JOURNAL OF BACTERIOLOGY, Nov. 1977, p. 590-595 Copyright g 1977 American Society for Microbiology
Vol. 132, No. 2 Printed in U.S.A.
Cleavage of Adenosine 5'-Monophosphate During Uptake by Streptomyces griseus JUAN F. MARTIN, AND ARNOLD L DEMAIN* Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication 31 May 1977
Unlabeled adenine brought about a (delayed) decrease in radioactivity that had been taken up by phosphate-limited resting cells of Streptomyces griseus from 114C]adenine-labeled adenosine 5'-monophosphate (AMP). Inorganic phosphate, on the other hand, stimulated adenine uptake from AMP, presumably by activating an energy-dependent active transport mechanism. Unlabeled phosphate rapidly diluted the uptake of radioactivity from [':2 PIAMP. Adenine inhibited uptake of [32 P]AMP but not that of r32P]orthophosphate; adenine is thought to act by inhibiting the cleavage of AMP. The uptake of :2P and 14C from double-labeled AMP showed marked differences; :12P was taken up much faster into both cells and nucleic acids. These data indicate that uptake of AMP components takes place after extracellular dephosphorylation of the nucleotide. MATERIALS AND METHODS While studying the regulation of candicidin biosynthesis by Streptomyces griseus, we obPreparation of the resting-cell system. We used served that antibiotic formation was inhibited S. griseus IMRU 3570, the strain producing the by exogenous phosphate (16) and by cyclic and polyene macrolide antibiotic candicidin. Cultures noncyclic nucleotides but not by the correspond- were grown in a glucose-soya peptone medium (17), ing nucleosides or by the bases themselves (J. which supports high antibiotic production, in 250F. Martin and A. L. Demain, Can. J. Micro- ml baffled flasks on a rotary shaker at 32°C and 250 Under these conditions cells started producing biol., in press). Whether intact nucleotides rpm. candicidin 18 h of growth. Cells were collected could enter the cell or whether they must be after 20 h after growth, washed, and suspended at 5 dephosphorylated before uptake was not mg of cellsof(dry weight) per ml in a phosphateknown. Although most phosphorylated com- limited, chemically defined medium as described pounds are hydrolyzed before uptake, some- previously (18). Under phosphate limitation, the such as L-a-glycerol phosphate (7), glucose 6- dry weight of the cells did not increase, but candiciphosphate (5). glucose 1-phosphate (3), and din was produced linearly with respect to time (19). Uptake experiments. Radioactive compounds other hexose phosphates (20)-are taken up in their intact form via active transport mecha- were added to the phosphate-limited cell system nisms. Evidence shows that, in Escherichia previously supplemented with either unlabeled phosphate (2.5 mM) or AMP (2.5 mM). The concencoli, hydrolysis of adenosine 5'-monophosphate trations and specific radioactivities of these com(AMP) and cytidine 5'-monophosphate takes pounds are indicated in the figure legends and in place before uptake (1, 13, 21). The enzyme 5'- this section, respectively. At short intervals, 250-gl nucleotidase (uridine diphosphate sugar hydro- samples were placed in 5 ml of ice-cold unlabeled lase, EC 3.1.3.5), which cleaves AMP to adeno- phosphate (5 mM) or AMP 1 mM) in a Buchnersine and phosphate, seems to be a periplasmic Millipore filter apparatus. The suspension was immediately filtered under vacuum through Whatman enzyme in this organism (21). No information is available on nucleotide GF/A glass-fiber filters. The filters were washed uptake in Streptomyces. We have found that with 20 ml of either of the unlabeled solutions, and counted in a Beckman LS-230 scintillaexogenous nucleotides inhibit antibiotic syn- dried, tion counter by the use of a naphthalene-dioxanethesis immediately after their addition, sug- 2,5-diphenyloxazole (10:100:0.8, vol/vol/wt) scintilgesting a rapid uptake of either the intact lation fluid. In the double-label experiments, the nucleotide or some component. We undertook filtrates were collected in tubes placed inside the this study to examine nucleotide assimilation vacuum flasks and 100-,41 fractions were counted. in S. griseus. Double-label determinations were made by the Hash method (6). For studies dealing with incorporation into nucleic acid, 100 ,ul of the resting cell culture was taken into 5 ml of ice-cold trichloroacetic acid. After 3 h the precipitate was filtered
t Present address: Instituto de Microbiologia Bioquimica,
CSIC, Departamento de Microbiologia, Facultad de Ciencias, Universidad de Salamanca, Salamanca, Spain.
