JOURNAL OF BACTEmOLOGY, Aug. 1976, p. 770-779
Vol. 127, No. 2 Printed in U.S.A.
Copyright © 1976 American Society for Microbiology
Regulation of Molybdate Transport by Clostridium pasteurianum BARBARA B. ELLIOTT AND L. E. MORTENSON* Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Received for publication 12 April 1976
The regulation of the molybdate (MoO42-) transport activity of Clostridium pasteurianum has been studied by observing the effects of NH:, carbamyl phosphate, MoO42-, and chloramphenicol on the ability of cells to take up MoO42-. Compared with cells fixing N2, cells grown in the presence of 1 mM NH3 are greater than 95% repressed for MoO42- transport. Uptake activity begins to increase just before NH3 exhaustion (under Ar or N2) and continues to increase throughout the lag period as cells shift from NH3-growing to N2-fixing conditions. When cells are shifted from N2-fixing to NH3-growing conditions the transport activity per fixed number of cells decreases by increase of cells in absence of transport synthesis. Carbamyl phosphate (.15 mM) but not NH3 inhibits 58% of the in vitro uptake activity. When 1 mM carbamyl phosphate is added just before the exhaustion of NH3, the transport activity, measured 2 h later, is 100% repressed. Cells grown in the presence of high MoO42- (1 mM) are 80% repressed for MoO42- transport. Synthesis of the MoO42- transport system is also completely stopped when chloramphenicol (300 ,ug/ml) is added just before the exhaustion of NH3 from the medium. These findings demonstrate that the ability of cells to transport MoO42- is dependent upon new protein synthesis and can be repressed by high levels of substrate. The regulation of MoO42- uptake by NH3 or carbamyl phosphate closely parallels the regulation of nitrogenase activity. Activity of neither nitrogenase component (Fe protein or MoFe protein) was detected even 3 h after the exhaustion of the NH3 if either MoO42- was absent or if W042- was present in place of MoO42-. The duration of the diauxic lag increases with decreasing concentration of MoO42- in the medium. If no MoO,2- is present the lag continues indefinitely. If MoO42- is added late in the lag period, growth under N2-fixing conditions resumes but only after a normal induction period. It has long been known that molybdenum is an essential element for biological dinitrogen fixation (3), and it is now known that molybde-
num is a constituent of the MoFe protein component of the nitrogenase complex (5, 10, 12, 16). Recently, attempts have been made to characterize the role of molybdenum in the catalytic reactions of the nitrogenase components (2, 7). Elliott and Mortenson have characterized the MoO,2- transport system in Clostridium pasteurianum (11). The lack of Mo has been shown to prevent the synthesis of nitrogenase (4, 7), but little detail is available on how Mo regulates or on the regulation of the molybdate transport system. The synthesis of the N2-fixing system of C. pasteurianum is regulated by NH3 (9). The regulation of nitrogenase activity by NH3 may occur partially via carbamyl phosphate which acts as an end-product inhibitor as well as a repressor of synthesis (17, 19). The present com-
munication describes the regulation of the molybdate transport system of C. pasteurianum and demonstrates that its regulation is closely associated with the regulation of nitrogenase activity in this organism. C. pasteurianum exhibits a diauxic growth pattern where during the first phase of exponential growth in the presence of ammonia and No, it utilizes ammonia as the nitrogen source, and no nitrogenase activity can be detected (9, 20). When the culture consumes all of the ammonium, a lag period ensues, the duration of which can be varied by several parameters (9). The paper also demonstrates that the length of the diauxic lag increases with decreasing MoO42- concentrations in the growth medium. MATERIALS AND METHODS Cell growth. A chemostat culture of C. pasteurianum W5, a N2-fixing anaerobe, was grown in a medium described by Daesch and Mortenson (9) con770
VOL. 127, 1976
REGULATION OF MOLYBDATE TRANSPORT
