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Control of biotin synthesis in Escherichia coli by exogenous biotin
PRELIMINARY NOTES
I59
P~ 1166
Control of biotin synthesis in Escherichia coli by exogenous biotin The results of numerous investigations have demonstrated conclusively that the biosynthesis of amino acids, purines, and pyrimidines in microorganisms is under strict physiological control via feedback or repression mechanisms 1 5. In contrast to such compounds which are subsequently incorporated into macromolecules of the cells, vitamins and coenzymes which are required only in catalytic amounts have been reported to be overproduced, suggesting a lack or inefficiency of biosynthetic control 6`v. Recently, however, there have been ~ndications that the synthesis of vitamins and coenzymes m a y also be subject to physiological control in microorganisms 8 10. During studies on the kinetics of biotin synthesis b y bacteria, data were accumulated demonstrating that the presence of exogenous vitamin affects markedly the amount of biotin produced by the cells. The Crookes strain of Escherichia colt (ATCC 8739) was employed and was grown in a glucose mineral salts medium (glucose, o.j=°/'/o, ammonium sulphate, o.4%" potassium dihydrogen phosphate, titpotassium hydrogen phosphate, and sodium chloride, o.I°o; magnesium sulphate, 0.07% ; sodium citrate, 0.05 %) at p H 6.8, 37 °, on a shaking machine to achieve aerobic conditions. The inoculunl was prepared from bacterial cells washed twice with saline and added to 20o ml of medium with or without supplement of biotin contained in 5oo-ml Erlenmeyer flasks. After incubation for 5 h, Io-ml aliquots of the cultures were removed and centrifuged immediately at 4 ° . The cell-free supernatants were stored in a refrigerator and the recovered cells washed once with saline in a refrigerated centrifuge at 4 °. The washed bacterial cells were then suspended in 2 ml of distilled water and boiled for 20 rain to extract free biotin. The supernatants were collected by centrifugation after the boiled cell suspensions had been diluted to io ml with distilled water. The boiled cells were then hydrolyzed with 0.5 nil of 6 N H2SO 4 at 121 ° for 60 rain for the liberation of bound biotin. The culture supernatants, boiled cell extracts, and the cell hydrolysates were analyzed for biotin content by microbiological assay employing Saccharomvces cerevisiae (Fleischman strain 139 ) . An examination of the data presented (Table I) reveals that the control culture (grown in the absence of exogenous biotin) synthesized large quantities of biotin TA]3LE I SYNTHESIS
OF
BIOTIN
BY
Escherichia colt
l*g biotin )< lO */ml culture
Zero time
After 5 h
medium
cells
total
o 14.8 24.2 32.3 38.4 48.7
1. 5 i. 5 1.5 1.5 1.5 1.5
1. 5 16. 3 25.7 33 .8 39.9 50.2
medium
134.o 83. 5 52.8 33.3 26.1 27.5
cells
total
28.8 28.1 25.3 27.3 28.0 28.o
162.8 111.6 78.1 60.6 54.1 55.5
Total biotin synthesized
161. 3 95-3 52.4 26.8 14.2 5.3
t~iochim. B i o p h y s . A c t a , 65 (1962) 159-16o
16o
PRELIMINARY NOTES
with the bulk appearing in the medium. Moreover, regardless of the concentration of exogenous biotin present in the medium at zero time, the amount of biotin retained b y the bacterial cells, the bulk of which was in a bound form, was remarkably constant. This is in agreement with the results of THOMPSONs who found that the amount of a particular B vitamin retained within the bacterial cell was relatively constant among different species, while the amount found in the medium varied widely. Of particular interest was the finding that the total amount of biotin synthesized by E. coli varied inversely with the amount of biotin present in the culture medium prior to the inoculation of bacteria. Thus, the presence of as little as 14.8. lO -4 ~g of biotin per ml of medium reduced significantly the amount of vitamin synthesized by thebacterialcells, while the addition of 48. 7 • lO -4/zg/ml resulted in almost complete inhibition of biotin synthesis. Such data are consistent with an efficient physiological control mechanism by the bacterial cell over the production of this vitamin. Moreover, the fact that the intracellular level of biotin remained relatively constant also suggests the existence of physiological control since the bound form of the vitamin is considered to be the coenzyme formll, 12. The mechanism of the control of biotin synthesis in E. coli is under investigation at the present time. This study was supported in part by the National Science Foundation (G-I 9 578) and by the Office of Naval Research (NRIo3-555).