590
VOL. 132, 1977
AMP CLEAVAGE DURING UPTAKE
through Whatman GF/A filters as before, and the filters were washed with 20 ml of cold trichloroacetic acid. Chemicals and radiochemicals. The crystalline sodium salt of AMP was obtained from Sigma Chemical Co., St. Louis, Mo. 18-'4C]adenosine 5'monophosphate ammonium salt (60 mCi/mmol) and adenosine 5'-monol72 P]phosphate ammonium salt (500 mCi/mmol) were purchased from the Radiochemical Centre, Amersham/Searle Corp., Arlington Heights, Ill. Disodium 132Plphosphate was obtained from New England Nuclear Corp., Boston, Mass. Adenine and all other chemicals were of reagent quality.
RESULTS Effect of unlabeled adenine on the uptake of label from adenine-labeled AMP. To establish whether AMP was cleaved to adenine, excess (5 mM) unlabeled phosphate or adenine was added to flasks that were supplemented with V14C]adenine-labeled AMP. When unlabeled adenine was added, radioactivity continued to be incorporated for 3 min, but the amount of label soon decreased (Table 1). Phosphate, on the other hand, stimulated label uptake. Similar experiments were carried out in which 5 mM unlabeled adenine was added to the culture immediately before the addition of 1 4C]AMP. Although the initial uptake was high, cold adenine inhibited the uptake of radioactivity from 114C]AMP after 5 min. Stimulation by phosphate of label uptake from adenine-labeled AMP. The data shown in Table 1 suggest that phosphate might have a stimulatory effect on the uptake of label from I1l C]adenine-labeled AMP. To further check this point, we added phosphate (5 mM) to a
I14C]AMP-supplemented
culture before addi-
tion of the label. Phosphate supplementation resulted in a drastic increase in label uptake in the first 10 min (Fig. 1). This result was probably caused by activation of an active transport mechanism (see Discussion). Dilution effect of unlabeled phosphate on label uptake from P2 PIAMP. Using TABLE 1. Effect of phosphate
or
adenine
on
[14C]AMP uptakea
Addition
4C uptakeb at indicated time (min) after addition:
0 None (control) ......... 6,813 5 mM phosphate ....... 6,364 5 mM adenine ......... 6,454
3 7,353 7,378 7,490
11
18
7,612 9,047 6,185
8,115 9,284 5,417
Excess unlabeled adenine (5 mM) or phosphate (5 mM) added 13 min after addition of [14C]AMP. b Expressed as counts per minute per milligram of cell
a
was
(dry weight).
591
4 --l
+
PHOSPHATE
~~(5 mM)
3_ a-
I
2 2
I/
CONTROL *
1rI
L
m
0
A
/
*~~ -~
J
LLJ y
O Q
0
,
5
I
I_
10 15 20 TIME (min.)
25
FIG. 1. Stimulation by phosphate of the uptake of label from [14C]adenine-labeled AMP. Cultures were supplemented with 2.5 mM unlabeled AMP for 20 min before addition of ["ClAMP (0.5 ,Cilml of culture). Excess phosphate (5 mM) was added immediately before addition of the radioactive AMP. Radioactivity was measured in counts per minute per microgram (dry weight).
312P]phosphate-labeled AMP, we conducted a dilution experiment in which 5 mM unlabeled phosphate was added during label uptake. Excess unlabeled phosphate diluted the uptake of radioactivity from [3'2P]AMP (Fig. 2), suggesting again that AMP is dephosphorylated before uptake. Inhibitory effect of adenine on label uptake from [312P]AMP. Excess adenine would not be expected to have a dilution effect on the uptake of label from 1312 P]phosphate-labeled AMP. However, 5 mM adenine clearly inhibited the uptake of [3'2P]phosphate from AMP (Fig. 2). There are two possible reasons for this phenomenon: adenine inhibits the activity of the enzyme that cleaves AMP, or adenine inhibits the uptake of phosphate itself after the AMP has been cleaved.
