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Utilization of uracil by a strain of Escherichia coli
Utilization of Uracil by a Strain of Escherichiu coli A. M. Moore and Joyce B. Boylen From the Biology
Division Atomic Energy of Canada, Ltd., Chalk River, Ontario, Canada Received February 16, 1954
Although for several years there has been in the literature evidence suggesting a role for free uracil in the metabolism of certain bacteria and higher plants, this evidence has been largely of an indirect nature, The ability of uracil to reverse the inhibitory effects of certain pyrimidine analogs (l-3) and to maintain the growth of some pyrimidine-requiring strains of bacteria (4, 5) may be cited. However, in none of these cases was the function of the uracil established. The only tracer studies until recently have been done with the rat as test organism, and the results in this case (6, 7) seem to rule out any considerable metabolic function for uracil-in particular, it does not appear to act as a precursor of nucleic acid pyrimidines although erotic acid does (8, 9). Indirect evidence of a new type has recently been advanced that uracil may be utilized for pyrimidine nucleotide synthesis in Escherichia c&i strain B (10) and in a strain of Serratia murcescens (11). The experiments involve a demonstration of the ability of added uracil to suppress the fixation of a portion of the CO2normally incorporated into the nucleic acids of the growing bacteria. The present investigation was undertaken in order to determine the fate of CY4-labeleduracil in a pyrimidine-requiring strain of E. coli (63-86). The growth of this organism, like that of other strains of E. coli is inhibited by 5-bromouracil, the inhibition being reversed by uracil. It seemedpossible therefore that this study might help to clarify the situation with respect to two of the types of indirect evidence already mentioned . While this work was in progress, Wright and Miller (12) reported a similar study using Lactobacillus bulgaricus 09. They found that erotic 312
UTILIZATION TABLE
Effect of Various Pyrimidine GP3Wth Uracil Uridine Uridylic Cytidine Cytidylic
0 Each compound
I
Supplements~ on E. coli 6.9-86 No Growth Cytosine Thymine Thymidylic acid Orotic acid 4.Methyluracil Isocytosine Hydrouracil
acid acid
was tested
313
OF URACIL
at a concentration
of 10-a M.
acid was utilized by it for the synthesis of its uridylic acids. Uracil was also utilized, but less readily.
and cytidylic
EXPERIMENTAL The pyrimidine-requiring strain of E. coli 63-86 which we have used was obtained through the kindness of Dr. Bernard Davis. It can be grown in liquid culture in the simple synthetic medium described by Lederberg (13) supplemented with a suitable pyrimidine compound. The specificity of the latter requirement in our experiments is illustrated in Table I. Growth of the cultures was followed by measuring their optical densities in a spectrophotometer at a wavelength of 610 rnp. For the radioactive runs the organism was grown at 37°C. with uracil-2-Cl4 (10-d J.1) as the sole pyrimidine supplement. A slow air stream, bubbled through t,he culture and through an NaOH t,rap in series, served to keep the culture suspended and aerated and, at the same time, to entrain any radioactive COZ that might be liberated. After the final density reading had been made and samples taken for viable plate counts, the cultures were centrifuged, washed repeatedly with water at, 5°C. until the supernatants contained negligible activity, and bacterial samples were taken for CL4 estimat,ion. The remainder was hydrolyzed for 2 hr. at 100°C. with I N HCl in 10 N formic acid. In test hydrolyses using ribonucleic acid (RNA) of yeast and deoxyribonucleic acid (DNA) of calf thymus, this procedure was found to yield products similar to those obtained with 1 N HCl alone. That is to say, the purines adenine and guanine were split from both RNA and DNA: the pyrimidines of the RNA were liberated as uridylic and cyt,idylic acids whereas those of DNA remained as a complex which did not migrate from the origin of the paper chromatograms. It may be noted that free thymine was not detected in these hydrolyzates although, as will be seen in the following, it was found in the bacterial hydrolyzates. The formic acid was included because it great,ly facilitates the dissolution of the bacteria. The products were fractionated by repeated paper chromatography with several solvents, using washed Whstman No. 1 filt,er paper. The pyrimidine nucleotides and the purines were isolated and estimated chemically hg means of their ultraviolet, absorpt,ion spectra, and nliquot,s were plated for Cl4 estimation.