taining 10 IMM Na2MoO4 and 22 ,M SO42-. For each experiment 600 ml of cells from a chemostat was used to inoculate 5,400 ml of fresh medium with no added molybdate. When the optical density reached 1.3 (550 nm with 1-cm light path in a Bausch and Lomb Spectronic 20 spectrophotometer) 800 ml of this culture was used to inoculate 7,200 ml of fresh medium without added molybdate. Five additional transfers were made until the calculated concentration in the final culture was 10- ,uM MoO42- (undetectable by any of the most sensitive analytical methods available). High purity sucrose was used to minimize contaminating MoO42. For batch cultures growing first on ammonia and then on N2, 1 mM ammonium acetate and 1 atm of N2 were used as nitrogen sources. For batch cultures growing first on N2 and then on ammonia, ammonium acetate was added at the appropriate time to make the final concentration 8 mM. Cells were harvested in a Sorval model RC-2 refrigerated centrifuge at 13,200 x g for 10 min. The collected cells were washed twice by suspending them in cold degassed minimal medium (11), followed by anaerobic centrifugation at 0°C. For the preparation of crude extracts, cells were grown in 1 mM ammonium acetate and 1 atm of N2 in a 160-liter steel fermentor maintained at pH 5.9. The MoO42- concentration was calculated to be 10-6 AM. Additions of 100 AM MoO42 and 100 MM W042were made at the indicated times. Three hours after the onset of the diauxic lag, cells were harvested, washed and dried under vacuum, and stored at -20°C until needed. Crude extracts. Cell extracts were prepared anaerobically by first suspending the cells in 4 volumes of 0.05 M tris(hydroxymethyl)aminomethane(Tris)hydrochloride, pH 8.5, previously degassed and equilibrated with H2. Then to 5 ml of cell suspension, 1 mg of lysozyme and 0.1 mg each of deoxyribonuclease I and ribonuclease A were added. The mixture was kept under an atmosphere of H2 and gently agitated at room temperature for 1 h. The extracts were centrifuged at 37,000 x g for 30 min (0C) under N2 or Ar in 6-ml centrifuge tubes fitted with a Teflon top and rubber serum stopper. All transfers of extracts were performed with a gas-tight syringe that was previously rinsed with deoxygenated 0.05 M Tris buffer, pH 7.0. Nitrogenase activity. Whole-cell acetylene reduction activity was assayed in a 8-ml serum vial containing 0.2 atm of C2H2, 0.8 atm of H2, 1.9 ml of cells, and 0.1 ml of 2% sucrose solutions made of 0.05 M potassium phosphate buffer, pH 7.2. At 2-min intervals, 30 ,ul of gas was removed and analyzed for C2H4. MoFe protein and Fe protein activities in extracts were determined as previously described. To assume maximum activity each was titrated with its complementary purified component (21). Analytic methods. Protein was determined by the biuret and Lowry methods (13, 15). Molybdenum was determined by the method of Cardenas and Mortenson (6). Ammonia was measured by nesslerization after diffusion and absorption of the gas in 5 nM H2SO4 in Conway dishes (8).
771
Uptake assay. Uptake assays were performed as previously described (11). Chemicals. Na2 5'MoO4 (specific activity, 105 Ci/ g) was purchased from New England Nuclear Corp. Na2MoO4, Na2WO4, Na2SO4, sucrose, chloramphenicol, carbamyl phosphate, adenosine 5'-triphosphate, creatine phosphokinase (EC 2.7.3.2), creatine phosphate, deoxyribonuclease I (EC 3.1.4.5), ribonuclease A (EC 2.7.16), and Tris base were purchased from Sigma Chemical Co. (St. Louis, Mo.) and were of the highest purity available. RESULTS
Molybdate uptake during diauxic growth. Examinations of the acetylene reduction activity of nitrogenase and the ammonia concentration of the surrounding medium during various times of the diauxic growth of C. pasteurianum are illustrated in Fig. 1. A 160-liter culture medium supplemented with 3 mM ammonium acetate and 100 nM N,MoO4 was inoculated from an ammonia-grown culture (10-s MM Mo). N, gas was sparged into the culture continuously. At the times indL,ated on Fig. 1, (i) 2-ml samples of the culture were removed for measurement of acetylene reduction by whole cells, (ii) 10-ml samples were centrifuged, and the 3.0
2.0F
.32 t, -30 o z
1.0 -I
.8 .6
10
g .4
-0
E
I
o
I
.2
0 1 2 3 4 5 6 7 8 9 10 TIME ( hr)
FIG. 1. Growth of C. pasteurianum on limiting ammonia (3 mM) in the presence ofN. with low Mo
(100 nM) and S042- (22 pM). Optical density measurements (0), acetylene reduction activity (0), and ammonia concentration (A) were performed as described. At the times indicated by the arrows 8-liter samples were harvested and the cells were prepared for assay of uptake activity (see Fig. 2).
