(Anderson, 1946) containing sodium chloride, sodium-potassium phosphate. added to this basic medium in a concentration sufficient for maximum growth.

Transcription

1 THE EFFECTS OF A TRYPTOPHAN-HISTIDINE DEFICIENCY IN A MUTANT OF ESCHERICHIA COLI MARGOT K. SANDS AND RICHARD B. ROBERTS Carnegie Institution of Washington, Department of Terrestrial Magnetism, Washington, D. C. Received for publication September 24, 95 Cells placed in a favorable environment are capable of duplicating themselves indefinitely. This implies that the over-all reaction rates of each of the synthetic systems must be balanced as every component of the cell is duplicated in the same timeinterval. It is evident that the various systems must be somehow coupled together since completely independent systems could not maintain these balanced rates in various environmental conditions. Thus, a coupling between the carbohydrate metabolism and synthetic activities is demonstrated by the decrease in oxygen uptake when nitrogen is removed from the medium. At the same time there is a degree of independence among the various metabolic systems. For example, carbohydrate metabolism proceeds at a reduced rate even though growth is stopped by lack of nitrogen. Also, certain ion deficiencies have more pronounced effects on some synthetic activities than on others. We have attempted to examine the degree of coupling among the various systems by observing the metabolism of mutant cells of Escherichia coli which were prevented from duplicating by the lack of one or more required amino acids. This deficiency would be expected to block protein synthesis, but the immediate effect on other systems, in particular nucleic acid synthesis, could not be predicted. Radioactive carbon, sulfur, and phosphorus were utilized as indicators of synthesis; growth and respiration were observed by the usual techniques. On completing these experiments it became apparent that the method utilized in this work might also be of value in determining the mode of action of various rtoisons and antibiotics. METHODS A mutant of E. coli, strain B, requiring tryptophan and histidine (TH) was obtained by using the Davis technique (Davis, 949) on the mutant B/r/l, tryptophan (Anderson, 946). The same technique was used to isolate a lysine requiring mutant from B/r (Witkin, 947). To avoid difficulties from reversions to wild type most of the experiments were conducted with the tryptophanhistidine mutant. Cells were grown overnight on a synthetic medium, M-9, (Anderson, 946) containing sodium chloride, sodium-potassium phosphate buffer, ammonium chloride, magnesium sulfate, and sufficient glucose (.2 g per liter) to give g wet weight of cells per liter. The required amino acids were added to this basic medium in a concentration sufficient for maximum growth. Under these conditions, growth stops abruptly when the glucose supply is exhausted and resumes without a lag when the cells are washed and placed in a fresh complete medium. Before use in experiments, cells were harvested by centrifuging, then washed twice with.85 per cent sodium chloride. s5

2 56 MARGOT K. SANDS AND RICHARD B. ROBERTS [VOL. 63 The radioactive isotopes were obtained from the Atomic Energy Commission at Oak Ridge. The radioactive phosphorus, received as H3P'24, was hydrolyzed in HCl before use to convert any other phosphate ions which might be present as contaminants to the orthophosphate form. S" was used as sulfate. C'4 was received as barium carbonate and converted to NaHC'43. Approximately millicurie of tracer was used per liter of medium. This quantity is adequate for counting and should have no harmful effect on the cells since the total radiation received is less than 5 roentgens (/, of the mean lethal dose). Radioactive samples were measured with a Geiger counter correcting, if necessary, for selfabsorption. With p32 and S36 present simultaneously, each sample was measured with and without a cover of aluminum foil. The individual contributions of the two radioelements could be calculated since the foil absorbed 9 per cent of the radiation from the S36 and 5 per cent of the radiation from the p32. Cell concentrations were determined by measuring the optical densities of suspensions with a Beckman spectrophotometer at 65 m,u. NISTWDINE (MG/ML.) TABLE Growth of tryptophan-hi8tidine mutant of Escherichia coli TRYPTOPHAN (MG/Mn.) o.2.8* * Cells (millions) per liter medium after 7 hours' incubation. RESULTS Growth:' Test tubes containing 2 ml of M-9 and varying concentrations of tryptophan and histidine were lightly inoculated with a washed suspension of mutant cells. After 7 hours' incubation at 37 C without aeration the optical densities were measured. Table shows that growth is limited by a deficiency of either tryptophan or histidine. When large iliocula of washed cells (tryptophan-histidine) were placed in a medium lacking the required amino acids, there was a period of slow growth. During the first two hours the optical density increased at a rate of 6 per cent per hour. After four hours the rate dropped to 3 per cent per hour and after 6 hours there was no further increase. Plate counts showed that the number of viable cells increased by a factor of two during the first four hours. The observed increase in the mass and number of cells was due to the tryptophan-histidine mutants, as samples plated on M-9 showed that less than. per cent of the population was capable of forming colonies on media lacking histidine and tryptophan. "Growth" is used to indicate any increase in cell mass of the culture. "Duplicate" is used for the special type of growth where the cells duplicate themselves indefinitely.