314
A.
M.
MOORE
AND
JOYCE
B.
BOYLEN
The chromatographic solvents employed included the following: water-saturated n-butanol (14) n-butanol, water, formic acid (14) n-butanol, water, ammonia (14) water-saturated isoamyl alcohol n-propanol, water (70:30 by volume) n-propanol, water (70:30) 0.8 N with respect to HCl. isobutyric acid, ammonium isobutyrate, pH 3.65 (15) The identification of the compounds isolated rests in part on their chromatographic behavior with known markers in four or more different solvents, and also on their ultraviolet absorption spectra throughout the range 230-300 rnp. The spectra of the uridylic acid fractions were examined at pH’s 7.0 and 11.0, and those of cytidylic acid at pH’s 7.0 and 1.0. The characteristic changes in absorption which these compounds exhibit over these ranges of pH are further checks of their identity and purity. Extracts of appropriate blank areas of the chromatograms served as references for the absorption spectra. The uracil-2-Cl4 used in this work was synthesized by a modification of the method of Davidson and Baudisch (16) using urea-C4 which had been synthesized from barium carbonate by the method of Zbarsky and Fischer (17). 04 estimations were made by plating samples as uniform, “weightless” layers on steel disks which were then counted in a windowless methane proportional counter.
--1oooo
-
f E i
so00
-
6000
-
4000
-
2 : E z 5 F M
a “RlDYLlC ACID 0 CYTIDYLC AClD 0 ACENINE .
I IO
5 AMOUNT
FIG.
1. Activities
found
?
in the isolated
k.I) I3 (mols
4a.a -20 I IO’ )
I nno 25
fractions in a series of runs in which as pyrimidine supplement. The straight the activity of the uracil supplied in the me-
E. eoli 63-86 was grown with uracil-2-C4 line drawn in the figure represents dium (6.12 X 107 counts/min./mmole).
0
ISOLATED
GUANINE
UTILIZATION TABLE Distribution .-i
~~-
315
OF URACIL II
of Cl4 in Isolated
Fractions
~ ~~~Per cent of total
I
activity
in hydrolyzate
Fraction 2
Uridylic acid Cytidylic acid Cytosinea Cytidinea Thymine Adenine Guanine Subtotal Unidentifiedb Recovery
35.1 35.5 3.85 1.32 9.48 0.44 1.32
27.3 31 .o 3.37 2.00 8.97 0.73 0.44 _-__87.0 73.8 5.15 92.0
-
-
3
4
26.9
26.9
34.1 3.93 1.48 9.48 0.72 0.68
37.2
77.3 8.48 85.8
4.76 1 8.54 1 .Ol 1.03 -_ 79.4 6.49 85.9
MWl
29.0 34.4 5.18 9.12 0.72 0.87 _-79.4 6.71 88.0
0 Identification based on chromatographic properties only. b This fraction represents material which remained at the origin of the original chromatogram and presumably includes part of the pyrimidines of the DNA. RESULTS
In the radioactive runs the mean activity found in the washed bacteria was (3.3 f O.OS)l X 1OP counts/min. per bacterium. Assuming this to be all present as labeled pyrimidine with the same molecular activity as the labeled uracil supplied to the organisms-an assumption which will be seen in the following to be essentially true-this activity corresponds to about 5.4 X 1P moles of pyrimidine per organism in the saturated culture. The results of the specific activity determinations on the isolated purines and pyrimidines are summarized in Fig. 1 where the activity in each sample is plotted against the number of moles of compound isolated from the bacteria. The straight line drawn in the figure represents the activity of the uracil supplied to the bacteria: 6.12 X lo7 counts/min./ mmole. It is evident that the uridylic and cytidylic acids have an activity per mole very close to that of the original uracil. The adenine and guanine, on the contrary, show no significant activity. In Table II are given the results for four parallel runs showing the 1 Standard error of mean.
