Inc.) per liter of medium. The liver extract was sterilized by filtration

Transcription

1 MUTATION OR VARIATION OF ESCHERICHIA COLI WITH RESPECT TO GROWTH REQUIREMENTS RAYMOND R. ROEPKE, RAYMOND L. LIBBY AND MARGARET H. SMALL American Cyanamid Company, Stamford, Connecticut Received for publication May 1, 1941 Beadle and Tatum (1941) and Tatum and Beadle (1942) have reported the isolation, from cultures of the ascomycete Neurospora exposed to x-rays, of mutant strains which require certain specific growth factors for normal growth. This report is concerned with the isolation and study of mutant strains of Escherichia coli. Although it has not been possible to establish a genetic basis for the growth-factor-requiring strains of E. coli, as Beadle and Tatum were able to do in the case of the fungi, the similarity between the results of the two studies leads us to consider the different strains of E. coli as mutants. Haberman and Ellsworth (1940), in a study concerned primarily with the effect of x-rays on colony morphology of bacteria, obtained several variants or mutants which differed in certain physiological activities from the parent strain. Although they did not obtain mutants requiring specific growth factors, they refer to a variant of different morphology which they could not isolate. It is possible that they were concerned with a mutant strain requiring an essential growth factor which was not present in their culture medium but which could be synthesized by the parent strain. METHODS Cultivation of normal and mutant strains A stock culture of E. coli was selected for use in this study. Although this strain usually grows well in a synthetic medium containing only inorganic salts and glucose, growth appears to be sensitive to slight changes in the medium. The addition of asparagine assures full growth after hours of incubation at 37 C with relatively small inocula. Thus, the basal, synthetic medium used was that described by MacLeod (1940) with the following composition: NaCl, 5.0 g; (NH4)2SO4, 4.72 g; KH2PO4, 2.72 g; glucose, 2.0 g; asparagine, 2.0 g; 1 ml of a solution containing 1 g of each of FeCl2, MgCl2 and CaCl2 in 600 ml; distilled water to make 1 L. The ph is adjusted to 7.0 with n NaOH, which requires approximately 15 ml. The "complete" medium used for growth of the treated cultures consisted of Difco A.C. Broth Experimental, with the addition of 0.2 ml of a crude liver extract (supplied through the courtesy of Dr. Y. SubbaRow of Lederle Laboratories, Inc.) per liter of medium. The liver extract was sterilized by filtration and added to the autoclaved broth. Although A.C. broth alone is sufficient for the growth of the more fastidious bacteria, the liver extract was added to assure as complete a medium as possible. In determining the growth requirements of the mutant strains, the test sub- 401

2 -~~~~ 402 RAYMOND R. ROEPKE, RAYMOND L. LIBBY AND MARGARET H. SMALL stances and the concentrations (mg per L of basal medium) at which they were used included: 1. Nicotinamide, 2 2. Calcium d(+) pantothenate, 1 3. Riboflavin, 1 4. Pyridoxine, 1 5. Thiamine HCI, p-aminobenzoic acid, Biotin (free acid), Folic acid, Choline, Inositol, Uracil, Cytosine, Thymine, Guanine HCl, Adenine sulfate, Xanthine, Pimelic acid, j3-alanine, Glutamine, 10 With the exception of dl-methionine, the following amino acids were tested at concentrations of 10 mg of the naturally occurring isomer or 20 mg of the racemic mixture per liter of medium: dl-methionine (10 mg per L) Glycine dl-alanine l(-)histidine 2 HCl dl-leucine dl-isoleucine dl-lysine HCl dl-valine dl-phenylalanine dl-serine dl-threonine l(+)arginine HC1 l(-)proline l(-)hydroxypyroline l(-)cystine l(-)tyrosine l(-)glutamic acid l(-)tryptophane The extent of growth was determined with a Photronreflectometer (Libby, 1938). The galvanometer deflection was calibrated in terms of total cell count with the aid of a Petroff-Hauser counting chamber. X-RAY TECHNIQUE A water-cooled x-ray tube with a copper target and beryllium window was used in all experiments in which cultures of Ewcherichia coli were exposed to x-rays. For exposure to hard (40 kv) x-rays, approximately 2.5 ml of the cul-- ture were transferred to a sterile alminum chamber fitted with an aluminum window of an inch thick. The chamber had an internal diameter and a depth of I inch and was placed in line with the x-ray beam 13 inches from the window of the x-ray tube. A smaller chamber, with an internal diatneter of i and a depth of X inch, was used for exposing cultures to soft (7 kv) x-rays. Water-proof cellophane inch thick was used for the window instead of aluminum in order to reduce the absorption of the soft x-rays. In order to assure uniform exposure of the cells to the soft x-rays, the culture was stirred continuously by means of a small nichrome wire attached to a rotating rod entering through the back of the chamber. Leakage around the rotating rod was prevented by the use of graphite packing. The small chamber was placed so that the cellophane window was approximately t inch from the beryllium window of the x-ray tube. The chambers were sterilized by heating in an oven at 155 C for 90 minutes.

