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Regulation of Nitrite Reductase Cellular Levels in the Cyanobiont Nostoc ANTH
Biochem. Physiol. Pflanzen 188, 241-246 (1992) Gustav Fischer Verlag lena
Regulation of Nitrite Reductase Cellular Levels in the Cyanobiont Nostoc ANTH S. SINGH North-Eastern Hill University, Shillong, India Key Term Index: Nitrate reductase; nitrite reductase; regulation; cyanobiont; Nostoc ANTH
Summary The cellular nitrite reductase actlVlty in response to different nitrogen sources has been studied in the cyanobiont Nostoc ANTH isolated from the liverwort Anthoceros. De novo protein synthesis of nitrite reductase occurred even in the absence of added nitrogen source, although enzyme activity was higher when nitrite or nitrate served as the sole nitrogen source. In tungstate-treated cells, nitrate did not show any positive effect on nitrite reductase activity, suggesting that the stimulatory effect was not due to nitrate itself but it requires its reduction to nitrite. Thus, nitrite may be the actual inducer in this case. Ammonium-grown cells showed reduced level of nitrite reductase activity. The nitrite reductase activity was freed from ammonium repression by L-methionine-DL-sulphoximine (MSX), an irreversible inhibitor of glutamine synthetase (GS). Thus ammonium metabolism through GS is required for the ammonium repression of nitrite reductase to occur.
Introduction
Cyanobacteria are oxygenic photosynthetic prokaryotes. Most of them are able to utilize nitrate, nitrite or ammonium as the sole source of nitrogen for growth (FoGG et al. 1973). Nitrate is the nitrogen source which is most widely utilized by the cyanobacteria. The assimilation of nitrate in cyanobacteria involves two successive steps, first nitrate is reduced to nitrite by nitrate reductase and the resulting nitrite is then reduced to ammonium by nitrite reductase (GUERRERO et al. 1981; FLORES et al. 1983). This process is supposed to be the predominant biological one for the production of reduced nitrogen from the oxidized inorganic precursors; however; its regulation is not yet fully understood in cyanobacteria. Nitrate reductase synthesis is significantly influenced by the nitrogen sources available during growth (HERRERO et al. 1985). The regulation of nitrate reductase is mainly achieved through ammonium promoted repression, with ammonium metabolism through glutamine synthetase (GS) being required for repression to occur (HERRERO et al. 1981). The available informations concerning the regulation of nitrite reductase in cyanobacteria are scarce and controversial. It is found to be ammonium repressible in Anacystis nidulans (MANZANO et al. 1976; HERRERO and GUERRERO 1986) and Abbreviations: GS, glutamine synthetase; MOPS, 3-(N-morpholine) propane sulphonic acid; MSX, L-methionine-DL-sulphoximine 16
BPP 188 (1992) 4
241
Anabaena sp . 7119 (MENDEZ et al. 1981) whereas it is induced by nitrite but not by nitrate in Anabaena cylindrica (OHMORI and HATTORI 1970). The present paper reports the role of nitrate, nitrite, ammonium and chloramphenicol in the regulation of cellular nitrate reductase activity in the cyanobiont Nostoc ANTH, a free-living isolate of the liverwort Anthoceros punctatus.
