APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1978, P. 533-539 0099-2240/78/0035-0533$02.00/0 Copyright i) 1978 American Society for Microbiology
Vol. 35, No. 3
Printed in U.S.A.
Relation Between Radiation Resistance and Salt Sensitivity of Spores of Five Strains of Clostridium botulinum Types A, B, and E I.
KISS,' C.
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RHEE,t N. GRECZ,2* T. A. ROBERTS,' AND J. FARKAS3
Microbial Biophysics Laboratory, Biology Department, Illinois Institute of Technology, Chicago, Illinois 60616'; Meat Research Institute Langford, Bristol, BS18 7DY, U.K.'; and Central Food Research Institute, Herman Otto ut 15, H-1525 Budapest, Hungary'
Received for publication 12 September 1977
The NaCl tolerance of different strains of Clostridium botulinum varies over a wide range, and the patterns of NaCl inhibition differ distinctly and characteristically from strain to strain. The more radiation-resistant strains, such as 33A, 62A, and 7272A, are more resistant to NaCl, whereas the more radiation-sensitive strains, such as 51B and 1304E, are more sensitive to NaCl. This rule appears to hold irrespective of whether the spores were unirradiated controls or whether they were radiation damaged prior to exposure to NaCl in the recovery media. The data seem to indicate that radiation doses in the shoulder portion of the radiation survival curves did not noticeably sensitize the spores to NaCl, whereas radiation doses in the exponential-decline portion of the survival curve invariably produced a distinct sensitization. Thus, strains 33A and 62A were not sensitized to NaCl by 0.3 to 0.4 Mrad, i.e., in the shoulder portion of the survival curve. Radiation-sensitive strain 51B, which shows no distinct shoulder in its survival curve, was sensitized to NaCl by 0.1 Mrad, the lowest radiation dose employed in this study. These observations seem to suggest a possible relationship between deoxyribonucleic acid repair capacity and salt tolerance.
It is known that spores irradiated with increasing radiation doses become increasingly sensitive to sodium chloride in the recovery medium (16). The reason for this is not yet clear, although it has been suggested that the increased salt sensitivity may be due to increased permeability of cell membrane after irradiation (1). Assuming that membranes in the same species are essentially similar, one would expect that an equal dose of radiation should, in theory, sensitize equally all strains irrespective of their basic radiation resistance. However, preliminary observations in our laboratory (9) suggested that NaCl and curing ingredients in the recovery medium, such as bacon, may have a distinctly different effect on different strains depending on their basic radiation resistances. Thus, Clostridium botulinum spores of relatively high inherent radiation resistance (D,o M 0.3 Mrad, e.g., strains 33A, 36A, and 53B) were generally strongly sensitized, whereas strains of lower radiation resistance (D1o _ 0.2 Mrad, e.g., strains 9B and 1288A) were sensitized to a considerably lesser extent. In this connection, it was of interest to com-
pare the salt sensitivity of spores belonging to strains of widely differing radiation resistances in a model system. With this in mind, we have selected five well-studied strains of C. botulinum for the present investigation: strain 33A of high radiation resistance (D1o _ 0.33 Mrad), strain 62A (D,o _ 0.23 Mrad), strain 7272A (D10.4 1og _ 0.2), and strains 51B (D1o _ 0.11 Mrad) and 1304E of low radiation resistance (2, 17). D1o values for type E strains range within 0.07 to 0.18 Mrad (17, 18). MATERIALS AND METHODS Spore production. Spores of C. botulinum 33A and 62A were produced as described by Durban and Grecz (6), in 5% Trypticase-0.5% peptone broth plus 0.2% sodium thioglycolate. Spores of strain 51B were produced in Roberts basal medium containing 5% Trypticase, 0.5% peptone, 0.2% yeast extract, and 1% ammonium sulfate (17). Three serial 1-ml transfers in 10 ml of medium were carried out before sporulation to obtain an actively growing culture. The final flask was inoculated with a 10% inoculum, and spores were harvested by centrifugation after 4 days of incubation at 30°C. At this time, the spores were usually contained in their mother cells (sporangia). To obtain free spores, the cells were treated for 4 h with lysozyme (200 mg/ml) at 37°C, modified from Grecz et al. (10). Therefore, sodium lauryl sulfate was added to 1% final
t Present address: Department of Food Science and Technology, College of Agriculture, Chonnam National University, Kwang-ju 500, Korea. 533
534
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KISS ET AL.
concentration and incubated for 1 h, and the treatment was terminated by centrifugation. The free spores were washed twice in 0.14 M NaCl plus 0.4% sodium thioglycolate. Spores of strains 7272A and 1304E were produced as described by Roberts and Ingram (17). Determination of spore numbers. Direct microscopic counts were made with a phase-contrast microscope in a Petroff-Hausser chamber. For strains 33A, 62A, and 51B, viable colony counts were made in triplicate in TYT agar (3). Sterile 0.14 M NaCl was used to make decimal dilutions. To the bottom of each Prickett culture tube, 0.3 ml of 5% membrane filtersterilized (Millipore Corp., Bedford, Mass.) NaHCO3 was added followed by 0.1-ml portions of the dilutions of the spores. Melted agar at 45°C was poured over the inoculum in such a way as to achieve mixing and uniform distribution of spores. After the agar had solidified, an additional layer of ca. 2 cm of the same medium was overlaid to create anaerobic conditions throughout the whole length of the tube. For strains 7272A and 1304E, viable counts were made in duplicate after decimal dilution in maintenance medium (0.9% sodium chloride, 0.1% peptone) using pour plates in 5-cm-diameter plastic petri dishes (Sterilin Ltd., Richmond upon Thames, England). The plates were incubated in anaerobic jars (Baird & Tatlock Ltd., Chadwell Heath, Essex, England) at 37°C. Gamma radiation. Spore suspensions of strains 33A, 62A, and 51B were heat activated at 80°C for 10 min and irradiated by 137CS gamma rays. To obtain an oxygen-free atmosphere, the suspension was bubbled with purified nitrogen gas through the whole course of irradiation in a Cesium-137 Gammator (Radiation Machinery Corp., Parsippany, N.J.). Irradiation was in an ice bath at a dose rate of 100 krads/h. Spores of 7272A and 1304E were heat activated at 60°C for 30 min. Samples (0.1 ml) of aqueous suspensions were distributed using Plastipak tuberculin syringes in 0.5-ml freeze-drying ampoules (BSS795,1961 type L) and sealed in air, and then treated in a Hotspot gamma irradiation facility. Salt sensitivity. To determine the NaCl sensitivity of heat-activated, unirradiated, and irradiated spores, count tubes containing the desired concentrations of NaCl were incubated for 6 to 8 days at 30°C in TYT agar. Colony counts were made at 2-day intervals. In the case of strains 7272A and 1304E, colony counts were determined in Oxoid RCA medium with different added NaCl levels (duplicate plates at each dilution level) at 37°C.