3 MUTATION AND GROWTH OF ESCHERICHIA COLI 403 ISOLATION OF MUTANT STRAINS The mutant strains were isolated from poured, broth-agar plates. By means of a small wire loop, an inoculum was transferred from each isolated colony first into the basal medium (MacLeod's) and then into the broth medium, using tubes containing approximately 4.5 ml of the basal, and 2 ml of the broth, medium. The corresponding broth cultures of those colonies which failed to give full growth in the basal medium after hours of incubation were washed with saline buffer (0.5% NaCl and 0.2% KH2P04 adjusted to ph 7 with NaOH) and the washed cells tested for growth in the basal medium. These cells were also stained and examined microscopically to detect contaminating bacteria. Cultures which failed to give normal growth in the basal medium and which appeared microscopically similar to Escherichia coli were considered to be mutants of E. coli. The growth requirements of the mutant strains were then determined by inoculation into the basal medium plus various growth factors, singly and in combination. The inoculations were made with small loops which transfer approximately 1.2 mm3 of culture. The size of the inoculum transferred from a colony to the basal medium varied appreciably. Growth was often delayed in the basal medium, i.e., full growth was obtained after hours of incubation. Usually this was due to the transfer of too -small an inoculum from a colony derived from normal E. coli. However, since the mutant strains were capable of reverting to the parent strain, a large inoculum from a colony derived from a mutant cell may contain a sufficient number of revert or normal cells to give a slightly delayed growth in the basal medium. Thus, if an inoculum of the cells of the corresponding broth culture gave growth in the basal medium, the broth culture was plated out on brot hagar and 6-12 colonies isolated and tested in a similar manner before discarding the culture as normal E. coli. RESULTS AND DISCUSSION Irradiation of cultures of Escherichia coli with hard x-rays In seven experiments, young (4-6 hours) and relatively old (23-25 hours) single-colony cultures were exposed to x-rays of 40 kv and 20 ma for 1540 mi, killing per cent of the cells. From the treated cultures, a total of 4,220 colonies were isolated and tested for growth in the basal medium. No mutant cultures were obtained. Although eight of the isolated colonies failed to grow in the basal medium, these were considered to be contaminating bacteria. Two were gram-positive cocci and six were large gram-positive rods. The colonies of the gram-positive rods were all obtained from the same culture, from which a total of 1,001 colonies were isolated. Irradiation of cultures of Escherichia coli with soft x-rays during several periods of growth in a complete medium In order to increase the probability of isolating mutant strains, cultures of E. coli were exposed to x-rays during several periods of growth in the complete