Materials and Methods Organism and Culture Conditions Axenic batch cultures of Nostoc ANTH, isolated from the gametophyte thalli of Anthoceros punctatus, were grown in BO-Ilo medium (N 2) (RIPPKA et aI . 1979) at 25°C and illuminated with day-light fluorescent tubes having a photon fluence rate of 50 !lmol m- 2 S-I. Assay of Enzyme Activities Nitrate reductase activity was assayed with dithionite-reduced methyl viologen as reductant in the cells permeabilized with mixed alkyltrimethyl ammonium bromide in a reaction mixture at a final concentration of 100llg m1 1 - (HERRERO et al. 1984). Nitrite reductase activity was also assayed in permeabilized cells. For this assay, cell suspension containing 400-500 Ilg protein was added to a reaction mixture containing the following reagents in a final volume of 1 ml : mixed alkyltrimethyl ammonium bromide, 100 !lg; MOPS/NaOH buffer , pH 7.2, 25 !lmol ; KN02, 0.5 !lmol; methyl viologen, 5 !lmol and Na2S204, 20 !lmol in 0.1 ml of 0.3 M NaHC0 3 . The reaction mixture was incubated at 30°C for 10 min and nitrite was estimated in the corresponding media freed of cells. Blanks in which the reaction was stopped at zero time were also prepared. Activity units corresponds to nmol N0 2 removed min - I . Analytical Methods Nitrite was estimated by the method of SNELL and SNELL (1949) whereas cellular protein was estimated by the method of LOWRY et al. (1951) using bovine serum albumin as the s tandard. Chemicals Mixed alkyltrimethyl ammonium bromide, L-methionine-DL-sulphoximine (MSX), chloramphenicol, methyl viologen , sodium dithionite and 3-(N-morpholine) propane sulphonic acid (MOPS) were obtained from Sigma Chern. Co. (USA) . Other chemicals were used as highest purity available from BDH, Poole.
Results and Discussion The data in Fig. 1 show that nitrite-grown cells had high nitrite reductase activity whereas ammonium-grown cells had negligible activity. When ammonium-grown cells were transferred to nitrate or nitrite media, the activity of nitrite reductase attained its normal level within 5 to 6 h (Fig. 1). Nitrate-grown cells supported the nitrite reductase activity only after a lag period of I h which also supports the contention of earlier report (RAI and SINGH 1982). This time lag was consistent with the necessity for nitrate to be reduced to nitrite which may be the actual inducer in this case. This derepression in nitrite reductase activity on transfer from ammonium 242
BPP 188 (1992) 4
TI ME (h)
Fig. 1. Kinetics of the derepression of nitrite reductase activity in Nosto c ANTH ceLLs on transfer of the ammonium-grown ceLLs to KN0 2 (2 mM) (0) ; or KN0 3 (2 mM) (e); or KN0 2 + chloramphenicol (6); or KN0 3 + chloramphenicol (.) . Chloramphenicol (50 Ilg ml- 1) was added to the cultures 12 h prior the addition of KNO z or KN0 3 so as to allow the action of the chemical .
to nitrate or nitrite medium was prevented by the addition of chloramphenicol (50 f.lg rnl- J), a protein synthesis inhibitor, suggesting that it probably resulted from de novo protein synthesis, rather than the activation of preformed enzyme. To study the response of various nitrogen sources on the activity levels of nitrite reductase , the Nostoc ANTH cells were grown in N2 , NO ]" , N02" and NHt medium and the changes in cellular nitrite reductase activity levels were examined. From the data of Table 1 it is evident that significant nitrite reductase activity was found even if no nitrogen source was available (Nz-medium), and maximum values were recorded if nitrite served as the sole source of nitrogen (Table I, data without MSX). Nitrate-grown cells also showed higher enzyme activity to that of Nrgrown cells. The presence of ammonium in the medium always led to negligible nitrite reductase activity even if nitrate or nitrite were simultaneously available to the cells. These results clearly indicate that nitrite reductase activity in Nostoc ANTH is ammonium-repressible and the negative effect exerted by ammonium cannot be mitigated either by nitrate or nitrite. The data in Fig. I and Table 1 (without MSX) together support the idea that the regulation of nitrite reductase in cyanobiont Nostoc ANTH in response to nitrogen source is mainly exerted at the transcriptional level, through repression determined by the ammonium in the medium like Anacystis nidulans (HERRERO and GUERRERO 1986). To determine whether the inhibition of nitrite reductase synthesis by ammonium could either be exerted by ammonium itself or by a product of its assimilation, MSX, a 16*
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243
Table 1. Effect of nitrogen source and MSX on cellular nitrite reductase activity in Nostoc ANTH Nitrogen Source
Nz KN0 3 KNOz N~Cl
KN0 3 + NH4Cl KNO z + NH4Cl
Nitrite reductase activity (nmol NO z removed (flg protein)-I min-I) -MSX
+MSX
9.7 23.7 36.2 0.5 0.5 1.3
3.9 46.7 57.3 12.7 34.5 51.3
Nostoc ANTH cells grown on Nz-medium were transferred to media containing KN0 3 (2 mM), or KNO z (2 mM) or NH4Cl (l mM). MSX (50 flM) was added to the cultures and incubated for 2 h under normal growth conditions. The data in each columns are the means of two independent observations and obtained by in situ assay of nitrite reductase activity in permeabilized cells.