RESULTS Comparative radiation resistance of Clostridium botulinum strains. Figure 1 compares the radiation survival curves of the five strains of C. botulinum studied in this project. Among these, strains 33A, 62A, and 7272A exhibited relatively high radiation resistances and distinct shoulders in their survival curves. On the other hand, strains 51B and 1304E were extremely sensitive and exhibited no detectable shoulder in their radiation survival curves. The
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survival curve of strain 51B is in essential agreement with several previous reports on this strain (2, 5, 9, 11, 12); however, it is interesting that strain 51B is significantly less resistant than 1304E in spite of the fact that generally C. botulinum type E strains are among the most sensitive sporeformers known. It should be noted that the tests summarized in Fig. 1 were done under a cooperative project in two different laboratories, and that test conditions were different in each laboratory. In essence, strains 33A, 62A, and 51B were irradiated under strictly anaerobic conditions, whereas strains 1304E and 7272A were irradiated with no anaerobic precautions. Therefore, any comparison of strains in this paper should take into account this difference in irradiation conditions. As will become evident from subsequent discussion, the data from the two experiments essentially amplify and extend the basic conclusions about the relationship between radiation injury and sodium chloride sensitivity of bacterial spores, even though the irradiation conditions were substantially different in the two experiments.
Salt sensitivity of unirradiated spores. Figure 2 illustrates the sensitivity to sodium chloride of unirradiated spores of the five strains of C. botulinum described in this study. Comparing Fig. 2 with Fig. 1, it is apparent that generally the more radiation-resistant strains 33A, 62A, and 7272A were also more
RADIATION VERSUS SALT SENSITIVITY OF SPORES
VOL. 35, 1978
resistant to sodium chloride than the less radiation-resistant strains 51B and 1304E. Strains 33A and 62A, which exhibited an extensive shoulder in their radiation survival curves in Fig. 1, showed also an extensive "shoulder" in their tolerance of increasing NaCl concentrations in Fig. 2. On the other hand, strains
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535
51B and 1304E, which exhibited no shoulder in their radiation survival curves in Fig. 1, showed also little or no shoulder in their tolerance of increasing NaCl concentrations in Fig. 2. Strain 7272A displayed an intermediate pattern of salt tolerance between that of resistant strains 33A and 62A and sensitive strains 51B and 1304E. Of the strains tested here, spores of strain 33A have been shown to contain one genome per spore, whereas spores of strains 62A and 51B contain two genomes (14; T. W. Kang, Ph.D. thesis, Illinois Institute of Technology, Chicago, 1976). Interestingly, the digenomic spores of 62A, which show a lower radiation resistance than the unigenomic 33A, emerged in Fig. 2 as having the same or slightly higher salt tolerance as 33A. Delay of colony development by NaCl in the medium. Figure 3 shows the colony counts of strains 33A, 62A, and 51B after 2 to 8 days of incubation at 30°C in TYT agar containing 0 to 8% added NaCl. It can be seen that the initial count of strain 33A without added NaCl reached a maximum number of visible colonies after 4 days, although the count after 2 days was only slightly, perhaps insignificantly, lower than that at 4 and 6 days. At NaCl concentrations above 1%, there was a dual effect: (i) a significant delay in development of visible colonies between 2 and 4 days, although the 4- and 6-day counts were at all NaCl concentrations virtually identical, and (ii) a progressive reduction in the number of colonies as a function of increasing NaCl concentration with complete inhibition at 6% NaCl. Additional counts after 8 and 10 days of incubation (data not shown in Fig. 3) revealed no further increase in colony number or colony size over that obtained after 4 to 6 days. Even after 6 to 10 days of incubation, the colonies formed 420 A . Jot. 5Z] o 2 o3 0* aI
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536
KISS ET AL.
in TYT agar containing 1 to 4% NaCl were noticeably smaller than those in the absence of NaCl. The pattern of salt tolerance of unirradiated spores of C. botulinum 62A (Fig. 3) was remarkably different from those of strains 33A and 51B. Concentrations of NaCl up to 4% had no effect on strain 62A; there was also not much difference in the size of the colonies or the rate of colony development. There appeared to be a sharp cutoff point at 4% NaCl, for no colonies were found in 5% NaCl after 2 and 4 days. However, after 6 days 67% of the colonies did appear in 5% NaCl, although no growth was detected in 6% NaCl even after 8 to 10 days. We have no explanation for this anomalous behavior of this strain. Strain 51B (Fig. 3) normally forms very small radiation-sensitive spores. This strain showed such slow growth that after 2 days at 30°C the colonies were practically not countable; therefore, the first counts were taken at 3 days. Note the conspicuous absence of any shoulder, indicating absence of any tolerance to even the lowest NaCl concentration (1%) tested in this study. Incubation for up to 10 days yielded only a very slight, perhaps insignificant increase in the number of colonies after 8 days of incubation in NaCl-containing tubes. The size of colonies of 51B is generally relatively small, and no difference in colony size was noted in the presence or absence of NaCl in the recovery medium. Effect of increasing radiation dose on NaCl sensitivity of spores. Figure 4 shows recovery of irradiated spores in TYT medium with and without 4% NaCl. The concentration of NaCl to be added to the recovery medium was selected on the basis of preliminary experiments summarized in Fig. 3. Figure 4 presents the 6-day colony counts, i.e., at the time of incubation when maximum counts were obtained. It is apparent from Fig. 4 that radiation did not essentially affect salt sensitivity of the spores of 33A up to 0.4 Mrad, i.e., in the shoulder portion of the radiation survival curve. However, in the exponential decline portion of the radiation survival curve, i.e., between 0.5 and 1.2 Mrad, a distinct sensitization was noted, yielding counts in the 4% NaCl medium that were 0.5 log cycle lower than those without NaCl. All irradiated spores, including those at 1.2 Mrad, produced countable colonies in media without added NaCl after 2 days and a maximum count after 4 days. However, in media with added 4% NaCl the colony development was noticeably delayed so that countable colonies could only be obtained after 4 and 6 days. It should be noted that radiation resistance of C. botulinum spores produced in the same sporulation medium frequently varies from crop to
APPL. ENVIRON. MICROBIOL.
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FIG. 4. Effect of NaCI in the recovery medium on survival of irradiated spores of C. botulinum 33A (0), 62A (A), and 51B (O). Open symbols are recoveries in media without added NaCl, closed symbols are recoveries in media with 4% added NaCl, except for salt-sensitive strain 51B (O), where only 1% NaCl (O) and 2% NaCl (-) was added.