4 404 RAYMOND R. ROEPKE, RAYMOND L. LIBBY AND MARGARET H. SMALL medium. A mutant cell produced in the early period of growth is likely to be carried over with the inoculum to the next culture if it reproduces at the same rate as cells of the parent strain. Thus, there would tend to be an accumulation of at least some of the mutant strains produced in the different periods of treatment. The cultures were treated with soft x-rays since it has been suggested by Wyckoff (1930) that an absorbed quantum of soft x-rays may affect a smaller portion of the cell than a quantum of -hard x-rays. The cultures were treated during growth at room temperature with x-rays of approximately 7 kv and 9 ma. The results obtained in four experiments, with the use of soft x-rays are summarized in table 1. In the first of these experiments, the growing culture was exposed in the larger chamber (containing 2.5 ml of culture) with the aluminum-window replaced with one of cellophane. In this chamber approximately 35 per cent of the cells suspended in saline buffer were kiled by an exposure of 17 hours. In the last three experiments, the cultur TABLE 1 Summary of results of treatment of E8cherichia coli with soft x-rays during several period8 of growth in the complete medium X-RAY TREATMENT N.OPDF zx!. NO. X-RAY 3ATNT NO. OF COLO- NO. OF NO. OF MUTANT NTO. OFDIM REP. NO. -MNIES ISOLATED CONTAIUNANTS COLONIES S No. of periods Total time stns hours , * 139* 1, , Total. 6, * The seventh x-ray treated culture of experiment 138 was exposed for seven-additional periods'of approximately eight hours each. were exposed in the smaller chamber (containing 0.04 ml of culture). In this chamber, approximately 70 per cent of the cells were killed during 15 hours of exposure. Mutant strain # (experiment 138, table 1) gave normal growth in the basal medium.containing nicotinamide. Five of the eight mutant colonies isolated in experiment 171 required methionine, and the remaining three colonies apparently contained mixtures of two different strains. Single-colony cultures derived from # gave normal growth with lysine but frequently gave delayed growth with thiamine and with methion-ine. Single-cell isolation yielded a culture which failed to give visible growth in the basal medium plus lysine, but gave normal growth in the basal medium plus thiamine. Methionerequing as well as thiamine-requiring cultures were obtained by serial; singlecolony isolation of # , the original culture of which gave normal or slightly delayed growth in the basal medium. Thus, # 44M-171 gave normal growth in the basal medium with methionine and no visible growth with thiamine (after 40 hours of incubation), whereas # 44T-171 gave normal growth with thiamine

5 MUTATION AND GROWTH OF ESCEERICHIA COLI 40An5 and no visible growth with methionine. The original culture of # frequently gave full growth in the basal medium after 40 hours of incubation, but gave full growth at 16 hours on the addition of thiamine. An attempt to obtain a pure culture of the mutant strain by single-colony isolation resulted in a culture which gave normal growth with methionine but no visible growth with thiamine even after 40 hours of incubation. Thus, three of the colonies isolated from the treated culture of experiment 171 contained a thiamine-requiring strain, but in each case this appeared to be associated with another mutant strain. Four of the 33 mutant colonies isolated in experiment 230 gave no visible growth in the basal medium, but gave normal growth on the addition of cystine. The remaining 29 colonies gave a very light growth in the basal medium after 16 hours and the growth increased appreciably on further incubation. No visible growth was obtained when recrystallized asparagine was used in the basal medium. Full growth was obtained by the addition of either glycine or serine, although growth appeared to be slower than that of the parent strain even with the addition of 50 mg of glycine or 100 mg of dl-serine per liter of basal medium. Several cultures of these colonies as well as the parent strain gave growth with the production of gas in lactose broth after 18 hours at 45 C. There remains the possibility that these colonies were derived from a contaminating strain of E. coli with more exacting growth requirements than that of our stock culture. The natural mutation of Escherichia coli during growth in a complete medium In order to determine whether the mutants isolated from the x-ray-treated cultures were produced as a result of the x-ray treatment or were naturally occurring mutations, it was deemed advisable to isolate and test a number of colonies from a culture which had been transferred serially in the complete medium without ray treatment. This is particularly advisable in view of the fact that Kohn and Harris (1942) found that methionine became an essential growth factor for E. coli when the original strain was transferred serially in a synthetic medium containing sulfanilamide and an amino-acid purine mixture which included methionine. After 30 transfers, the culture was found to grow in the basal medium only after the addition of methionine. In one experiment of this type, a single-colony culture of E. coli was transferred 25 times in the complete medium. Each transfer was incubated at room temperature for 7-18 hours under conditions similar to those used in the experiments with soft x-rays. The 25th culture was plated out and, of the 1,994 colonies which were isolated, six were mutant colonies requiring four different growth factors. Colonies # , # 1, , and # 1, required arginine, # required threonine, # required methionine and # 1, required tryptophane. The original culture of # also appeared to contain mutant cells requiring thiamine, since the washed cells gave no growth in the basal medium, but frequently gave growth (usually slightly delayed) with thiamine as well as normal growth with threonine. After further purification of this culture by single-colony isolation, several cultures were obtained which gave normal growth with threonine and no visible growth with thiamine. From the results thus far obtained, no definite conclusions can be made con-

