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Evidence for a functional pyridine nucleotide cycle in Escherichia coli
SHORT COMMUNICATIONS
539
of peptide-cysteine observed earlier in this organism3. The changes in peptide-cysteine were similar to those reported10 for GSH during the cell cycle of C. eZZi;bsoidea. When the level of total cellular acid-soluble 35S is calculated as percent of total cellular N from the data of JOHNSON AND SCHMIDT~ and is plotted along with the cellular levels (T/Ototal cellular N) of acid-soluble free, peptide, and free j&s peptide sulfur amino acids during the cell cycle (Fig. 2), the plot of acid-soluble 35s is identical to the combined plots of the free and peptide sulfur amino acids except between 0.4 and 0.9 of the cell cycle. During this time interval, the peptide sulfur amino acids (predominantly cysteine of GSH) continued to increase while the total 3% of the trichloroacetic acid extract either decreased or remained constant. Thus, the increase in GSH between 0.4 and 0.7 of the cell cycle appears to be occurring at the expense of endogenous sulfur (presumably protein sulfur) assimilated in the previous cell cycle. This investigation was supported in part by the National Science Foundation Grant GB-4682 and by Public Health Service Grant GM-12042 from the National Institute of General Medical Sciences. Thanks are extended to Dr. H. A. Hopkins for his critical review of the manuscript. Department T’irginia
T. A. HARE
of Bioclzemistry and Nutrition, Polytechnic Institute,
Blacksburg,
ROBERT
R. SCHMIDT
Va. 24 061 (U.S.A.)
I R. A. JOHNSON AND R. R. SCHMIDT, Biochim. Biophys. Acta. 74 (1963) 428. 2 R. R. SCHMIDT AND H. T. SPENCER, J. Cellular Comp. Physiol., 64 (1964) 249. 3 T. A. HARE AND R. R. SCHMIDT, J. Cellular Physiol.. in the press. 4 R. R. SCHMIDT, in 1. L. CAMERON AND G. M. PADILLA, Cell Synchrony-Studies in Biosynthetic Regulation, Academic Press, New York, 1966, p. 189. 5 F. L. LIPMANN AND L. C. TUTTLE, J. Biol. Chem., 159 (x945) 21. 6 E. HASE, S. MIHARA, H. OTSUKA AND H. TAMIYA. Biochim. Biophys. Acta, 32 (1959) 298. 7 E. HASE, S. MIHARA, H. OTSUKA AND H. TAMIYA, Arch. Biochem. Biophys., 83 (1959) 170. 8 P. B. HAMILTON, Anal. Chem., 30 (1958) 914. 9 3%.CALVIN, in S. COLOWICK, A. LAZAROW, E. RACKER, D. R. SCHWARZ, E. STADTMAN AND H. WAELSCH, Glutathione: A Symposium, Academic Press, New York, 1954. p. 3. IO T. KANAZAWA, Plant Cell Physiol. Tokyo, 5 (1964) 333. II T. A. HARE AND R. R. SCHMIDT, Appl. Microbial., 16 (1968) 496.
Received
August
r8th,
1969 Biochim.
Biophys.
Acta,
192 (1969) 537-539
nnA 23 549 Evidence
for a functional
pyridine
nucleotide
cycle in Escherichia co/i
The de novo synthesis of NAD in Escherichia coli occurs through the synthesis of quinolinate from aspartate and a 3-carbon compound (Reaction a, Scheme I)~ and its subsequent conversion into NAD (Reactions b, c, and d), but without the occurrence of nicotinate as a free intermediate2. In addition to the de nova pathway, E. coli can also utilize nicotinate or nicotinamide sp4 for NAD synthesis by employing a salvage pathway (Reactions f and g). More recently5, it has also been demonstrated that this pathway is essential for the utilization of exogenous NAD. Biochim.
Biophys.