316
A. M. MOORE AND JOYCE B. BOYLEN
amounts of activity recovered in each fraction. Traces of cytosine, cytidine, and thymine which were found on the chromatograms are included here. By pooling the material from several runs it was possible to obtain sufficient thymine to compare it with a known standard in five different chromatographic solvents and also to check its absorption spectrum at two pH’s (7.0 and 11.0). Its Cl4 activity was 5.7 X lo7 counts/min./ n-mole, or 93 Y0of that of the uracil stock. DISCUSSION
The ability of the pyrimidine-requiring E. coli strain 63-86 to grow on any one of the compounds listed in Table I, col. 1, is most easily understood if all these compounds are interconvertible in the organism and are used for the same purpose. The tracer experiments verify that all the uridylic and cytidylic acids of the organism may be synthesized without dilution from the uracil supplied, thus supporting the hypothesis of interconvertibility. The failure of free cytosine to support growth of the mutant suggests that the interconversion occurs at the nucleoside or nucleotide level. The finding of labeled thymine also with the same activity per mole as the original uracil demonstrates the ability of the organism to methylate uracil (or one of its derivatives) at position 5. In the face of this, the failure of thymine or thymidylic acid to support growth of the mutant suggests either that the methylation is irreversible or that free thymine is not an intermediate. The five pyrimidine compounds listed in Table II together account for 78 % of the total activity in the original hydrolyzate (88 % of the recovered activity). Thus incorporation into RNA and, probably, into DNA represents by far the major fate of the absorbed uracil. The absence of appreciable activity in the purine fractions rules out any appreciable degradation of the uracil to form labeled carbonate since this has been shown to give rise to labeling of both purines and pyrimidines in E. co& (10). The E. coli strain 63-86 differs from L. bulgaricus 09 and from the rat both in its inability to utilize erotic acid and in its ready utilization of uracil for nucleic acid synthesis. This mutant was derived from an irradiated culture of the wild type “Waksman” straiq2 and in all probability differs from it in a single genetic character. It would seem, there%Bernard Davis, personal communication.
UTILIZATION
OF URACIL
fore, that the findings with regard to uracil utilization should also be largely true for the wild type.
317 described here
SUMMARY
The fate of uracil-2-Cl4 in a pyrimidine-requiring strain of E. coli (63-86) has been investigated. Uridylic acid, cytidylic acid, and thymine were isolated from the organism and found to have the same Cl4 activity per mole as the uracil supplied. It is concluded that in this strain of E. coli uracil is capable of serving as the precursor of all the pyrimidines of its ribonucleic acid and possibly of its deoxyribonucleic acid. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
STRANDSKOV, F., AND WYSS, O., J. Bacterial. 62, 675 (1946). BER, A., Ezperientia 6. 455 (1949). TOTTER, W. R., Nature 164, 63 (1949). SMILEY, K. L., NIVEN, C. F., JR., AND SHERMAN, J. M., J. Bacterial. 46, 445 (1943). FEENEY, R. E., MUELLER, J. H., AND MILLER, P. A., J. Bacterial. 46, 659 (1943). PLENTL, A. A., AND SCHOENHEIMER, R., J. Biol. Chem. 163, 203 (1944). BENDICH, A., GETLER, H., AND BROWN, G. B., J. Biol. Chem. 177, 565 (1949). ARVIDSON, H., ELIASSON, N. A., HAMMARSTEN, E., REICHARD, P., VON UBISCH, H., AND BERGSSTR~M, S., J. Biol. Chem. 179, 169 (1949). WEED, L. L., AND WILSON, D. W., J. Biol. Chem. 169,435 (1951). BOLTON, E. J., ABELSON, P. H., AND ALDOUS, E., J. Biol. Chem. 196,179 (1952). MCLEAN, D. J., AND PURDIE, E. F., J. Biol. Chew 197, 539 (1952). WRIGHT, 1,. D., AND MILLER, C. S., Proc. Sot. Exptl. Biol. Med. 81, 131 (1952). LEDERBERG, J., Genetics 32, 505 (1947). MARKHAM, R., AND SMITH, J. Il., Biochem. J. 46, 294 (1949). MAGASANIK, B., VISCHER, E., DONIGER, R., ELSON, D., AND CHARGAFF, E., J. Biol. Chem. 186, 37 (1950). DAVIDSON, D., AND BAUDISCH, O., J. Am. Chem. Sot. 48, 2379 (1926). ZBARSKY, S. H., AND FISCHER, I., Can. J. Research B27, 81 (1949).
