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Interpretation of inactivation kinetics of spores of Bacillus megatherium
ARCHIVES
OF
BIOCHEMISTRY
Interpretation
AND
BIOPHYSICS
74, 28-45 (1958)
of Inactivation Kinetics of Bacillus mega therium
of Spores
C. R. Woese From the Biophysics
Department, Received
Yale University, May
New Haven,
Connecticut
6, 1957
The bacterial spore has several unique properties which make it an interesting system for study. It has very few enzyme systems, little or no metabolism, and it contains a relatively small amount of water (1). It seems reasonable, therefore, that its relatively simple nature would make the bacterial spore a fruitful system for elucidating the mechanism of the action of radiation on the cell. The lack of water in the spore could conceivably minimize or eliminate indirect radiation action, and the small number of spore enzyme systems would narrow down the possible number of sites upon which radiation can act. There have been a few studies concerned with the action of ionizing radiation on bacterial spores (2, 3), but none of these has been particularly complete. Lea (2) reports the inactivation of Bacillus mesentericus and Bacillus megatherium spores to be single hit, and shows that, for B. mesentericus, radiation of high ion density is four times as effective in killing the spore as is radiation of lower ion density. This ion density effect is not noticed for vegetative forms of bacteria, but is noticed for fern spores, fungus spores, unicellular algae, and yeast (3). In this paper the effect of ionizing radiation of varying ion densities on spores of B. megatherium is reported. MATERIALS
AND METHODS
Spores The spores used in this study were of B. megatherium 899 (which produces a bacteriophage) and a derivative of this strain called D (which does not produce detectable bacteriophage). Spores were grown on potato agar plants at pH 7.2.
28
INACTIVATION
OF
BACTERIAL
SPORES
29
Media The medium used to assay the spores for ability to produce colonies was composed of 15 g. Bacto-agar, 12 g. yeast extract (Difco), and 4 g. Trypticase per 1000 ml. of distilled water; pH was adjusted to 7.4 with NaOH. Broth used in wet irradiations consisted of the same formula as did the above with the exception that the agar was omitted.
Wet irradiations were done in broth at 4°C. using filtered (1 mm. aluminum) 250-kv. x-rays from a General Electric “Maxitron 250” machine. For dry irradiations, distilled water suspensions of spores were freeze-dried on glass cover slips over a layer of gelatin and irradiated with x-rays, protons (from the Brookhaven National Laboratory cyclotron), deuterons, or a-particles (from the Yale cyclotron), using the technique developed by Pollard et al. (4). The rate of energy loss of the particles was varied by passing them through aluminum foils of known centimeter-air equivalence (4). Dose measurement in the case of x-rays was done by means of a Victoreen ionization chamber. Dosimetry for cyclotron bombardments is discussed fully by Pollard et al. (4).
Assay Procedures Spores were resuspended in or diluted into 0.9% NaCl solutions and diluted serially by factors of 10. Resuspended spores are not clumped, as seen in the microscope; 0.1 ml. of the appropriate dilutions were put on agar plates, 2.5 ml. of melted agar (45”C.), was layered over this, and the plates were incubated at 35°C. overnight and scored the next day. In order to improve the accuracy of the assay for survivals of B. megatherium 899 spores in the survival range from 60 to loo’%, the following modification of the above assay procedure was used. B. megatherium 899 spores (or vegetative cells) when plated in the presence of a suitable sensitive strain under proper conditions will form the same number of plaques as they would have colonies were they plated without sensitive strain.1 Each plaque contains one characteristic center colony (a colony of B. megatherium 899) which is easily recognizable. When spores of B. megatherium 899 are irradiated and plated in the manner just described, they do not lose their ability to form plaques (i.e., produce virus) for radiation doses which reduce the colony count by at least one factor of 10 (5); these plaques (obviously) have no center colonies. Therefore, by counting the number of plaques without center colonies and comparing this to the total number of plaques, one has a direct measure of the number of spores inactivated, not merely the number of spores surviving. For colony survivals between 60 and loo%, counting of the per cent inactivated (number of plaques without center colonies) is clearly more accurate statistically than counting the per cent surviving (number of plaques with center colonies, or what is experimentally shown to be equiv1 P. Cowles, personal communication.
30
WOESE
alent, the number of colonies on a corresponding tive strain).
agar plate not seeded with sensa-
RESULTS
When spores are irradiated either in broth at 4°C. or dry at room temperature using x-rays as the source of ionizing radiation and plated to determine colony-forming ability, it is found that there is no difference between the shape of the survival curve or the dose necessary to give a certain per cent survival in the two cases. However, when spores are irradiated in saline suspensions at 4°C. there is a slight increase in the sensitivity to x-rays (roughly 15% less dose needed to produce a given
I
IO
KS
5 IO
IC
IC
,, 0.2 0.4
FIG. 1. Survival
08
I.0 I.2 4.0
I.6
2.4 x-v1.n-7 2.0 8.0xIOSn
of B. megatherium 899 spores as a function dose. Irradiation carried out in broth at 4°C.
of x-ray
INACTIVATION
OF
BACTERIAL
SPORES
31
I
IO-
I6
$I IO
IO
IO I
I t4
1 O!
I 1.2 I2
I 1.6 16
I I 2.0 20x0 ,a particles/cm2 2.4 X=SD.n-7
Fro. 2. Survival of B. megatherium 899 spores as a function of dose of deuterons having an energy loss of 220 e.v./lOO A.
amount of inactivation), while the shape of the survival curve remains practically unchanged. Although Lea (2) reported the inactivation of B. megatherium spores to be single hit (i.e., a straight line on a semilogarithmic plot), we fmd the inactivation by x-rays with this particular strain to be decidedly not single hit (see Fig. 1). The curve shown in the figure is theoretical and till be discussed below. Furthermore, the same shape curve is obtained whether the spores used are B. megatherium 899 (lysogenic) or its derivative, D (nonphage producing). When dried spores are irradiated with protons, deuterons, and cr-partitles of varying ion densities, the shape of the survival curves is the
32
WOESE I
IO-
lo-
-f 2 10-z
lo’
IO-
FIG.
0.4
08
1.2 1.6 2.0 ’o 2.4 X=SD 4 8 x Id”particleS’/7cm~ 3. Survival of B. megatherium 899 spores as a function of dose of deuterons having an energy loss of 430 e.v./lOO A.
same as that obtained with x-rays; it does not change as a function of ion density. This implies that radiation destroys the same spore structures at all ion densities. Figures 2, 3, and 4 show survival of B. megathe&urn spores as a function of dose of deuterons whose rate of energy loss is 220 e.v./lOO A. protein, deuterons whose energy loss is 430 e.v./lOO A. protein, and a-particles whose energy loss is 970 e.v./lOO A. protein, respectively. Figure 5 shows the “cross section parameter” (see Discussion below) for survival curves at various ion densities on a log-log plot. The slope of this curve is the same for both lysogenic B. megatherium 899 and nonlysogenic B. megatherium D.
