Bethe8da, Maryland Received for publication September 23, 1948

Transcription

1 STUDIES OF A PURINE-REQUIRING MUTANT STRAIN OF ESCHERICHIA COLI ROBERT GUTHRIE Experimental Biology and Medicine Institute, National Institutes of Health, Bethe8da, Maryland Received for publication September 23, 1948 The production and isolation of mutant strains of Escherichia coli requiring for growth an exogenous source of an amino acid, B vitamin, purine, or pyrimidine have been reported by several investigators. The results have been summarized in a recent review (Tatum, 1946). Such strains have been used recently for the assay of amino acids (Lampen and Jones, 1947; Lampen, Jones, and Perkins, 1947). The present paper deals with a purine-requiring strain of E. coli, , which was isolated following treatment with ultraviolet light. Under the conditions described herein, growth of this organism occurred only when any one of the purine bases, adenine, guanine, xanthine, or hypoxanthine, was present. Within limits, the amount of growth was related to the amount of purine added. Various purine bases have been described as either stimulatory substances or as essential growth substances for a number of bacteria (Peterson and Peterson, 1945). Pappenheimer and Hottle (1940) have described a strain of group A hemolytic streptococcus with a requirement for purines that is qualitatively similar to that of E. coli Tatum (1946) has produced a mutant strain of E. coli with nitrogen mustard that requires purine. Any one of the four purine bases listed above is active. Mitchell and Houlahan (1946) have employed a mutant of the mold, Neurospora, for the bioassay of adenine plus hypoxanthine in tissue extracts. It is not intended in this report to propose the use of E. coli in the routine assay of purine bases. However, it is felt that the nature of the purine requirement of this organism appeared to be such as to warrant further investigation of its use for assay of natural materials for purine content. The stability of the requirement was such that little difficulty was encountered in maintaining this mutant strain in the laboratory. Studies of the conditions under which reversion to a purine-independent type occurs, are described. METHODS Isolation of organism. An arginine-requiring strain of E. coli ATC9661, isolated by Roepke, Libby, and Small (1944), was used for the production of nutritionally deficient daughter strains. For the isolation of nutritional mutants from a suspension that had been irradiated with an ultraviolet germicidal lamp, the "layer-plate" technique of Lederberg and Tatum (1946) was employed. E. coli , isolated in this manner, was found by a modification of the "auxanographic" plate method 39

2 40 ROBERT GUTHRIE [VOL. 57 (Lederberg, 1946) to require purine for growth in the "minimal" glucose salts medium (medium A, table 1). Adenine, guanine, xanthine, or hypoxanthine. each permitted growth when added separately to this medium. At a later date it was discovered that this strain did not require arginine. The reason for the loss of the arginine requirement in is not known. However, a culture of the parent strain, 9661, lost its requirement for arginine on two different occasions during transfer on meat infusion agar slants in this laboratory. When comparison with the wild-type E. coli was desired, strain ATC9723 was used, since it is this strain from which strain 9661 was produced. CONSTITUENT TABLE 1 Composition of basal media A* and B AMOUNT PER 500 ML OF DOUBLE- STRENGTH MEDIUM CONSTITUENT AMOUNT PER 5W ML OF DOUBLE- STRENGTH MEDIU NHClt g Uracil I mg NH4NO g Thymine mg Na2SO g Thiamine** mg MgSO4-7H2O g Riboflavin** mg K2HPO g Nicotinic acid** mg KH2PO g Calcium pantothenate** mg Glucose (anhydrous, Merck) 5.0 g Biotin** mg Trace element... solutiont 1.0 ml Pteroylglutamic acid** mg Acid-hydrolized vitamin-free p-aminobenzoic acid** 0.01 mg casein, 10% ml Pyridoxine** mg L-Cystinell mg Inositol** mg DL-Tryptophanll mg ph adjusted to * The first 8 ingredients constitute medium A. t All salts were of cp reagent quality. $ Horowitz, N. H., and Beadle, G. W., 1943, J. Biol. Chem., 150, 325. Snell, E. E., 1947, Biol. Symposia, 12, 189. Solutions of 10 mg per ml were used. Solutions of 1 mg per ml were used. * A single solution containing all of the vitamins was prepared in such a manner that 10 ml is equivalent to the amounts in the table. This was kept at 5 C and renewed monthly. No differences were found between strains 9723, 9661, and when tested by the usual bacteriological criteria for E. coli-communior, including tests for ability to ferment 14 carbohydrates. Stock cultures and inocula. The stock cultures were maintained on horse meat infusion agar slants. For each experiment, a stock culture kept in the cold room was transferred twice on agar slants. After an incubation period of 16 to 20 hours, cells from the second slant culture were washed once in saline and resuspended in 10 ml. Under these condtions, the turbidity of the inoculum usually gave a reading of the order of on the galvanometer scale of the colorimeter (Evelyn, 660 filter). One drop of the inoculum was pipetted into each tube. Media. Two basal media were used for the experiments. Medium A is a simple glucose salts mixture (Gray and Tatum, 1944) but medium B is a more complex purine-free medium (table 1).