6 406 RAYMOND R. ROEPKE, RAYMOND L. LIBBY AND MARGARET H. SALL cerning the effect of x-rays on the rate of mutation of bacteria to growth-factorrequiring strains. The relative growth rates of mutant and normal Escherichia coli cells in a complete medium The results obtained by Lewis (1934) in a study of bacterial variation indicate that variation or mutation occurs, in some cases at least, independently of environmental influences, and that the selective action of the medium determines whether or not the variant or mutant cells will outgrow the parent cells. We are concerned with mutations to strains which have more exacting growth requirements than the parent strain; and such mutants might not be expected to outgrow the parent strain even in media containing an abundance of the required growth factor or factors. Lankford et al. (1943) isolated from patients certain strains of the gonococcus which require a thermolabile growth factor present in yeast extract. These strains were capable of mutating to the "normal" strain which did not require the growth factor, and the authors found that approximately one out of 50,000 cells grew when plated in a deficient medium. Although they were unable to reverse the process and obtain a deficient strain from the "normal" strain, they reported that "this phenomenon has been observed in vivo in a hospitalized patient." This might be interpreted as indicating that the deficient strain is capable of growings in vivo at a slightly greater rate than that of the "normal" strain. The fact that four or more colonies of the same mutant strain of E. coli were isolated from the same culture, whereas none were obtained from similarly treated cultures (table 1), suggests that some strains may be capable of growing at a slightly greater rate than the parent strain in the complete medium. A comparison of the growth rate and of the maxmum growth attained by single-colony cultures of the various strains in the complete medium indicate that at least three of the mutant strains may be capable of outgrowing the parent strain in this medium. In order to obtain inocula of equal size and viability, the single-colony cultures were inoculated into 10 ml of the complete medium and incubated until a given turbidity reading was obtained (approximately one-fourth of maximum growth). At this point the growth was stopped by placing the tubes in ice water. From these cultures, inoculations were made into a second series of tubes and incubated at 36 C. The relative growth rate of each culture was calculated from the time required to obtain a given turbidity reading (approximately one-half of maxmum growth). As shown by the results of three experiments given in tables 2 and 3, two of the mutant strains (s and # ) consistently grew -at a greater rate than did the parent strain, whereas a third strain (# ) gave a greater maximum growth. The parent strain may continue to synthesize at least some of the particular growth substance required by a mutant strain, even though the medium supplies an abundance of that substance. If this is true, then the cells of the parent strain must transform a greater amount of energy during growth in a complete

7 MUTATION AND GROWTH OF ESCHERICHIA COLI 407 medium than do cells of the mutant strain and, if energy transformation is a factor limiting the rate of growth, the mutant strain will tend to outgrow the parent strain. TABLE 2 A comparison of the growth rate of single-colony cultures of normal Escherichia coli and of mutant strains in the complete medium CULTURE NO. REQUIRED GROWTH FACTOR PER CENT DIFFERENCE FROM THE AVERAGE GROWTH RATE OF NORML E. COLI* I II III Averaget E. coli % E. coli % T-171 Thiamine Nicotinamide Glycine or serine Methionine Cystine Tryptophane Arginine * A + sign indicates a higher and a - sign a lower growth rate than the average of the control cultures (normal E. coli). t Algebraic&average of the values obtained in the three experiments. TABLE 3 A comparison of the maximum growth obtained by single-colony cultures of normal Escherichia coli and mutant strains in the complete medium CULTURE NO. PER CENT DIFFERENCE IN FINAL TURBIDITY READING FROM AVERAGE OF NORMAL E. COLI* I II III Averaget E. coli # E. coli S T * The final turbidity readings in the three experiments were made after incubation at 36 C for 10 hours and 45 minutes, 7 hours and 30 minutes and 7 hours and 20 minutes, respectively. t Algebraic average of the results obtained in the three experiments. The procedures with which we have thus far been successful in obtaining growth-factor-requiring strains obviously favor the isolation of those strains which tend to outgrow the parent strain in the complete medium. More extensive attempts are underway to obtain growth-factor-requiring strains by radia-