3 952 TRYPTOPHAN-HISTIDINE DEFICIENCY 57 Repration: The oxygen uptake of the tryptophan-histidine mutant was observed using a Warburg respirometer. Table 2 shows that the respiration rate was increased by the addition of ammonium chloride even though the required amino acids were not present. When both ammonium chloride and the amino acids were added, there was a further increase in the respiration rate. Sulfur: Synthesis of protein was measured by the incorporation of radioactive sulfur. Washed cells were distributed to tubes containing M-9 and radiosulfate TABLE 2 Relative rates of respiration of tryptophan-histidine mutant of Escherichia coli in various media SUPPLEMNTS TO NITROCOEN-PKEE MEDIU No supplement Tryptophan and histidine, Ammonium chloride, Tryptophan and histidine.2 mg/ml.4 mg/ml + ammonium chloride TABLE 3 Radiosulfur uptake (counts/second) by tryptophan-histidine mutant of Escherichia coli SUPPLEMNTS To BASAL MEIUM-.2 mo ZAC PEE ML flux () (2) (3) (4) (Ha) Histidine Tryptophan Histidine and tryp. c/s % of (4) c/s % of (4) c/s % of (4) (tc/a Cold trichloroacetic acid 2 3 soluble 8 (6) 23 (2) 6 (5) 2 35 (5) 3 (3) 33 (4) 236 Trichloroacetic acid insol uble (protein) 2 (5.8) 22 (6) 26 (7) (5.5) 48 (8.2) 36 (6.2) 582 Cells (millions) per liter Concentration of cells and aerated at 37 C. At intervals, samples were removed and the optical density was measured. These samples were washed with.85 per cent NaCl, centrifuged, and the dry pellets of cells frozen. At the end of each experiment, the frozen pellets were extracted for one-half hour with cold 5 per cent trichloroacetic acid and centrifuged. The trichloroacetic acid precipitate contained the sulfur incorporated into proteins (Bolton, Cowie, and Sands, 952). Table 3 shows that in the absence of either required amino acid, protein synthesis is reduced to less than one tenth of the normal rate. Phosphorus: Radioactive phosphorus was used to observe the synthesis of