glutamate analogue and inhibitor of GS, was used to distinguish between these possibilities. MSX effectively and irreversibly inhibits the ammonium assimilation in cyanobacteria (STEWART and ROWELL 1975). The supplementation of MSX to the medium permitted the development of nitrite reductase activity even in the presence of ammonium. The enzyme activity in the MSX-treated cells was also remarkably high in the presence of nitrate and nitrite (Table 1). These results indicate that ammonium metabolism through GS is required for ammonium repression of nitrite reductase activity in Nostoc ANTH. Thus, the repression of nitrite reductase in Nostoc ANTH is not exerted by ammonium itself or, at least not by ammonium alone, but it requires the operation of GS and probably involves the participation of some organic-nitrogen containing metabolites. The data with MSX reaffirmed the positive effect of nitrate or nitrite on nitrite reductase activity, even when ammonium was simultaneously available to the cells. To further assess the possibilities whether the development of nitrite reductase activity could either be exerted by nitrate itself or by nitrite resulting from its reduction, cells devoid of functional nitrate reductase have been used. Cells of Nostoc ANTH devoid of functional nitrate reductase, but with full activity of other components of nitrate assimilating systems were obtained by treatment with tungstate (HERRERO and GUERRERO 1986). Molybdenum is a prosthetic group of cyanobacterial nitrate reductase with an essential role in catalysis (MIKAMI and IDA 1984). As with nitrate reductase from other sources (GUERRERO et al. 1981), tungsten, a structural analogue of molybdenum, can substitute for molybdenum in Nostoc ANTH enzyme, leading to the formation of inactive molecules (SINGH 1992). Nostoc ANTH cells, grown on ammonium when transferred to nitrate medium without molybdate but with tungstate, showed negligible nitrate reductase activity (Fig. 2A). The tungstate-treated cells with non functional nitrate reductase did not show the nitrate stimulation of nitrite reductase activity, indicating that nitrate itself is 244
BPP 188 (1992) 4
Fig. 2. Effect of tungstate on the derepression of nitrate reductase (A) and nitrate reductase (B) activities of Nostoc ANTH when ammonium-grown cells were transferred to nitrate-containing medium. Nostoc ANTH cells grown on ammonium medium (without molybdate) were incubated in nitrate-medium lacking molybdate, but with 300 ~M sodium tungstate, for 12 h. The medium was then supplemented with ammonium chloride (5 mM). After 24 h the cells were harvested and resuspended in nitrate-medium with 4 ~M sodium molybdate (0); or + 300 ~M sodium tungstate (e).
not the inducer of nitrite reductase but the reduction of nitrate to nitrite through nitrate reductase is required for positive effect. This is in contrasts with the findings obtained with A. nidulans (HERRERO and GUERRERO 1986) in which nitrate induces the nitrite reductase without being reduced to nitrite. Thus, the differential responses of nitrite reductase to the nitrogen sources may represent a characteristic feature of the regulation of nitrate assimilation in cyanobacteria.
