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Role of glutamine synthetase, glutamine and NH4+ in the regulation of NO2− uptake in the cyanobiont nostoc ANTH
J. Plant Physiol. Vol.
142. pp. 403-406 (1993)
Role of Glutamine Synthetase, Glutamine and NH4 + in the Regulation of N0 2 - Uptake in the Cyanobiont Nostoc ANTH SURENDRA SINGH'~ Department of Biochemistry, North-Eastern Hill University, Shillong-793014, India Received November 10, 1993 . Accepted April 23, 1993
Summary
The role of glutamine synthetase (GS), glutamine and NH4 + in the regulation of NO z- uptake was studied in the cyanobiont Nostoc ANTH, a free-living isolate of the liverwort Anthoceros punctatus. The uptake of NO z- was repressed in the presence of NH4 + and glutamine. L-methionine-DL-sulfoximine (MSX) which inhibited the GS activity, could not inhibit the NO z- uptake, however, it prevented the NH4 + inhibition of NO z- uptake. Repression of the NO z- uptake by NH4 + did not require de novo protein synthesis but it required GS activity, suggesting that NH4 + itself is not the repressor signal but its metabolism via GS is required for the NH4 + repression of NO z- uptake to occur. The derepression of the NO z- uptake system, however, required de novo protein synthesis but it did not require GS activity.
Key words: Nostoc A NTH, Cyanobiont, N0 2 - uptake. Abbreviations: GS = glutamine synthetase; HEPES = 4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid; MSX = L-methionine-DL-sulfoximine. Introduction
A better understanding of nitrogen metabolism is a prerequisite for enhancing the potential use of diazotrophic cyanobacteria in biotechnology. Studies made on N0 2 - metabolism of this group of organisms have shown that the process of NO z- assimilation comprises at least three consecutive steps; uptake, followed by reduction of NO z- into NH3 and incorporation of NH3 to carbon skeletons yielding amino acids. However, the role of glutamine synthetase (GS) activity, glutamine and NH4 + in the regulation of N0 2 - uptake is not yet fully understood in cyanobacteria. GS plays a key role in NH4 + assimilation in cyanobacteria (Stewart 1980). MSX, a glutamate analogue and an irreversible inhibitor of GS (Stewart and Rowell 1975) has been widely used to probe the relationship between GS and other processes of nitrogen metabolism, including nitrogenase derepression (Stewart and Rowell 1975, Singh 1991). Using Nostoc ANTH, a free-living isolate from the liverwort Anthoceros punctatus and its MSX-resistant mutant
* Department of Microbiology, Barkatullah University, Bhopal462026, (M.P.), India. © 1993 by Gustav Fischer Verlag, Stuttgart
strain (Msxr), I have studied the role of GS, glutamine and NH4 + in the regulation of NO z- uptake and provide evidence here to show that (1) the NO z- uptake system in Nostoe ANTH is NH4 +-repressible and that NH4 + itself is not the repressor signal but its metabolism via GS is required; (2) while the repression of N0 2 - uptake by NH4 + requires GS activity, it does not require de novo protein synthesis; and (3) the derepression of the N0 2 - uptake requires de novo protein synthesis but it does not require GS activity.
Materials and Methods
Organisms and Culture Conditions Nostoc ANTH (isolated from the gametophyte of Anthoceros punctatus) was axenically grown in BG-ll o medium (Rippka et al. 1979). MSX' mutant strains were isolated earlier (Singh 1992 b) and were maintained in N 2-medium supplemented with 2 mM glutamine. The cultures were incubated at 24 ± 1 °C and illuminated with day-light fluorescent tubes having a photon fluence rate of 50I-lmolm-2s-1. When needed NH 4CI and glutamine were added to the medium in the final concentration of 2 mM. The medium was buffered to pH 7.5 with 10 mM HEPES/NaOH.