4 58 MARGOT K. SANDS AND RICHXRD B. ROBERTS [VOL. 63 nucleic acids. The initial procedure was the same as with radiosulfur; in addition the trichloroacetic acid precipitate was further fractionated by a modification of TABLE 4 Radiophosphorus uptake (counts/second) by tryptophan-histidine mutant of Escherichia coli SUPPLEMENTS TO BASAL MEDIUM-.2 IG EACH PEz ML T(E) (BR). () I / 4) (2) Histidine c/s 4)f (3) Tryptophan c 4) (4) Histidine and tryptothan Desoxypentose nucleic acid Pentose nucleic acid (23) (28) (3) (22) (33) (38) (7) (24) (23) (28) (5) (24) Alcohol soluble (lipids) (5) (3) Cells (millions) per liter 2 32 (6) (44) 3 (32) (42) Concentration of cells ~~~ TABLE 5 Radiocarbon uptake (counts/second) by tryptophan-hiatidine mutant of Escherichia ccli SauJPLENT TO BASAL med.m-o.2 YG EAcH PzE ml () ~~~(2) (3) (4) () Histidine Tryptophan Histidine and tryptcophan c/s % of (4) c/s % of (4) c/s % of (4) (c/s) Hot trichloroacetic acid soluble (nu- 65 (8) 99 (27) 84 (23) 358 cleic acid) Trichloroacetic acid insoluble (pro- 59 () 76 (4) 59 () 536 tein) Alcohol soluble 4 (2) 6 (35) 45 (225) 2 Cells (millions) per liter Concentration of cells hr the Schmidt-Thannhauser method (945). After one hour extraction with 95 per cent ethanol at 2 C to remove the lipids, the precipitate was digested for 8 hours with N KOH. Finally, precipitation with trichloroacetic acid separated the desoxypentose nucleic acid from the pentose nucleic acid. Table 4 shows that when either required amino acid is omitted from the me-

5 952] TRYPTOPHAN-HISTIDINE DEFICIENCY 59 dium, the incorporation of phosphorus into nucleic acids is reduced to roughly one fourth. Sulfur-phosphorus: Experiments with sufur and phosphorus in parallel cultures consistently showed that the omission of one of the required amino acids reduced protein synthesis by a factor of ten and synthesis of nucleic acid by a factor of four. To ensure identity of conditions, radiosulfur and radiophosphorus were both added to the same tubes. The procedure followed was the same as in the radiophosphorus experiments. Although these radioactivity measurements are less accurate, the results were the same as those of tables 3 and 4. Carbon: The syntheses of nucleic acids and protein were also studied by measuring the incorporation of radiocarbon. Washed cells were shaken for one hour at 37 C in the presence of C42. Following this, the cells were washed and the lipids and carbohydrates removed by extraction with hot 8 per cent ethanol and an ethanol-ether mixture. The ethanol precipitate was then treated with 5 per cent trichloroacetic acid for twenty miniutes at C; the soluble fraction contained nucleic acid while the trichloroacetic acid precipitate contained the proteins. This completely independent method gives results (table 5) which confirm those obtained with radiosulfur and radiophosphorus in showing that the amino acid deficiency causes a greater reduction of protein synthesis than of nucleic acid synthesis. Altemnation of amino acids: In one experiment the cells were incubated for one hour with tryptophan and then washed and incubated for the second hour with histidine. The phosphorus uptake was the same as during two hours' incubation with tryptophan alone. Evidently, there is very little storage of free amino acids or peptides, and both amino acids are required simultaneously (Swanson and Clark, 95). Exchange: Mutants grown in the presence of radiophosphorus and radiosulfur and subsequently harvested, washed, and incubated for two hours in M-9 showed a loss of radioactivity. The trichloroacetic acid soluble p32 decreased by 5 per cent while the nucleic acid p32 decreased by 5 per cent. The protein bound S's showed a similar decrease of 5 per cent. The loss of P32 from the trichloroacetic acid soluble fraction is smaller than the loss observed in normally grown cells. This could be expected from the observed lower rate of oxygen uptake. However, the 5 per cent loss of nucleic acid p32 and protein SI' is greater than the loss observed with normally growing cells which show no loss from these fractions (Roberts and Roberts, 95; Cowie, Bolton, and Sands, 95). Consequently, this loss probably is due to the degradation of cellular components. Lysine mutant: The lysine deficient mutant gave similar results in experiments on growth, respiration, and the incorporation of sulfur and phosphorus. In addition, the rate of adaptation to a new energy source, arabinose, was observed. This adaptation process required the same time whether or not lysine or ammonium chloride was present. DISCUSSION The mutants used in these experiments do not duplicate when they lack a required amino acid. However, in a dense culture, some of their metabolic activities