592
J. BACTERIOL.
MARTIN AND DEMAIN
fect on the uptake of phosphate label from AMP, it did not affect the uptake of free phosphate (Fig. 4). Therefore, excess adenine probably acts by inhibiting AMP cleavage rather than by interfering with the phosphate transport system. Nature of intracellular phosphate: incorporation of phosphate and AMP into nucleic acids. To determine whether the rapid uptake of free phosphate resulted in a more rapid labeling of nucleic acids (as compared with the incorporation of label from :12 P]AMP), we studied the incorporation of:12 P from labeled inorganic phosphate and AMP into nucleic acids (cold trichloroacetic acid-precipitable material). Our results (Fig. 4) show that free phosphate labeled nucleic acid much faster than phosphate from AMP. The data also indicate that,
I'
12
aa0
CLi -J
m LLI
0
wIw
20 TIME (min.) FIG. 2. Dilution effect of phosphate and inhibitory effect of adenine on the uptake of label from phosphate-labeled AMP. Cultures were supplemented with 0.5 mM unlabeled AMP before addition of f32P]AMP (5 x 105 cpmlmlb of culture). Adenine and phosphate at 5 mM (final concentration) were added at the times indicated by the arrows. Radioactivity was measured in counts per minute per microgram (dry weight).
To determine whether adenine had any effect on the uptake of phosphate, we compared the uptake of free phosphate to that of AMP labeled in the phosphate moiety, both in the presence and absence of added 5 mM adenine. The results (Fig. 3) indicated that S. griseus incorporated free phosphate at a much faster rate than it did the label from AMP. This finding suggests that, if AMP is cleaved before phosphate uptake, the hydrolysis of the 5'-nucleotide is the rate-limiting step. Almost all of the added labeled phosphate was taken up in the first minutes of incubation. Such a rapid uptake of free phosphate is to be expected in phosphate-limited resting cells. A high-affinity transport of phosphate, which involves de novo protein synthesis, has been found in Neurospora under conditions of phosphorus starvation; this mechanism occurs in addition to the constitutive, low-affinity phosphate transport system (14). Although adenine had a clear inhibitory ef-
a-Icn 0
z
aa-
2 0 U)
a-
a-
pi
2
0
0
LL.
LL -J
-J Lb
m
w
IL
-J
0
0
LI y
w y
a.
a-
m
TIME (min.) FIG. 3. Comparative uptake of label from inorganic f32Pjphosphate and [3]PjAMP and the effect of adenine on these processes. Duplicate series of flasks were supplemented with unlabeled AMP (2.5 mM) or disodium phosphate (2.5 mM). After 15 min, radioactiuity (5 x 10- cpmlml) from either [32P]AMP or [32P]disodium phosphate, respectively, was added. Excess unlabeled adenine (5 mM) was added to [32P]AMP-supplemented flasks at 5 min after addition of the radioactive compound and to [32P]phosphate-supplemented flasks 10 min before addition of radioactivity. Radioactivity was measured in counts per minute per microgram (dry weight).
VOL. 132, 1977
AMP CLEAVAGE DURING UPTAKE
593
disappearance from the medium, is shown in Fig. 5. Uptake of P label was rapid ^ A and that of the adenine moiety (14C label) was A A very slow. Up to 30 min after AMP addition, A TOTAL} only the phosphate label was removed from Xthe medium, whereas 'IC label remained enUPTAKE FROM / tirely in the medium. We found similar results ~~~~~INORGANIC PHOSPHATE when we studied the incorporation of label from double-labeled AMP into nucleic acid (Fig. 6). S NUCLEIC ACIDS In, / ^_X^DISCUSSION / , _ -The results of this work indicate that, in S. X griseus (as in E. coll 1211 and Salmonella l typhimurium [ 11]), uptake of AMP components into the cell took place after the nucleotide was as their
*-
80 XL
Vol. 132, No. 2 Printed in U.S.A.