404
SURENDIlA SINGH
Table 1: Effect of nitrogen source and MSX on N0 2 - uptake and GS activity in the cyanobiont Nostoc ANTH. Nitrogen source
Nz
N0 2 NH.+ Glutamine
-MSX
+MSX
N0 2 - uptake (a)
Gs activity (b)
N02 - uptake (a)
GS activity (b)
165.7 165.1 41.3 4.9
1625.7 1545.9 874.5 1575.3
241.3 246.7 219.5 4.7
0.0 0.0 0.0 0.0
Nostoc ANTH was grown on N z, NO z-, NH. +and g1utamine-containing media. MSX (250 J-LM) was added to the cultures and incubated for 2 h under normal growth conditions. NO z- uptake and GS activity were assayed as described in Materials and Methods. The concentration of nitrogen sources were as follows: KNO z, 2 mM; NH.CI, 2 mM; and glutamine, 2 mM. The data in each column are means of two independent experiments. (a) nmol N02 - J-Lg- 1 protein h- I (b) nmol 'Y-g1utamyl hydroxamate formed mg- I protein min-I
Uptake Assay
Uptake of N02 - was assayed by measuring its depletion from the external medium. Exponentially growing cells were harvested by centrifugation at 10,000 x g for 15 min, washed with Tricinel NaOH buffer (PH 8.1) and resuspended in the same buffer to a final density of 200 J-Lg protein mL -I. Assays were carried out in open flasks under aerobic conditions with continuous shaking at 28°C and a photon fluence rate of 50J-Lmolm- 2 s- 1 (from day-light fluorescent tubes). After 30 min stabilization, the experiments were started by the addition of KN0 2 (1 mM final concentration) to the cell suspension at zero time. From the samples withdrawn at time intervals, the cells were separated by centrifugation and cell-free supernatants were analyzed for the residual N0 2 -. When needed the medium was supplemented with NH.CI (5 mM) for repression studies. MSX (250 J-LM) and streptomycin (20 J-Lg mL -I) were added to the flasks 2 h prior to the addition of N02 - and were present during the experiment. The linear portions of the curves (uptake during 0- 30 min) were used to calculate the uptake rates. Glutamine Synthetase Activity
GS transferase activity was measured following the method of Sampaio et al. (1979). Analytical Methods
N0 2 - was estimated following the method of Snell and Snell (1949) whereas cellular protein was estimated by the method of Lowry et al. (1951) standardized with bovine serum albumin. Chemicals
MSX, streptomycin, tncme and HEPES were obtained from Sigma Chern. Co. (USA). Other chemicals were used as highest purity available from BDH, Poole.
Results and Discussion Table 1 presents data on the effects of different nitrogen sources on the NO z- uptake and GSactivity levels in the cyanobiont Nostoc ANTH in the presence and absence of MSX. The NO z- uptake activity was rather similar in N z and N02 --grown cells. The presence of NH4+ in the medium led to a reduced level of NO z- uptake. The data thus, indicate that NO z- uptake in Nostoc ANTH is NH4 +-repres-
sible. The addition of MSX which resulted in the complete inhibition of GS activity, could not inhibit the NO z- uptake, however, it prevented the NH4 + inhibition of N0 2 uptake, suggesting that the catalytic function of GS is not necessarily required for the short-term uptake of NO z- but it is required for the NH4 + inhibition of NO z- uptake. Thus, NH4 + itself is not the inhibitor of NO z- uptake but the metabolism of NH4 + through GS is being required for repression to occur. The NO z- uptake was very low in glutamine-grown cells (Table 1). Since, glutamine partially prevented the MSX uptake in cyanobacteria (Haselkorn et al. 1980) MSX was allowed for 2 h to enter the cells and to inactivate GS before the addition of glutamine. Glutamine also inhibited the N0 2 - uptake even in the presence of MSX and the degree of inhibition was similar to that of normal untreated cells (Table 1). These results suggests that unlike NH4 +, glutamine inhibits NO z- uptake both in untreated and in MSX-treated cells and therefore, it is suggested that an organic nitrogenous compound which may be glutamine itself or a glutamine derivative seems to act as a repressor of NO z- uptake in the cyanobiont Nostoc ANTH. Since, MSX increased the NO z- uptake and glutamine inhibited the NO z- uptake, it is suggested that the catalytic function of GS has a major role in the regulation of NO z- uptake in the cyanobiont Nostoc ANTH. Further to ascertain and to determine the role of GS and de novo protein synthesis in the repression/derepression of N0 2 - uptake system, the NO z- uptake was studied in the presence and absence of MSX and streptomycin (Fig. 1). For repression study, the N z-grown cells were transferred to NH4 +-medium. Starting at zero time, the cells were harvested at regular time intervals and assayed for NO z- uptake. It is evident from Fig. 1 that NO z- uptake remained unaltered in the presence of NH4 + upto 1 h and then declined to a negligible level as the time of preincubation in the NH4 +-medium was increased. This time lag was consistent with the necessity for NH4 + to be converted to glutamine (or related products) which might be the actual repressor in this case. MSX prevented the NH4 + inhibition of NO z- uptake whereas streptomycin, a protein synthesis inhibitor could not do so. This suggests that repression of NOz - uptake by NH4 + requires the operation of GS i.e .. NH4 + assimilation but it does not require de novo protein synthesis. These results further suggest that NH4 + itself is