Acta,
192 (1969) 539-541
SHORT
540
COMMUNICATIOSS
Several lines of indirect evidence have been offered supporting the concept that NAD turnover occurs in various organisms and that the nicotinamide formed can be recvcled by the salvage pathway, resulting in the resynthesis of NAD3. Other studies, however, have led to the conclusion that no significant NAD turnover occurs in E. coliG~‘. The present communication offers direct evidence for NAD turnover and the presence of a functional pyridine nucleotide cycle in E. coli. Quinolinate
-
Nfcotinoty mononucleotlde
(b)
(c)
Deamido
-NAD
t 0)
Ig:f
1 Aspartate
Nicotinate
\
~
JD
+ i3
Scheme
Nicotinomide
I. NAD
biosynthesis
in E. coli.
Evidence indicating that this cycle is operative in E. coli was obtained in crossfeeding (syntrophism) experiments. When cells containing mutations in the deamidase locus only (Reaction f), e.g. RS-2, were streaked on unsupplemented agar plates at right angles to cells which utilize either nicotinate or nicotinamide (126, Reaction b or W-3899, Reaction a) growth of the latter type of cells was observed. However, when the same type of cross-feeding experiment was attempted with auxotrophs which utilize nicotinate but not nicotinamide (RS-126, Reactions b and f or V-3S95 Namer, Reactions a and f), growth of these mutants was not observed. Neither class of mutants (126 or RS-126) was cross-fed by wild-type E. coli K-12 cells. These results indicate that RS-2 cells excrete nicotinamide and suggest that in the normal cell the pyridine nucleotide cycle is operative but with an active nicotinamide deamidase present the nicotinamide formed as a consequence of NAD turnover is reutilized and is not excreted into the medium. To confirm this hypothesis, the following experiments were conducted: Strains 126, KS-126 and RS-2 were grown in medium9 supplemented with 2.14 ,uM y-l”C]nicotinate. At appropriate intervals aliquots were centrifuged free of cells, the radioisotope containing supernatant was resolved by high-voltage paper electrophoresis and paper chromatography9 and the nicotinate and nicotinamide content determined by measuring the radioactivity with a Nuclear-Chicago Actigraph III. The data presented (Table I) show that during the course of active growth cells of nicotinateauxotroph 126 depleted the medium of nicotinate and did not excrete nicotinamide, whereas RS-126 cells lacking nicotinamide deamidase5 (double-mutant derived from 126) excreted nicotinamide. RS-z cells also demonstrated nicotinate uptake despite an intact de novo NAD biosynthetic pathway, but lacking nicotinamide deamidase, actively excreted nicotinamide. The growth of wild-type cells, as in the case of 126, resulted in the uptake of nicotinate without nicotinamide excretion. From the evidence presented it can be concluded that NAD turnover does occur in actively growing nicotinateauxotroph and prototroph cells of E. coli, but is only evident when the salvage pathway is blocked, resulting in the excretion of a product typical of turnover, e.g. nicotinamide. Conversely, the presence of a functional pyridine nucleotide cycle allows normal cells to utilize turnover products for NAD Biochim.
Biophys.
Acta,
rgz
(1969)
53g-5+1
SHORT
541
COMMUNICATIONS
TABLE
I EXCRETION DURING GROWTH 0F E. coli
NICOTINATE UPTAKE AND NICOTINAMIDE
Time
(h)
Medium A 540em
Strain RS-2
Strain RS-126
Strain 126
Medium Nicotinate Nicotinamide A 54~nm (nmoleslml (nmoleslml (nmoles/ml medium) medium) medium) Nicotinate
0.1
2.14
0.2
I
.go
Nicotinamide (nmoleslml medium)
Medium Nicotinate A j40 nm (nmoles~ml medium)
Nicotinamide (nmoleslml medium)
0
0.1
2.14
0
0.2
2.14
0
0
0.2
1.80
0.*2
0.5
I.59
Trace
1.3
1.00
0.15
0.4
1.58
0
0.j
0.9
0.95
0
1.1
I.33 0.33
0.44 0.90
I.5
0.38
0
‘.7
0.22
1.32
2.6 3.0
0.45 0.41
0.35 0.62
2.2
0.18
0
2.3
0.16
I.51
3.1
O,43
0.71
2.7
0.19
0
2.6
0.16
I.69
resynthesis. These results also agree with earlier demonstrations of the key role played by nicotinamide deamidase in the utilization of exogenous nicotinamide and NAD by E. coli4s5. This investigation was supported in part by research grants from the National Science Foundation (GB-8112) and the U.S. Public Health Service (GM-100006). One of the authors (R. K. G.) is a Career Development Awardee 5-K3-GM-9252, National Institutes of Health, U.S. Public Health Service. Department of Chemistry, California State College, Los Angeles, Calif. 90032 Department
A.J. (U.S.A.)
of Biology, The University of Texas
R. K. GHOLSON* T. S. MATNEY
dd. D. Anderson
Houston,
ANDREOLI
T. GROVER
Hospital and Tumor Institute, Texas 77025 (U.S.A.)