crop (12). The spores of C. botulinum 33A used in this study had essentially the same radiation resistance as those reported by Anellis and Koch (2), i.e., Do _0.3 Mrad. Recovery data for C. botulinum strain 62A (Fig. 4) show again no significant inhibition of spore recovery by 4% NaCl, as noted in the shoulder portion of the radiation survival curve, i.e., up to ca. 0.3 Mrad. However, in the exponential inactivation portion of the survival curve, particularly between ca. 0.6 and 1.2 Mrad, 4% NaCl exerted a distinctly inhibitory effect on spore recovery. The effects of NaCl on strains 33A and 62A appeared to be distinctly different. In 62A, inhibition became progressively more pronounced at higher radiation doses, resulting in a distinct divergency of survival curves in media with and without added NaCl, starting from a barely detectable difference of counts at 0.2 to 0.4 Mrad and ending in ca. one log cycle difference in counts at 1.0 to 1.2 Mrad. In contrast, colony counts of strain 33A in the shoulder portion of the survival curve were essentially identical in media with or without added NaCl. However, in the exponential-decline portion, i.e., between 0.6 and 1.2 Mrad, the counts appeared to be uniformly ca. 0.5 log cycle lower in 4% NaCl than in media without added NaCl, resulting in parallel rather than divergent survival curves in the exponential-decline portion of the survival curve. Similar data for strain 51B are shown in Fig. 4. This strain is unique among known strains of C. botulinum for its extreme radiation sensitivity
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RADIATION VERSUS SALT SENSITIVITY OF SPORES
and for absence of any detectable shoulder in its radiation survival curve (11). The semilog radiation survival curve without an initial shoulder shown for 51B (Fig. 4) is in agreement with previous results regarding the radiation death kinetics of this strain (7). Because of the high salt sensitivity of strain 51B (Fig. 3), even the low NaCl concentration selected for this experiment (1%) had some inhibitory effect on the control unirradiated spores. At 2% NaCl, as compared with 0 and 1% NaCl, inhibition of irradiated spores exhibited a distinctly divergent pattern, starting with some 53% inhibition at 0 Mrad, 58% at 0.05 Mrad, and progressively increasing to some 99% at 0.4 Mrad. The D1o values for 0 and 1% NaCl were identical (D0o _ 0.110 Mrad), whereas the D1o value for spore recovery in 2% NaCl was considerably lower (D1o 0.089 Mrad). Sensitivity of irradiated spores to increasing concentrations of NaCl. Figure 5 illustrates the effect of sodium chloride concentrations from 0 to 7% on recovery of irradiated (0.4 Mrad) and unirradiated spores of C. botulinum 7272A, a relatively salt-tolerant and radiation-resistant strain, and C. botulinum 1304E, a relatively radiation-sensitive strain. With strain 7272A, the killing effect of 0.4 Mrad itself was ca. 0.5 log cycle reduction in counts. It is evident that 0.4 Mrad sensitized spores of 7272A even to the lowest salt concentration used in this experiment (2%), since recovery was ca. 10% lower in 2% NaCl than in media with no added salt. Further radiation sensitization was observed at 4.5 and 5.5% NaCl. The radiation-sensitive spores of strain 1304E lost 1.5 log viability on initial irradiation to 0.4 Mrad (Fig. 5). Unirradiated spores were relatively tolerant to 1.5% NaCl (85% recovery), but showed progressive inhibition by 2% NaCl and above. e.
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537
In contrast to unirradiated spores, irradiation to 0.4 Mrad caused the spores to lose practically
all their salt tolerance. Irradiated spores showed immediate decline in counts, with ca. 70% loss of viable counts at 1.5% NaCl. Even lower recovery was obtained in media with 2.5 and 3.0% added NaCl. Thus the radiation-sensitive strain 1304E showed a seeming similarity to the other radiation-sensitive strain, 51B, in that it exhibited (i) an extreme sensitivity to NaCl as well as (ii) as strong sensitization by 0.4 Mrad to NaCl in the recovery medium. Comparison of strains. The NaCl tolerance curves of Fig. 2 suggest that strains 62A and 1304E are populations relatively homogeneous in salt tolerance, i.e., the bulk of the population becomes inhibited across a relatively narrow concentration range, while strains 7272A and 51B are heterogeneous, since the slope of the tolerance curve is relatively shallower. It could then be predicted that strain 1304E spores after irradiation would develop a uniform sensitivity to irradiation. Figure 5 shows this to be the case; in 2% salt, spore recovery is 90% of the unirradiated population and only about 10% of the irradiated population. On the other hand, spores of strain 7272A, which are heterogeneous in NaCl resistance, show about the same spectrum of resistance after irradiation as before, i.e., about 75% of the unirradiated count at every salt level. This may argue that in strain 7272A salt resistance is somehow independent of radiation resistance. Since the experiments with strains 51B, 62A, and 33A are in a different format, a similar analysis cannot be done with these strains. DISCUSSION The observations reported in this paper seem to suggest a possible relation between DNA repair capacity and salt tolerance. Radiationresistant strains, such as 33A, have an efficient system for rejoining of DNA single-strand breaks (SSB), whereas radiation-sensitive strains, such as 51B, have a deficient SSB-rejoining system (7). The rejoining of SSB appears to be accomplished by a single enzyme, the DNA ligase (15), which requires no active metabolism in the cell for its activity. Therefore DNA ligase can function under essentially non-physiological conditions, i.e., at 0°C (1), in cells suspended in distilled water, in the presence of metabolic inhibitors (4), in cells at pH 2, in frozen cells at -16°C (M. Long, personal communication), and in bacterial spores prior to germination (7). An extensive statistical computer analysis of radiation survival curves of 15 strains of C. botulinum has recently brought out a significant relation an
538
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between the size of the shoulder in the radiation survival curve and the overall radiation resistance of a spore population of 104 to 106 spores of a particular strain; this is in contrast to the relative unimportance of the slopes, since they were essentially similar in the 14 strains of widely different basic radiation resistance (11). The coincident occurrence of DNA SSB rejoining and NaCl tolerance in the shoulder portion of the survival curve of C. botulinum strain 33A (and possibly strain 62A) suggests that a rejoined (or intact) DNA is essential for recovery in NaCl-containing media. Since in the shoulder portion the DNA SSB are successfully rejoined, it is attractive to speculate that the loss in recovery in the exponentialdecline portion of the radiation survival curve is due to failure of the DNA SSB-rejoining system or to accumulation of some other lesions than simple SSB. Therefore other repair mechanisms must be invoked by the cell for successful recovery, such as DNA excision resynthesis repair and perhaps recombinational repair. These recovery operations are apparently not as efficient as direct SSB rejoining. Furthermore, excision resynthesis would require the functional existence of a metabolic environment in the cell cytoplasm; i.e., spores must be germinated, their cytoplasm must be hydrated, the essential nucleases must be operative, and an essential precursor pool must be available. Since NaCl inhibits the cell in the exponential-decline portion of the survival curve, where metabolic rescue operations seem to be of paramount importance for recovery, it is logical to conclude that NaCl must interfere with some aspect(s) of cell metabolism while the cell is in the process of recovery from initial radiation injury. Once SSB are rejoined, the cell can grow in relatively high NaCl concentrations (>4%); however, in case of defective SSB rejoining, such as in strain 51B, even extremely low NaCl concentrations of the order of normal saline, i.e., 1%, are distinctly inhibitory. In this connection it may be important to reemphasize that characteristically for each individual strain the same radiation dose that prevents DNA SSB rejoining in the shoulder portion of the survival curve also noticeably reduces recovery in NaCl (see Fig. 3). The extreme sensitivity of strain 51B to NaCl even when not irradiated may perhaps be related to the fact that the chromosome of 51B spores is generally extremely fragile, as was observed under conditions of DNA extraction from the spores for sucrose gradient centrifugation (7, 12). Although a rejoined DNA seems to be essential for recovery in NaCl-containing media, it is not clear whether NaCl actually interferes with the process of direct rejoining of DNA SSB them-
APPL. ENVIRON. MICROBIOL.