Regulation of N0 2- uptake in Nostoe ANTH
405
Table 2: Effect of MSX on N0 2- uptake and GS transferase activity in MSX' strain of the cyanobiont Nostoe ANTH. +MSX
-MSX
Incubation period (min) 0 10 20 30
N0 2- uptake (a)
GS activity (b)
N0 2- uptake (a)
GS activity (b)
204.7 205.3 204.9 206.1
495.6 496.3 495.7 496.5
205.1 206.5 205.6 204.9
496.2 494.9 496.3 495.5
MSX (250 ~M) was added to the cell suspension and kept in light at the start of the incubation period. The data in each column are means of two independent experiments. (a) nmol N0 2- ~g-I protein h- I (b) nmoll'-glutamyl hydroxamate formed mg- I protein min- I
100
2 c:0 u
80
50
0 ~
w
""«
60
f-
0
"-
12
24
~
w f-
40
n: f-
z 20
o
12
24 TI ME
36 (h)
Fig. 1: Kinetics of repression/derepression of the N0 2- uptake system in Nostoe ANTH. 0, control; e, + MSX; 6, + streptomycin. The 100% N0 2- uptake corresponds to 165.7nmol N0 2- ~g-I proteinh- I. Inset shows the kinetics of the derepression of the N0 2- uptake on transfer of NH4 +-grown cells to the N 2-medium (details as above). not the repressor of N0 2- uptake and that its assimilation products (which may be glutamine itself) may have been involved. Furthermore, the cells treated with MSX, not only showed the recovery of NO z- uptake from NH4 + inhibition but exhibited an increased level of NO z- uptake (Fig. 1). Thus, the regulatory system modulating NO z- uptake via the active uptake system seems to involve products of NH4 + assimilation via GS. Analogous results were also observed in unicellular cyanobacterium Anacystis nidulans (Flores et al. 1987). To study the derepression of NO z- uptake system, the NH4 + incubated cells (for 24 h) were transferred to N z-medium and N0 2- uptake activity was regularly measured. It is evident from the data of Fig. 1 (inset) that the activity of the NO z- uptake system was restored to normal within 24 h. This derepression of the NO z- uptake system was prevented by the protein synthesis inhibitor, streptomycin. However,
the GS inhibitor, MSX could not prevent the derepression of the NO z- uptake system. This suggests that the derepression of the NO z- uptake system does not require GS activity but it requires de novo protein synthesis when NH4 + incubated cells were transferred to N 2-medium. Further, to ascertain and to obtain more conclusive evidence regarding the role of GS in N0 2- uptake, the NO zuptake was studied in MSX' mutant strain of Nostoe ANTH isolated by mutagenesis with NTG on media containing 50 IlM MSX (Singh 1992 b). The GS transferase activity of the MSX' was about 30 % to that of its wild type strain and was insensitive to MSX (Table 2). The MSX resistance was therefore, not due to failure of the inhibitor to enter the cells, as reported in a mutant of Anabaena variabilis (Chapman and Meeks 1983). The N0 2- uptake in the MSX' strain having defective GS was higher as compared to the wild type strain having normal GS and was unaffected by MSX (Table 2). These results suggest that MSX has no role in the shortterm uptake of N0 2- in the cyanobiont Nostoe ANTH. Since, in MSX' strain, both GS and NO z- uptake were unaffected by MSX in contrast to the wild type strain in which only GS was inhibited, it is suggested that MSX has a single target. This explanation is strengthened by the finding that MSX' strain liberates NH4 + in the external medium in the presence ofN0 2- (Singh 1992 b). Thus, overall it is concluded that (1) NO z- uptake in Nostoe ANTH is constitutive and NH4 + -repressible; (2) NH4 + itself is not the regulator of N02- uptake but a product of NH4 + assimilation via GS is being required; (3) while the repression of N02- uptake by NH4 + requires GS activity, it does not require de novo protein synthesis; and (4) the derepression of NO z- uptake requires new protein synthesis but it does not require GS activity. Acknowledgements
Financial assistance from DSTP, Council of Scientific & Industrial Research, New Delhi, India is gratefully acknowledged. References CHAPMAN, J. S. and ]. C. MEEKS: Glutamine and glutamate transport by Anabaena variabilis.]. Bacterio!' 156, 122-129 (1983). FLORES, E., A. HERRERO, and M. G. GUERRERO: Nitrite uptake and its regulation in the cyanobacterium Anacystis nidulans. Biochim. Biophys. Acta 896, 103 -108 (1987).