Division Atomic Energy of Canada, Ltd., Chalk River, Ontario, Canada Received February 16, 1954
Although for several years there has been in the literature evidence suggesting a role for free uracil in the metabolism of certain bacteria and higher plants, this evidence has been largely of an indirect nature, The ability of uracil to reverse the inhibitory effects of certain pyrimidine analogs (l-3) and to maintain the growth of some pyrimidine-requiring strains of bacteria (4, 5) may be cited. However, in none of these cases was the function of the uracil established. The only tracer studies until recently have been done with the rat as test organism, and the results in this case (6, 7) seem to rule out any considerable metabolic function for uracil-in particular, it does not appear to act as a precursor of nucleic acid pyrimidines although erotic acid does (8, 9). Indirect evidence of a new type has recently been advanced that uracil may be utilized for pyrimidine nucleotide synthesis in Escherichia c&i strain B (10) and in a strain of Serratia murcescens (11). The experiments involve a demonstration of the ability of added uracil to suppress the fixation of a portion of the CO2normally incorporated into the nucleic acids of the growing bacteria. The present investigation was undertaken in order to determine the fate of CY4-labeleduracil in a pyrimidine-requiring strain of E. coli (63-86). The growth of this organism, like that of other strains of E. coli is inhibited by 5-bromouracil, the inhibition being reversed by uracil. It seemedpossible therefore that this study might help to clarify the situation with respect to two of the types of indirect evidence already mentioned . While this work was in progress, Wright and Miller (12) reported a similar study using Lactobacillus bulgaricus 09. They found that erotic 312
UTILIZATION TABLE
Effect of Various Pyrimidine GP3Wth Uracil Uridine Uridylic Cytidine Cytidylic
0 Each compound
I
Supplements~ on E. coli 6.9-86 No Growth Cytosine Thymine Thymidylic acid Orotic acid 4.Methyluracil Isocytosine Hydrouracil
acid acid
was tested
313
OF URACIL
at a concentration
of 10-a M.
acid was utilized by it for the synthesis of its uridylic acids. Uracil was also utilized, but less readily.
and cytidylic
EXPERIMENTAL The pyrimidine-requiring strain of E. coli 63-86 which we have used was obtained through the kindness of Dr. Bernard Davis. It can be grown in liquid culture in the simple synthetic medium described by Lederberg (13) supplemented with a suitable pyrimidine compound. The specificity of the latter requirement in our experiments is illustrated in Table I. Growth of the cultures was followed by measuring their optical densities in a spectrophotometer at a wavelength of 610 rnp. For the radioactive runs the organism was grown at 37°C. with uracil-2-Cl4 (10-d J.1) as the sole pyrimidine supplement. A slow air stream, bubbled through t,he culture and through an NaOH t,rap in series, served to keep the culture suspended and aerated and, at the same time, to entrain any radioactive COZ that might be liberated. After the final density reading had been made and samples taken for viable plate counts, the cultures were centrifuged, washed repeatedly with water at, 5°C. until the supernatants contained negligible activity, and bacterial samples were taken for CL4 estimat,ion. The remainder was hydrolyzed for 2 hr. at 100°C. with I N HCl in 10 N formic acid. In test hydrolyses using ribonucleic acid (RNA) of yeast and deoxyribonucleic acid (DNA) of calf thymus, this procedure was found to yield products similar to those obtained with 1 N HCl alone. That is to say, the purines adenine and guanine were split from both RNA and DNA: the pyrimidines of the RNA were liberated as uridylic and cyt,idylic acids whereas those of DNA remained as a complex which did not migrate from the origin of the paper chromatograms. It may be noted that free thymine was not detected in these hydrolyzates although, as will be seen in the following, it was found in the bacterial hydrolyzates. The formic acid was included because it great,ly facilitates the dissolution of the bacteria. The products were fractionated by repeated paper chromatography with several solvents, using washed Whstman No. 1 filt,er paper. The pyrimidine nucleotides and the purines were isolated and estimated chemically hg means of their ultraviolet, absorpt,ion spectra, and nliquot,s were plated for Cl4 estimation.
314
A.
