(0-D-galactosidase) of Escherichia coli.

Transcription

1 EFFECTS OF X-RAYS ON THE FORMATION OF LACTASE IN A LYSOGENIC STRAIN AND A SENSITIVE STRAIN OF ESCHERICHIA COLI1 CHARLES YANOFSKY Department of Microbiology, Osborn Botanical Laboratory, Yale University, New Haven, Connecticut The process of adaptive enzyme formation has aroused considerable interest through the years. In view of the interesting results of several recent investigations of the effects of radiations on adaptive enzyme formation (Spiegelman et al., 1951; Swenson and Giese, 195; Brandt et al., 1951; Billen and Lichstein, 1952; Swenson, 195) it seemed desirable to investigate the effects of x-rays on the adaptive formation of the enzyme in which we are particularly interested, the lactase (-D-galactosidase) of Escherichia coli. During the course of this study a related problem was encountered, namely the relationship between adaptive enzyme formation and lysogenic phage production, and this also will be considered in this publication. METHODS Received for publication September 8, 1952 The strains of E. coli used in this investigation were K-12, shown to be lysogenic by Lederberg (1951), and W-1485,2 a strain derived from K-12 and sensitive to lambda, a phage liberated by K-12. The medium employed was the standard Gray and Tatum (1944) synthetic minimal medium, except that NH4N3 was omitted and 1.25 g of NH4Cl were added per liter; the carbon source was glucose (.8 per cent). Cells were grown in Fernbach flasks containing 5 ml of media. The flasks were shaken at 37 C for either 16 or 22 hours. The cells were harvested by centrifugation, washed once or twice with cold saline, resuspended in cold.5 M phosphate buffer at ph 7., and centrifuged in an International clinical centrifuge for 3 seconds. This short centrifugation removes a small quantity of easily 1 These investigations were supported in part by the Atomic Energy Commission contract (AT (3-1)-117) and in part by the Rockefeller Foundation. 'Kindly supplied by Dr. J. Lederberg of the University of Wisconsin. sedimentable brown material which is usually present in cultures grown on glucose. Optical density was determined in an Evelyn colorimeter equipped with a 66 filter. Suspensions of cells were irradiated with a Maxitron 25 x-ray machine3 at 25 kv and 3 ma with a 1 mm aluminum filter. The initial density of cells varied slightly in different experiments. Cells were placed in Erlenmeyer flasks (25 or 5 ml) containing buffer (.5 M phosphate buffer at ph 7.,.1 M with respect to Na) or buffer plus lactose (1.2 gm per ml) and shaken in a water bath maintained at 37 C. Aliquots were removed at regular intervals and their lactase activity assayed. When activity of the supernatant was to be determined, each aliquot from the flask was centrifuged for 25 minutes at 2, g to remove the majority of the cells. Lactase activity was determined by a modification of the method of Lederberg (195). Portions (2 ml) of a solution of 3 X 1- M o-nitrophenylp-d-galactoside in sodium phosphate buffer were added to tubes containing 1 ml of the test solution in the same buffer. The tubes then were incubated for 15 or 3 minutes at 37 C. Two ml of 1 M K2CO3 were added to stop the reaction and develop the color of the liberated o-nitrophenol. After 5 minutes the tubes were read in a Klett- Summerson colorimeter using a 42 filter. Cell turbidity blanks and a substrate blank were run with each assay. All results are expressed in terms of the MM of o-nitrophenyl-f-d-galactoside hydrolyzed per ml of sample during a 3 minute incubation period at 37 C. In some cases the activity is expressed in terms ofthe protein content of the sample assayed. Cellular extracts were prepared by shaking aliquots from the flasks with glass beads at 2 to 4 C as described by Lester and Bonner (1952) and removing the cellular debris by centrifugation. 'The author is indebted to Dr. N. H. Giles of Yale University for the use of the x-ray machine. 383