Department of Microbiology Medical School, University of Cincinnati, Cincinnati, Ohio (U.S.A.)
CHIK H. PAl HERMAN C. LICHSTEIN
E. UMBARGER AND B. BROWN, J. Biol. Chem., 233 (1958) 415 . S. GOTS, J. Biol. Chem., 228 (I957) 57. A. YATES AND A. B. PARDEE, J. Biol. Chem., 227 (1957) 677. MAGASANIK, Ann. Rev. Microbiol., II (1957) 221. • Cellular Regulatory Mechanisms, Cold Spring H a r b o r S y m p o s i a on Q u a n t i t a t i v e Biology, 26 (1961). R. C. THOMPSON, Univ. Texas Publ., 4237 (1942) 87. A. C. WILSON AND A. B. PARDEE, J. Gen. Microbiol., 28 (1962) 283. s V. H. CHELDELIN AND A. BAICH, Biochim. Biophys. Acta, 46 (1961) 6o7. 9 j . IMSANDE AND A. B. PARDEE, J. Biol. Chem., 237 (1962) 13o 5. 10 M. CHANG AND T. J. BOND, Biochim. Biophys. Acta, 54 (1961) 614. Xl y . KAZlRO, E. LEONE AND S. OCHOA, Proc. Natl. Acad. Sci. U.S., 46 (196o) 1319. 1, S. J. WAKIL AND D. M. GIBSON, Bioehim. Biophys. Acta, 41 (196o) 122. 1 H. J. 8 R. 4 B.
Received August Ioth, 1962 Biochim. Biophys. Acta, 65 (1962) 159-16o
PN 1 1 5 4
A new intermediate in aromatic biosynthesis A scheme outlined recently 1 for the biosynthesis of the aromatic amino acids suggested that the common pathway includes at least one substance (compound X) after shikimic-3-enolpyruvic ether-5-phosphate (compound Z 1 phosphate) ~. Such a scheme is shown in Fig. I. The available evidence also indicated that the bacterial vitamins, 4-aminobenzoic acid and 4-hydroxybenzoic acid could arise from the unknown intermediate. Biochim. Biophys. Acta, 65 (1962) i 6 o - i 6 3
I59
P~ 1166
Control of biotin synthesis in Escherichia coli by exogenous biotin The results of numerous investigations have demonstrated conclusively that the biosynthesis of amino acids, purines, and pyrimidines in microorganisms is under strict physiological control via feedback or repression mechanisms 1 5. In contrast to such compounds which are subsequently incorporated into macromolecules of the cells, vitamins and coenzymes which are required only in catalytic amounts have been reported to be overproduced, suggesting a lack or inefficiency of biosynthetic control 6`v. Recently, however, there have been ~ndications that the synthesis of vitamins and coenzymes m a y also be subject to physiological control in microorganisms 8 10. During studies on the kinetics of biotin synthesis b y bacteria, data were accumulated demonstrating that the presence of exogenous vitamin affects markedly the amount of biotin produced by the cells. The Crookes strain of Escherichia colt (ATCC 8739) was employed and was grown in a glucose mineral salts medium (glucose, o.j=°/'/o, ammonium sulphate, o.4%" potassium dihydrogen phosphate, titpotassium hydrogen phosphate, and sodium chloride, o.I°o; magnesium sulphate, 0.07% ; sodium citrate, 0.05 %) at p H 6.8, 37 °, on a shaking machine to achieve aerobic conditions. The inoculunl was prepared from bacterial cells washed twice with saline and added to 20o ml of medium with or without supplement of biotin contained in 5oo-ml Erlenmeyer flasks. After incubation for 5 h, Io-ml aliquots of the cultures were removed and centrifuged immediately at 4 ° . The cell-free supernatants were stored in a refrigerator and the recovered cells washed once with saline in a refrigerated centrifuge at 4 °. The washed bacterial cells were then suspended in 2 ml of distilled water and boiled for 20 rain to extract free biotin. The supernatants were collected by centrifugation after the boiled cell suspensions had been diluted to io ml with distilled water. The boiled cells were then hydrolyzed with 0.5 nil of 6 N H2SO 4 at 121 ° for 60 rain for the liberation of bound biotin. The culture supernatants, boiled cell extracts, and the cell hydrolysates were analyzed for biotin content by microbiological assay employing Saccharomvces cerevisiae (Fleischman strain 139 ) . An examination of the data presented (Table I) reveals that the control culture (grown in the absence of exogenous biotin) synthesized large quantities of biotin TA]3LE I SYNTHESIS