I N. OGASAWARA, J. L. R. CHANDLER, R. K. GHOLSON, R. J. ROSSER AND A. J. ANDREOLI,
Biochim. Biophys. Acta, 141 (1967) rgg. 2 A. J. ANDREOLI, M. IKEDA, Y. NISHIZUKA AND 0. HAYAISHI, Biochem. Biophys.
3 4
5 6 7 8 g
Res. Commun.,
12 (1963) 92. R. K. GHOLSON, Nature, 212 (1966) 933. T. K. SUNDARAM, Biochim. Biophys. Acta, 136 (1967) 586. R. K. GHOLSON, G. J. TRITZ, T. S. MATNEY AND A. J. ANDREOLI, J. Bacterial., gg (1969) 895. J. IMSANDE AND A. B. PARDEE, J. Biol. Chem., 237 (1962) 1305. M. IIZUKA AND D. MIZUNO, Biochim. Biophys. Acta, 148 (1967) 320. R. A. YATES AND A. B. PARDEE, J. Biol. Chem., 221 (1956) 743. R. E. SAXTON, V. ROCHA, R. J. ROSSER, A. J. ANDREOLI, M. SHIMOYAMA, A. KOSAKA, J. L. R. CHANDLER AND R. K. GHOLSON, Biochim. Biophys. Acta, 156 (1968) 77.
Received
September
* Permanent Okla., U.S.A.
8th, 1969
address: Department
of Biochemistry,
Oklahoma
State University.
Stillwater,
Biochim. Biophys. Acta, Igz (1969) 539-541
539
of peptide-cysteine observed earlier in this organism3. The changes in peptide-cysteine were similar to those reported10 for GSH during the cell cycle of C. eZZi;bsoidea. When the level of total cellular acid-soluble 35S is calculated as percent of total cellular N from the data of JOHNSON AND SCHMIDT~ and is plotted along with the cellular levels (T/Ototal cellular N) of acid-soluble free, peptide, and free j&s peptide sulfur amino acids during the cell cycle (Fig. 2), the plot of acid-soluble 35s is identical to the combined plots of the free and peptide sulfur amino acids except between 0.4 and 0.9 of the cell cycle. During this time interval, the peptide sulfur amino acids (predominantly cysteine of GSH) continued to increase while the total 3% of the trichloroacetic acid extract either decreased or remained constant. Thus, the increase in GSH between 0.4 and 0.7 of the cell cycle appears to be occurring at the expense of endogenous sulfur (presumably protein sulfur) assimilated in the previous cell cycle. This investigation was supported in part by the National Science Foundation Grant GB-4682 and by Public Health Service Grant GM-12042 from the National Institute of General Medical Sciences. Thanks are extended to Dr. H. A. Hopkins for his critical review of the manuscript. Department T’irginia
T. A. HARE
of Bioclzemistry and Nutrition, Polytechnic Institute,
Blacksburg,
ROBERT
R. SCHMIDT
Va. 24 061 (U.S.A.)