2 384 CHARLES YANOFSKY [VOL. 65 This procedure liberates more th._9 per cent of the original lactase activity. The protein content of extracts was determined by the method of Lowry et al. (1951). The agar layer technique (Adams, 195) was used for lambda assays with the modifications suggested by Weigle and Delbriick (1951). The number of K-12 cells induced to phage production by x-ray treatment was determined by plating aliquots of washed cells. For the assay of free lambda, aliquots of suspensions containing cells plus liberated phage were centrifuged at low speed to remove most of the cells without removing lambda. The supernatants were plated then with the sensitive strain. Viability counts are cells plus suspending solution) taken directly from culture flasks. Results obtained by this method (see table 2) appeared to disagree with the results of the extract analyses in that K-12 samples showed greater lactase activity as the x-ray dose was increased. However, determinations of lactase activity of K-12 supernatants provided the explanation for the apparent discrepancy. Extracts of lactose grown cells of strain K-12, as reported by Lederberg (195), have approximately twenty times the lactase activity (with o-nitrophenyl-,8-d-galactoside as substrate) as the cells themselves. Thus, if a portion of the K-12 cells which formed lactase lysed as a result of x-ray treatment, lactase would be released into TABLE 1 Effect of x-ray8 on viability and the formation of lactase by Escherichia coli FLASK NO. STRAIN X-RAY DOSE PER CENT SURVIVAL LACTOSE, pm/ml SM ONPO HYDROLYZED*t ROENTGENS MG EXTRACT PROTEIN 1 K (.4) 2 K (17) 3 K-12 5, (24) 4 K-12 1, (23) 5 K-12 2, (18) 6 W (.4) 7 W (2) 8 W , (21) 9 W , (16) 1 W , (16) * Data are for cells from a 16 hour old culture except the values in parentheses which are for cells from a 22 hour old culture. All flasks incubated for 3 hours at 37 C. t ONPG-o-nitrophenyl-,5-D-galactoside. based on the number of colonies appearing on nutrient agar plates after 48 hours at 37 C. RESUTS The effects of x-rays on lactase formation in glucose grown cells of strains K-12 and W-1485 were determined initially by measuring the specific activity of cellular extracts. As can be seen in table 1 x-ray doses which had a marked effect on the viability of each strain had only a slight effect on their ability to form lactase adaptively. Doses of 2, r or greater generally caused a decrease in lactase. This decrease can be accounted for, in part, by reasons which will become clear later. Lactase formation was followed also by assaying the lactase activity of aliquots (containing the suspending solution where it would show much greater activity. That the release of lactase into the suspending solution is the explanation for the increased lactase activity of samples containing irradiated K-12 cells can be seen in the last colurmn in table 2. In view of the fact that one major difference between strain K-12 and strain W-1485 is that strain K-12 is known to be lysogenic (Lederberg, 1951) and since Lwoff et al. (195) have reported that x-rays induce phage production in lysogenic cultures, it was assumed tentatively that phage production with subsequent lysis was responsible for the appearance of lactase in K-12 supernatants. This assumption implies that phage production and lactase formation occur in the same cells. To test this hypothesis experiments were performed in

3 1953] EFFECTS OF X-RAYS ON FORMATION OF LACTASE which samples were removed from cultures at various intervals and assayed for extracellular lactase activity and free phage. The results of one such experiment are illustrated in figure 1. It can be seen that the phage titer increased sharply during incubation and that correlated with the increase in free phage titer was the appearance of lactase in the suspending solution. Results similar to these have been obtained with ultraviolet light instead of x-rays. Figure 1 also shows that there was considerable lysis of K-12 cells exposed to a dose of 2, r. A portion of the lysed cells was responsible for the appearance of phage and lactase in supernatants; however, there was usually appreciable lysis before the marked increase in phage titer and before lactase appeared in the suspending medium. The reduction by early lysis of the number of cells which will form more lactase accounts, at least in part, for the lower actual lactase TABLE 2 The lactase activity of suspen8sion8 and supernatante of Escherichia coli, strains K-1S and W-1485 X-RAY DOSE OPTICAL pm ONPGt HYDROLYZED pm ONPGt HYDROLYZED FLASK NO. STRAIN 3 ROENTGENS LACTOSE, AM/ML DENSITY X S CIHANGE* MLSXIL SUPERNATANT 1 W W W , W , W , K K K-12 5, K-12 1, K-12 2, * Initial optical density of cellular suspension in each flask with strain K-12 was.465 and in each flask with strain W-1485,.41. Three hour incubation at 37 C. t ONPG-o-nitrophenyl-,j-D-galactoside. w Q o(5 L z C c-o.8.4 7u@ minutes Figure 1. Phage (-O) and lactase (@-@ appearance in the suspending medium of K-12 cells exposed to an x-ray dose of 2, r and then incubated with lactose. Optical density changes during the incubation period are shown in the upper left hand corner. 385 levels observed with K-12 cells exposed to a dose E- of 2, r (see table 1). DISCUSSION Results show that the viability of cells is much more susceptible to x-rays than is the ability to form lactase adaptively. This suggests that killing with x-rays involves some cellular constituent which is not a limiting factor in the formation of adaptive enzymes. These observations might be taken to indicate that the formation of adaptive enzymes can proceed in cells which cannot grow and divide. However, the only test of viability employed in this investigation was based on the ability of plated cells to form visible colonies on nutrient agar plates after incubation for 48 hours. This method does not distinguish between cells which cannot divide and cells which can only undergo a few divisions. It was found also that during the incubation of irradiated K-12 cells with lactose, lactase was