ELLIOTT AND MORTENSON 772 supernatant solution was frozen for later ammonia determination, and (iii) optical density (OD) measurements were made. The length of the diauxic lag in the experiment was long because of the number of generations the cells grew on ammonia, and because the cells were growing on low concentrations of MoO4-2 and SO42-. At the times indicated by the arrows, 8liter samples were harvested and washed, and uptake assays were performed. Figure 2 shows the uptake data for this experiment. The ability
of cells of C. pasteurianum to transport molybdate is >95% repressed in cultures growing on 2 mM ammonium acetate. Similar results are obtained for 8-liter cultures growing on 8 mM ammonia. Just before the onset of the diauxic lag, before the appearance of acetylene reduction activity, the cells begin to show a low level of uptake activity. As the cells progress through the diauxic lag period their ability to transport molybdate increases. The uptake ability at 5 h after the lag begins reaches 60% of the maximum seen in cultures growing solely on N2, whereas the acetylene reduction activity is only 25% of maximum. The maximum concentrations of the acetylene reduction and up-
J. BACTERIOL.
AA
400
8 . CP
, , °0 E
_ BV B b
o (
e/ A i E 200 w -
D D
b
g
a-
o E
o0
100
I ~~~~~~~~III 10 20 30 40 50 60
I
I
TIME (min) 5 6
z 300 _ ~3 / /
4
N
4n E
/ X / ^/
_
/b/"
FIG. 3. The dilution of MoO42- uptake activity after a shift from N2-fixing to NH3-growing conditions. Cells at time A were harvested at an OD of 0.6 just before the addition of ammonium acetate (final concentration, 8 mM). Letters A through E correspond to the following times and OD readings: (A) 12 h, 0.6; (B) 13 h, 0.9; (C) 14 h, 1.5; (D) 15 h, 2.2; (E)
15.5 h, 3.0.
take systems would be reached if growth was followed for a longer period. Dilution of MoO42- uptake activity during ;X a. shift from N2-fixing to NH3-growing condi8 h 0.1 165 0.7 120 1.0
90
10.0 60 100.0 60 1,000.0 60 10,000.0 No growth a Cells were grown in a medium with limiting (NH4)2SO4 (1 mM) void of detectable MoO42-. The MoO42- concentration was adjusted as described above. The length of the lag was measured from the time the (NH4)2SO4 was exhausted and cell growth stopped until the time growth resumed under N2fixing conditions.
10 uM the normal 60-min lag period was lengthened, reaching 165 min at 0.1 ,uM. Concentrations above 10 mM MoO42- completely inhibited cell growth. Fe protein and MoFe protein activity in the absence of MoO42-. Experiments were designed to deternine if MoO42- acts as an inducer of nitrogenase synthesis in C. pasteurianum. A 160-liter culture medium supplemented with 1 mM ammonium acetate with no detectable MoO42- present (calculated concentration
Vol. 127, No. 2 Printed in U.S.A.
Copyright © 1976 American Society for Microbiology
Regulation of Molybdate Transport by Clostridium pasteurianum BARBARA B. ELLIOTT AND L. E. MORTENSON* Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Received for publication 12 April 1976
The regulation of the molybdate (MoO42-) transport activity of Clostridium pasteurianum has been studied by observing the effects of NH:, carbamyl phosphate, MoO42-, and chloramphenicol on the ability of cells to take up MoO42-. Compared with cells fixing N2, cells grown in the presence of 1 mM NH3 are greater than 95% repressed for MoO42- transport. Uptake activity begins to increase just before NH3 exhaustion (under Ar or N2) and continues to increase throughout the lag period as cells shift from NH3-growing to N2-fixing conditions. When cells are shifted from N2-fixing to NH3-growing conditions the transport activity per fixed number of cells decreases by increase of cells in absence of transport synthesis. Carbamyl phosphate (.15 mM) but not NH3 inhibits 58% of the in vitro uptake activity. When 1 mM carbamyl phosphate is added just before the exhaustion of NH3, the transport activity, measured 2 h later, is 100% repressed. Cells grown in the presence of high MoO42- (1 mM) are 80% repressed for MoO42- transport. Synthesis of the MoO42- transport system is also completely stopped when chloramphenicol (300 ,ug/ml) is added just before the exhaustion of NH3 from the medium. These findings demonstrate that the ability of cells to transport MoO42- is dependent upon new protein synthesis and can be repressed by high levels of substrate. The regulation of MoO42- uptake by NH3 or carbamyl phosphate closely parallels the regulation of nitrogenase activity. Activity of neither nitrogenase component (Fe protein or MoFe protein) was detected even 3 h after the exhaustion of the NH3 if either MoO42- was absent or if W042- was present in place of MoO42-. The duration of the diauxic lag increases with decreasing concentration of MoO42- in the medium. If no MoO,2- is present the lag continues indefinitely. If MoO42- is added late in the lag period, growth under N2-fixing conditions resumes but only after a normal induction period. It has long been known that molybdenum is an essential element for biological dinitrogen fixation (3), and it is now known that molybde-
