in inactivation rates similar to that obtained with lambda. In this experiment, we have the result that the radiosensitive volume of lambda is about 4/11 that of T2, since the inactivation doses are 110,000 r. and 40,000 r. Therefore, if lambda fits in the category of viruses whose nucleic acid volume equals their radiosensitive volume, the nucleic acid contents should also be in the ratio 4/11. Siminovitch and Smith3 have studied the nucleic acid content of the virulent form of lambda and find that it contains 8 X 10-16 g. of nucleic acid per particle, as compared with their result of 2 X 10-16 g. of nucleic acid for T2. Thus, the nucleic acids are in the ratio 8/20 which is experimentally equal to the ratio 4/11 found by irradiat,ion. This equality of target and nucleic acid may be interpreted as showing that essentially only the nucleic acid is involved in the subsequent reproduction of the virus. Such an interpretation has already been shown by Hershey and Chase (4) to be closely correcb for T2. Despite this support o$ the suggested interpretation, the equality of target and nucleic a Siminovitch,

L., and Smith,

J., personal

communication.

LYSOGENIC

UACTEKIOPHAGI~:

30!1

acid volumes is surprising, for the previously mentioned hyperprotection studies indicate that the irradiations as carried out to determine target sizes (and including the work presented in this paper) are not done under conditions which give the smallest obtainable target size. It is true that we have compared target sizes obtained with viruses irradiated under entirely similar conditions, so that the ratio of the target sizes may well be correcat. However, it is still necessary to ask which is the “right” target,, if, indeed, any target size has any meaning in terms of a physical volume. It, might be said that one can only state that the radiosensitivty of a virus varies directly with its nucleic acid content, and the equality of target and nucleic acid volumes is a fictitious one arising from uncertainties in the experimental results and in the theory used to translate ina&ivation curves into target volumes. Since the explanations of the cyst&e and low-temperature hyperprotections have not been given by anyone, it seems unwise to offer further speculation without additional experimental results. SUMMARY

X-ray inactivation data for the temperate and virulent forms of the lysogenic phage lambda show that their radiosensitive volumes are about one-third that of T2 to which they are nearly equal in size; the respective inactivation doses are 102,000 r. and 120,000 r., for the temperate and virulent forms. The difference in inactivation doses is shown t,o be statistically highly significant. Thus, the loss of ability to lysogenize has been correlated with a loss of radiosensitive volume. Several possible implications of those findings are discussed. REFERENCES 1. 2. 3. 4.

WEIGLE, J. J., AND DELBR~~CB, M., J. Bacterial. 62, 301 (1951). EPSTEIN, H. T., Nature 171, 394 (1953). WATSON, J. D., J. Bacterial 60, 697 (1950). HERSHEY, A. D., AND CHASE, M., J. Gem Physiol. 36, 39 (1952).