3 1949] MUTANT STRAIN OF ESCHERICHIA COLI 41 Specificity of the Purine Requirement Several substances (other than the four purine bases) were tested separately in tubes of medium A and medium B for their effect upon the requirement of strain for purine. Uric acid, theobromine, theophylline, barbituric acid, caffeine, 5(4)-amino-4(5)-imidazolecarboximide,I cytosine, xanthopterin, histidine, arginine, alloxan, hydantoin, allantoin, creatinine, desoxyribose nucleic acid,2 and ribonucleic acid were unable to replace guanine, nor did they antagonize its utilization when tested separately in medium B containing 2.5 X 10-1 M guanine. Ribonucleic acid gave slight growth on occasion, which might have been due to the presence of traces of free purine. Acid hydrolyzates of the nucleic acids permitted good growth. The nucleic acids were tested in amounts of 2 mg per 10 ml; in the case of each of the other substances, 0.1 mg was used..% - iaj Growth Response to the Purine Bases Assay procedure. The usual microbiological assay techniques were employed. The double strength basal media were dispensed in 5-ml amounts in colorimeter tubes. Test solutions and water were added to make a final volume of 10 ml. All tubes were prepared in duplicate. The colorimeter tubes had previously been selected to agree within 2 units on the galvanometer scale. In all experiments the tubes were sterilized by autoclaving 15 minutes at 15 pounds pressure and, after inoculation, were incubated at 37 C. The results were usually recorded at several intervals from 12 to 24 hours, but the results after 16 hours' incubation were considered most reliable for medium B. For experiments with medium A an incubation period of at least 24 hours was usually required to ensure maximum response. Growth curnes. Figure 1 shows for strain the curves of growth response to each of the four purine bases, and to an equimolar mixture of all four substances, when these were added to medium B in the various amounts indicated in the figure. These growth curves are of the type exhibited by many assay organisms for required substances. Stability of the Purine Requiremnet Reversion in assay tubes. If the assay tubes containing medium B were allowed to undergo incubation beyond the period for assay, heavy growth frequently appeared in tubes that contained limiting concentrations of purine. Such behavior in other mutant strains of E. coli has been described as "back-mutation" (Ryan, 1946). It is assumed that this behavior is due to the occurrence in the population of mutant cells that have reverted to the wild type, in the sense that they appear to have regained the ability to synthesize purine. This belief is supported by experiments in which colonies appearing on plates of purine-free medium seeded with strain (table 5) were picked and shown to consist of cells 1 Kindly supplied by Dr. William Shive. Postulated by him as a possible precursor for biological synthesis of purines (Shive et al., 1947). ' Kindly supplied by Dr. J. P. Greenstein.

4 42 ROBERT GUTHRIE [VOL. 57 which grew on purine-free medium A in the same manner as strain 9723, the wild-type E. coli. Six such reverted colonies were picked and tested in the usual bacteriological media. All gave reactions typical for E. coli-communior. 40 (> 50 z z 60 GUANINE NTHINE IL ADENIN 2 70 MIXTURE 0 Z HPXNTHINE NOTE: INCUBATION4 so 8 TIME, 1S HR TEM1P,v PURINE, I0d5M Figure 1. Growth response of Escherichia coli to purines in medium B. GUANINE, 10- TABLE 2 Occurrence of reversion in ascay tubes containing medium B GALVANOMIETER READIhG AFTER HOURS OF INCUBATION INDICATED , 99 94, 98 55, 98 40, 98 41, , 95 93, 96 92, 95 92, 95 85, , 93 92, 93 92, 93 92, 93 82, , 90 87, 88 74, 78 57, 58 68, , 84 82, 82 80, 81 66, 78 62, , 82 75, 80 73, 79 72, 77 75, , 77 76, 77 71, 71 70, 72 73, , 68 72, 74 64, 65 64, 65 68, , 60 57, 57 58, 58 57, 58 61, 62 A value of 100 denotes complete absence of turbidity. Figures in italics denote occurrence of reversion. The frequency of reversion does not appear to be sufficiently great to affect the reliability of the assay results. This is indicated in table 2, which gives the data from a typical experiment in which galvanometer readings of tubes were recorded at frequent intervals during incubation. The italicized values denote C