I R. A. JOHNSON AND R. R. SCHMIDT, Biochim. Biophys. Acta. 74 (1963) 428. 2 R. R. SCHMIDT AND H. T. SPENCER, J. Cellular Comp. Physiol., 64 (1964) 249. 3 T. A. HARE AND R. R. SCHMIDT, J. Cellular Physiol.. in the press. 4 R. R. SCHMIDT, in 1. L. CAMERON AND G. M. PADILLA, Cell Synchrony-Studies in Biosynthetic Regulation, Academic Press, New York, 1966, p. 189. 5 F. L. LIPMANN AND L. C. TUTTLE, J. Biol. Chem., 159 (x945) 21. 6 E. HASE, S. MIHARA, H. OTSUKA AND H. TAMIYA. Biochim. Biophys. Acta, 32 (1959) 298. 7 E. HASE, S. MIHARA, H. OTSUKA AND H. TAMIYA, Arch. Biochem. Biophys., 83 (1959) 170. 8 P. B. HAMILTON, Anal. Chem., 30 (1958) 914. 9 3%.CALVIN, in S. COLOWICK, A. LAZAROW, E. RACKER, D. R. SCHWARZ, E. STADTMAN AND H. WAELSCH, Glutathione: A Symposium, Academic Press, New York, 1954. p. 3. IO T. KANAZAWA, Plant Cell Physiol. Tokyo, 5 (1964) 333. II T. A. HARE AND R. R. SCHMIDT, Appl. Microbial., 16 (1968) 496.
Received
August
r8th,
1969 Biochim.
Biophys.
Acta,
192 (1969) 537-539
nnA 23 549 Evidence
for a functional
pyridine
nucleotide
cycle in Escherichia co/i
The de novo synthesis of NAD in Escherichia coli occurs through the synthesis of quinolinate from aspartate and a 3-carbon compound (Reaction a, Scheme I)~ and its subsequent conversion into NAD (Reactions b, c, and d), but without the occurrence of nicotinate as a free intermediate2. In addition to the de nova pathway, E. coli can also utilize nicotinate or nicotinamide sp4 for NAD synthesis by employing a salvage pathway (Reactions f and g). More recently5, it has also been demonstrated that this pathway is essential for the utilization of exogenous NAD. Biochim.
Biophys.
Acta,
192 (1969) 539-541
SHORT
540
COMMUNICATIOSS
Several lines of indirect evidence have been offered supporting the concept that NAD turnover occurs in various organisms and that the nicotinamide formed can be recvcled by the salvage pathway, resulting in the resynthesis of NAD3. Other studies, however, have led to the conclusion that no significant NAD turnover occurs in E. coliG~‘. The present communication offers direct evidence for NAD turnover and the presence of a functional pyridine nucleotide cycle in E. coli. Quinolinate
-
Nfcotinoty mononucleotlde
(b)
(c)
Deamido
-NAD
t 0)
Ig:f
1 Aspartate
Nicotinate
\
~
JD
+ i3
Scheme
Nicotinomide
I. NAD
biosynthesis
in E. coli.
Evidence indicating that this cycle is operative in E. coli was obtained in crossfeeding (syntrophism) experiments. When cells containing mutations in the deamidase locus only (Reaction f), e.g. RS-2, were streaked on unsupplemented agar plates at right angles to cells which utilize either nicotinate or nicotinamide (126, Reaction b or W-3899, Reaction a) growth of the latter type of cells was observed. However, when the same type of cross-feeding experiment was attempted with auxotrophs which utilize nicotinate but not nicotinamide (RS-126, Reactions b and f or V-3S95 Namer, Reactions a and f), growth of these mutants was not observed. Neither class of mutants (126 or RS-126) was cross-fed by wild-type E. coli K-12 cells. These results indicate that RS-2 cells excrete nicotinamide and suggest that in the normal cell the pyridine nucleotide cycle is operative but with an active nicotinamide deamidase present the nicotinamide formed as a consequence of NAD turnover is reutilized and is not excreted into the medium. To confirm this hypothesis, the following experiments were conducted: Strains 126, KS-126 and RS-2 were grown in medium9 supplemented with 2.14 ,uM y-l”C]nicotinate. At appropriate intervals aliquots were centrifuged free of cells, the radioisotope containing supernatant was resolved by high-voltage paper electrophoresis and paper chromatography9 and the nicotinate and nicotinamide content determined by measuring the radioactivity with a Nuclear-Chicago Actigraph III. The data presented (Table I) show that during the course of active growth cells of nicotinateauxotroph 126 depleted the medium of nicotinate and did not excrete nicotinamide, whereas RS-126 cells lacking nicotinamide deamidase5 (double-mutant derived from 126) excreted nicotinamide. RS-z cells also demonstrated nicotinate uptake despite an intact de novo NAD biosynthetic pathway, but lacking nicotinamide deamidase, actively excreted nicotinamide. The growth of wild-type cells, as in the case of 126, resulted in the uptake of nicotinate without nicotinamide excretion. From the evidence presented it can be concluded that NAD turnover does occur in actively growing nicotinateauxotroph and prototroph cells of E. coli, but is only evident when the salvage pathway is blocked, resulting in the excretion of a product typical of turnover, e.g. nicotinamide. Conversely, the presence of a functional pyridine nucleotide cycle allows normal cells to utilize turnover products for NAD Biochim.