INACTIVATION
10-51
I 0.s
0.4
BACTERIAL
1.2 2
33
SPORES
I 2%
1.6
\I 2.4
x=so2w7
x IO particles/cm 4. Survival of B. megatherium 899 spores as a function of dose of m-particles having an energy loss of 970 e.v./lOO A. I
FIG.
OF
3
DISCUSSION
Since B. megatherium spores can be irradiated in cold broth or dry in a cyclotron vacuum with the same results, it is probable that the mechanism of inactivation is the same in both instances. In addition, the fact that irradiation in saline rather than broth does little to increase the radiosensitivity of the spores argues that free radicals, etc. (6) formed by irradiation of the solution external to the spore play only a small part in the inactivation process [since such radicals would be prevented from reacting with the spores in broth by broth proteins (S)]. It seems safe to infer, then, that the major part of the radiation
34
WOESE
Ion density FIG.
in ev/ 100 % of protein
5. Inactivation cross-section parameter spores as a function of ion density
for B. megatherium of radiation.
899
action on the spore occurs as the result of ionizations or excitations occurring within the spore itself, and that this action is not mediated through migration of free radicals, etc., since there is no change in radiosensitivity of the spore whether irradiated in solution or dry in vmuo . Shape of Survival Curves Turning to the shape of the survival curves, we ask whether simple target theory (assuming a single ionization in a target is capable of destroying the target’s function) (2, 4) can explain the shape of the survival curves reported here, and if so whether it can explain the shape of the ion-density curve (Fig. 5). It is evident from the shape of the curves that an assumption of one hit (ionization) in a single target will not serve as a model. Therefore, we have to consider such possibilities as more than one hit in a single target, hits in several targets of the same type, or several types of targets.
INACTIVATION
OF
BACTERIAL
SPORES
35
Consider first the model of more than one hit in a single target. One can calculate this family of curves from the Poisson distribution (7). These have the general formula N/N0 = e-z(l + II: +2/Z!
+ ..a + zn/n!)
where N is the number of survivors for a given 5, No is the number present for x = 0, n + 1 is the minimum number of hits required to inactivate, and x = SD, where S is a “target size” constant in appropriate units and D is the dose of radiation. If we take instead as our model n equal-sized targets and require that there is survival until each of these targets has received at least one hit, the mathematical form of the survival curve is (5) N/No = 1 - (1 - e-%)%= eBZ[n - n(n - 2)e-%/2! + . . . f e-(n-l)z] where the symbols have the same meaning as above with the exception that n is the number of targets. The experimental curve together with the family of one-or-more-hitsin-n-targets curves is shown in Fig. 6 on a semilogarithmic plot. On such a plot this family of curves reduced to (5) In N/No = --2 + ln[n - n(n - 2)e-“/2! + . .. f e+l)] and for large x this approaches lnN/No
= -x+lnn
which i.s merely a straight line with slope - 1 and intercept In n. The n-hits-in-one-target curves are very similar to those of Fig. 6 (5, 7) so are not shown here. Neither of these two types of theoretical curves fit the experimental curve with sufficient accuracy. Curves for n = 2 or 4 approximate the experimental down to 20% or 1% survival, respectively (see Fig. S), but deviate widely for lower survivals. Curves for n = 4 which approximate the experimental below 20% survival give too high a theoretical survival in the region above 40%. A second objection to curves of the n-hits-in-one-target type on a simple target model is that with increasing ion density the shape of the curve should approach a linear curve on a semilogarithmic plot because one highly ionizing particle will be capable of producing the requisite number of ionizations in the target (4). (This is contrary to experimental evidence.) While the above-mentioned theoretical curves do not fit the data, some of them come close to doing so in certain regions of the experimental survival curve; this suggests that if still another adjustable parameter is added to the system, the experimental curve could be fit. Several
36
WOESE
2.0 I2
4.0 24
36
6.0 4 8
;
40 100 n.4's: 5 IOl-l=2n =8,16
6. Experimental inactivation curve of B. megatherium 899 compared to theoretical “one-or-more-hits in TZtargets” curves (see text for explanation).
FIG.
combinations of two of the above-mentioned one-hit-in each of n targets curves using targets of different radiation sizes and/or different multiplicities should give adequate fit to the data. The fact that the iondensity change does not change the shape of the survival curves would, however, add the constraint that the ratio of the volumes of the two populations of targets should be equal to the ratio of their areas (4, 7). This would mean that the targets, if spherical, would have to be the same size, or they could be something like cylinders of the same radius with no constraint on their relative length. For instance, the combination of either one or more hits in each of two targets or one or more hits in each of 16 targets with ratios of target sizes of about 2: 1, respectively,
INACTIVATION
OF BACTERIAL
SPORES
37
gives sufficiently good fit to the data to prevent ruling this out as a possible model. The last model to be discussed has the appeal of being simple in comparison to the two types of target model discussed above. There are several considerations that point to the fact that “two-hit” mechanisms play a role in inactivation of the spore of B. megatherium. First, the inactivation curve presented in Fig. 1 can be approximated by a simple two-target model for survivals between about 30 and 100 % (see Fig. 6). Secondly, the radiation destruction of plaque formation by spores of lysogenic B. megatherium is a simple two-hit curve of the form N/N,, = e-%(2 - ewz).l Th ese facts suggest the following model. Consider a system composed of 2n equal-sized targets grouped into n pairs, with the condition for inactivation being that at least two members of at least one pair receive at least one hit each; in other words, as long as one member of each pair has not been hit there is survival. This survival curve has been derived by Atwood and Norman (5) and is expressed by the formula N/No = e-%1(2 - e-z)” where all the terms have been definea previously. On a semilogarithmic plot the curve takes the form In N/No = -nx
+ n In (2 - e-2)
and for large x this approaches InNIN,,
= -nx+nInZ
which is a straight line of slope -n and intercept n In 2. Figure 7 shows these curves for n = 3, 5, 10, 80, and 500 with parameters adjusted to give the best possible fit to the experimental data. It will be seen that for moderate changes in n (for n greater than about 5) the shape of the curve changes very little when only N/N,, 2 1OP is considered. The best fit of the experimental data is obtained for n between 5 and 10. The ion-density curve of Fig. 5 is drawn from experimental data interpreted on this model with n = 7, x = SD [where S is the crosssection parameter, having dimensions of area, and D is dose of radiation in particle&q. cm. (4)]. It is not necessary to require that the n pairs of targets all be of the same size. Though the shape of the inactivation curve is changed by
38
WOESE
iq-
FIG. 7. Inactivation of B. megatherium 899 (dots) compared to theoretical curves derived on the “n pairs of targets” model (see text for explanation).
not requiring this, curves of this more general formulation usually fit within the limits shown in Fig. 7 (unpublished calculations) and therefore need not be considered in further detail. The particular shape of the survival curve observed here is not unique in biological literature. Zirkle and Tobias (8) and Wood (9) have observed this for diploid yeast (but not haploid yeast). These authors also invoked the model of “n pairs of targets” to explain their survivalcurve shapes. On this basis they were able to unify the mechanism of the action of radiation on haploid and diploid yeast. The parameter “n,” which determines the number of pairs, they found to be between the bounds of 20 and 64, with 30 a best choice. The value of n determined in the present study is between 5 and 10.