Cleavage of Adenosine 5'-Monophosphate During Uptake by Streptomyces griseus JUAN F. MARTIN, AND ARNOLD L DEMAIN* Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication 31 May 1977
Unlabeled adenine brought about a (delayed) decrease in radioactivity that had been taken up by phosphate-limited resting cells of Streptomyces griseus from 114C]adenine-labeled adenosine 5'-monophosphate (AMP). Inorganic phosphate, on the other hand, stimulated adenine uptake from AMP, presumably by activating an energy-dependent active transport mechanism. Unlabeled phosphate rapidly diluted the uptake of radioactivity from [':2 PIAMP. Adenine inhibited uptake of [32 P]AMP but not that of r32P]orthophosphate; adenine is thought to act by inhibiting the cleavage of AMP. The uptake of :2P and 14C from double-labeled AMP showed marked differences; :12P was taken up much faster into both cells and nucleic acids. These data indicate that uptake of AMP components takes place after extracellular dephosphorylation of the nucleotide. MATERIALS AND METHODS While studying the regulation of candicidin biosynthesis by Streptomyces griseus, we obPreparation of the resting-cell system. We used served that antibiotic formation was inhibited S. griseus IMRU 3570, the strain producing the by exogenous phosphate (16) and by cyclic and polyene macrolide antibiotic candicidin. Cultures noncyclic nucleotides but not by the correspond- were grown in a glucose-soya peptone medium (17), ing nucleosides or by the bases themselves (J. which supports high antibiotic production, in 250F. Martin and A. L. Demain, Can. J. Micro- ml baffled flasks on a rotary shaker at 32°C and 250 Under these conditions cells started producing biol., in press). Whether intact nucleotides rpm. candicidin 18 h of growth. Cells were collected could enter the cell or whether they must be after 20 h after growth, washed, and suspended at 5 dephosphorylated before uptake was not mg of cellsof(dry weight) per ml in a phosphateknown. Although most phosphorylated com- limited, chemically defined medium as described pounds are hydrolyzed before uptake, some- previously (18). Under phosphate limitation, the such as L-a-glycerol phosphate (7), glucose 6- dry weight of the cells did not increase, but candiciphosphate (5). glucose 1-phosphate (3), and din was produced linearly with respect to time (19). Uptake experiments. Radioactive compounds other hexose phosphates (20)-are taken up in their intact form via active transport mecha- were added to the phosphate-limited cell system nisms. Evidence shows that, in Escherichia previously supplemented with either unlabeled phosphate (2.5 mM) or AMP (2.5 mM). The concencoli, hydrolysis of adenosine 5'-monophosphate trations and specific radioactivities of these com(AMP) and cytidine 5'-monophosphate takes pounds are indicated in the figure legends and in place before uptake (1, 13, 21). The enzyme 5'- this section, respectively. At short intervals, 250-gl nucleotidase (uridine diphosphate sugar hydro- samples were placed in 5 ml of ice-cold unlabeled lase, EC 3.1.3.5), which cleaves AMP to adeno- phosphate (5 mM) or AMP 1 mM) in a Buchnersine and phosphate, seems to be a periplasmic Millipore filter apparatus. The suspension was immediately filtered under vacuum through Whatman enzyme in this organism (21). No information is available on nucleotide GF/A glass-fiber filters. The filters were washed uptake in Streptomyces. We have found that with 20 ml of either of the unlabeled solutions, and counted in a Beckman LS-230 scintillaexogenous nucleotides inhibit antibiotic syn- dried, tion counter by the use of a naphthalene-dioxanethesis immediately after their addition, sug- 2,5-diphenyloxazole (10:100:0.8, vol/vol/wt) scintilgesting a rapid uptake of either the intact lation fluid. In the double-label experiments, the nucleotide or some component. We undertook filtrates were collected in tubes placed inside the this study to examine nucleotide assimilation vacuum flasks and 100-,41 fractions were counted. in S. griseus. Double-label determinations were made by the Hash method (6). For studies dealing with incorporation into nucleic acid, 100 ,ul of the resting cell culture was taken into 5 ml of ice-cold trichloroacetic acid. After 3 h the precipitate was filtered
t Present address: Instituto de Microbiologia Bioquimica,
CSIC, Departamento de Microbiologia, Facultad de Ciencias, Universidad de Salamanca, Salamanca, Spain.