5 1949] MUTANT STRAIN OF ESCHERICHIA COLI 43 reversion. It is apparent that the results of an assay may be recorded after 16 hours of incubation without interference due to reversion of the assay organism. Similar results were obtained repeatedly with each of the four purine bases, as well as for a mixture of them, when used to supplement medium B. Reversion was found to be extremely rare in medium A, as compared with medium B. This difference in the two media is shown in table 3. TABLE 3 Difference in frequency of reversion in tubes containing medium A and medium B* TUBES SHOWING TOTA TUBES PER CENT REVERSION TOA UBS RVERSION Medium A Medium B * Table includes data for all concentrations of purine at which reversion occurred (0-4 X 10-6 m), and for each of the four purine bases. TABLE 4 Turbidity produced during serial transfer in medium A containing a suboptimal concentration of guanine (0.9 X 10-'M) TRANSFER NO. GALVANOMETER READING AFTER 48 HOURS STRAIN STRAIN 9723 it * The tubes were kept at room temperature for a period of about 1 month after the 2- day incubation period. Increased growth was never observed in any of the tubes. t A large loopful from a fresh agar slant culture was used as an inoculum; therefore the turbidity was not recorded. For the succeeding transfers 1.0 ml of the preceding culture was used. Transfers were made at approximately 48-hour intervals. When strain was subjected to serial transfer of a heavy inoculum in suboptimal concentration of a required metabolite, heavy growth occurred upon the second transfer in medium B. In this process, 1.0 ml of a 10-ml liquid culture containing 20 jig of guanine (0.9 X 10-6 M) was transferred to a second tube of the same medium. A parallel series was conducted with strain However, the same experiment with medium A gave completely negative results even after the eighth transfer (table 4). These results agree with the low frequency of reversion encountered in tube assays with medium A. The very low incidence of reversion to purine synthesis in tubes of medium A with limiting concentrations of purine, as compared to frequent reversion in

6 44 ROBERT GUTHRIE [VOL. 57 medi-um B under the same conditions, may be explained either on the basis of different selection pressures in the two media or to an actual difference in "reverse mutation" rates. The selective effects and the reverse mutation rates in the two media are being studied and will be the subject of a subsequent report. An attempt will also be made to elucidate the component, or components, of medium B that are responsible for the higher reversion frequency in this medium. Frequency of reverted cells. Several attempts were made to estimate the frequency of reverted cells in the stock meat infusion medium inoculated with strain by using a plate technique similar to that described elsewhere (Lewis, 1934; Lederberg and Tatum, 1946; Ryan, 1946). A large number of washed cells were diluted by multiples of ten and seeded into a purine-free agar medium and also into a meat infusion agar medium. From a comparison of the number of colonies that appeared in the plates of the complete medium with the number in the purine-free medium, the incidence of purine-synthesizing cells was calculated (table 5) as approximately TABLE 5 Estimation of tih frequency of reverted cells in strain cultures on meat infusion agar slant8 TOTAL NUMBER OF NUMBER 01 PROPORTION 0F HISTORY 01 CULTURE TESTED CELLS TESTED* REVERTED CELLSt REVERTED CELLS Stock culture from cold room X X i0-12th daily serial transfer on stock agar. slants X X 10-8 Isolated by layer-plate "purification" (see text) X X 108 Plate counts of cell suspensions made with meat infusion medium in plates. t Plate counts of same suspensions made with purine-free medium A in plates. As may be seen in table 5, the frequency of reverted cells appeared to be of the same order of magnitude in cells derived from either a stock culture kept in the cold room, a fresh culture with a history of 12 consecutive daily transfers on the agar slant medium, or from a "purified" culture that was freshly isolated from a Lederberg-Tatum layer plate (Lederberg and Tatum, 1946), wherein the colony from which the culture was isolated had appeared only after the addition of punine to the plate. Reversion in stock cultures. Two instances of stock agar slant cultures in which the proportion of purine-independent cells seemed to be greater than is usually the case have been encountered. This was shown in both cases by a decreased incubation time, after inoculation into medium B, before the appearance of heavy growth in tubes that contained a low concentration of purine. Such a stock culture may be easily subjected to "purification" by the layer-plate technique (Lederberg and Tatum, 1946) with the object of obtaining strains in which the incidence of pune-synthesizing cells may be reduced to the usual level (10s). This technique might be employed with any strain of bacteria used in micro.

