THE CHEMICAL REVERSAL OF ULTRAVIOLET EFFECTS ON BACTERIA

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1 THE CHEMICAL REVERSAL OF ULTRAVIOLET EFFECTS ON BACTERIA SOLON A. ELLISON, BERNARD F. ERLANGER, AND PETER ALLEN Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York Received for publication October 2, 1954 It is now well established that the effects of ultraviolet radiations on a variety of cells and cell process can be reversed in part by exposure to strong illumination in the visble or near ultraviolet range (Kelner, 1949; Errara, 1953). Both lethal and mutagenic effects on bacteria are photoreversible although to different degrees. When high ultraviolet dosages are employed, induced mutations are photostable although reactivation still occurs in the expected manner (Newcombe and Whitehead, 1951). Heinmets and his co-workers have shown that the inactivation of E. coli strain B/r by ultraviolet radiation (Heinmets, 1953; Heinmets et al., 1954b) and by other physical or chemical agents (Heinmets et al., 1954a) could be reversed to a significant degree by certain metabolites related to the citric acid cycle. These authors found it necessary to suspend the irradiated culture in buffer containing the metabolite prior to plating; they were unable to obtain reactivation by plating directly in a defined medium supplemented by the chemicals employed. The results reported below demonstrate that both the lethal and the mutagenic effects of ultraviolet radiation can be reversed by plating in nutrient agar containing acetate. METHODS The bacteria employed were Corynebacteium bovis,1 Escherichia coli strain B, and an ultraviolet resistant mutant (E. coli strain B/15-17) derived from the latter by a procedure similar to that by which Witkin (1947) selected E. coli strain B/r. All three are photoreactivable. E. coli strain B/15-17 is similar to E. coli strain B/r in its radiation resistance. The two E. coli strains were grown in M-9 medium (Anderson, 1946) with aeration, usually for 18 hours, but occasionally for other time periods when necessary for specific experiments. 1 Obtained from Dr. E. L. Stokstad, Lederle Laboratories. C. bovis was grown in nutrient broth. Before irradiation the organisms were thoroughly washed and resuspended, at an approximate concentration of 6 X 18 per ml, in M-9 medium without glucose. Four ml aliquots were measured into petri dishes and agitated by hand 25 cm from a "mineralite V-41" lamp equipped with a filter peaked at 254 A. Assays were made in duplicate or triplicate by plating appropriate dilutions as pour plates in 1 per cent nutrient agar supplemented by various amounts of sodium acetate. Colony counts were made after hours of incubation at 37 C. The procedure for determining the effect on mutagenesis was patterned after that of Newcombe and Whitehead (1951). The basal layers employed in the petri dishes were either 1 per cent nutrient agar or the same medium supplemented by 25 mg per ml of sodium acetate. Counts were made after 48 hours. The mutation studied was that to streptomycin resistance. A GE AH-5 lamp was employed for photoreactivation. All operations were carried out in a darkened room with illumination provided by two 25 watt photographic safety lamps. RESULTS Figure 1 represents graphically the survival curve of E. coli strain B, E. coli strain B/15-17, and C. bovis. The decreased susceptibility of E. coli strain B/15-17 as compared to E. coli strain B is readily apparent. The sensitivity of C. bovis is similar to that of E. coli strain B/ The sensitivity to ultraviolet radiation (Demerec and Latarjet, 1946) and the capacity to be reactivated by acetate were somewhat dependent on the growth phase of the culture as shown in table 1. Organisms in the stationary phase were employed in all experiments for easier comparison with the results of other investigators. Figure 2 illustrates the relation between acetate concentration and bacterial survival. 536

2 19]5 CHEMICAL REVERSAL OF ULTRAVIOLET EFFECTS ON BACTERIA 537 ( Co,~~ ~ ~~~~ Cl V/S\ It mg/mi ' \~~~~~\~ \~~~N.1. COL.. V \ 2 {-- lt Figure 1. Survival curves of Escherichia coli strain B, E. coli strain B/15-17, and Corynebacterium bovis. The survival curves of E. coli strain B in nutrient agar containing sodium acetate indicate that as the acetate concentration increased, longer exposures were required for a similar killing effect. In figure 3, additional data are plotted to show the percentage of survivors as a function of the acetate concentration. Each line represents a single ultraviolet dose. No effect was observed on the unirradiated controls. It may be seen that the number of survivors was proportional to the acetate concentration for all ultraviolet TABLE 1 Relation of culture phase to ultraviolet inactivation and acetate reactivation of Bscherichia coli strain B Phase % Survivors after 15 sec UV Sodium Acetate Concentration (mg per ml) Logarithmic Stationary Figure S. The effect of acetate on the survival curve of irradiated Escherichia coli strain B. sn 11 a: vw o.. --o' I* -11C OP.4 4 oe SODIUM ACETATE MG/ML Figure S. The relation between acetate concentration and survival of irradiated Escherichia coli strain B. 3