M.
MOORE
AND
JOYCE
B.
BOYLEN
The chromatographic solvents employed included the following: water-saturated n-butanol (14) n-butanol, water, formic acid (14) n-butanol, water, ammonia (14) water-saturated isoamyl alcohol n-propanol, water (70:30 by volume) n-propanol, water (70:30) 0.8 N with respect to HCl. isobutyric acid, ammonium isobutyrate, pH 3.65 (15) The identification of the compounds isolated rests in part on their chromatographic behavior with known markers in four or more different solvents, and also on their ultraviolet absorption spectra throughout the range 230-300 rnp. The spectra of the uridylic acid fractions were examined at pH’s 7.0 and 11.0, and those of cytidylic acid at pH’s 7.0 and 1.0. The characteristic changes in absorption which these compounds exhibit over these ranges of pH are further checks of their identity and purity. Extracts of appropriate blank areas of the chromatograms served as references for the absorption spectra. The uracil-2-Cl4 used in this work was synthesized by a modification of the method of Davidson and Baudisch (16) using urea-C4 which had been synthesized from barium carbonate by the method of Zbarsky and Fischer (17). 04 estimations were made by plating samples as uniform, “weightless” layers on steel disks which were then counted in a windowless methane proportional counter.
--1oooo
-
f E i
so00
-
6000
-
4000
-
2 : E z 5 F M
a “RlDYLlC ACID 0 CYTIDYLC AClD 0 ACENINE .
I IO
5 AMOUNT
FIG.
1. Activities
found
?
in the isolated
k.I) I3 (mols
4a.a -20 I IO’ )
I nno 25
fractions in a series of runs in which as pyrimidine supplement. The straight the activity of the uracil supplied in the me-
E. eoli 63-86 was grown with uracil-2-C4 line drawn in the figure represents dium (6.12 X 107 counts/min./mmole).
0
ISOLATED
GUANINE
UTILIZATION TABLE Distribution .-i
~~-
315
OF URACIL II
of Cl4 in Isolated
Fractions
~ ~~~Per cent of total
I
activity
in hydrolyzate
Fraction 2
Uridylic acid Cytidylic acid Cytosinea Cytidinea Thymine Adenine Guanine Subtotal Unidentifiedb Recovery
35.1 35.5 3.85 1.32 9.48 0.44 1.32
27.3 31 .o 3.37 2.00 8.97 0.73 0.44 _-__87.0 73.8 5.15 92.0
-
-
3
4
26.9
26.9
34.1 3.93 1.48 9.48 0.72 0.68
37.2
77.3 8.48 85.8
4.76 1 8.54 1 .Ol 1.03 -_ 79.4 6.49 85.9
MWl
29.0 34.4 5.18 9.12 0.72 0.87 _-79.4 6.71 88.0
0 Identification based on chromatographic properties only. b This fraction represents material which remained at the origin of the original chromatogram and presumably includes part of the pyrimidines of the DNA. RESULTS
In the radioactive runs the mean activity found in the washed bacteria was (3.3 f O.OS)l X 1OP counts/min. per bacterium. Assuming this to be all present as labeled pyrimidine with the same molecular activity as the labeled uracil supplied to the organisms-an assumption which will be seen in the following to be essentially true-this activity corresponds to about 5.4 X 1P moles of pyrimidine per organism in the saturated culture. The results of the specific activity determinations on the isolated purines and pyrimidines are summarized in Fig. 1 where the activity in each sample is plotted against the number of moles of compound isolated from the bacteria. The straight line drawn in the figure represents the activity of the uracil supplied to the bacteria: 6.12 X lo7 counts/min./ mmole. It is evident that the uridylic and cytidylic acids have an activity per mole very close to that of the original uracil. The adenine and guanine, on the contrary, show no significant activity. In Table II are given the results for four parallel runs showing the 1 Standard error of mean.