6 5 MARGOT K. SANDS AND RICHARD B. ROBERTS [VOL. 63 proceed at reduced rates even in the absence of the required amino acids. For several hours, the cell mass increases; the number of cells also increases indicating cell division. Synthesis of cellular components continues as shown by the incorporation of radioactive indicators. Respiration also continues although at a reduced rate. These processes are not due to traces of the required amino acids remaining from the original protein media, as the rates remain constant during the first two hours. Neither can they be ascribed to bacterial contaminants as this possibility is ruled out by plate counts. Growth eventually stops after six hours probably because the balance among the various components of the cell is too severely disturbed. In the absence of the required amino acids, per cent or less of the normal protein synthesis takes place. This small residual synthetic activity is probably due to the utilization of tryptophan and histidine released from degraded proteins. The 536 released by the cells indicates that 5 per cent of the proteins are degraded; this quantity should be sufficient to supply the tryptophan and histidine required for the observed synthesis. The rates of synthesis and degradation appear to be equal with the total protein remaining constant. The C4 radioactivity observed in the alcohol soluble fraction was actually higher when the amino acids were omitted. Free amino acids were not found in the medium, and Taylor (947) has shown that they are not accumulated in E. coli. Consequently, it seems that when the amino acids are not utilized for protein synthesis they are no longer formed, and their precursors accumulate. When the required amino acids are missing, nucleic acid synthesis as measured by phosphorus or carbon is reduced by a factor of four. As tryptophan and histidine are not recognized as precursors of nucleic acid, it would appear that this reduction in the nucleic acid synthesis is a secondary effect of the primary blockage in protein synthesis. Protein synthesis and nucleic acid synthesis are, however, somewhat independent since the conditions which reduce the rate of protein synthesis by a factor of ten reduce nucleic acid synthesis by a factor of four. The residual growth and the oxygen uptake observed when the required amino acids are missing indicate again that some of the synthetic activities of the cells oontinue at reduced rates even when the protein synthesis is blocked. SUMMARY The metabolism of a mutant of Escherichia coli requiring tryptophan and histidine was compared in media with and without these amino acids. Synthesis of cellular components was observed using C4, S36 and p32 as indicators. In the absence of the amino acids, protein synthesis is reduced to per cent of normal while nucleic acid synthesis is less affected being 25 per cent of normal. Protein synthesis and nucleic acid synthesis are thus partially independent. REFERENCES ANDERSON, E. H. 946 Growth requirements of virus-resistant mutants of Escherichia coli strain "B". Proc. Natl. Acad. Sci., 32, BOLTON, E. T., COWIE, D. B., AND SANDS, M. K. 952 Sulfur metabolism in Escherichia coli. III. The metabolic fate of sulfate sulfur. J. Bact., 63,

7 952 TRYPTOPHAN-HISTIDINE DEFICIENCY 5 COWIE, D. B., BOLTON, E. T., AND SANDS, M. K. 95 Sulfur metabolism in Escherichia coli. I. Sulfate metabolism of normal and mutant cells. J. Bact., 6, DAVIS, B. D. 949 Isolation of biochemically deficient mutants of bacteria by means of penicillin. Proc. Natl. Acad. Sci., 35, -. ROBERTS, R. B., AND ROBERTS, I. Z. 95 Potassium metabolism in Eacherichia coli. III. Interrelationship of potassium and phosphorus metabolism. J. Cellular Comp. Physiol., 36, 5-4. SCHMIDT, T., AND THANNHAUSER, S. J. 945 A method for the determination of desoxyribonucleic acid, ribose nucleic acid, and phosphoproteins in animal tissues. J. Biol. Chem., 6, SWANSON, P. P., AND CLARK, H. E. 95 Metabolism of proteins and amino acids. Ann. Rev. Biochem., 9, TAYLOR, E. S. 947 Assimilation of amino acids by bacteria. 3. Concentration of free amino acids in internal environment of various bacteria and yeasts. J. Gen. Microbiol.,, WITKIN, E. M. 947 Genetics of resistance to radiation in Escherichia coli. Genetics, 32,