selves. In the present study, the spores were irradiated in ice water with no NaCl. Since it is known that under these conditions DNA breaks are rejoined relatively rapidly, i.e., within 20 to 40 min (12), sufficient time was available for the spores to complete DNA rejoining during irradiation and subsequent preparation of spore dilutions prior to exposure to recovery media. Previous reports indicate that there is no effect when NaCl is added to the irradiation menstrum but not to the recovery medium (16). From this, one may argue that NaCl in concentrations of 3 to 5% has no effect on DNA SSB rejoining in bacterial spores, but that intact (or rejoined) DNA strands are in some way a prerequisite for recovery of cells in NaCl-containing media. Our conclusion that active metabolism is required for expression of the NaCl block of cell recovery is further supported by the photomicrographic data of Gould (8), who has reported that at 4 to 7% NaCl, germination of Bacillus spores occurs apparently normally, the spore wall is shed, and the developing cells elongate slowly, but cell multiplication is prevented. In the light of current understanding of spore germination and outgrowth (13), the fact that the cells elongate and grow out suggests that RNA transcription and protein synthesis must be operative at the NaCl concentrations that subsequently inhibit colonial development. It is not possible, however, without further experiments, to make a positive statement as to which exact metabolic operation is inhibited by NaCl, although several may be suspect, particularly DNA excision resynthesis, DNA replication, cell division, etc. So far, it is only possible to say that the NaCl blocks some essential metabolic event(s) occurring prior to the first cell division. We see no reasonable evidence at this time to invoke damage to membrane and loss of permeability characteristics in explaining the inhibitory action of NaCl against radiation-damaged cells. In fact, the observation that extremely small NaCl concentrations of the order ofnormal saline, i.e., 1% NaCl, may act in an inhibitory manner in radiation-sensitive strains argues against the idea of membrane permeability being responsible for the NaCl block. Furthermore, the timing of the expression of NaCl block after germination, initiation of RNA and protein synthesis, and essential completion of growth of the first cell seems not to be consistent with the idea of defective cell membranes. ACKNOWLEDGMENTS This work was carried out in part under the United StatesHungary Cooperative Science Program, FHR 03/147, sponsored by the Office of International Relations of the U.S. National Science Foundation and the Hungarian Institute for
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RADIATION VERSUS SALT SENSITIVITY OF SPORES
Cultural Relations. Financial research support was provided by the U.S. Army Research Office, grant no. DAHCO 4-75-G0112. C.O.R. was supported by International Atomic Energy Agency fellowship SC/202/KOR/7312. LITERATURE CITED 1. Altman, K. I., G. B. Gerber, and S. Okada. 1970. Radiation biochemistry, vol. 1. Academic Press Inc., New York. 2. Anellis, A., and R. B. Koch. 1962. Comparative resistance of strains of Clostridium botulinum to gamma rays. Appl. Microbiol. 10:326-330. 3. Anellis, A., E. Shattuck, D. B. Rowley, E. W. Ross, Jr., D. N. Whaley, and V. R. Dowell, Jr. 1975. Lowtemperature irradiation of beef and methods for evaluation of a radappertization process. Appl. Microbiol. 30:811-320. 4. Driedger, A. A., and M. J. Grayston. 1971. Demonstration of two types of DNA repair in X-irradiated Micrococcus radiodurans. Can. J. Microbiol. 17:495-499. 5. Durban, E., E. Durban, and N. Greez. 1974. Production of spore spheroplasts of Clostridium botulinum and DNA extraction for density gradient centrifugation. Can. J. Microbiol. 20:353-358. 6. Durban, E., and N. Grecz. 1969. Resistance of spores of Clostridium botulinum 33A to combinations of ultraviolet and gamma rays. Appl. Microbiol. 18:44-50. 7. Durban, E., N. Grecz, and J. Farkas. 1974. Direct enzymatic repair of deoxyribonucleic acid single-strand breaks in dormant spores. J. Bacteriol. 118:129-138. 8. Gould, G. W. 1964. Effect of food preservation on the growth of bacteria from spores, p. 17-24. In N. Molin and A. Erichsen (ed.), Microbial inhibitors in food. 4th International Symposium on Food Microbiology: the action, use and natural occurrence of microbial inhibitors in foods, June 1964, Goiteborg, Sweden. Almqvist & Wiksell, Stockholm.
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9. Greez, N. 1965. Biophysical aspects of Clostridia. J. Appl. Bacteriol. 28:17-35. 10. Greez, N., A. Anellis, and M. D. Schneider. 1962. Procedure for cleaning of Clostridium botulinum spores. J. Bacteriol. 84:552-558. 11. Grecz, N., H. Lo, T. W. Kang, and J. Farkas. 1977. Characteristics of radiation survival curves of spores of Clostridium botulinum strains p. 603-630. In A. N. Barker, J. Wolf, D. J. Ellar, G. J. Dring, and G. W. Gould (ed.), Spores 1976, vol. 2. Academic Press Inc., New York. 12. Grecz, N., H. Lo, E. J. Kennedy, and E. Durban. 1973. Gamma radiation studies of Clostridium botulinum types A, B, and E: biological aspects, p. 177-191. In Radiation preservation of food. International Atomic Energy Agency, Vienna. 13. Hansen, J. N., G. Spiegelman, and H. 0. Halvorson. 1970. Bacterial spore outgrowth-its regulation. Science 168:1291-1298. 14. Kang, T. W., and N. Greez. 1975. Chromosome segregation patterns during germination of Clostridium botulinum spores, p. 513-516. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff (ed.), Spores VI. American Society for Microbiology, Washington, D.C. 15. Lehmann, L R. 1974. DNA ligase: structure, mechanism and function. Science 186:790-797. 16. Roberts, T. A., P. J. Ditchett, and M. Ingram. 1965. Effect of sodium chloride on radiation resistance and recovery of irradiated anaerobic spores. J. Appl. Bacteriol. 28:336-348. 17. Roberts, T. A., and M. Ingram. 1965. Radiation resistance of spores of Clostridium species in aqueous suspension. J. Food Sci. 30:879-885. 18. Schmidt, C. F., W. K. Nank, and R. V. Lechnowich. 1962. Radiation sterilization of food. II. Some aspects of the growth, sporulation and radiation resistance of spores of C. botulinum type E. J. Food Sci. 27:77-84.
Vol. 35, No. 3
Printed in U.S.A.
Relation Between Radiation Resistance and Salt Sensitivity of Spores of Five Strains of Clostridium botulinum Types A, B, and E I.
KISS,' C.
0.