Acknowledgements This work was supported by DSTP, Council of Scientific & Industrial Research (Govt. of ' India), New Delhi. BPP 188 (1992) 4
245
References FLORES , E., RAMOS , J. L. , HERRERO , A. , and GUERRERO, M. G .: Nitrate assimilation by cyanobacteria. Photosynthetic Prokaryotes : Cell Differentiation and Function (Eds.): PAPAGEORGIOU, G. c., and PACKER , K .) pp. 363-387. Elsevier, New York 1983. FOGG, G. E., STEWART, W. D. P., FAY , P., and WALSBY , A. E.: The Blue-green Algae. Academic Press, London 1973. GUERRERO, M. G., VEGA, J. M. , and LOSADA, M.: The assimilatory nitrate reducing system and its regulation. Annu. Rev. Plant Physiol. 32, 169-204 (1981) . HERRERO , A., FLORES, E., and GUERRERO , M. G.: Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. strain 7119, and Nostoc sp. strain 6719. J. Gen. Microbiol. 145, 175-180 (1981) . HERRERO , A., FLORES , E. , and GUERRERO , M. G.: Regulation of nitrate reductase level in Anacystis nidulans: activity decay under nitrogen stress . Arch . Biochem. Biophys. 234 , 454-459 (1984) . HERRERO , A., FLORES, E. , and GUERRERO, M. G.: Regulation of nitrate reductase cellular levels in the cyanobacteria Anabaena variabilis and Synechocystis sp. FEMS Microbial. Lett. 26, 21-25 (1985). HERRERO, A., and GUERRERO , M . G.: Regulation of nitrite reductase in the cyanobacterium Anacystis nidulans. J. Gen. Microbiol. 132, 2463- 2468 (1986). LOWRY , D . H., ROSEBROUGH, N. J. , FARR , A. L., and RANDALL, R . J.: Protein measurement with the falin-phenol reagent. J. BioI. Chern. 193,265-275 (1951). MANZANO, c. , CANDAV , P. , GOMEZ-MoRENO, c. , RELIMPIO, A . M., and LOSADA, M.: Ferredoxin-dependent photosynthetic reduction of nitrate and nitrite by particles of Anacystis nidulans. Mol. Cell. Biochem . 10, 161-169 (1976). MENDEZ , 1. M. , and VEGA, J . M.: Purification and molecular properties of nitrite reductase from Anabaena sp. 7119 . Physiol. Plant. 52, 7-14 (1981). MIKAMI, B., and IDA, S.: Purification and properties of ferredoxin-nitrate reductase from the cyanobacterium Plectonema boryanum. Biochim. Biophys . Acta 791,294-304 (1984). OHMORI, K., and HATTORI, A.: Induction of nitrate and nitrite reductases in Anabaena cylindrica. Plant Cell Physiol. 11,873- 878 (1970). RAI, A . K ., and SINGH, S.: Regulation of nitrate uptake in Nostoc muscorum by glutamine synthetase. FEMS Microbial. Lett . 14,303-306 (1982). RIPPKA, R., DERUELLES, J., WATERBURY, J. B., HERDMAN , M., and STANIER, R. Y.: Generic assignment, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111 , 1-61 (1979) . SINGH , S.: Regulation of nitrite uptake in the cyanobiont Nostoc ANTH. Indian J. Exp. BioI. 30, 288-291 (1992). SNELL , F. D., and SNELL , C. T .: Colorimetric Methods of Analysis , vol. 3, pp . 804-805. Van Nostrand, New York 1949. STEWART, W. D. P., and ROWELL , P.: Effect of L-methionine-DL-sulphoximine on the assimilation of newly fixed NH), acetylene reduction and heterocyst production in Anabaena cylindrica. Biochem. Biophys. Res. Commun. 65 , 846-856 (1975).
Received June 3,1991; revised form accepted April 8, 1992 Author's address: Dr. SVRENDRA SINGH, Department of Biochemistry , School of LifeSciences, North-Eastern Hill University , Shillong-793014 , India.
246
BPP 188 (1992) 4
Regulation of Nitrite Reductase Cellular Levels in the Cyanobiont Nostoc ANTH S. SINGH North-Eastern Hill University, Shillong, India Key Term Index: Nitrate reductase; nitrite reductase; regulation; cyanobiont; Nostoc ANTH
Summary The cellular nitrite reductase actlVlty in response to different nitrogen sources has been studied in the cyanobiont Nostoc ANTH isolated from the liverwort Anthoceros. De novo protein synthesis of nitrite reductase occurred even in the absence of added nitrogen source, although enzyme activity was higher when nitrite or nitrate served as the sole nitrogen source. In tungstate-treated cells, nitrate did not show any positive effect on nitrite reductase activity, suggesting that the stimulatory effect was not due to nitrate itself but it requires its reduction to nitrite. Thus, nitrite may be the actual inducer in this case. Ammonium-grown cells showed reduced level of nitrite reductase activity. The nitrite reductase activity was freed from ammonium repression by L-methionine-DL-sulphoximine (MSX), an irreversible inhibitor of glutamine synthetase (GS). Thus ammonium metabolism through GS is required for the ammonium repression of nitrite reductase to occur.