590
VOL. 132, 1977
AMP CLEAVAGE DURING UPTAKE
through Whatman GF/A filters as before, and the filters were washed with 20 ml of cold trichloroacetic acid. Chemicals and radiochemicals. The crystalline sodium salt of AMP was obtained from Sigma Chemical Co., St. Louis, Mo. 18-'4C]adenosine 5'monophosphate ammonium salt (60 mCi/mmol) and adenosine 5'-monol72 P]phosphate ammonium salt (500 mCi/mmol) were purchased from the Radiochemical Centre, Amersham/Searle Corp., Arlington Heights, Ill. Disodium 132Plphosphate was obtained from New England Nuclear Corp., Boston, Mass. Adenine and all other chemicals were of reagent quality.
RESULTS Effect of unlabeled adenine on the uptake of label from adenine-labeled AMP. To establish whether AMP was cleaved to adenine, excess (5 mM) unlabeled phosphate or adenine was added to flasks that were supplemented with V14C]adenine-labeled AMP. When unlabeled adenine was added, radioactivity continued to be incorporated for 3 min, but the amount of label soon decreased (Table 1). Phosphate, on the other hand, stimulated label uptake. Similar experiments were carried out in which 5 mM unlabeled adenine was added to the culture immediately before the addition of 1 4C]AMP. Although the initial uptake was high, cold adenine inhibited the uptake of radioactivity from 114C]AMP after 5 min. Stimulation by phosphate of label uptake from adenine-labeled AMP. The data shown in Table 1 suggest that phosphate might have a stimulatory effect on the uptake of label from I1l C]adenine-labeled AMP. To further check this point, we added phosphate (5 mM) to a
I14C]AMP-supplemented
culture before addi-
tion of the label. Phosphate supplementation resulted in a drastic increase in label uptake in the first 10 min (Fig. 1). This result was probably caused by activation of an active transport mechanism (see Discussion). Dilution effect of unlabeled phosphate on label uptake from P2 PIAMP. Using TABLE 1. Effect of phosphate
or
adenine
on
[14C]AMP uptakea
Addition
4C uptakeb at indicated time (min) after addition:
0 None (control) ......... 6,813 5 mM phosphate ....... 6,364 5 mM adenine ......... 6,454
3 7,353 7,378 7,490
11
18
7,612 9,047 6,185
8,115 9,284 5,417
Excess unlabeled adenine (5 mM) or phosphate (5 mM) added 13 min after addition of [14C]AMP. b Expressed as counts per minute per milligram of cell
a
was
(dry weight).
591
4 --l
+
PHOSPHATE
~~(5 mM)
3_ a-
I
2 2
I/
CONTROL *
1rI
L
m
0
A
/
*~~ -~
J
LLJ y
O Q
0
,
5
I
I_
10 15 20 TIME (min.)
25
FIG. 1. Stimulation by phosphate of the uptake of label from [14C]adenine-labeled AMP. Cultures were supplemented with 2.5 mM unlabeled AMP for 20 min before addition of ["ClAMP (0.5 ,Cilml of culture). Excess phosphate (5 mM) was added immediately before addition of the radioactive AMP. Radioactivity was measured in counts per minute per microgram (dry weight).
312P]phosphate-labeled AMP, we conducted a dilution experiment in which 5 mM unlabeled phosphate was added during label uptake. Excess unlabeled phosphate diluted the uptake of radioactivity from [3'2P]AMP (Fig. 2), suggesting again that AMP is dephosphorylated before uptake. Inhibitory effect of adenine on label uptake from [312P]AMP. Excess adenine would not be expected to have a dilution effect on the uptake of label from 1312 P]phosphate-labeled AMP. However, 5 mM adenine clearly inhibited the uptake of [3'2P]phosphate from AMP (Fig. 2). There are two possible reasons for this phenomenon: adenine inhibits the activity of the enzyme that cleaves AMP, or adenine inhibits the uptake of phosphate itself after the AMP has been cleaved.