4 386 CHARLES YANOFSKY [VOL. 65 released into the suspending solution. In addition to the appearance of lactase in the suspending solution, phage also appeared. These observations indicate that lactase was formed in cells which formed phage and was released from the cells during lysis. This conclusion leads to the question-are phage and lactase being formed simultaneously in the same cells? If it is assumed that phage formation starts from the moment of induction or soon thereafter, then it follows that lactase and phage are being formed in cells at the same time. Borek (1952) has shown, however, that the formation of phage in strain K-12 is delayed or prevented when specifically required substances are limiting. Thus, it is conceivable that the production of phage does not start in glucose grown cells until the cells contain enough lactase to utilize the only available carbon source, lactose, at a substantial rate. This possibility must be considered although there does not seem to be any reason to assume that lactose utilization limits phage production more than it does lactase formation. In a recent study, Jacob (1951) has found that adaptation can still proceed in lysogenic bacteria induced to phage production. Monod and Wollman (1947) had demonstrated previously that adaptive enzyme formation was inhibited completely by a phage infection. There are good reasons to suspect that production of lysogenic phage might be a somewhat different matter than production of nonlysogenic phage, particularly in view of the genetic investigations of Lederberg and Lederberg (1953) which have shown that lambda segregation in strain K-12 is under strict nuclear control. It is conceivable that lambda formation does not compete with adaptive enzyme formation because normal bacterial genetic material regulates both processes. Infection with a nonlysogenic phage, on the other hand, introduces a foreign competing genetic system which apparently prevents the normal cellular processes of the host bacterium. It is possible also that whether or not adaptation and phage production can proceed in the same cell at the same time depends upon the length of the latent period of phage development. ACKNOWLEDGMENT The author is indebted to Mrs. M. Bonner and Mrs. C. Dillingham for their technical assistance. SUI(ARY The effects of x-rays on the adaptive formation of lactase in two strains of Escherichia coli were investigated. It was found that x-ray doses which have a marked effect on the viability of the two strains have only a slight effect on their ability to form lactase adaptively. One of the strains studied, K-12, releases lactase into the suspending medium when it is incubated with lactose following irradiation. The release of lactase into the suspending medium was shown to be due to the lysis of cells in which the production of phage was induced by irradiation. The appearance of appreciable lactase activity in the suspending medium of irradiated K-12 cells incubated with lactose indicates that lactase was formed in the cells which lysed and liberated phage. All the evidence obtained is consistent with the view that the formation of lactase and lysogenic phage production can occur simultaneously in the same cells. REFERENCES ADAMS, M. H. 195 Methods of study of bacterial viruses. In Methods in medical re- 8earch. Vol. 2, The Year Book Publishers, Chicago. BILLEN, D., AND LICHSTEIN, H. C The effect of x radiation on the adaptive formation of formic hydrogenlyase in Escherichia coli. J. Bact., 63, BOREK, E Factors controlling aptitude and phage development in a lysogenic Escherichia coli K-12. Biochim. et Biophys. Acta, 8, BRANDT, C. L., FREEMAN, P. J., AND SWENSON, P. A The effect of radiations on galactozymase formation in yeast. Science, 113, GRAY, C. H., AND TATUM, E. L X-ray induced growth factor requirements in bacteria. Proc. Natl. Acad. Sci., 3, JACOB, F Adaptation enzymatique pendant le d6veloppement du bacteriophage chez Pseudomonas pyocyanea. Compt. rend., 232, LEDERBERG, ESTHER M Lysogenicity in E. coli K-12. Genetics, 36, 56. LEDERBERG, ESTHER M., AND LEDERBERG, J Genetic studies of lysogenicity in Escherichia coli. In press. LEDERBERG, J. 195 The,-D-galactosidase of Escherichia coli, strain K-12. J. Bact., 6, LESTER, G., AND BONNER, D. M The oc-

5 1953] EFFECTS OF X-RAYS ON FORMATION OF LACTASE 387 currence of -galactosidase in Escherichia coli. J. Bact., 63, LOWRY,. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, LWOFF, A., SIMINOVITCH, L., AND KJELDGAARD, N. 195 Induction de production de phage dans une bact6rie lysog6ne. Ann. inst. Pasteur, 79, MONOD, J., AND WOLLMAN, E L'inhibition de la croissance et de l'adaptation enzymatique chez les bact6ries infect6es par le bact6riophage. Ann. inst. Pasteur, 73, SPIEGELMAN, S., BARON, L. S., AND QUASTLER, H Enzymatic adaptation in non-viable cells. Federation Proc., 1, SWENSON, P. A. 195 The action spectrum of the inhibition of galactozymase production by ultraviolet light. Proc. Natl. Acad. Sci., 36, SWENSON, P. A., AND GIESE, A. C. 195 Photoreactivation of galactozymase formation in yeast. J. Cellular Comp. Physiol., 36, WEIGLE, J. J., AND DELBRIjCK, M Mutual exclusion between an infecting phage and a carried phage. J. Bact., 62, Downloaded from on December 12, 218 by guest