Introduction
Cyanobacteria are oxygenic photosynthetic prokaryotes. Most of them are able to utilize nitrate, nitrite or ammonium as the sole source of nitrogen for growth (FoGG et al. 1973). Nitrate is the nitrogen source which is most widely utilized by the cyanobacteria. The assimilation of nitrate in cyanobacteria involves two successive steps, first nitrate is reduced to nitrite by nitrate reductase and the resulting nitrite is then reduced to ammonium by nitrite reductase (GUERRERO et al. 1981; FLORES et al. 1983). This process is supposed to be the predominant biological one for the production of reduced nitrogen from the oxidized inorganic precursors; however; its regulation is not yet fully understood in cyanobacteria. Nitrate reductase synthesis is significantly influenced by the nitrogen sources available during growth (HERRERO et al. 1985). The regulation of nitrate reductase is mainly achieved through ammonium promoted repression, with ammonium metabolism through glutamine synthetase (GS) being required for repression to occur (HERRERO et al. 1981). The available informations concerning the regulation of nitrite reductase in cyanobacteria are scarce and controversial. It is found to be ammonium repressible in Anacystis nidulans (MANZANO et al. 1976; HERRERO and GUERRERO 1986) and Abbreviations: GS, glutamine synthetase; MOPS, 3-(N-morpholine) propane sulphonic acid; MSX, L-methionine-DL-sulphoximine 16
BPP 188 (1992) 4
241
Anabaena sp . 7119 (MENDEZ et al. 1981) whereas it is induced by nitrite but not by nitrate in Anabaena cylindrica (OHMORI and HATTORI 1970). The present paper reports the role of nitrate, nitrite, ammonium and chloramphenicol in the regulation of cellular nitrate reductase activity in the cyanobiont Nostoc ANTH, a free-living isolate of the liverwort Anthoceros punctatus.
Materials and Methods Organism and Culture Conditions Axenic batch cultures of Nostoc ANTH, isolated from the gametophyte thalli of Anthoceros punctatus, were grown in BO-Ilo medium (N 2) (RIPPKA et aI . 1979) at 25°C and illuminated with day-light fluorescent tubes having a photon fluence rate of 50 !lmol m- 2 S-I. Assay of Enzyme Activities Nitrate reductase activity was assayed with dithionite-reduced methyl viologen as reductant in the cells permeabilized with mixed alkyltrimethyl ammonium bromide in a reaction mixture at a final concentration of 100llg m1 1 - (HERRERO et al. 1984). Nitrite reductase activity was also assayed in permeabilized cells. For this assay, cell suspension containing 400-500 Ilg protein was added to a reaction mixture containing the following reagents in a final volume of 1 ml : mixed alkyltrimethyl ammonium bromide, 100 !lg; MOPS/NaOH buffer , pH 7.2, 25 !lmol ; KN02, 0.5 !lmol; methyl viologen, 5 !lmol and Na2S204, 20 !lmol in 0.1 ml of 0.3 M NaHC0 3 . The reaction mixture was incubated at 30°C for 10 min and nitrite was estimated in the corresponding media freed of cells. Blanks in which the reaction was stopped at zero time were also prepared. Activity units corresponds to nmol N0 2 removed min - I . Analytical Methods Nitrite was estimated by the method of SNELL and SNELL (1949) whereas cellular protein was estimated by the method of LOWRY et al. (1951) using bovine serum albumin as the s tandard. Chemicals Mixed alkyltrimethyl ammonium bromide, L-methionine-DL-sulphoximine (MSX), chloramphenicol, methyl viologen , sodium dithionite and 3-(N-morpholine) propane sulphonic acid (MOPS) were obtained from Sigma Chern. Co. (USA) . Other chemicals were used as highest purity available from BDH, Poole.