592
J. BACTERIOL.
MARTIN AND DEMAIN
fect on the uptake of phosphate label from AMP, it did not affect the uptake of free phosphate (Fig. 4). Therefore, excess adenine probably acts by inhibiting AMP cleavage rather than by interfering with the phosphate transport system. Nature of intracellular phosphate: incorporation of phosphate and AMP into nucleic acids. To determine whether the rapid uptake of free phosphate resulted in a more rapid labeling of nucleic acids (as compared with the incorporation of label from :12 P]AMP), we studied the incorporation of:12 P from labeled inorganic phosphate and AMP into nucleic acids (cold trichloroacetic acid-precipitable material). Our results (Fig. 4) show that free phosphate labeled nucleic acid much faster than phosphate from AMP. The data also indicate that,
I'
12
aa0
CLi -J
m LLI
0
wIw
20 TIME (min.) FIG. 2. Dilution effect of phosphate and inhibitory effect of adenine on the uptake of label from phosphate-labeled AMP. Cultures were supplemented with 0.5 mM unlabeled AMP before addition of f32P]AMP (5 x 105 cpmlmlb of culture). Adenine and phosphate at 5 mM (final concentration) were added at the times indicated by the arrows. Radioactivity was measured in counts per minute per microgram (dry weight).
To determine whether adenine had any effect on the uptake of phosphate, we compared the uptake of free phosphate to that of AMP labeled in the phosphate moiety, both in the presence and absence of added 5 mM adenine. The results (Fig. 3) indicated that S. griseus incorporated free phosphate at a much faster rate than it did the label from AMP. This finding suggests that, if AMP is cleaved before phosphate uptake, the hydrolysis of the 5'-nucleotide is the rate-limiting step. Almost all of the added labeled phosphate was taken up in the first minutes of incubation. Such a rapid uptake of free phosphate is to be expected in phosphate-limited resting cells. A high-affinity transport of phosphate, which involves de novo protein synthesis, has been found in Neurospora under conditions of phosphorus starvation; this mechanism occurs in addition to the constitutive, low-affinity phosphate transport system (14). Although adenine had a clear inhibitory ef-
a-Icn 0
z
aa-
2 0 U)
a-
a-
pi
2
0
0
LL.
LL -J
-J Lb
m
w
IL
-J
0
0
LI y
w y
a.
a-
m
TIME (min.) FIG. 3. Comparative uptake of label from inorganic f32Pjphosphate and [3]PjAMP and the effect of adenine on these processes. Duplicate series of flasks were supplemented with unlabeled AMP (2.5 mM) or disodium phosphate (2.5 mM). After 15 min, radioactiuity (5 x 10- cpmlml) from either [32P]AMP or [32P]disodium phosphate, respectively, was added. Excess unlabeled adenine (5 mM) was added to [32P]AMP-supplemented flasks at 5 min after addition of the radioactive compound and to [32P]phosphate-supplemented flasks 10 min before addition of radioactivity. Radioactivity was measured in counts per minute per microgram (dry weight).
VOL. 132, 1977
AMP CLEAVAGE DURING UPTAKE
593
disappearance from the medium, is shown in Fig. 5. Uptake of P label was rapid ^ A and that of the adenine moiety (14C label) was A A very slow. Up to 30 min after AMP addition, A TOTAL} only the phosphate label was removed from Xthe medium, whereas 'IC label remained enUPTAKE FROM / tirely in the medium. We found similar results ~~~~~INORGANIC PHOSPHATE when we studied the incorporation of label from double-labeled AMP into nucleic acid (Fig. 6). S NUCLEIC ACIDS In, / ^_X^DISCUSSION / , _ -The results of this work indicate that, in S. X griseus (as in E. coll 1211 and Salmonella l typhimurium [ 11]), uptake of AMP components into the cell took place after the nucleotide was as their
*-
80 XL