INACTIVATION
OF BACTERIAL
Shape of Ion-Density
SPORES
39
Curves
Not only are the survival curves of B. megatherium spores similar to those for diploid yeast, but the cross-section parameter, X, varies with ion density in the same fashion. Figure 5 shows that on a log-log plot S vs. ion density is a straight line for ion densities above about 200 e.v./lOO A. protein, up to the highest values of ion density used. For ion densities less than 200 e.v./lOO A. there is a deviation from this straight line. For B. megatherium, X varies as the 1.4-power (7) of ion density. Data of Donnellan and Morowitz (10) on spores of Bacillus subtilis also show the same type of ion-density curve as shown here, with S varying as the 1.5-power (5) of ion density. When the data of Zirkle and Tobias (8) are analyzed in this fashion, it is found that for ion densities greater than 200 e.v./lOO A. tissue, the logarithm of crosssection parameter varies as the 1.3-power’ of logarithm of ion density. Simple target theory (2, 4) demands that the cross-section parameter vary as the rth power of ion density, where r goes from a maximum of 1.0 at low ion densities to 0.0 at high ion densities (i.e., S approaches a maximum value). Therefore, in all these cases simple target theory cannot explain ion-density effects. It should be mentioned here, however, in connection with the yeast results that Sayeg (11) studying the effect of radiations of varying ion density on haploid yeast, could not repeat all the results of Zirkle and Tobias. Although there is systematic deviation from linearity in the log-log ion-density plot below about 200 e.v./lOO A. for B. megatherium spores (see Fig. 5), and also for B. subtilis spores (lo), the data in both cases are not sufficient to determine the slope of the line in this region. However, the data of Zirkle and Tobias for yeast (8) show this portion of the ion-density curve to have a slope of about 1.O (1.04 as calculated from their graphs). A slope of 1.0 is not incompatible with simple target theory providing the thickness of the target is less than the average distance between primary ionizations on a particle track, so that usually only one primary ionization per particle track occurs within a target (4). This distance for a 200-e.v./lOO-A. particle is about 50 A. The figure of 50 A. is not an unreasonable upper bound for the thickness of a target, especially since there is a possibility that the target is nucleic acid [see discussion of this by Zirkle and Tobias (S)]. Simple target theory might hold in these cases for ion densities below 200 e.v./lOO A., or, in other words, when no more than one primary ionization occurs within a target. The similarities between ion-density curves published by Zirkle and
40
WOESE
Tobias for yeast and those for B. megatherium and B. subtilis spores are quite remarkable. However, there is one point on which spore and yeast ion-density curves differ, this being the difference in absolute values of the cross-section parameter, which means a vertical displacement of the ion-density curve on a log-log plot. For example, at 1000 e.v./lOO A., B. megatherium spore has a cross section, nX, of about 6 X lo-r0 sq. cm., while yeast has 80 X lo-lo sq. cm. cross section. This difference of a factor of about 13 could be dispensed with by saying that the actual target in yeast is 13X that in B. megatherium, as yeast cells are much larger than spores. If simple target theory cannot account for the ion-density data for spores and perhaps yeast, are there any reasonable modifications of it which will do so? Simple target theory assumes a spatially well-defined target and that any ionization (but not excitations) occurring within this target destroys its biological function (2, 4), whereas ionizations occurring outside of the boundaries of the target have no effect on its biological function. A rather reasonable assumption which would make target theory more adaptable is to hypothesize that any given ionization occurring within the target does not inactivate it with certainty, but had only a probability, p, of doing so. It is known (4) that ionizing particles, such as high-energy deuterons, do not produce ionizations that are randomly distributed along their track, but, instead, a primary ionizing event caused by the passage of the particle can lead to any number of secondary ionizing events in the near vicinity of the primary event, thus producing a cluster of ionizing events. The relative frequencies of occurrence of the total number of ionizations per primary ionizing event are known. Thus, in this modification of simple target theory we can assign a probability pl to inactivation of a target by a primary ionizing event causing only one ionization within the target, a probability pz to inactivation of a target by a primary ionizing event causing two ionizations within a target, etc. When one deals with highly ionizing particles (e.g., 1 m.e.v. alpha particle), there is a very good chance that a single particle will cause more than one primary ionizing event within a target (of the usual biological size) ; in this case, the total number of ionizations within the target, which determines pm , will be the sum of the ionizations from all clusters occurring within the target. Usually assumed in this type of treatment is that the ionizations must occur within an extremely short time of each other [otherwise, unless the p values are directly proportional to the
INACTIVATION
OF
BACTERIAL
41
SPORES
number of ionizations, the survival curves are not linear on a semilogarithmic plot to begin with and change shape as a function of ion density (4)]. It is evident that if one desires to obtain ion-density curves on this model which differ as much as possible from those predicted by simple target theory, one should assign to all values of p, for m less than a,certain value the probability 0, and unit probabilities form greater than this value. This amounts to saying that less than a certain number of simultaneous ionizations fail to inactivate the target, while equal to or greater than this number always do so. Also, the larger the minimum number of ionizations requirement is, the greater the deviation of the ion-density curve from that predicted by simple target theory. This “all or none” modification of simple target theory is developed by Pollard el al. (4). The modification has enabled them to account successfully for the action of radiation on several enzymes, where simple target theory has failed to do so; minimum ionization requirements of about three ionizations were used. In order to compare the results of the above model to the experimental data, the predictions of the model regarding the cross-section parameter as a function of ion density are plotted on a log-log plot in Fig. 8. In the figure i is ion density, t is target thickness (a constant), m + 1 is the minimum number of ionizations occurring in the target required to inactivate it, and Pm+ is the probability that m + 1 or greater ionizations will occur in the target simultaneously. (P,+1 multiplied by a suitable constant gives the cross-section parameter). The general formula used to determine these curves is [(4) and unpublished calculations]:
P m+l == 1 - e-%[I + qm.z + A,-1(xz/2!) + B,-2(x3/3
!) +
cm-3(x4/4!)