3 538 S. A. ELLISON, B. F. ERL4NGER, AND P. ALLEN [VOL. 69 exposures tted and that the maximal effect was produced by 25 to 3 mg per ml of sodium acetate. However, it may be noted that the growth rate was somewhat slower as the acetate concentration was increased. Indeed, at concentrations greater than 3 mg per ml, growth was quite slow, and the colonies were considerably smaller than the controls, thus rendering their enumeration difficult. The apparent maximum may therefore merely reflect this inhibition. No significant alteration of the ph of the medium resulted from the addition of sodium acetate in the concentrations employed. When experiments similar to the foregoing were done using E. coli strain B/15-17, no reactivation was noted. It was considered that the radiation resistance of E. coli strain B/15-17 might result from increased local production of acetate as compared with the wild-type organism. I w = SODIUM ACETATE TABLE 2 The reversal of induced mutation of Escherichia coli strain B by acetate Survivors ~~~Sodium acetate UV No acetate Dose Noacetate (25 mg per ml) Sr or Sr or Sr or Sr or Total/mi Sd*/ Sd/1 TotaVml Sd/ Sd/1s ml viable ml viable SJC O 7.6X1' X X X X X OX ,6 1. 1X OX , 1. 1 X ,3 * Sr = Streptomycin resistant; Sd = streptomycin dependent. If this were true, the lack of effect of additional " u-v acetate would be readily understandable. There- - * fore determinations were made of volatile acid production (Niederl and Niederl, 1942) by E. coli so"u-v strain B and E. coli strain B/15-17 in aerated and nonaerated cultures in M-9 medium. No 5 uv- * difference between the two strains was detected. Since the ultraviolet sensitivity of C. bovis was similar to that of E. coli strain B/15-17, the effect of acetate on it was studied. Typical results are Shown in figure 4. Although growth of this species was slightly inhibited at arl acetate to uit concentrations tested, it is readily apparent that 2 reactivation occurred at both ultraviolet dosages,f,.employed., "-V u The effect of acetate on induced mutation of E. coli strain B was investigated since visible light revers the mutagenic effect of ultraviolet light on bacteria. Plating in acetate agar did not affect the rates of either spontaneous mutation or of induced mutations following low ultraviolet doses of less than 2 seconds (Elison et al., 1954). In other experiments, as described in table 2 and figure 5, definite evidence of the reversal of mutagenesis was obtained. All colonies which appeared on agar containing 'o5 streptomycin were presumed to be mutants. It MG/ML was not considered necessary, and no attempt was Figure 4. The reactivation of Corynebacteniumn made, to distinguish between streptomycin bovis by acetate. The broken linles represent un- resistant and streptomycin dependent bacteria. corrected counts. The solid lines represent counts Not only was the proportion of mutants to the corrected for the inhibitory effe4 et of acetate on total population decreased, but the absolute the unirradiated control. number of mutants present was considerably less