8 If 408 RAYMOND R. ROEPKE, R&YMOND L. LIBBY AND MARGART. L tive treatment without serial passage in the complete medium in order to increa the possibility of obtaining mutants which may not be able to grow as rapidly as the parent strain in the complete medium. Characteristics and staility of the mutant strains The7mutant cells have a tendency to revert or mutate back to the parent strain during growth. Reversion becomes apparent during incubation of an inoculum -J 0. Z0C ) ~2O A1 NiCOTNIC ACID -MICROGRAMS PER L O%f so TURUDITY --DEOCASE IN ph OF MEDIUM (ditially AT ph ) P IX K--411 r NICOTINICACIDE NICOTINAMIDE-MICROGRAMS PER L j z I A 0.4 _ Jo FIG. 1. GROWTH AT 17 Houns oi MUTANT STRAN s IN BASAL MEDIuM PLuS NICOTINBAMID OR NICoTNIc ACID P 2 in the basal medium or in media containing suboptimum amounts of the re-' quired growth factor. Under such conditions, the revert or normal oells outgrow the mutant cells and the culture then gives normal growth in the basal medium. However, with most mutant strains of Escherichia coli it is a relatively simple

9 MUTATION AND GROWTH OF EBCHERICHIA COLI matter, by single-colony isolation, to obtain cultures sufficiently pure so that inoculation into a large number of tubes of medium containing the required growth factor in suboptimum concentration gives full growth only in a small percentage of the tubes even after incubation for 48 hours or longer, whereas inocula of the same size into the basal medium plus optimum concentration of the growth factor give full growth in 16 hours. With larger inocula, a greater percentage of the tubes show full growth with suboptimum concentrations of the required growth factor. An9 TABLE 4 The effect of ph on the growth of mutant strain # in the basal medium plus nicotinic acid INITIAL ph OF TIUE OF EXPT. NO. MEDIUM INCUBATION MINMUM CONCENTRATION OF NICOTINIC ACID GIVING VISIBLE GROWTH 'Total molar X 108 Un-ionized8 molar X 10' hours (>244 ) (>0.835) (>406 ) (>1.45) t * The concentration of un-ionized nicotinic acid was calculated at the original ph of the medium. With the basal medium at ph 8, the ph decreases measurably during incubation even in the tubes which show no growth (see t). t At the end of 40 hours of incubation the average ph in three tubes which showed no growth was 7.91 ( ). At this ph the concentration of un-ionized nicotinic acid (Ka = 1.4 X 10-') was calculated to be 1.78 X 10- molar. With media at ph 6.00, the ph of the control tubes remained unchanged at the end of 40 hours. Several of the mutant strains were tested for stability during serial transfer in the basal medium containing optimum concentrations of the required growth factor. After 24 hours of incubation, each serial culture was tested for reversion by inoculation into the basal medium and incubation for 40 hours. With three single-colony cultures of the nicotinamide-requiring strain (# ), reversion was noted after 5 to' 10 serial transfers in the basal medium containing nicotinamide. With four different single-colony, thiamine-requiring cultures (derived from # 15T-171, # 44T-171 and # 754T-171), reversion became apparent after the second to the seventh serial transfer in the basal medium plus thiamine. Two methionine-requiring cultures (# and # ) were carried through 30 transfers (in duplicate) in the basal medium plus methionine. None of 30 cultures in each of the four series gave visible growth when inoculated

10 410 RAYMOND R. ROEPKE, RAYMOND L. LIBBY AND MARGARET H. SMALL into the basal medium and incubated for 40 hours. The stability of a mutant strain as determined in this manner is dependent on the relative growth rates of the mutant and revert strains as well as on the rate of reversion. Cultures of strains requiring nicotinamide, thiamine, and methionine have been kept on broth-agar slants in a refrigerator for 14 weeks without noticeable reversion. 600.J 500 * 754T-T ) T-171 S300 Pie THIAMINE- MICROGRAMS PER L. GRoWTH AT 16 HouRs OF THIAMINE-REQUIRING CULTURES IN THE BASAL MEDIUM PLUS THIAMiNE All of the mutant strains thus far isolated fail to give visible growth in the basal medium unless reversion has occurred, in which case the growth is equivalent to that of the parent strain. The amino-acid-requiring strains give a graded growth response with increasing concentrations of the required amino acid, and studies are under way to determine the suitability of these strains for use as test organisms in the microbiological assay for amino acids. The nicotinamide-requiring strain gives a typical growth curve with increasing concentrations of nicotinamide within 17 hours, but there is a sudden break in