+
. . -1.
where the symbols which have not been defined as yet have the following meanings : z = it Q,,,= probability that a primary ionization will give m or less ionizations in the target (ion cluster) p, = probability that a primary ionization will give exactly m ionizations in the target Am-, = paa-1 + pzqm-z + . -. + pm-lql Bm-2 = p,A,2
+ p2Aw-3 +
- - . + pm-2A1
Cm-3
+
. . . +
=
p&n-3
p&,-4
+
pm-3&
42
WOESE
0. x=iT FIG.
8. Ion-density curves predicted by “ionization model (see text) compared to experimental
requirement” curve.
It can be seen from Fig. 8 that there is a portion of each curve which is approximately a straight line with a slope greater than unity. The figure shows that for m + 1 = 3, this slope is 1.10, and for m + 1 = 8, the slope is 1.34. Recalling the corresponding slopes for the experimental ion-density curve (Fig. 5) and those for B. subtilis (10) (1.47 and 1.58, respectively), one sees that only when the minimum ionization requirement exceeds 8 does the slope of the resulting theoretical curve approach the experimental. It is felt that an assumption that up to eight ionizations can occur within a target with no effect, whereas over this number is effective is a very artificial one, so we conclude that this modification of target theory does not satisfactorily account for the experimental data on spores. An indirect mechanism of radiation action involving migration of free radicals, etc., was invoked by Zirkle and Tobias (8) and by Sayeg (11) to account for the action of radiation on yeast. As mentioned previously, we feel that indirect action (in the sense of migration of radicals, etc.),
INACTIVATIOX
OF
BACTERIAL
SPORES
43
cannot apply to spores because spores have very little water (l), if any, and the experimental results reported here are the same whether the spores are irradiated in cold broth or dry in a cyclotron vacuum (which would remove any water if present). A model which we feel accounts for the data presented here follows. The model is very general and, at present, has little sound theoretical basis. This model is an indirect action type in that energy is transferred from the site of radiation action to a “target,” but the transfer does not involve migration of radicals, etc. Simply stated it is this: Assume a target which is a biological unit (such as an enzyme molecule or a chromosome, etc.) ; any ionization occurring within the unit will destroy its biological function, whereas any single ionization occurring outside the unit has very low probability of doing so. However, if many ionizations occur simultaneously outside the unit, there is a finite probability (which is distance dependent and a function of target size) that energy will be transferred to the unit, thus inactivating it. Since there are very broad limits on the choice of this probability (as a function of biological unit size, of ion density, and distance), the data can obviously be fit. Lea (2) has developed a model which is formally quite similar to the above one; he assumes that the probability of an ionization’s inactivating a target decreases exponentially with distance from the “target center,” so that as ion density increases, the observed “radius” of the target. increases. The importance of the model proposed here lies in its biological implications: namely, that there are basic biological units, defined in terms of their function and physical measurements, which can be inactivated by single ionizations. In other words, radiation of low ion density does measure the size of a biological unit. But when radiation of high ion densit,y is used, one has to take into account the fact that in a suitable environment energy can be transferred from ionizations occurring outside of the unit to it, thus inactivating it. Finally, we must review any supporting evidence for the radiation action concept proposed here. First, there is a great weight of evidence in favor of the hypothesis that one ionization occurring within a basic biological unit serves to inactivate it, and that in the dry state only ionizations occurring within the unit can inactivate it. This evidence has been summarized by Pollard et al. (4). That there can be transfer of energy produced by an ionization in the dry state under special conditions is implied by the data of Setlow (12), who studied the effect of
44
WOESE
irradiation on an enzyme-substrate complex, and also by Alexander and Charlesby (13). That there is energy transfer (at high ion densities) in the spore rests on the following argument. We have demonstrated2 that when plaque formation of lysogenic strains of B. megatherium is inactivated by irradiation of spores with relatively high doses of x-ray, the prophage is what is destroyed, and it is highly probable that the prophage is a piece of deoxyribonucleic acid (DNA) corresponding to about 50 % of the analogous free virus DNA. When spores are irradiated at low ion densities, the size of the prophage determined by radiation is one half that of the free virus irradiated at the same ion density. Further, we have demonstrated2 that, in all probability, at any ion density plaque formation is destroyed by destruction of the prophage (for the cross sections of different prophages remain in the same ratio at all ion densities). However, at high ion densities the ratio of prophage size to corresponding free virus size (same ion density) is at least 2.5: 1 (it is 1: 2 at low ion densities). This apparent increase in size of the prophage with increasing ion density can most satisfactorily be explained in terms of the energy-transfer mechanism discussed above. In the light of the above argument the author considers there to be a good case in favor of the proposed energy-transfer mechanism operating in spores. If all measurable properties of the spore behaved similarly to those reported above with ion density, there would be strong evidence in favor of energy transfer, providing the “biological units” responsible for some of the processes could be purified and their radiation sizes in the purified state determined. ACKNOWLEDGMENTS The author is indebted to Dr. R. B. Setlow icism in the preparation of this manuscript.
and Dr. H. P. Rappaport
for crit-
SUMMARY
Radiation inactivation of the ability of Bacillus megatherium spores to produce colonies was investigated. The shape of the survival curve was determined and found to be independent of radiation conditions (cold broth vs. dry in vacua) and ion density of radiation. Theoretical implications of the inactivation curve are discussed. 2 C. R. Woese, manuscript
in preparation.
INACTIVATION
OF BACTERIAL SPORES
45
REFERENCES 1. POWELL, J., AND STRANGE, R. E., Biochem. J. 64, 205 (1953). 2. LEA, D. E., “Action of Radiation on Living Cells.” Cambridge (Eng.) University Press, 1955. 3. ZIRHLE, R. E., in “Radiation Biology. I” (Hollaender, ed.). McGraw-Hill, New York, 1954. 4. POLLARD, E. C., GUILD, W. R., HUTCHINSON, F., AND SETLOW, R. B., Prog. in Biophys. and Biophys. Chem. 6, 72 (1955). 5. ATWOOD, K. C., AND NORMAN, A., Proc. N. Y. Acad. Sci. 36(12), 696 (1949). 6. DALE, W. M., in “Radiation Biology. I” (Hollaender, ed.). McGraw-Hill,
New York, 1954. 7. TIMOFEEV-RESOVSKII, N. V., AND ZIMMER, K. G., “Das Trefferprinzip in der Biologie”. Hirzel, Lepzig, 1947. 8. ZIRKLE, R., AND TOBIAS, C., Arch. Biochem. Biophys. 47, 282 (1953). 9. WOOD, T., Proc. Sot. Exptl. Biol. Med. 84, 446 (1953). 10. DONNELLAN, J. E., AND MOROWITZ, H. J., Radiation Research 7, 71 (1957). 11. SAYEG, J. A., Ph.D. Thesis, University of California, 1954. 12. SETLOW, R. B., AND DOYLE, B. Radiation Research 2, 15 (1955). 13. ALEXANDER, P., AND CHARLESBY, A., Nature 173,578 (1954).