RHEE,t N. GRECZ,2* T. A. ROBERTS,' AND J. FARKAS3
Microbial Biophysics Laboratory, Biology Department, Illinois Institute of Technology, Chicago, Illinois 60616'; Meat Research Institute Langford, Bristol, BS18 7DY, U.K.'; and Central Food Research Institute, Herman Otto ut 15, H-1525 Budapest, Hungary'
Received for publication 12 September 1977
The NaCl tolerance of different strains of Clostridium botulinum varies over a wide range, and the patterns of NaCl inhibition differ distinctly and characteristically from strain to strain. The more radiation-resistant strains, such as 33A, 62A, and 7272A, are more resistant to NaCl, whereas the more radiation-sensitive strains, such as 51B and 1304E, are more sensitive to NaCl. This rule appears to hold irrespective of whether the spores were unirradiated controls or whether they were radiation damaged prior to exposure to NaCl in the recovery media. The data seem to indicate that radiation doses in the shoulder portion of the radiation survival curves did not noticeably sensitize the spores to NaCl, whereas radiation doses in the exponential-decline portion of the survival curve invariably produced a distinct sensitization. Thus, strains 33A and 62A were not sensitized to NaCl by 0.3 to 0.4 Mrad, i.e., in the shoulder portion of the survival curve. Radiation-sensitive strain 51B, which shows no distinct shoulder in its survival curve, was sensitized to NaCl by 0.1 Mrad, the lowest radiation dose employed in this study. These observations seem to suggest a possible relationship between deoxyribonucleic acid repair capacity and salt tolerance.
It is known that spores irradiated with increasing radiation doses become increasingly sensitive to sodium chloride in the recovery medium (16). The reason for this is not yet clear, although it has been suggested that the increased salt sensitivity may be due to increased permeability of cell membrane after irradiation (1). Assuming that membranes in the same species are essentially similar, one would expect that an equal dose of radiation should, in theory, sensitize equally all strains irrespective of their basic radiation resistance. However, preliminary observations in our laboratory (9) suggested that NaCl and curing ingredients in the recovery medium, such as bacon, may have a distinctly different effect on different strains depending on their basic radiation resistances. Thus, Clostridium botulinum spores of relatively high inherent radiation resistance (D,o M 0.3 Mrad, e.g., strains 33A, 36A, and 53B) were generally strongly sensitized, whereas strains of lower radiation resistance (D1o _ 0.2 Mrad, e.g., strains 9B and 1288A) were sensitized to a considerably lesser extent. In this connection, it was of interest to com-
pare the salt sensitivity of spores belonging to strains of widely differing radiation resistances in a model system. With this in mind, we have selected five well-studied strains of C. botulinum for the present investigation: strain 33A of high radiation resistance (D1o _ 0.33 Mrad), strain 62A (D,o _ 0.23 Mrad), strain 7272A (D10.4 1og _ 0.2), and strains 51B (D1o _ 0.11 Mrad) and 1304E of low radiation resistance (2, 17). D1o values for type E strains range within 0.07 to 0.18 Mrad (17, 18). MATERIALS AND METHODS Spore production. Spores of C. botulinum 33A and 62A were produced as described by Durban and Grecz (6), in 5% Trypticase-0.5% peptone broth plus 0.2% sodium thioglycolate. Spores of strain 51B were produced in Roberts basal medium containing 5% Trypticase, 0.5% peptone, 0.2% yeast extract, and 1% ammonium sulfate (17). Three serial 1-ml transfers in 10 ml of medium were carried out before sporulation to obtain an actively growing culture. The final flask was inoculated with a 10% inoculum, and spores were harvested by centrifugation after 4 days of incubation at 30°C. At this time, the spores were usually contained in their mother cells (sporangia). To obtain free spores, the cells were treated for 4 h with lysozyme (200 mg/ml) at 37°C, modified from Grecz et al. (10). Therefore, sodium lauryl sulfate was added to 1% final
t Present address: Department of Food Science and Technology, College of Agriculture, Chonnam National University, Kwang-ju 500, Korea. 533
534
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KISS ET AL.
concentration and incubated for 1 h, and the treatment was terminated by centrifugation. The free spores were washed twice in 0.14 M NaCl plus 0.4% sodium thioglycolate. Spores of strains 7272A and 1304E were produced as described by Roberts and Ingram (17). Determination of spore numbers. Direct microscopic counts were made with a phase-contrast microscope in a Petroff-Hausser chamber. For strains 33A, 62A, and 51B, viable colony counts were made in triplicate in TYT agar (3). Sterile 0.14 M NaCl was used to make decimal dilutions. To the bottom of each Prickett culture tube, 0.3 ml of 5% membrane filtersterilized (Millipore Corp., Bedford, Mass.) NaHCO3 was added followed by 0.1-ml portions of the dilutions of the spores. Melted agar at 45°C was poured over the inoculum in such a way as to achieve mixing and uniform distribution of spores. After the agar had solidified, an additional layer of ca. 2 cm of the same medium was overlaid to create anaerobic conditions throughout the whole length of the tube. For strains 7272A and 1304E, viable counts were made in duplicate after decimal dilution in maintenance medium (0.9% sodium chloride, 0.1% peptone) using pour plates in 5-cm-diameter plastic petri dishes (Sterilin Ltd., Richmond upon Thames, England). The plates were incubated in anaerobic jars (Baird & Tatlock Ltd., Chadwell Heath, Essex, England) at 37°C. Gamma radiation. Spore suspensions of strains 33A, 62A, and 51B were heat activated at 80°C for 10 min and irradiated by 137CS gamma rays. To obtain an oxygen-free atmosphere, the suspension was bubbled with purified nitrogen gas through the whole course of irradiation in a Cesium-137 Gammator (Radiation Machinery Corp., Parsippany, N.J.). Irradiation was in an ice bath at a dose rate of 100 krads/h. Spores of 7272A and 1304E were heat activated at 60°C for 30 min. Samples (0.1 ml) of aqueous suspensions were distributed using Plastipak tuberculin syringes in 0.5-ml freeze-drying ampoules (BSS795,1961 type L) and sealed in air, and then treated in a Hotspot gamma irradiation facility. Salt sensitivity. To determine the NaCl sensitivity of heat-activated, unirradiated, and irradiated spores, count tubes containing the desired concentrations of NaCl were incubated for 6 to 8 days at 30°C in TYT agar. Colony counts were made at 2-day intervals. In the case of strains 7272A and 1304E, colony counts were determined in Oxoid RCA medium with different added NaCl levels (duplicate plates at each dilution level) at 37°C.
RESULTS Comparative radiation resistance of Clostridium botulinum strains. Figure 1 compares the radiation survival curves of the five strains of C. botulinum studied in this project. Among these, strains 33A, 62A, and 7272A exhibited relatively high radiation resistances and distinct shoulders in their survival curves. On the other hand, strains 51B and 1304E were extremely sensitive and exhibited no detectable shoulder in their radiation survival curves. The
l Oo/u/n7&m
14
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A2
dut (lfimd) FIG. 1. Radiation survival curves of C. botulinum spores, types A, B, and E. The survival curves were determined in two different laboratories; strains 33A, 62A, and 51B were irradiated anaerobically, whereas strains 7272A and 1304E were irradiated with no anaerobic precautions.
survival curve of strain 51B is in essential agreement with several previous reports on this strain (2, 5, 9, 11, 12); however, it is interesting that strain 51B is significantly less resistant than 1304E in spite of the fact that generally C. botulinum type E strains are among the most sensitive sporeformers known. It should be noted that the tests summarized in Fig. 1 were done under a cooperative project in two different laboratories, and that test conditions were different in each laboratory. In essence, strains 33A, 62A, and 51B were irradiated under strictly anaerobic conditions, whereas strains 1304E and 7272A were irradiated with no anaerobic precautions. Therefore, any comparison of strains in this paper should take into account this difference in irradiation conditions. As will become evident from subsequent discussion, the data from the two experiments essentially amplify and extend the basic conclusions about the relationship between radiation injury and sodium chloride sensitivity of bacterial spores, even though the irradiation conditions were substantially different in the two experiments.