406
SURENDRA SINGH
HASELKORN, R., B. MAZUR, J. ORR, D. RICE, N. WOOD, and R. RIPPKA: Heterocyst differentiation and nitrogen fixation in cyanobacteria. In: NEWTON, W. E. and W. H. JOHNSON (eds.): Nitrogen Fixation, pp. 259-278, University Park Press, Baltimore (1980). LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: Protein measurement with the folin-phenol reagent. J. BioI. Chern. 193, 265-275 (1951). RIPPKA, R., J. DERUELLES, J. B. WATERBURY, M. HERDMAN, and R. Y. STANIER: Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111, 1-61 (1979). SAMPAIO, M. J. A. M., P. ROWELL, and W. D. P. STEWART: Purification and some properties of glutamine synthetase from nitrogen fixing cyanobacteria Anabaena cylindrica and Nostoc sp. J. Gen. Microbiol. 111, 181-191 (1979).
SINGH, S.: Involvement of ammonium assimilation in ammonium inhibition of nitrogenase activity in the cyanobacterium Nostoc ANTH. IndianJ. Exp. BioI. 29, 496-497 (1991). - Regulation of nitrite uptake in the cyanobiont Nostoc ANTH. Indian J. Exp. BioI. 30,288-291 (1992 a). - Photobiological production of ammonia by an L-methionineDL-sulfoximine resistant mutant of cyanobiont Nostoc ANTH. Indian J. Exp. BioI. 30, 284 - 287 (1992 b). SNELL, F. D. and C. T. SNELL: Colorimetric Methods of Analysis, Vol. 3, pp. 804-805, Van Nostrand, New York (1949). STEWART, W. D. P.: Some aspects of structure and function in N 2 fixing cyanobacteria. Ann. Rev. Microbiol. 34, 497 - 536 (1980). STEWART, W. D. P. and P. ROWELL: Effect of L-methionine-DL-sulphoximine on the assimilation of newly fixed NH), acetylene reduction and heterocyst production in Anabaena cylindrica. Biochern. Biophys. Res. Commun. 65, 846-856 (1975).
142. pp. 403-406 (1993)
Role of Glutamine Synthetase, Glutamine and NH4 + in the Regulation of N0 2 - Uptake in the Cyanobiont Nostoc ANTH SURENDRA SINGH'~ Department of Biochemistry, North-Eastern Hill University, Shillong-793014, India Received November 10, 1993 . Accepted April 23, 1993
Summary
The role of glutamine synthetase (GS), glutamine and NH4 + in the regulation of NO z- uptake was studied in the cyanobiont Nostoc ANTH, a free-living isolate of the liverwort Anthoceros punctatus. The uptake of NO z- was repressed in the presence of NH4 + and glutamine. L-methionine-DL-sulfoximine (MSX) which inhibited the GS activity, could not inhibit the NO z- uptake, however, it prevented the NH4 + inhibition of NO z- uptake. Repression of the NO z- uptake by NH4 + did not require de novo protein synthesis but it required GS activity, suggesting that NH4 + itself is not the repressor signal but its metabolism via GS is required for the NH4 + repression of NO z- uptake to occur. The derepression of the NO z- uptake system, however, required de novo protein synthesis but it did not require GS activity.