4 19551 CHEMICAL REVERSAL OF ULTRAVIOLET EFFECTS ON BACTERIA 539 ". -i ~- a Figure 5. The effect of acetate on ultraviolet induced mutation of Escherichia coli strain B. The broken lines represent bacterial survival. The solid lines represent the number of mutants. Sr = streptomycin resistant; Sd = streptomycin dependent. in the cultures grown on acetate agar after exposure to the higher ultraviolet doses. The possibilities must be considered either that the effect observed was the result of an increased sensitivity of the mutants to inhibition by acetate, or that the phenotypic lag was increased by acetate. No data referring to the first point are presently available. Growth in acetate agar, although less rapid than the control, was fast enough so that the six hours allowed for phenotypic expression were probably adequate. DISCUSSION The decision to study the effect of acetate on irradiated bacteria was based on the following reasoning. Photoreactivation seemed analogous to photosynthesis in the initial step utilizing radiant energy. Calvin had proposed that thioctic acid (6, 8-dithiooctanoic acid) might be the photodynamic substance concerned in photosynthesis (Calvin and imassini, 1952). Thioctic acid was known to be widely distributed in bacteria and, indeed, to be an essential growth factor for some species. In some instances, acetate in high concentrations could be substituted for it (Stokstad et al., 195). Since thioctic acid was not available to us when these experiments were begun, acetate was employed in its stead. However, when a sample of thioctic acid was recently tested (obtained from Dr. E. L. Patterson, Lederle Laboratories), no clearly defined evidence of activity in photoreactivation was obtained. Further investigation of this point is planned. There do not appear to be any irreconcilable contradictions between our results and those of Heinmets et al. (1954b). Their methods consisted in suspending irradiated bacteria in a solution of the metabolite to be tested, and counting viable organisms after various intervals. Although no reactivation was obtained on plates, this may have been due to the use of E. coli strain B/r as the test organism, as well as the concentrations of metabolites employed, since these were substantially lower than those which we have found effective. One obvious point of interest is the relation, if any, of the effects described here to those produced by visible light. Attempts have been made to ascertain whether light and acetate act through related mechanisms by determining whether the effects of combined treatment of E. coli strain B were additive or synergistic. Variation in the test organism made it difficult to interpret the results, but, under some circumstances at least, the two appeared synergistic (Ellison, unpublished observations). These experiments are being continued. The failure to reactivate E. coli strain B/15-17 by acetate, despite its ability to be photoreactivated, indicates that the two phenomena are dissociable and perhaps denotes a fundamental dissimilarity in their mechanisms. In addition, the findings tend to confirm the conclusion of Roberts and Aldous (1949) that qualitatively different mechanisms are involved in the killing of E. coli strain B and E. coli strain B/r by ultraviolet radiation. SUMMARY The lethal and mutagenic effects of ultraviolet radiation on Escherichia coli strain B were reversed by plating the irradiated bacteria in

5 54 S. A. ELLISON, B. F. ERLANGER, AND P. ALLEN [VOL. 69 nutrient agar to which sodium acetate had been added. The degree of reactivation was directly proportional to the acetate concentration and appeared to be maximal at about 25 mg per ml of sodium acetate. Reactivation of irradiated Corynebacterium bovis was obtained by the same procedure. REFERENCES ANDERSON, E. H Growth requirements of virus-resistant mutants of Eschewrichia coli strain "B". Proe. Natl. Acad. Sci. U. S., 32, CALVIN, M., AND MASSINI, P The path of carbon in photosynthesis. Experientia, 8, DEMEREC, M., AND LATARJET, R 'Mutations in bacteria induced by radiations. Cold Spring Harbor Symposia Quant. Biol., 11, ELLISON, S. A., ERLANGER, B. F., AND ALLEN, P. Z Chemical reactivation of ultraviolet irradiated bacteria. Federation Proc., 13, 491. ERRARA, M Mechanisms of biological action of ultraviolet and visible radiations. Progr. Biophys. and Biophys. Chem., 3, HEINMETS, F Reactivation of ultraviolet inactivated Escherichia coli by pyruvate. J. Bacteriol., 66, HEINMETS, F., TAYLOR, W. W., AND LEHMAN, J. J. 1954a The use of metabolites in the restoration of the viability of heat and chemically inactivated Escherichia coli. J. Bacteriol., 67, HEINMETS, F., LEHMAN, J. J., TAYLOR, W. W., AND KATHAN, R. H. 1954b The study of factors which influence metabolic reactivation of the ultraviolet inactivated Escherichia coli. J. Bacteriol., 67, KELNER, A Photoreactivation of ultraviolet irradiated Escherichia coli, with special reference to the dose-reduction principle and to ultraviolet induced mutants. J. Bacteriol., 58, NEWCOMBE, H. B., AND WHITEHEAD, H. A Photoreversal of ultraviolet-induced mutagenie and lethal effects in Escherichia coli. J. Bacteriol., 61, NIEDERL, J. B., AND NIEDERL, V Micromethods of quantitative organic analysis. 2nd ed. John Wiley & Sons, New York. ROBERTS, R. B., AND ALDOUS, E Recovery from ultraviolet irradiation in Escherichia coli. J. Bacteriol., 57, STOKSTAD, E. L. R., HOFFMAN, C. E., AND BELT, M. 195 Role of protogen in the nutrition of an unidentified Corynebacterium. Proc. Soc. Exptl. Biol. NMed., 74, WITKIN, E. M Genetics of resistance to radiation in E. coli. Genetics, 32, Downloaded from on July 24, 218 by guest