316
A. M. MOORE AND JOYCE B. BOYLEN
amounts of activity recovered in each fraction. Traces of cytosine, cytidine, and thymine which were found on the chromatograms are included here. By pooling the material from several runs it was possible to obtain sufficient thymine to compare it with a known standard in five different chromatographic solvents and also to check its absorption spectrum at two pH’s (7.0 and 11.0). Its Cl4 activity was 5.7 X lo7 counts/min./ n-mole, or 93 Y0of that of the uracil stock. DISCUSSION
The ability of the pyrimidine-requiring E. coli strain 63-86 to grow on any one of the compounds listed in Table I, col. 1, is most easily understood if all these compounds are interconvertible in the organism and are used for the same purpose. The tracer experiments verify that all the uridylic and cytidylic acids of the organism may be synthesized without dilution from the uracil supplied, thus supporting the hypothesis of interconvertibility. The failure of free cytosine to support growth of the mutant suggests that the interconversion occurs at the nucleoside or nucleotide level. The finding of labeled thymine also with the same activity per mole as the original uracil demonstrates the ability of the organism to methylate uracil (or one of its derivatives) at position 5. In the face of this, the failure of thymine or thymidylic acid to support growth of the mutant suggests either that the methylation is irreversible or that free thymine is not an intermediate. The five pyrimidine compounds listed in Table II together account for 78 % of the total activity in the original hydrolyzate (88 % of the recovered activity). Thus incorporation into RNA and, probably, into DNA represents by far the major fate of the absorbed uracil. The absence of appreciable activity in the purine fractions rules out any appreciable degradation of the uracil to form labeled carbonate since this has been shown to give rise to labeling of both purines and pyrimidines in E. co& (10). The E. coli strain 63-86 differs from L. bulgaricus 09 and from the rat both in its inability to utilize erotic acid and in its ready utilization of uracil for nucleic acid synthesis. This mutant was derived from an irradiated culture of the wild type “Waksman” straiq2 and in all probability differs from it in a single genetic character. It would seem, there%Bernard Davis, personal communication.
UTILIZATION
OF URACIL
fore, that the findings with regard to uracil utilization should also be largely true for the wild type.
317 described here
SUMMARY
The fate of uracil-2-Cl4 in a pyrimidine-requiring strain of E. coli (63-86) has been investigated. Uridylic acid, cytidylic acid, and thymine were isolated from the organism and found to have the same Cl4 activity per mole as the uracil supplied. It is concluded that in this strain of E. coli uracil is capable of serving as the precursor of all the pyrimidines of its ribonucleic acid and possibly of its deoxyribonucleic acid. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
STRANDSKOV, F., AND WYSS, O., J. Bacterial. 62, 675 (1946). BER, A., Ezperientia 6. 455 (1949). TOTTER, W. R., Nature 164, 63 (1949). SMILEY, K. L., NIVEN, C. F., JR., AND SHERMAN, J. M., J. Bacterial. 46, 445 (1943). FEENEY, R. E., MUELLER, J. H., AND MILLER, P. A., J. Bacterial. 46, 659 (1943). PLENTL, A. A., AND SCHOENHEIMER, R., J. Biol. Chem. 163, 203 (1944). BENDICH, A., GETLER, H., AND BROWN, G. B., J. Biol. Chem. 177, 565 (1949). ARVIDSON, H., ELIASSON, N. A., HAMMARSTEN, E., REICHARD, P., VON UBISCH, H., AND BERGSSTR~M, S., J. Biol. Chem. 179, 169 (1949). WEED, L. L., AND WILSON, D. W., J. Biol. Chem. 169,435 (1951). BOLTON, E. J., ABELSON, P. H., AND ALDOUS, E., J. Biol. Chem. 196,179 (1952). MCLEAN, D. J., AND PURDIE, E. F., J. Biol. Chew 197, 539 (1952). WRIGHT, 1,. D., AND MILLER, C. S., Proc. Sot. Exptl. Biol. Med. 81, 131 (1952). LEDERBERG, J., Genetics 32, 505 (1947). MARKHAM, R., AND SMITH, J. Il., Biochem. J. 46, 294 (1949). MAGASANIK, B., VISCHER, E., DONIGER, R., ELSON, D., AND CHARGAFF, E., J. Biol. Chem. 186, 37 (1950). DAVIDSON, D., AND BAUDISCH, O., J. Am. Chem. Sot. 48, 2379 (1926). ZBARSKY, S. H., AND FISCHER, I., Can. J. Research B27, 81 (1949).