num is a constituent of the MoFe protein component of the nitrogenase complex (5, 10, 12, 16). Recently, attempts have been made to characterize the role of molybdenum in the catalytic reactions of the nitrogenase components (2, 7). Elliott and Mortenson have characterized the MoO,2- transport system in Clostridium pasteurianum (11). The lack of Mo has been shown to prevent the synthesis of nitrogenase (4, 7), but little detail is available on how Mo regulates or on the regulation of the molybdate transport system. The synthesis of the N2-fixing system of C. pasteurianum is regulated by NH3 (9). The regulation of nitrogenase activity by NH3 may occur partially via carbamyl phosphate which acts as an end-product inhibitor as well as a repressor of synthesis (17, 19). The present com-
munication describes the regulation of the molybdate transport system of C. pasteurianum and demonstrates that its regulation is closely associated with the regulation of nitrogenase activity in this organism. C. pasteurianum exhibits a diauxic growth pattern where during the first phase of exponential growth in the presence of ammonia and No, it utilizes ammonia as the nitrogen source, and no nitrogenase activity can be detected (9, 20). When the culture consumes all of the ammonium, a lag period ensues, the duration of which can be varied by several parameters (9). The paper also demonstrates that the length of the diauxic lag increases with decreasing MoO42- concentrations in the growth medium. MATERIALS AND METHODS Cell growth. A chemostat culture of C. pasteurianum W5, a N2-fixing anaerobe, was grown in a medium described by Daesch and Mortenson (9) con770
VOL. 127, 1976
REGULATION OF MOLYBDATE TRANSPORT
taining 10 IMM Na2MoO4 and 22 ,M SO42-. For each experiment 600 ml of cells from a chemostat was used to inoculate 5,400 ml of fresh medium with no added molybdate. When the optical density reached 1.3 (550 nm with 1-cm light path in a Bausch and Lomb Spectronic 20 spectrophotometer) 800 ml of this culture was used to inoculate 7,200 ml of fresh medium without added molybdate. Five additional transfers were made until the calculated concentration in the final culture was 10- ,uM MoO42- (undetectable by any of the most sensitive analytical methods available). High purity sucrose was used to minimize contaminating MoO42. For batch cultures growing first on ammonia and then on N2, 1 mM ammonium acetate and 1 atm of N2 were used as nitrogen sources. For batch cultures growing first on N2 and then on ammonia, ammonium acetate was added at the appropriate time to make the final concentration 8 mM. Cells were harvested in a Sorval model RC-2 refrigerated centrifuge at 13,200 x g for 10 min. The collected cells were washed twice by suspending them in cold degassed minimal medium (11), followed by anaerobic centrifugation at 0°C. For the preparation of crude extracts, cells were grown in 1 mM ammonium acetate and 1 atm of N2 in a 160-liter steel fermentor maintained at pH 5.9. The MoO42- concentration was calculated to be 10-6 AM. Additions of 100 AM MoO42 and 100 MM W042were made at the indicated times. Three hours after the onset of the diauxic lag, cells were harvested, washed and dried under vacuum, and stored at -20°C until needed. Crude extracts. Cell extracts were prepared anaerobically by first suspending the cells in 4 volumes of 0.05 M tris(hydroxymethyl)aminomethane(Tris)hydrochloride, pH 8.5, previously degassed and equilibrated with H2. Then to 5 ml of cell suspension, 1 mg of lysozyme and 0.1 mg each of deoxyribonuclease I and ribonuclease A were added. The mixture was kept under an atmosphere of H2 and gently agitated at room temperature for 1 h. The extracts were centrifuged at 37,000 x g for 30 min (0C) under N2 or Ar in 6-ml centrifuge tubes fitted with a Teflon top and rubber serum stopper. All transfers of extracts were performed with a gas-tight syringe that was previously rinsed with deoxygenated 0.05 M Tris buffer, pH 7.0. Nitrogenase activity. Whole-cell acetylene reduction activity was assayed in a 8-ml serum vial containing 0.2 atm of C2H2, 0.8 atm of H2, 1.9 ml of cells, and 0.1 ml of 2% sucrose solutions made of 0.05 M potassium phosphate buffer, pH 7.2. At 2-min intervals, 30 ,ul of gas was removed and analyzed for C2H4. MoFe protein and Fe protein activities in extracts were determined as previously described. To assume maximum activity each was titrated with its complementary purified component (21). Analytic methods. Protein was determined by the biuret and Lowry methods (13, 15). Molybdenum was determined by the method of Cardenas and Mortenson (6). Ammonia was measured by nesslerization after diffusion and absorption of the gas in 5 nM H2SO4 in Conway dishes (8).