OF
BIOTIN
BY
Escherichia colt
l*g biotin )< lO */ml culture
Zero time
After 5 h
medium
cells
total
o 14.8 24.2 32.3 38.4 48.7
1. 5 i. 5 1.5 1.5 1.5 1.5
1. 5 16. 3 25.7 33 .8 39.9 50.2
medium
134.o 83. 5 52.8 33.3 26.1 27.5
cells
total
28.8 28.1 25.3 27.3 28.0 28.o
162.8 111.6 78.1 60.6 54.1 55.5
Total biotin synthesized
161. 3 95-3 52.4 26.8 14.2 5.3
t~iochim. B i o p h y s . A c t a , 65 (1962) 159-16o
16o
PRELIMINARY NOTES
with the bulk appearing in the medium. Moreover, regardless of the concentration of exogenous biotin present in the medium at zero time, the amount of biotin retained b y the bacterial cells, the bulk of which was in a bound form, was remarkably constant. This is in agreement with the results of THOMPSONs who found that the amount of a particular B vitamin retained within the bacterial cell was relatively constant among different species, while the amount found in the medium varied widely. Of particular interest was the finding that the total amount of biotin synthesized by E. coli varied inversely with the amount of biotin present in the culture medium prior to the inoculation of bacteria. Thus, the presence of as little as 14.8. lO -4 ~g of biotin per ml of medium reduced significantly the amount of vitamin synthesized by thebacterialcells, while the addition of 48. 7 • lO -4/zg/ml resulted in almost complete inhibition of biotin synthesis. Such data are consistent with an efficient physiological control mechanism by the bacterial cell over the production of this vitamin. Moreover, the fact that the intracellular level of biotin remained relatively constant also suggests the existence of physiological control since the bound form of the vitamin is considered to be the coenzyme formll, 12. The mechanism of the control of biotin synthesis in E. coli is under investigation at the present time. This study was supported in part by the National Science Foundation (G-I 9 578) and by the Office of Naval Research (NRIo3-555).
Department of Microbiology Medical School, University of Cincinnati, Cincinnati, Ohio (U.S.A.)
CHIK H. PAl HERMAN C. LICHSTEIN
E. UMBARGER AND B. BROWN, J. Biol. Chem., 233 (1958) 415 . S. GOTS, J. Biol. Chem., 228 (I957) 57. A. YATES AND A. B. PARDEE, J. Biol. Chem., 227 (1957) 677. MAGASANIK, Ann. Rev. Microbiol., II (1957) 221. • Cellular Regulatory Mechanisms, Cold Spring H a r b o r S y m p o s i a on Q u a n t i t a t i v e Biology, 26 (1961). R. C. THOMPSON, Univ. Texas Publ., 4237 (1942) 87. A. C. WILSON AND A. B. PARDEE, J. Gen. Microbiol., 28 (1962) 283. s V. H. CHELDELIN AND A. BAICH, Biochim. Biophys. Acta, 46 (1961) 6o7. 9 j . IMSANDE AND A. B. PARDEE, J. Biol. Chem., 237 (1962) 13o 5. 10 M. CHANG AND T. J. BOND, Biochim. Biophys. Acta, 54 (1961) 614. Xl y . KAZlRO, E. LEONE AND S. OCHOA, Proc. Natl. Acad. Sci. U.S., 46 (196o) 1319. 1, S. J. WAKIL AND D. M. GIBSON, Bioehim. Biophys. Acta, 41 (196o) 122. 1 H. J. 8 R. 4 B.
Received August Ioth, 1962 Biochim. Biophys. Acta, 65 (1962) 159-16o
PN 1 1 5 4
A new intermediate in aromatic biosynthesis A scheme outlined recently 1 for the biosynthesis of the aromatic amino acids suggested that the common pathway includes at least one substance (compound X) after shikimic-3-enolpyruvic ether-5-phosphate (compound Z 1 phosphate) ~. Such a scheme is shown in Fig. I. The available evidence also indicated that the bacterial vitamins, 4-aminobenzoic acid and 4-hydroxybenzoic acid could arise from the unknown intermediate. Biochim. Biophys. Acta, 65 (1962) i 6 o - i 6 3