Key words: Nostoc A NTH, Cyanobiont, N0 2 - uptake. Abbreviations: GS = glutamine synthetase; HEPES = 4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid; MSX = L-methionine-DL-sulfoximine. Introduction
A better understanding of nitrogen metabolism is a prerequisite for enhancing the potential use of diazotrophic cyanobacteria in biotechnology. Studies made on N0 2 - metabolism of this group of organisms have shown that the process of NO z- assimilation comprises at least three consecutive steps; uptake, followed by reduction of NO z- into NH3 and incorporation of NH3 to carbon skeletons yielding amino acids. However, the role of glutamine synthetase (GS) activity, glutamine and NH4 + in the regulation of N0 2 - uptake is not yet fully understood in cyanobacteria. GS plays a key role in NH4 + assimilation in cyanobacteria (Stewart 1980). MSX, a glutamate analogue and an irreversible inhibitor of GS (Stewart and Rowell 1975) has been widely used to probe the relationship between GS and other processes of nitrogen metabolism, including nitrogenase derepression (Stewart and Rowell 1975, Singh 1991). Using Nostoc ANTH, a free-living isolate from the liverwort Anthoceros punctatus and its MSX-resistant mutant
* Department of Microbiology, Barkatullah University, Bhopal462026, (M.P.), India. © 1993 by Gustav Fischer Verlag, Stuttgart
strain (Msxr), I have studied the role of GS, glutamine and NH4 + in the regulation of NO z- uptake and provide evidence here to show that (1) the NO z- uptake system in Nostoe ANTH is NH4 +-repressible and that NH4 + itself is not the repressor signal but its metabolism via GS is required; (2) while the repression of N0 2 - uptake by NH4 + requires GS activity, it does not require de novo protein synthesis; and (3) the derepression of the N0 2 - uptake requires de novo protein synthesis but it does not require GS activity.
Materials and Methods
Organisms and Culture Conditions Nostoc ANTH (isolated from the gametophyte of Anthoceros punctatus) was axenically grown in BG-ll o medium (Rippka et al. 1979). MSX' mutant strains were isolated earlier (Singh 1992 b) and were maintained in N 2-medium supplemented with 2 mM glutamine. The cultures were incubated at 24 ± 1 °C and illuminated with day-light fluorescent tubes having a photon fluence rate of 50I-lmolm-2s-1. When needed NH 4CI and glutamine were added to the medium in the final concentration of 2 mM. The medium was buffered to pH 7.5 with 10 mM HEPES/NaOH.
404
SURENDIlA SINGH
Table 1: Effect of nitrogen source and MSX on N0 2 - uptake and GS activity in the cyanobiont Nostoc ANTH. Nitrogen source
Nz
N0 2 NH.+ Glutamine
-MSX
+MSX
N0 2 - uptake (a)
Gs activity (b)
N02 - uptake (a)
GS activity (b)
165.7 165.1 41.3 4.9
1625.7 1545.9 874.5 1575.3
241.3 246.7 219.5 4.7
0.0 0.0 0.0 0.0
Nostoc ANTH was grown on N z, NO z-, NH. +and g1utamine-containing media. MSX (250 J-LM) was added to the cultures and incubated for 2 h under normal growth conditions. NO z- uptake and GS activity were assayed as described in Materials and Methods. The concentration of nitrogen sources were as follows: KNO z, 2 mM; NH.CI, 2 mM; and glutamine, 2 mM. The data in each column are means of two independent experiments. (a) nmol N02 - J-Lg- 1 protein h- I (b) nmol 'Y-g1utamyl hydroxamate formed mg- I protein min-I
Uptake Assay
Uptake of N02 - was assayed by measuring its depletion from the external medium. Exponentially growing cells were harvested by centrifugation at 10,000 x g for 15 min, washed with Tricinel NaOH buffer (PH 8.1) and resuspended in the same buffer to a final density of 200 J-Lg protein mL -I. Assays were carried out in open flasks under aerobic conditions with continuous shaking at 28°C and a photon fluence rate of 50J-Lmolm- 2 s- 1 (from day-light fluorescent tubes). After 30 min stabilization, the experiments were started by the addition of KN0 2 (1 mM final concentration) to the cell suspension at zero time. From the samples withdrawn at time intervals, the cells were separated by centrifugation and cell-free supernatants were analyzed for the residual N0 2 -. When needed the medium was supplemented with NH.CI (5 mM) for repression studies. MSX (250 J-LM) and streptomycin (20 J-Lg mL -I) were added to the flasks 2 h prior to the addition of N02 - and were present during the experiment. The linear portions of the curves (uptake during 0- 30 min) were used to calculate the uptake rates. Glutamine Synthetase Activity
GS transferase activity was measured following the method of Sampaio et al. (1979). Analytical Methods
N0 2 - was estimated following the method of Snell and Snell (1949) whereas cellular protein was estimated by the method of Lowry et al. (1951) standardized with bovine serum albumin. Chemicals
MSX, streptomycin, tncme and HEPES were obtained from Sigma Chern. Co. (USA). Other chemicals were used as highest purity available from BDH, Poole.