Biophys.
Acta,
rgz
(1969)
53g-5+1
SHORT
541
COMMUNICATIONS
TABLE
I EXCRETION DURING GROWTH 0F E. coli
NICOTINATE UPTAKE AND NICOTINAMIDE
Time
(h)
Medium A 540em
Strain RS-2
Strain RS-126
Strain 126
Medium Nicotinate Nicotinamide A 54~nm (nmoleslml (nmoleslml (nmoles/ml medium) medium) medium) Nicotinate
0.1
2.14
0.2
I
.go
Nicotinamide (nmoleslml medium)
Medium Nicotinate A j40 nm (nmoles~ml medium)
Nicotinamide (nmoleslml medium)
0
0.1
2.14
0
0.2
2.14
0
0
0.2
1.80
0.*2
0.5
I.59
Trace
1.3
1.00
0.15
0.4
1.58
0
0.j
0.9
0.95
0
1.1
I.33 0.33
0.44 0.90
I.5
0.38
0
‘.7
0.22
1.32
2.6 3.0
0.45 0.41
0.35 0.62
2.2
0.18
0
2.3
0.16
I.51
3.1
O,43
0.71
2.7
0.19
0
2.6
0.16
I.69
resynthesis. These results also agree with earlier demonstrations of the key role played by nicotinamide deamidase in the utilization of exogenous nicotinamide and NAD by E. coli4s5. This investigation was supported in part by research grants from the National Science Foundation (GB-8112) and the U.S. Public Health Service (GM-100006). One of the authors (R. K. G.) is a Career Development Awardee 5-K3-GM-9252, National Institutes of Health, U.S. Public Health Service. Department of Chemistry, California State College, Los Angeles, Calif. 90032 Department
A.J. (U.S.A.)
of Biology, The University of Texas
R. K. GHOLSON* T. S. MATNEY
dd. D. Anderson
Houston,
ANDREOLI
T. GROVER
Hospital and Tumor Institute, Texas 77025 (U.S.A.)
I N. OGASAWARA, J. L. R. CHANDLER, R. K. GHOLSON, R. J. ROSSER AND A. J. ANDREOLI,
Biochim. Biophys. Acta, 141 (1967) rgg. 2 A. J. ANDREOLI, M. IKEDA, Y. NISHIZUKA AND 0. HAYAISHI, Biochem. Biophys.
3 4
5 6 7 8 g
Res. Commun.,
12 (1963) 92. R. K. GHOLSON, Nature, 212 (1966) 933. T. K. SUNDARAM, Biochim. Biophys. Acta, 136 (1967) 586. R. K. GHOLSON, G. J. TRITZ, T. S. MATNEY AND A. J. ANDREOLI, J. Bacterial., gg (1969) 895. J. IMSANDE AND A. B. PARDEE, J. Biol. Chem., 237 (1962) 1305. M. IIZUKA AND D. MIZUNO, Biochim. Biophys. Acta, 148 (1967) 320. R. A. YATES AND A. B. PARDEE, J. Biol. Chem., 221 (1956) 743. R. E. SAXTON, V. ROCHA, R. J. ROSSER, A. J. ANDREOLI, M. SHIMOYAMA, A. KOSAKA, J. L. R. CHANDLER AND R. K. GHOLSON, Biochim. Biophys. Acta, 156 (1968) 77.
Received
September
* Permanent Okla., U.S.A.
8th, 1969
address: Department
of Biochemistry,
Oklahoma
State University.
Stillwater,
Biochim. Biophys. Acta, Igz (1969) 539-541