7 1949] MUTANT STRAIN OF E5CHERICHU COLI 45 biological asays that has a tendency to lose a given nutrient requirement during subculture. Reversion as related to purine concentration. During several experiments it was observed that when only those tubes of medium B which contained definitely suboptimal concentrations of purine (less than 2.5 X 10-1 M) were considered, the proportion of tubes showing reversion to purine independence (heavy turbidity) appeared to be greater at the lowest concentration of purine. This was true after the tubes were incubated several days beyond the usual period of 48 to 72 hours. The data are shown in table 6. The frequency of reversion at the lower concentrations of purine is significantly greater by the X2 test than the frequency at the higher concentrations. It is interesting to compare the results in table 6 with those obtained by Ryan (1946) for the leucineless Neurospora strai A. Ryan found that, contrary to his expectation, "adaptation" frequency at limiting concentrations of leucine varied inversely with the amount of leucine present in the medium. TABLE 6 Reversion as related to purine concentration in medium B 10' M GUANINE NUMBER OF TUBES NUMBER OF REVERSIONS PER CENT REVERSION He found that the reverted mycelium was that of a heterokaryon containing leucineless and leucine-synthesizing nuclei, and that the growth of this heterokaryon was suppressed by added leucine. SUMMARY A purine-requiring mutant strain of Escherichia coli has been isolated. Among compounds tested, only the purine bases, adenine, guanine, xanthine, or hypoxanthine, permitted growth of this organism. Within limits, growth was proportional to the amount of added purine base. This was the case for each compound added separately, or for a mixture of all four. The stability of the purine requirement was such that little difficulty was encountered in maintaining the mutant strain. Reversion to purine independence occurred, but upon the basis of present results it would appear that this could not seriously affect the use of the organism for the assay of purines. The frequency,of reverted cells on the stock culture medium was studied and estimated to be of the order of 108. This frequency was not significantly altered by serial transfer on this medium. The frequency of reversion was found to be related inversely to the concentration of purine. Upon prolonged incubation at limiting concentrations of purine, reversion was frequent in a medium containing hydrolyzed casein, B vitamins, and pyrimidines

8 46 ROBERT GUTHRIE [VOL. 57 but was rare in a simple glucose salts medium. to exist in a serial transfer experiment. This difference was also found REFERENCES GRAY, C. H., AND TATUM, E. L X-ray induced growth factor requirements in bacteria. Proc. Natl. Acad. Sci. U. S., 30, LAmPEN, J. O., AND JONES, M. J Relation between the rate of growth of a mutant strain of Escherichia coli and the efficiency of its utilization of arginine. J. Bact., 53, LAMPEN, J. O., JONES, M. J., ANJ9 PERKINS, A. B Studies on the sulfur metabolism of Escherichia coli. I. The growth characteristics and metabolism of a mutant strain requiring methionine. Arch. Biochem., 13, LEDERBERG, J Studies in bacterial genetics. J. Bact., 52, 503. LEDERBERG, J., AND TATUM, E. L Detection of biochemical mutants of microorganisms. J. Biol. Chem., 165, LEWIS, I. M Bacterial variation with special reference to behavior of some mutable strains of colon bacteria in synthetic medium. J. Bact., 28, MITCHELL, H. K., AND HOULAAN, M. B Adenine-requiring mutants of Neuro8pora cra8sa. Federation Proc., 3, PAPPENHEIMER, A. M., AND HOTTLE, G. A Effect of certain purines, and C02 on growth of a strain of group A hemolytic streptococcus. Proc. Soc. Exptl. Biol. Med., 44, PETERSON, W. H., AND PETERSON, M. S Relation of bacteria to vitamins and other growth factors. Bact. Revs., 9, ROEPKE, R. R., LIBBY, R. L., AND SMALL, M. H Mutation or variation of E8cherichia coli with respect to growth requirements. J. Bact., 48, RYAN, F. J Back-mutation and adaptation of nutritional mutants. Cold Spring Harbor Symposia Quant. Biol., 11, SMIIVE, W., ACKERMAN, W. W., GORDON, M., GETZENDANDER, M. E., AND EAKIN, R. E (4)-Amino-4(5)-imidazolecarboxamide, a precursor of purines. J. Am. Chem. Soc., 69, TATUM, E. L Induced biochemical mutations in bacteria. Cold Spring Harbor Symposia Quant. Biol., 11,