Salt sensitivity of unirradiated spores. Figure 2 illustrates the sensitivity to sodium chloride of unirradiated spores of the five strains of C. botulinum described in this study. Comparing Fig. 2 with Fig. 1, it is apparent that generally the more radiation-resistant strains 33A, 62A, and 7272A were also more
RADIATION VERSUS SALT SENSITIVITY OF SPORES
VOL. 35, 1978
resistant to sodium chloride than the less radiation-resistant strains 51B and 1304E. Strains 33A and 62A, which exhibited an extensive shoulder in their radiation survival curves in Fig. 1, showed also an extensive "shoulder" in their tolerance of increasing NaCl concentrations in Fig. 2. On the other hand, strains
I'
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8
Ns !Itnw,/,'e/i&o' ('/-' FIG. 2. NaCI tolerance of C. botulinum strains germinating and growing out from the spore state in agar media under anaerobic conditions. Strains 33A, 62A, and 51B were grown in TYT agar containing added NaCI in the indicated concentrations. Strains 7272A and 1304E were grown in Oxoid RCA agar with the indicated NaCI additions. Recovery data are those after 6 days of incubation at 30°C.
C. 00/. 33A
C. MoI.
535
51B and 1304E, which exhibited no shoulder in their radiation survival curves in Fig. 1, showed also little or no shoulder in their tolerance of increasing NaCl concentrations in Fig. 2. Strain 7272A displayed an intermediate pattern of salt tolerance between that of resistant strains 33A and 62A and sensitive strains 51B and 1304E. Of the strains tested here, spores of strain 33A have been shown to contain one genome per spore, whereas spores of strains 62A and 51B contain two genomes (14; T. W. Kang, Ph.D. thesis, Illinois Institute of Technology, Chicago, 1976). Interestingly, the digenomic spores of 62A, which show a lower radiation resistance than the unigenomic 33A, emerged in Fig. 2 as having the same or slightly higher salt tolerance as 33A. Delay of colony development by NaCl in the medium. Figure 3 shows the colony counts of strains 33A, 62A, and 51B after 2 to 8 days of incubation at 30°C in TYT agar containing 0 to 8% added NaCl. It can be seen that the initial count of strain 33A without added NaCl reached a maximum number of visible colonies after 4 days, although the count after 2 days was only slightly, perhaps insignificantly, lower than that at 4 and 6 days. At NaCl concentrations above 1%, there was a dual effect: (i) a significant delay in development of visible colonies between 2 and 4 days, although the 4- and 6-day counts were at all NaCl concentrations virtually identical, and (ii) a progressive reduction in the number of colonies as a function of increasing NaCl concentration with complete inhibition at 6% NaCl. Additional counts after 8 and 10 days of incubation (data not shown in Fig. 3) revealed no further increase in colony number or colony size over that obtained after 4 to 6 days. Even after 6 to 10 days of incubation, the colonies formed 420 A . Jot. 5Z] o 2 o3 0* aI
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FIG. 3. Development of visible colonies from unirradiated spores of C. botulinum 33A, 62A, and 51B as a function of increasing NaCl concentration in the recovery medium after 2 to 8 days of incubation at 30°C.
536
KISS ET AL.
in TYT agar containing 1 to 4% NaCl were noticeably smaller than those in the absence of NaCl. The pattern of salt tolerance of unirradiated spores of C. botulinum 62A (Fig. 3) was remarkably different from those of strains 33A and 51B. Concentrations of NaCl up to 4% had no effect on strain 62A; there was also not much difference in the size of the colonies or the rate of colony development. There appeared to be a sharp cutoff point at 4% NaCl, for no colonies were found in 5% NaCl after 2 and 4 days. However, after 6 days 67% of the colonies did appear in 5% NaCl, although no growth was detected in 6% NaCl even after 8 to 10 days. We have no explanation for this anomalous behavior of this strain. Strain 51B (Fig. 3) normally forms very small radiation-sensitive spores. This strain showed such slow growth that after 2 days at 30°C the colonies were practically not countable; therefore, the first counts were taken at 3 days. Note the conspicuous absence of any shoulder, indicating absence of any tolerance to even the lowest NaCl concentration (1%) tested in this study. Incubation for up to 10 days yielded only a very slight, perhaps insignificant increase in the number of colonies after 8 days of incubation in NaCl-containing tubes. The size of colonies of 51B is generally relatively small, and no difference in colony size was noted in the presence or absence of NaCl in the recovery medium. Effect of increasing radiation dose on NaCl sensitivity of spores. Figure 4 shows recovery of irradiated spores in TYT medium with and without 4% NaCl. The concentration of NaCl to be added to the recovery medium was selected on the basis of preliminary experiments summarized in Fig. 3. Figure 4 presents the 6-day colony counts, i.e., at the time of incubation when maximum counts were obtained. It is apparent from Fig. 4 that radiation did not essentially affect salt sensitivity of the spores of 33A up to 0.4 Mrad, i.e., in the shoulder portion of the radiation survival curve. However, in the exponential decline portion of the radiation survival curve, i.e., between 0.5 and 1.2 Mrad, a distinct sensitization was noted, yielding counts in the 4% NaCl medium that were 0.5 log cycle lower than those without NaCl. All irradiated spores, including those at 1.2 Mrad, produced countable colonies in media without added NaCl after 2 days and a maximum count after 4 days. However, in media with added 4% NaCl the colony development was noticeably delayed so that countable colonies could only be obtained after 4 and 6 days. It should be noted that radiation resistance of C. botulinum spores produced in the same sporulation medium frequently varies from crop to
APPL. ENVIRON. MICROBIOL.