OF
BIOCHEMISTRY
Interpretation
AND
BIOPHYSICS
74, 28-45 (1958)
of Inactivation Kinetics of Bacillus mega therium
of Spores
C. R. Woese From the Biophysics
Department, Received
Yale University, May
New Haven,
Connecticut
6, 1957
The bacterial spore has several unique properties which make it an interesting system for study. It has very few enzyme systems, little or no metabolism, and it contains a relatively small amount of water (1). It seems reasonable, therefore, that its relatively simple nature would make the bacterial spore a fruitful system for elucidating the mechanism of the action of radiation on the cell. The lack of water in the spore could conceivably minimize or eliminate indirect radiation action, and the small number of spore enzyme systems would narrow down the possible number of sites upon which radiation can act. There have been a few studies concerned with the action of ionizing radiation on bacterial spores (2, 3), but none of these has been particularly complete. Lea (2) reports the inactivation of Bacillus mesentericus and Bacillus megatherium spores to be single hit, and shows that, for B. mesentericus, radiation of high ion density is four times as effective in killing the spore as is radiation of lower ion density. This ion density effect is not noticed for vegetative forms of bacteria, but is noticed for fern spores, fungus spores, unicellular algae, and yeast (3). In this paper the effect of ionizing radiation of varying ion densities on spores of B. megatherium is reported. MATERIALS
AND METHODS
Spores The spores used in this study were of B. megatherium 899 (which produces a bacteriophage) and a derivative of this strain called D (which does not produce detectable bacteriophage). Spores were grown on potato agar plants at pH 7.2.
28
INACTIVATION
OF
BACTERIAL
SPORES
29
Media The medium used to assay the spores for ability to produce colonies was composed of 15 g. Bacto-agar, 12 g. yeast extract (Difco), and 4 g. Trypticase per 1000 ml. of distilled water; pH was adjusted to 7.4 with NaOH. Broth used in wet irradiations consisted of the same formula as did the above with the exception that the agar was omitted.
Wet irradiations were done in broth at 4°C. using filtered (1 mm. aluminum) 250-kv. x-rays from a General Electric “Maxitron 250” machine. For dry irradiations, distilled water suspensions of spores were freeze-dried on glass cover slips over a layer of gelatin and irradiated with x-rays, protons (from the Brookhaven National Laboratory cyclotron), deuterons, or a-particles (from the Yale cyclotron), using the technique developed by Pollard et al. (4). The rate of energy loss of the particles was varied by passing them through aluminum foils of known centimeter-air equivalence (4). Dose measurement in the case of x-rays was done by means of a Victoreen ionization chamber. Dosimetry for cyclotron bombardments is discussed fully by Pollard et al. (4).
Assay Procedures Spores were resuspended in or diluted into 0.9% NaCl solutions and diluted serially by factors of 10. Resuspended spores are not clumped, as seen in the microscope; 0.1 ml. of the appropriate dilutions were put on agar plates, 2.5 ml. of melted agar (45”C.), was layered over this, and the plates were incubated at 35°C. overnight and scored the next day. In order to improve the accuracy of the assay for survivals of B. megatherium 899 spores in the survival range from 60 to loo’%, the following modification of the above assay procedure was used. B. megatherium 899 spores (or vegetative cells) when plated in the presence of a suitable sensitive strain under proper conditions will form the same number of plaques as they would have colonies were they plated without sensitive strain.1 Each plaque contains one characteristic center colony (a colony of B. megatherium 899) which is easily recognizable. When spores of B. megatherium 899 are irradiated and plated in the manner just described, they do not lose their ability to form plaques (i.e., produce virus) for radiation doses which reduce the colony count by at least one factor of 10 (5); these plaques (obviously) have no center colonies. Therefore, by counting the number of plaques without center colonies and comparing this to the total number of plaques, one has a direct measure of the number of spores inactivated, not merely the number of spores surviving. For colony survivals between 60 and loo%, counting of the per cent inactivated (number of plaques without center colonies) is clearly more accurate statistically than counting the per cent surviving (number of plaques with center colonies, or what is experimentally shown to be equiv1 P. Cowles, personal communication.
30
WOESE
alent, the number of colonies on a corresponding tive strain).
agar plate not seeded with sensa-
RESULTS
When spores are irradiated either in broth at 4°C. or dry at room temperature using x-rays as the source of ionizing radiation and plated to determine colony-forming ability, it is found that there is no difference between the shape of the survival curve or the dose necessary to give a certain per cent survival in the two cases. However, when spores are irradiated in saline suspensions at 4°C. there is a slight increase in the sensitivity to x-rays (roughly 15% less dose needed to produce a given
I
IO
KS
5 IO
IC
IC
,, 0.2 0.4
FIG. 1. Survival
08
I.0 I.2 4.0
I.6
2.4 x-v1.n-7 2.0 8.0xIOSn
of B. megatherium 899 spores as a function dose. Irradiation carried out in broth at 4°C.
of x-ray
INACTIVATION
OF
BACTERIAL
SPORES
31
I
IO-
I6
$I IO
IO
IO I
I t4
1 O!
I 1.2 I2
I 1.6 16
I I 2.0 20x0 ,a particles/cm2 2.4 X=SD.n-7
Fro. 2. Survival of B. megatherium 899 spores as a function of dose of deuterons having an energy loss of 220 e.v./lOO A.
amount of inactivation), while the shape of the survival curve remains practically unchanged. Although Lea (2) reported the inactivation of B. megatherium spores to be single hit (i.e., a straight line on a semilogarithmic plot), we fmd the inactivation by x-rays with this particular strain to be decidedly not single hit (see Fig. 1). The curve shown in the figure is theoretical and till be discussed below. Furthermore, the same shape curve is obtained whether the spores used are B. megatherium 899 (lysogenic) or its derivative, D (nonphage producing). When dried spores are irradiated with protons, deuterons, and cr-partitles of varying ion densities, the shape of the survival curves is the
32
WOESE I
IO-
lo-
-f 2 10-z
lo’
IO-
FIG.
0.4
08
1.2 1.6 2.0 ’o 2.4 X=SD 4 8 x Id”particleS’/7cm~ 3. Survival of B. megatherium 899 spores as a function of dose of deuterons having an energy loss of 430 e.v./lOO A.
same as that obtained with x-rays; it does not change as a function of ion density. This implies that radiation destroys the same spore structures at all ion densities. Figures 2, 3, and 4 show survival of B. megathe&urn spores as a function of dose of deuterons whose rate of energy loss is 220 e.v./lOO A. protein, deuterons whose energy loss is 430 e.v./lOO A. protein, and a-particles whose energy loss is 970 e.v./lOO A. protein, respectively. Figure 5 shows the “cross section parameter” (see Discussion below) for survival curves at various ion densities on a log-log plot. The slope of this curve is the same for both lysogenic B. megatherium 899 and nonlysogenic B. megatherium D.