771
Uptake assay. Uptake assays were performed as previously described (11). Chemicals. Na2 5'MoO4 (specific activity, 105 Ci/ g) was purchased from New England Nuclear Corp. Na2MoO4, Na2WO4, Na2SO4, sucrose, chloramphenicol, carbamyl phosphate, adenosine 5'-triphosphate, creatine phosphokinase (EC 2.7.3.2), creatine phosphate, deoxyribonuclease I (EC 3.1.4.5), ribonuclease A (EC 2.7.16), and Tris base were purchased from Sigma Chemical Co. (St. Louis, Mo.) and were of the highest purity available. RESULTS
Molybdate uptake during diauxic growth. Examinations of the acetylene reduction activity of nitrogenase and the ammonia concentration of the surrounding medium during various times of the diauxic growth of C. pasteurianum are illustrated in Fig. 1. A 160-liter culture medium supplemented with 3 mM ammonium acetate and 100 nM N,MoO4 was inoculated from an ammonia-grown culture (10-s MM Mo). N, gas was sparged into the culture continuously. At the times indL,ated on Fig. 1, (i) 2-ml samples of the culture were removed for measurement of acetylene reduction by whole cells, (ii) 10-ml samples were centrifuged, and the 3.0
2.0F
.32 t, -30 o z
1.0 -I
.8 .6
10
g .4
-0
E
I
o
I
.2
0 1 2 3 4 5 6 7 8 9 10 TIME ( hr)
FIG. 1. Growth of C. pasteurianum on limiting ammonia (3 mM) in the presence ofN. with low Mo
(100 nM) and S042- (22 pM). Optical density measurements (0), acetylene reduction activity (0), and ammonia concentration (A) were performed as described. At the times indicated by the arrows 8-liter samples were harvested and the cells were prepared for assay of uptake activity (see Fig. 2).
ELLIOTT AND MORTENSON 772 supernatant solution was frozen for later ammonia determination, and (iii) optical density (OD) measurements were made. The length of the diauxic lag in the experiment was long because of the number of generations the cells grew on ammonia, and because the cells were growing on low concentrations of MoO4-2 and SO42-. At the times indicated by the arrows, 8liter samples were harvested and washed, and uptake assays were performed. Figure 2 shows the uptake data for this experiment. The ability
of cells of C. pasteurianum to transport molybdate is >95% repressed in cultures growing on 2 mM ammonium acetate. Similar results are obtained for 8-liter cultures growing on 8 mM ammonia. Just before the onset of the diauxic lag, before the appearance of acetylene reduction activity, the cells begin to show a low level of uptake activity. As the cells progress through the diauxic lag period their ability to transport molybdate increases. The uptake ability at 5 h after the lag begins reaches 60% of the maximum seen in cultures growing solely on N2, whereas the acetylene reduction activity is only 25% of maximum. The maximum concentrations of the acetylene reduction and up-
J. BACTERIOL.
AA
400
8 . CP
, , °0 E
_ BV B b
o (
e/ A i E 200 w -
D D
b
g
a-
o E
o0
100
I ~~~~~~~~III 10 20 30 40 50 60
I
I
TIME (min) 5 6
z 300 _ ~3 / /
4
N
4n E
/ X / ^/
_
/b/"
FIG. 3. The dilution of MoO42- uptake activity after a shift from N2-fixing to NH3-growing conditions. Cells at time A were harvested at an OD of 0.6 just before the addition of ammonium acetate (final concentration, 8 mM). Letters A through E correspond to the following times and OD readings: (A) 12 h, 0.6; (B) 13 h, 0.9; (C) 14 h, 1.5; (D) 15 h, 2.2; (E)
15.5 h, 3.0.
take systems would be reached if growth was followed for a longer period. Dilution of MoO42- uptake activity during ;X a. shift from N2-fixing to NH3-growing condi8 h 0.1 165 0.7 120 1.0
90
10.0 60 100.0 60 1,000.0 60 10,000.0 No growth a Cells were grown in a medium with limiting (NH4)2SO4 (1 mM) void of detectable MoO42-. The MoO42- concentration was adjusted as described above. The length of the lag was measured from the time the (NH4)2SO4 was exhausted and cell growth stopped until the time growth resumed under N2fixing conditions.
10 uM the normal 60-min lag period was lengthened, reaching 165 min at 0.1 ,uM. Concentrations above 10 mM MoO42- completely inhibited cell growth. Fe protein and MoFe protein activity in the absence of MoO42-. Experiments were designed to deternine if MoO42- acts as an inducer of nitrogenase synthesis in C. pasteurianum. A 160-liter culture medium supplemented with 1 mM ammonium acetate with no detectable MoO42- present (calculated concentration