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FIG. 4. Effect of NaCI in the recovery medium on survival of irradiated spores of C. botulinum 33A (0), 62A (A), and 51B (O). Open symbols are recoveries in media without added NaCl, closed symbols are recoveries in media with 4% added NaCl, except for salt-sensitive strain 51B (O), where only 1% NaCl (O) and 2% NaCl (-) was added.
crop (12). The spores of C. botulinum 33A used in this study had essentially the same radiation resistance as those reported by Anellis and Koch (2), i.e., Do _0.3 Mrad. Recovery data for C. botulinum strain 62A (Fig. 4) show again no significant inhibition of spore recovery by 4% NaCl, as noted in the shoulder portion of the radiation survival curve, i.e., up to ca. 0.3 Mrad. However, in the exponential inactivation portion of the survival curve, particularly between ca. 0.6 and 1.2 Mrad, 4% NaCl exerted a distinctly inhibitory effect on spore recovery. The effects of NaCl on strains 33A and 62A appeared to be distinctly different. In 62A, inhibition became progressively more pronounced at higher radiation doses, resulting in a distinct divergency of survival curves in media with and without added NaCl, starting from a barely detectable difference of counts at 0.2 to 0.4 Mrad and ending in ca. one log cycle difference in counts at 1.0 to 1.2 Mrad. In contrast, colony counts of strain 33A in the shoulder portion of the survival curve were essentially identical in media with or without added NaCl. However, in the exponential-decline portion, i.e., between 0.6 and 1.2 Mrad, the counts appeared to be uniformly ca. 0.5 log cycle lower in 4% NaCl than in media without added NaCl, resulting in parallel rather than divergent survival curves in the exponential-decline portion of the survival curve. Similar data for strain 51B are shown in Fig. 4. This strain is unique among known strains of C. botulinum for its extreme radiation sensitivity
VOL. 35, 1978
RADIATION VERSUS SALT SENSITIVITY OF SPORES
and for absence of any detectable shoulder in its radiation survival curve (11). The semilog radiation survival curve without an initial shoulder shown for 51B (Fig. 4) is in agreement with previous results regarding the radiation death kinetics of this strain (7). Because of the high salt sensitivity of strain 51B (Fig. 3), even the low NaCl concentration selected for this experiment (1%) had some inhibitory effect on the control unirradiated spores. At 2% NaCl, as compared with 0 and 1% NaCl, inhibition of irradiated spores exhibited a distinctly divergent pattern, starting with some 53% inhibition at 0 Mrad, 58% at 0.05 Mrad, and progressively increasing to some 99% at 0.4 Mrad. The D1o values for 0 and 1% NaCl were identical (D0o _ 0.110 Mrad), whereas the D1o value for spore recovery in 2% NaCl was considerably lower (D1o 0.089 Mrad). Sensitivity of irradiated spores to increasing concentrations of NaCl. Figure 5 illustrates the effect of sodium chloride concentrations from 0 to 7% on recovery of irradiated (0.4 Mrad) and unirradiated spores of C. botulinum 7272A, a relatively salt-tolerant and radiation-resistant strain, and C. botulinum 1304E, a relatively radiation-sensitive strain. With strain 7272A, the killing effect of 0.4 Mrad itself was ca. 0.5 log cycle reduction in counts. It is evident that 0.4 Mrad sensitized spores of 7272A even to the lowest salt concentration used in this experiment (2%), since recovery was ca. 10% lower in 2% NaCl than in media with no added salt. Further radiation sensitization was observed at 4.5 and 5.5% NaCl. The radiation-sensitive spores of strain 1304E lost 1.5 log viability on initial irradiation to 0.4 Mrad (Fig. 5). Unirradiated spores were relatively tolerant to 1.5% NaCl (85% recovery), but showed progressive inhibition by 2% NaCl and above. e.
O
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2
A/gCe Cno0r7Mn'a/o0 n (¼/o) FIG. 5. Effect of increasing concentrations of NaCI in the medium on recovery of unirradiated and irradiated spores of C. botulinum 7272A and 1304E after 6 days of incubation at 30°C.
537
In contrast to unirradiated spores, irradiation to 0.4 Mrad caused the spores to lose practically
all their salt tolerance. Irradiated spores showed immediate decline in counts, with ca. 70% loss of viable counts at 1.5% NaCl. Even lower recovery was obtained in media with 2.5 and 3.0% added NaCl. Thus the radiation-sensitive strain 1304E showed a seeming similarity to the other radiation-sensitive strain, 51B, in that it exhibited (i) an extreme sensitivity to NaCl as well as (ii) as strong sensitization by 0.4 Mrad to NaCl in the recovery medium. Comparison of strains. The NaCl tolerance curves of Fig. 2 suggest that strains 62A and 1304E are populations relatively homogeneous in salt tolerance, i.e., the bulk of the population becomes inhibited across a relatively narrow concentration range, while strains 7272A and 51B are heterogeneous, since the slope of the tolerance curve is relatively shallower. It could then be predicted that strain 1304E spores after irradiation would develop a uniform sensitivity to irradiation. Figure 5 shows this to be the case; in 2% salt, spore recovery is 90% of the unirradiated population and only about 10% of the irradiated population. On the other hand, spores of strain 7272A, which are heterogeneous in NaCl resistance, show about the same spectrum of resistance after irradiation as before, i.e., about 75% of the unirradiated count at every salt level. This may argue that in strain 7272A salt resistance is somehow independent of radiation resistance. Since the experiments with strains 51B, 62A, and 33A are in a different format, a similar analysis cannot be done with these strains. DISCUSSION The observations reported in this paper seem to suggest a possible relation between DNA repair capacity and salt tolerance. Radiationresistant strains, such as 33A, have an efficient system for rejoining of DNA single-strand breaks (SSB), whereas radiation-sensitive strains, such as 51B, have a deficient SSB-rejoining system (7). The rejoining of SSB appears to be accomplished by a single enzyme, the DNA ligase (15), which requires no active metabolism in the cell for its activity. Therefore DNA ligase can function under essentially non-physiological conditions, i.e., at 0°C (1), in cells suspended in distilled water, in the presence of metabolic inhibitors (4), in cells at pH 2, in frozen cells at -16°C (M. Long, personal communication), and in bacterial spores prior to germination (7). An extensive statistical computer analysis of radiation survival curves of 15 strains of C. botulinum has recently brought out a significant relation an
538
KISS ET AL.
between the size of the shoulder in the radiation survival curve and the overall radiation resistance of a spore population of 104 to 106 spores of a particular strain; this is in contrast to the relative unimportance of the slopes, since they were essentially similar in the 14 strains of widely different basic radiation resistance (11). The coincident occurrence of DNA SSB rejoining and NaCl tolerance in the shoulder portion of the survival curve of C. botulinum strain 33A (and possibly strain 62A) suggests that a rejoined (or intact) DNA is essential for recovery in NaCl-containing media. Since in the shoulder portion the DNA SSB are successfully rejoined, it is attractive to speculate that the loss in recovery in the exponentialdecline portion of the radiation survival curve is due to failure of the DNA SSB-rejoining system or to accumulation of some other lesions than simple SSB. Therefore other repair mechanisms must be invoked by the cell for successful recovery, such as DNA excision resynthesis repair and perhaps recombinational repair. These recovery operations are apparently not as efficient as direct SSB rejoining. Furthermore, excision resynthesis would require the functional existence of a metabolic environment in the cell cytoplasm; i.e., spores must be germinated, their cytoplasm must be hydrated, the essential nucleases must be operative, and an essential precursor pool must be available. Since NaCl inhibits the cell in the exponential-decline portion of the survival curve, where metabolic rescue operations seem to be of paramount importance for recovery, it is logical to conclude that NaCl must interfere with some aspect(s) of cell metabolism while the cell is in the process of recovery from initial radiation injury. Once SSB are rejoined, the cell can grow in relatively high NaCl concentrations (>4%); however, in case of defective SSB rejoining, such as in strain 51B, even extremely low NaCl concentrations of the order of normal saline, i.e., 1%, are distinctly inhibitory. In this connection it may be important to reemphasize that characteristically for each individual strain the same radiation dose that prevents DNA SSB rejoining in the shoulder portion of the survival curve also noticeably reduces recovery in NaCl (see Fig. 3). The extreme sensitivity of strain 51B to NaCl even when not irradiated may perhaps be related to the fact that the chromosome of 51B spores is generally extremely fragile, as was observed under conditions of DNA extraction from the spores for sucrose gradient centrifugation (7, 12). Although a rejoined DNA seems to be essential for recovery in NaCl-containing media, it is not clear whether NaCl actually interferes with the process of direct rejoining of DNA SSB them-
APPL. ENVIRON. MICROBIOL.