Results and Discussion Table 1 presents data on the effects of different nitrogen sources on the NO z- uptake and GSactivity levels in the cyanobiont Nostoc ANTH in the presence and absence of MSX. The NO z- uptake activity was rather similar in N z and N02 --grown cells. The presence of NH4+ in the medium led to a reduced level of NO z- uptake. The data thus, indicate that NO z- uptake in Nostoc ANTH is NH4 +-repres-
sible. The addition of MSX which resulted in the complete inhibition of GS activity, could not inhibit the NO z- uptake, however, it prevented the NH4 + inhibition of N0 2 uptake, suggesting that the catalytic function of GS is not necessarily required for the short-term uptake of NO z- but it is required for the NH4 + inhibition of NO z- uptake. Thus, NH4 + itself is not the inhibitor of NO z- uptake but the metabolism of NH4 + through GS is being required for repression to occur. The NO z- uptake was very low in glutamine-grown cells (Table 1). Since, glutamine partially prevented the MSX uptake in cyanobacteria (Haselkorn et al. 1980) MSX was allowed for 2 h to enter the cells and to inactivate GS before the addition of glutamine. Glutamine also inhibited the N0 2 - uptake even in the presence of MSX and the degree of inhibition was similar to that of normal untreated cells (Table 1). These results suggests that unlike NH4 +, glutamine inhibits NO z- uptake both in untreated and in MSX-treated cells and therefore, it is suggested that an organic nitrogenous compound which may be glutamine itself or a glutamine derivative seems to act as a repressor of NO z- uptake in the cyanobiont Nostoc ANTH. Since, MSX increased the NO z- uptake and glutamine inhibited the NO z- uptake, it is suggested that the catalytic function of GS has a major role in the regulation of NO z- uptake in the cyanobiont Nostoc ANTH. Further to ascertain and to determine the role of GS and de novo protein synthesis in the repression/derepression of N0 2 - uptake system, the NO z- uptake was studied in the presence and absence of MSX and streptomycin (Fig. 1). For repression study, the N z-grown cells were transferred to NH4 +-medium. Starting at zero time, the cells were harvested at regular time intervals and assayed for NO z- uptake. It is evident from Fig. 1 that NO z- uptake remained unaltered in the presence of NH4 + upto 1 h and then declined to a negligible level as the time of preincubation in the NH4 +-medium was increased. This time lag was consistent with the necessity for NH4 + to be converted to glutamine (or related products) which might be the actual repressor in this case. MSX prevented the NH4 + inhibition of NO z- uptake whereas streptomycin, a protein synthesis inhibitor could not do so. This suggests that repression of NOz - uptake by NH4 + requires the operation of GS i.e .. NH4 + assimilation but it does not require de novo protein synthesis. These results further suggest that NH4 + itself is
Regulation of N0 2- uptake in Nostoe ANTH
405
Table 2: Effect of MSX on N0 2- uptake and GS transferase activity in MSX' strain of the cyanobiont Nostoe ANTH. +MSX
-MSX
Incubation period (min) 0 10 20 30
N0 2- uptake (a)
GS activity (b)
N0 2- uptake (a)
GS activity (b)
204.7 205.3 204.9 206.1
495.6 496.3 495.7 496.5
205.1 206.5 205.6 204.9
496.2 494.9 496.3 495.5
MSX (250 ~M) was added to the cell suspension and kept in light at the start of the incubation period. The data in each column are means of two independent experiments. (a) nmol N0 2- ~g-I protein h- I (b) nmoll'-glutamyl hydroxamate formed mg- I protein min- I
100
2 c:0 u
80
50
0 ~
w
""«
60
f-
0
"-
12
24
~
w f-
40
n: f-
z 20
o
12
24 TI ME
36 (h)