INACTIVATION
10-51
I 0.s
0.4
BACTERIAL
1.2 2
33
SPORES
I 2%
1.6
\I 2.4
x=so2w7
x IO particles/cm 4. Survival of B. megatherium 899 spores as a function of dose of m-particles having an energy loss of 970 e.v./lOO A. I
FIG.
OF
3
DISCUSSION
Since B. megatherium spores can be irradiated in cold broth or dry in a cyclotron vacuum with the same results, it is probable that the mechanism of inactivation is the same in both instances. In addition, the fact that irradiation in saline rather than broth does little to increase the radiosensitivity of the spores argues that free radicals, etc. (6) formed by irradiation of the solution external to the spore play only a small part in the inactivation process [since such radicals would be prevented from reacting with the spores in broth by broth proteins (S)]. It seems safe to infer, then, that the major part of the radiation
34
WOESE
Ion density FIG.
in ev/ 100 % of protein
5. Inactivation cross-section parameter spores as a function of ion density
for B. megatherium of radiation.
899
action on the spore occurs as the result of ionizations or excitations occurring within the spore itself, and that this action is not mediated through migration of free radicals, etc., since there is no change in radiosensitivity of the spore whether irradiated in solution or dry in vmuo . Shape of Survival Curves Turning to the shape of the survival curves, we ask whether simple target theory (assuming a single ionization in a target is capable of destroying the target’s function) (2, 4) can explain the shape of the survival curves reported here, and if so whether it can explain the shape of the ion-density curve (Fig. 5). It is evident from the shape of the curves that an assumption of one hit (ionization) in a single target will not serve as a model. Therefore, we have to consider such possibilities as more than one hit in a single target, hits in several targets of the same type, or several types of targets.
INACTIVATION
OF
BACTERIAL
SPORES
35
Consider first the model of more than one hit in a single target. One can calculate this family of curves from the Poisson distribution (7). These have the general formula N/N0 = e-z(l + II: +2/Z!
+ ..a + zn/n!)
where N is the number of survivors for a given 5, No is the number present for x = 0, n + 1 is the minimum number of hits required to inactivate, and x = SD, where S is a “target size” constant in appropriate units and D is the dose of radiation. If we take instead as our model n equal-sized targets and require that there is survival until each of these targets has received at least one hit, the mathematical form of the survival curve is (5) N/No = 1 - (1 - e-%)%= eBZ[n - n(n - 2)e-%/2! + . . . f e-(n-l)z] where the symbols have the same meaning as above with the exception that n is the number of targets. The experimental curve together with the family of one-or-more-hitsin-n-targets curves is shown in Fig. 6 on a semilogarithmic plot. On such a plot this family of curves reduced to (5) In N/No = --2 + ln[n - n(n - 2)e-“/2! + . .. f e+l)] and for large x this approaches lnN/No
= -x+lnn
which i.s merely a straight line with slope - 1 and intercept In n. The n-hits-in-one-target curves are very similar to those of Fig. 6 (5, 7) so are not shown here. Neither of these two types of theoretical curves fit the experimental curve with sufficient accuracy. Curves for n = 2 or 4 approximate the experimental down to 20% or 1% survival, respectively (see Fig. S), but deviate widely for lower survivals. Curves for n = 4 which approximate the experimental below 20% survival give too high a theoretical survival in the region above 40%. A second objection to curves of the n-hits-in-one-target type on a simple target model is that with increasing ion density the shape of the curve should approach a linear curve on a semilogarithmic plot because one highly ionizing particle will be capable of producing the requisite number of ionizations in the target (4). (This is contrary to experimental evidence.) While the above-mentioned theoretical curves do not fit the data, some of them come close to doing so in certain regions of the experimental survival curve; this suggests that if still another adjustable parameter is added to the system, the experimental curve could be fit. Several
36
WOESE
2.0 I2
4.0 24
36
6.0 4 8
;
40 100 n.4's: 5 IOl-l=2n =8,16
6. Experimental inactivation curve of B. megatherium 899 compared to theoretical “one-or-more-hits in TZtargets” curves (see text for explanation).
FIG.
combinations of two of the above-mentioned one-hit-in each of n targets curves using targets of different radiation sizes and/or different multiplicities should give adequate fit to the data. The fact that the iondensity change does not change the shape of the survival curves would, however, add the constraint that the ratio of the volumes of the two populations of targets should be equal to the ratio of their areas (4, 7). This would mean that the targets, if spherical, would have to be the same size, or they could be something like cylinders of the same radius with no constraint on their relative length. For instance, the combination of either one or more hits in each of two targets or one or more hits in each of 16 targets with ratios of target sizes of about 2: 1, respectively,
INACTIVATION
OF BACTERIAL
SPORES
37
gives sufficiently good fit to the data to prevent ruling this out as a possible model. The last model to be discussed has the appeal of being simple in comparison to the two types of target model discussed above. There are several considerations that point to the fact that “two-hit” mechanisms play a role in inactivation of the spore of B. megatherium. First, the inactivation curve presented in Fig. 1 can be approximated by a simple two-target model for survivals between about 30 and 100 % (see Fig. 6). Secondly, the radiation destruction of plaque formation by spores of lysogenic B. megatherium is a simple two-hit curve of the form N/N,, = e-%(2 - ewz).l Th ese facts suggest the following model. Consider a system composed of 2n equal-sized targets grouped into n pairs, with the condition for inactivation being that at least two members of at least one pair receive at least one hit each; in other words, as long as one member of each pair has not been hit there is survival. This survival curve has been derived by Atwood and Norman (5) and is expressed by the formula N/No = e-%1(2 - e-z)” where all the terms have been definea previously. On a semilogarithmic plot the curve takes the form In N/No = -nx
+ n In (2 - e-2)
and for large x this approaches InNIN,,
= -nx+nInZ
which is a straight line of slope -n and intercept n In 2. Figure 7 shows these curves for n = 3, 5, 10, 80, and 500 with parameters adjusted to give the best possible fit to the experimental data. It will be seen that for moderate changes in n (for n greater than about 5) the shape of the curve changes very little when only N/N,, 2 1OP is considered. The best fit of the experimental data is obtained for n between 5 and 10. The ion-density curve of Fig. 5 is drawn from experimental data interpreted on this model with n = 7, x = SD [where S is the crosssection parameter, having dimensions of area, and D is dose of radiation in particle&q. cm. (4)]. It is not necessary to require that the n pairs of targets all be of the same size. Though the shape of the inactivation curve is changed by
38
WOESE
iq-
FIG. 7. Inactivation of B. megatherium 899 (dots) compared to theoretical curves derived on the “n pairs of targets” model (see text for explanation).
not requiring this, curves of this more general formulation usually fit within the limits shown in Fig. 7 (unpublished calculations) and therefore need not be considered in further detail. The particular shape of the survival curve observed here is not unique in biological literature. Zirkle and Tobias (8) and Wood (9) have observed this for diploid yeast (but not haploid yeast). These authors also invoked the model of “n pairs of targets” to explain their survivalcurve shapes. On this basis they were able to unify the mechanism of the action of radiation on haploid and diploid yeast. The parameter “n,” which determines the number of pairs, they found to be between the bounds of 20 and 64, with 30 a best choice. The value of n determined in the present study is between 5 and 10.