11 MUTATION AND GROWTH OF ESCHERICHIA COLI 411 the growth curve when nicotinic acid is substituted for nicotinamide (figure 1). Also, the concentration of nicotinic acid required to give full growth is greater than that of nicotinamide. The decrease in ph of the culture media is roughly proportional to the growth. Since growth in the basal medium results in a decrease in ph, it was suspected that the break in the growth-concentration curve obtained with nicotinic acid might be due to an effect of ph on the utilization of nicotinic acid by this mutant strain. That such is the case is indicated by the results given in table 4. From these results it is evident that, with limiting concentrations of nicotinic acid, the growth rate increases when the growth is sufficient to bring about a decrease in the ph of the culture medium. Thus, except perhaps with prolonged incubation, this mutant strain would give a sigmoid growth curve with increasing concentrations of nicotinic acid. In attempting to explain the effect of ph of the media on the bacteriostatic activity of the sulfonamides, Cowles (1942) has suggested that the sulfonamides may enter the bacterial cell only in the un-ionized form. Brueckner (1943) has suggested that the same condition may exist with Escherichia coli and p-aminobenzoic acid, and with Staphylococcus aureus and nicotinic acid. The results given in table 4 indicate a correlation between the concentration of un-ionized nicotinic acid and the rate of growth of the mutant strain in media of different ph. The thiamine-requiring mutant cultures also give a sigmoid growth curve in the basal medium with increasing concentrations of thiamine (figure 2); however, with this strain, the utilization of thiamine is not affected noticeably by the ph of the medium. It appears that a critical concentration of thiamine must be exceeded in order to obtain significant growth, although the critical concentration is not the same for all cultures. Inoculation from cultures of this strain grown in the basal medium plus thiamine into the basal medium without added thiamine usually fails to give visible growth even with prolonged incubation, indicating that growth in the presence of thiamine is not due to adaptation, or reversion to the parent strain. SUMMARY Eight different mutant strains were obtained by single-colony isolation from cultures of Escherichia coli which had been transferred serially in a complete medium with and without x-ray treatment. These strains appear to have lost the ability to synthesize nicotinamide, thiamine, methionine, lysine, cystine, arginine, threonine or tryptophane. A ninth strain with less sharply defined growth requirements was also isolated from one of the treated cultures. This strain gives growth in the basal (synthetic) medium only after the addition of either glycine or serine. Evidence is presented to show that several of the growth-factor-requiring strains may be capable of outgrowing the parent strain in a complete medium, thus facilitating the isolation of these particular mutant strains. Although the mutant strains tend to revert to the parent strain, the rate of reversion is not sufficiently great to interfere seriously with the use of these strains as test organisms.

12 412 RAYMOND R. ROEPKE, RAYMOND L. LIBBY AND MARGARET H. SL ACKNOWLEDGMENT The authors are indebted to Dr. Dan McLachlan, Jr. for assistance in experiments involving x-ray treatment of the bacterial cultures, and to Dr. J 0. Lampen for single-cell isolations. REFERENCES BEADLE, G. W., AND TATUM, E. L Genetic control of biochemical reactions in Neurospora. Proc. Natl. Acad. Sci., 27, BRUECKNER, A. H Sulfonamide activity as influenced by variation in ph of culture media. Yale J. Biol. Med., 15, COWLEs, P. B The possible r6le of ionization in the bacteriostatic action of the sulfonamides. Yale J. Biol. Med., 14, HABERMAN, S., AND ELLSWORTH, L. D Lethal and dissociative effectssof x-rays on bacteria. J. Bact., 40, KOHN, H. I., AND HARRis, J. S Methionine made an essential growth factor by cultivationofe. coliinthe presence of methionineandsulfanilamide. J.Bact.,44, LANKFORD,C.E.,SCOTT,V.,Cox, M. F., AND COOKE,W. R Some aspects of nutritional variation of the gonococcus. J. Bact., 45, LEWIS, I. M Bacterial variation with special reference to behavior of some mutabile strains of colon bacteria in synthetic media. J. Bact., 28, LIBBY, R. L The photronreflectometer-an instrument for the measurement of turbid systems. J. Immunol., 34, MAcLEOD, C. M The inhibition of the bacteriostatic action of sulfonamide drug by substances of animal and bacterial origin. J. Exptl. Med., 72, TATUM, E. L., AND BEADLE, G. W Genetic control of biochemical reactions in Neurospora: ain "aminobenzoicless" mutant. Proc. Natl. Acad. Sci., 28, WYCKOFF, R. W. G The killing of colon bacilli by x-rays of different wave lengths. J. Exptl. Med., 52,