selves. In the present study, the spores were irradiated in ice water with no NaCl. Since it is known that under these conditions DNA breaks are rejoined relatively rapidly, i.e., within 20 to 40 min (12), sufficient time was available for the spores to complete DNA rejoining during irradiation and subsequent preparation of spore dilutions prior to exposure to recovery media. Previous reports indicate that there is no effect when NaCl is added to the irradiation menstrum but not to the recovery medium (16). From this, one may argue that NaCl in concentrations of 3 to 5% has no effect on DNA SSB rejoining in bacterial spores, but that intact (or rejoined) DNA strands are in some way a prerequisite for recovery of cells in NaCl-containing media. Our conclusion that active metabolism is required for expression of the NaCl block of cell recovery is further supported by the photomicrographic data of Gould (8), who has reported that at 4 to 7% NaCl, germination of Bacillus spores occurs apparently normally, the spore wall is shed, and the developing cells elongate slowly, but cell multiplication is prevented. In the light of current understanding of spore germination and outgrowth (13), the fact that the cells elongate and grow out suggests that RNA transcription and protein synthesis must be operative at the NaCl concentrations that subsequently inhibit colonial development. It is not possible, however, without further experiments, to make a positive statement as to which exact metabolic operation is inhibited by NaCl, although several may be suspect, particularly DNA excision resynthesis, DNA replication, cell division, etc. So far, it is only possible to say that the NaCl blocks some essential metabolic event(s) occurring prior to the first cell division. We see no reasonable evidence at this time to invoke damage to membrane and loss of permeability characteristics in explaining the inhibitory action of NaCl against radiation-damaged cells. In fact, the observation that extremely small NaCl concentrations of the order ofnormal saline, i.e., 1% NaCl, may act in an inhibitory manner in radiation-sensitive strains argues against the idea of membrane permeability being responsible for the NaCl block. Furthermore, the timing of the expression of NaCl block after germination, initiation of RNA and protein synthesis, and essential completion of growth of the first cell seems not to be consistent with the idea of defective cell membranes. ACKNOWLEDGMENTS This work was carried out in part under the United StatesHungary Cooperative Science Program, FHR 03/147, sponsored by the Office of International Relations of the U.S. National Science Foundation and the Hungarian Institute for
VOL. 35, 1978
RADIATION VERSUS SALT SENSITIVITY OF SPORES
Cultural Relations. Financial research support was provided by the U.S. Army Research Office, grant no. DAHCO 4-75-G0112. C.O.R. was supported by International Atomic Energy Agency fellowship SC/202/KOR/7312. LITERATURE CITED 1. Altman, K. I., G. B. Gerber, and S. Okada. 1970. Radiation biochemistry, vol. 1. Academic Press Inc., New York. 2. Anellis, A., and R. B. Koch. 1962. Comparative resistance of strains of Clostridium botulinum to gamma rays. Appl. Microbiol. 10:326-330. 3. Anellis, A., E. Shattuck, D. B. Rowley, E. W. Ross, Jr., D. N. Whaley, and V. R. Dowell, Jr. 1975. Lowtemperature irradiation of beef and methods for evaluation of a radappertization process. Appl. Microbiol. 30:811-320. 4. Driedger, A. A., and M. J. Grayston. 1971. Demonstration of two types of DNA repair in X-irradiated Micrococcus radiodurans. Can. J. Microbiol. 17:495-499. 5. Durban, E., E. Durban, and N. Greez. 1974. Production of spore spheroplasts of Clostridium botulinum and DNA extraction for density gradient centrifugation. Can. J. Microbiol. 20:353-358. 6. Durban, E., and N. Grecz. 1969. Resistance of spores of Clostridium botulinum 33A to combinations of ultraviolet and gamma rays. Appl. Microbiol. 18:44-50. 7. Durban, E., N. Grecz, and J. Farkas. 1974. Direct enzymatic repair of deoxyribonucleic acid single-strand breaks in dormant spores. J. Bacteriol. 118:129-138. 8. Gould, G. W. 1964. Effect of food preservation on the growth of bacteria from spores, p. 17-24. In N. Molin and A. Erichsen (ed.), Microbial inhibitors in food. 4th International Symposium on Food Microbiology: the action, use and natural occurrence of microbial inhibitors in foods, June 1964, Goiteborg, Sweden. Almqvist & Wiksell, Stockholm.
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9. Greez, N. 1965. Biophysical aspects of Clostridia. J. Appl. Bacteriol. 28:17-35. 10. Greez, N., A. Anellis, and M. D. Schneider. 1962. Procedure for cleaning of Clostridium botulinum spores. J. Bacteriol. 84:552-558. 11. Grecz, N., H. Lo, T. W. Kang, and J. Farkas. 1977. Characteristics of radiation survival curves of spores of Clostridium botulinum strains p. 603-630. In A. N. Barker, J. Wolf, D. J. Ellar, G. J. Dring, and G. W. Gould (ed.), Spores 1976, vol. 2. Academic Press Inc., New York. 12. Grecz, N., H. Lo, E. J. Kennedy, and E. Durban. 1973. Gamma radiation studies of Clostridium botulinum types A, B, and E: biological aspects, p. 177-191. In Radiation preservation of food. International Atomic Energy Agency, Vienna. 13. Hansen, J. N., G. Spiegelman, and H. 0. Halvorson. 1970. Bacterial spore outgrowth-its regulation. Science 168:1291-1298. 14. Kang, T. W., and N. Greez. 1975. Chromosome segregation patterns during germination of Clostridium botulinum spores, p. 513-516. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff (ed.), Spores VI. American Society for Microbiology, Washington, D.C. 15. Lehmann, L R. 1974. DNA ligase: structure, mechanism and function. Science 186:790-797. 16. Roberts, T. A., P. J. Ditchett, and M. Ingram. 1965. Effect of sodium chloride on radiation resistance and recovery of irradiated anaerobic spores. J. Appl. Bacteriol. 28:336-348. 17. Roberts, T. A., and M. Ingram. 1965. Radiation resistance of spores of Clostridium species in aqueous suspension. J. Food Sci. 30:879-885. 18. Schmidt, C. F., W. K. Nank, and R. V. Lechnowich. 1962. Radiation sterilization of food. II. Some aspects of the growth, sporulation and radiation resistance of spores of C. botulinum type E. J. Food Sci. 27:77-84.