INACTIVATION
OF BACTERIAL
Shape of Ion-Density
SPORES
39
Curves
Not only are the survival curves of B. megatherium spores similar to those for diploid yeast, but the cross-section parameter, X, varies with ion density in the same fashion. Figure 5 shows that on a log-log plot S vs. ion density is a straight line for ion densities above about 200 e.v./lOO A. protein, up to the highest values of ion density used. For ion densities less than 200 e.v./lOO A. there is a deviation from this straight line. For B. megatherium, X varies as the 1.4-power (7) of ion density. Data of Donnellan and Morowitz (10) on spores of Bacillus subtilis also show the same type of ion-density curve as shown here, with S varying as the 1.5-power (5) of ion density. When the data of Zirkle and Tobias (8) are analyzed in this fashion, it is found that for ion densities greater than 200 e.v./lOO A. tissue, the logarithm of crosssection parameter varies as the 1.3-power’ of logarithm of ion density. Simple target theory (2, 4) demands that the cross-section parameter vary as the rth power of ion density, where r goes from a maximum of 1.0 at low ion densities to 0.0 at high ion densities (i.e., S approaches a maximum value). Therefore, in all these cases simple target theory cannot explain ion-density effects. It should be mentioned here, however, in connection with the yeast results that Sayeg (11) studying the effect of radiations of varying ion density on haploid yeast, could not repeat all the results of Zirkle and Tobias. Although there is systematic deviation from linearity in the log-log ion-density plot below about 200 e.v./lOO A. for B. megatherium spores (see Fig. 5), and also for B. subtilis spores (lo), the data in both cases are not sufficient to determine the slope of the line in this region. However, the data of Zirkle and Tobias for yeast (8) show this portion of the ion-density curve to have a slope of about 1.O (1.04 as calculated from their graphs). A slope of 1.0 is not incompatible with simple target theory providing the thickness of the target is less than the average distance between primary ionizations on a particle track, so that usually only one primary ionization per particle track occurs within a target (4). This distance for a 200-e.v./lOO-A. particle is about 50 A. The figure of 50 A. is not an unreasonable upper bound for the thickness of a target, especially since there is a possibility that the target is nucleic acid [see discussion of this by Zirkle and Tobias (S)]. Simple target theory might hold in these cases for ion densities below 200 e.v./lOO A., or, in other words, when no more than one primary ionization occurs within a target. The similarities between ion-density curves published by Zirkle and
40
WOESE
Tobias for yeast and those for B. megatherium and B. subtilis spores are quite remarkable. However, there is one point on which spore and yeast ion-density curves differ, this being the difference in absolute values of the cross-section parameter, which means a vertical displacement of the ion-density curve on a log-log plot. For example, at 1000 e.v./lOO A., B. megatherium spore has a cross section, nX, of about 6 X lo-r0 sq. cm., while yeast has 80 X lo-lo sq. cm. cross section. This difference of a factor of about 13 could be dispensed with by saying that the actual target in yeast is 13X that in B. megatherium, as yeast cells are much larger than spores. If simple target theory cannot account for the ion-density data for spores and perhaps yeast, are there any reasonable modifications of it which will do so? Simple target theory assumes a spatially well-defined target and that any ionization (but not excitations) occurring within this target destroys its biological function (2, 4), whereas ionizations occurring outside of the boundaries of the target have no effect on its biological function. A rather reasonable assumption which would make target theory more adaptable is to hypothesize that any given ionization occurring within the target does not inactivate it with certainty, but had only a probability, p, of doing so. It is known (4) that ionizing particles, such as high-energy deuterons, do not produce ionizations that are randomly distributed along their track, but, instead, a primary ionizing event caused by the passage of the particle can lead to any number of secondary ionizing events in the near vicinity of the primary event, thus producing a cluster of ionizing events. The relative frequencies of occurrence of the total number of ionizations per primary ionizing event are known. Thus, in this modification of simple target theory we can assign a probability pl to inactivation of a target by a primary ionizing event causing only one ionization within the target, a probability pz to inactivation of a target by a primary ionizing event causing two ionizations within a target, etc. When one deals with highly ionizing particles (e.g., 1 m.e.v. alpha particle), there is a very good chance that a single particle will cause more than one primary ionizing event within a target (of the usual biological size) ; in this case, the total number of ionizations within the target, which determines pm , will be the sum of the ionizations from all clusters occurring within the target. Usually assumed in this type of treatment is that the ionizations must occur within an extremely short time of each other [otherwise, unless the p values are directly proportional to the
INACTIVATION
OF
BACTERIAL
41
SPORES
number of ionizations, the survival curves are not linear on a semilogarithmic plot to begin with and change shape as a function of ion density (4)]. It is evident that if one desires to obtain ion-density curves on this model which differ as much as possible from those predicted by simple target theory, one should assign to all values of p, for m less than a,certain value the probability 0, and unit probabilities form greater than this value. This amounts to saying that less than a certain number of simultaneous ionizations fail to inactivate the target, while equal to or greater than this number always do so. Also, the larger the minimum number of ionizations requirement is, the greater the deviation of the ion-density curve from that predicted by simple target theory. This “all or none” modification of simple target theory is developed by Pollard el al. (4). The modification has enabled them to account successfully for the action of radiation on several enzymes, where simple target theory has failed to do so; minimum ionization requirements of about three ionizations were used. In order to compare the results of the above model to the experimental data, the predictions of the model regarding the cross-section parameter as a function of ion density are plotted on a log-log plot in Fig. 8. In the figure i is ion density, t is target thickness (a constant), m + 1 is the minimum number of ionizations occurring in the target required to inactivate it, and Pm+ is the probability that m + 1 or greater ionizations will occur in the target simultaneously. (P,+1 multiplied by a suitable constant gives the cross-section parameter). The general formula used to determine these curves is [(4) and unpublished calculations]:
P m+l == 1 - e-%[I + qm.z + A,-1(xz/2!) + B,-2(x3/3
!) +
cm-3(x4/4!)