Fig. 1: Kinetics of repression/derepression of the N0 2- uptake system in Nostoe ANTH. 0, control; e, + MSX; 6, + streptomycin. The 100% N0 2- uptake corresponds to 165.7nmol N0 2- ~g-I proteinh- I. Inset shows the kinetics of the derepression of the N0 2- uptake on transfer of NH4 +-grown cells to the N 2-medium (details as above). not the repressor of N0 2- uptake and that its assimilation products (which may be glutamine itself) may have been involved. Furthermore, the cells treated with MSX, not only showed the recovery of NO z- uptake from NH4 + inhibition but exhibited an increased level of NO z- uptake (Fig. 1). Thus, the regulatory system modulating NO z- uptake via the active uptake system seems to involve products of NH4 + assimilation via GS. Analogous results were also observed in unicellular cyanobacterium Anacystis nidulans (Flores et al. 1987). To study the derepression of NO z- uptake system, the NH4 + incubated cells (for 24 h) were transferred to N z-medium and N0 2- uptake activity was regularly measured. It is evident from the data of Fig. 1 (inset) that the activity of the NO z- uptake system was restored to normal within 24 h. This derepression of the NO z- uptake system was prevented by the protein synthesis inhibitor, streptomycin. However,
the GS inhibitor, MSX could not prevent the derepression of the NO z- uptake system. This suggests that the derepression of the NO z- uptake system does not require GS activity but it requires de novo protein synthesis when NH4 + incubated cells were transferred to N 2-medium. Further, to ascertain and to obtain more conclusive evidence regarding the role of GS in N0 2- uptake, the NO zuptake was studied in MSX' mutant strain of Nostoe ANTH isolated by mutagenesis with NTG on media containing 50 IlM MSX (Singh 1992 b). The GS transferase activity of the MSX' was about 30 % to that of its wild type strain and was insensitive to MSX (Table 2). The MSX resistance was therefore, not due to failure of the inhibitor to enter the cells, as reported in a mutant of Anabaena variabilis (Chapman and Meeks 1983). The N0 2- uptake in the MSX' strain having defective GS was higher as compared to the wild type strain having normal GS and was unaffected by MSX (Table 2). These results suggest that MSX has no role in the shortterm uptake of N0 2- in the cyanobiont Nostoe ANTH. Since, in MSX' strain, both GS and NO z- uptake were unaffected by MSX in contrast to the wild type strain in which only GS was inhibited, it is suggested that MSX has a single target. This explanation is strengthened by the finding that MSX' strain liberates NH4 + in the external medium in the presence ofN0 2- (Singh 1992 b). Thus, overall it is concluded that (1) NO z- uptake in Nostoe ANTH is constitutive and NH4 + -repressible; (2) NH4 + itself is not the regulator of N02- uptake but a product of NH4 + assimilation via GS is being required; (3) while the repression of N02- uptake by NH4 + requires GS activity, it does not require de novo protein synthesis; and (4) the derepression of NO z- uptake requires new protein synthesis but it does not require GS activity. Acknowledgements
Financial assistance from DSTP, Council of Scientific & Industrial Research, New Delhi, India is gratefully acknowledged. References CHAPMAN, J. S. and ]. C. MEEKS: Glutamine and glutamate transport by Anabaena variabilis.]. Bacterio!' 156, 122-129 (1983). FLORES, E., A. HERRERO, and M. G. GUERRERO: Nitrite uptake and its regulation in the cyanobacterium Anacystis nidulans. Biochim. Biophys. Acta 896, 103 -108 (1987).
406
SURENDRA SINGH
HASELKORN, R., B. MAZUR, J. ORR, D. RICE, N. WOOD, and R. RIPPKA: Heterocyst differentiation and nitrogen fixation in cyanobacteria. In: NEWTON, W. E. and W. H. JOHNSON (eds.): Nitrogen Fixation, pp. 259-278, University Park Press, Baltimore (1980). LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: Protein measurement with the folin-phenol reagent. J. BioI. Chern. 193, 265-275 (1951). RIPPKA, R., J. DERUELLES, J. B. WATERBURY, M. HERDMAN, and R. Y. STANIER: Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111, 1-61 (1979). SAMPAIO, M. J. A. M., P. ROWELL, and W. D. P. STEWART: Purification and some properties of glutamine synthetase from nitrogen fixing cyanobacteria Anabaena cylindrica and Nostoc sp. J. Gen. Microbiol. 111, 181-191 (1979).
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