+
. . -1.
where the symbols which have not been defined as yet have the following meanings : z = it Q,,,= probability that a primary ionization will give m or less ionizations in the target (ion cluster) p, = probability that a primary ionization will give exactly m ionizations in the target Am-, = paa-1 + pzqm-z + . -. + pm-lql Bm-2 = p,A,2
+ p2Aw-3 +
- - . + pm-2A1
Cm-3
+
. . . +
=
p&n-3
p&,-4
+
pm-3&
42
WOESE
0. x=iT FIG.
8. Ion-density curves predicted by “ionization model (see text) compared to experimental
requirement” curve.
It can be seen from Fig. 8 that there is a portion of each curve which is approximately a straight line with a slope greater than unity. The figure shows that for m + 1 = 3, this slope is 1.10, and for m + 1 = 8, the slope is 1.34. Recalling the corresponding slopes for the experimental ion-density curve (Fig. 5) and those for B. subtilis (10) (1.47 and 1.58, respectively), one sees that only when the minimum ionization requirement exceeds 8 does the slope of the resulting theoretical curve approach the experimental. It is felt that an assumption that up to eight ionizations can occur within a target with no effect, whereas over this number is effective is a very artificial one, so we conclude that this modification of target theory does not satisfactorily account for the experimental data on spores. An indirect mechanism of radiation action involving migration of free radicals, etc., was invoked by Zirkle and Tobias (8) and by Sayeg (11) to account for the action of radiation on yeast. As mentioned previously, we feel that indirect action (in the sense of migration of radicals, etc.),
INACTIVATIOX
OF
BACTERIAL
SPORES
43
cannot apply to spores because spores have very little water (l), if any, and the experimental results reported here are the same whether the spores are irradiated in cold broth or dry in a cyclotron vacuum (which would remove any water if present). A model which we feel accounts for the data presented here follows. The model is very general and, at present, has little sound theoretical basis. This model is an indirect action type in that energy is transferred from the site of radiation action to a “target,” but the transfer does not involve migration of radicals, etc. Simply stated it is this: Assume a target which is a biological unit (such as an enzyme molecule or a chromosome, etc.) ; any ionization occurring within the unit will destroy its biological function, whereas any single ionization occurring outside the unit has very low probability of doing so. However, if many ionizations occur simultaneously outside the unit, there is a finite probability (which is distance dependent and a function of target size) that energy will be transferred to the unit, thus inactivating it. Since there are very broad limits on the choice of this probability (as a function of biological unit size, of ion density, and distance), the data can obviously be fit. Lea (2) has developed a model which is formally quite similar to the above one; he assumes that the probability of an ionization’s inactivating a target decreases exponentially with distance from the “target center,” so that as ion density increases, the observed “radius” of the target. increases. The importance of the model proposed here lies in its biological implications: namely, that there are basic biological units, defined in terms of their function and physical measurements, which can be inactivated by single ionizations. In other words, radiation of low ion density does measure the size of a biological unit. But when radiation of high ion densit,y is used, one has to take into account the fact that in a suitable environment energy can be transferred from ionizations occurring outside of the unit to it, thus inactivating it. Finally, we must review any supporting evidence for the radiation action concept proposed here. First, there is a great weight of evidence in favor of the hypothesis that one ionization occurring within a basic biological unit serves to inactivate it, and that in the dry state only ionizations occurring within the unit can inactivate it. This evidence has been summarized by Pollard et al. (4). That there can be transfer of energy produced by an ionization in the dry state under special conditions is implied by the data of Setlow (12), who studied the effect of
44
WOESE
irradiation on an enzyme-substrate complex, and also by Alexander and Charlesby (13). That there is energy transfer (at high ion densities) in the spore rests on the following argument. We have demonstrated2 that when plaque formation of lysogenic strains of B. megatherium is inactivated by irradiation of spores with relatively high doses of x-ray, the prophage is what is destroyed, and it is highly probable that the prophage is a piece of deoxyribonucleic acid (DNA) corresponding to about 50 % of the analogous free virus DNA. When spores are irradiated at low ion densities, the size of the prophage determined by radiation is one half that of the free virus irradiated at the same ion density. Further, we have demonstrated2 that, in all probability, at any ion density plaque formation is destroyed by destruction of the prophage (for the cross sections of different prophages remain in the same ratio at all ion densities). However, at high ion densities the ratio of prophage size to corresponding free virus size (same ion density) is at least 2.5: 1 (it is 1: 2 at low ion densities). This apparent increase in size of the prophage with increasing ion density can most satisfactorily be explained in terms of the energy-transfer mechanism discussed above. In the light of the above argument the author considers there to be a good case in favor of the proposed energy-transfer mechanism operating in spores. If all measurable properties of the spore behaved similarly to those reported above with ion density, there would be strong evidence in favor of energy transfer, providing the “biological units” responsible for some of the processes could be purified and their radiation sizes in the purified state determined. ACKNOWLEDGMENTS The author is indebted to Dr. R. B. Setlow icism in the preparation of this manuscript.
and Dr. H. P. Rappaport
for crit-
SUMMARY
Radiation inactivation of the ability of Bacillus megatherium spores to produce colonies was investigated. The shape of the survival curve was determined and found to be independent of radiation conditions (cold broth vs. dry in vacua) and ion density of radiation. Theoretical implications of the inactivation curve are discussed. 2 C. R. Woese, manuscript
in preparation.
INACTIVATION
OF BACTERIAL SPORES
45
REFERENCES 1. POWELL, J., AND STRANGE, R. E., Biochem. J. 64, 205 (1953). 2. LEA, D. E., “Action of Radiation on Living Cells.” Cambridge (Eng.) University Press, 1955. 3. ZIRHLE, R. E., in “Radiation Biology. I” (Hollaender, ed.). McGraw-Hill, New York, 1954. 4. POLLARD, E. C., GUILD, W. R., HUTCHINSON, F., AND SETLOW, R. B., Prog. in Biophys. and Biophys. Chem. 6, 72 (1955). 5. ATWOOD, K. C., AND NORMAN, A., Proc. N. Y. Acad. Sci. 36(12), 696 (1949). 6. DALE, W. M., in “Radiation Biology. I” (Hollaender, ed.). McGraw-Hill,
New York, 1954. 7. TIMOFEEV-RESOVSKII, N. V., AND ZIMMER, K. G., “Das Trefferprinzip in der Biologie”. Hirzel, Lepzig, 1947. 8. ZIRKLE, R., AND TOBIAS, C., Arch. Biochem. Biophys. 47, 282 (1953). 9. WOOD, T., Proc. Sot. Exptl. Biol. Med. 84, 446 (1953). 10. DONNELLAN, J. E., AND MOROWITZ, H. J., Radiation Research 7, 71 (1957). 11. SAYEG, J. A., Ph.D. Thesis, University of California, 1954. 12. SETLOW, R. B., AND DOYLE, B. Radiation Research 2, 15 (1955). 13. ALEXANDER, P., AND CHARLESBY, A., Nature 173,578 (1954).