Results and Discussion The data in Fig. 1 show that nitrite-grown cells had high nitrite reductase activity whereas ammonium-grown cells had negligible activity. When ammonium-grown cells were transferred to nitrate or nitrite media, the activity of nitrite reductase attained its normal level within 5 to 6 h (Fig. 1). Nitrate-grown cells supported the nitrite reductase activity only after a lag period of I h which also supports the contention of earlier report (RAI and SINGH 1982). This time lag was consistent with the necessity for nitrate to be reduced to nitrite which may be the actual inducer in this case. This derepression in nitrite reductase activity on transfer from ammonium 242
BPP 188 (1992) 4
TI ME (h)
Fig. 1. Kinetics of the derepression of nitrite reductase activity in Nosto c ANTH ceLLs on transfer of the ammonium-grown ceLLs to KN0 2 (2 mM) (0) ; or KN0 3 (2 mM) (e); or KN0 2 + chloramphenicol (6); or KN0 3 + chloramphenicol (.) . Chloramphenicol (50 Ilg ml- 1) was added to the cultures 12 h prior the addition of KNO z or KN0 3 so as to allow the action of the chemical .
to nitrate or nitrite medium was prevented by the addition of chloramphenicol (50 f.lg rnl- J), a protein synthesis inhibitor, suggesting that it probably resulted from de novo protein synthesis, rather than the activation of preformed enzyme. To study the response of various nitrogen sources on the activity levels of nitrite reductase , the Nostoc ANTH cells were grown in N2 , NO ]" , N02" and NHt medium and the changes in cellular nitrite reductase activity levels were examined. From the data of Table 1 it is evident that significant nitrite reductase activity was found even if no nitrogen source was available (Nz-medium), and maximum values were recorded if nitrite served as the sole source of nitrogen (Table I, data without MSX). Nitrate-grown cells also showed higher enzyme activity to that of Nrgrown cells. The presence of ammonium in the medium always led to negligible nitrite reductase activity even if nitrate or nitrite were simultaneously available to the cells. These results clearly indicate that nitrite reductase activity in Nostoc ANTH is ammonium-repressible and the negative effect exerted by ammonium cannot be mitigated either by nitrate or nitrite. The data in Fig. I and Table 1 (without MSX) together support the idea that the regulation of nitrite reductase in cyanobiont Nostoc ANTH in response to nitrogen source is mainly exerted at the transcriptional level, through repression determined by the ammonium in the medium like Anacystis nidulans (HERRERO and GUERRERO 1986). To determine whether the inhibition of nitrite reductase synthesis by ammonium could either be exerted by ammonium itself or by a product of its assimilation, MSX, a 16*
BPP 188 (1992) 4
243
Table 1. Effect of nitrogen source and MSX on cellular nitrite reductase activity in Nostoc ANTH Nitrogen Source
Nz KN0 3 KNOz N~Cl
KN0 3 + NH4Cl KNO z + NH4Cl
Nitrite reductase activity (nmol NO z removed (flg protein)-I min-I) -MSX
+MSX
9.7 23.7 36.2 0.5 0.5 1.3
3.9 46.7 57.3 12.7 34.5 51.3
Nostoc ANTH cells grown on Nz-medium were transferred to media containing KN0 3 (2 mM), or KNO z (2 mM) or NH4Cl (l mM). MSX (50 flM) was added to the cultures and incubated for 2 h under normal growth conditions. The data in each columns are the means of two independent observations and obtained by in situ assay of nitrite reductase activity in permeabilized cells.
glutamate analogue and inhibitor of GS, was used to distinguish between these possibilities. MSX effectively and irreversibly inhibits the ammonium assimilation in cyanobacteria (STEWART and ROWELL 1975). The supplementation of MSX to the medium permitted the development of nitrite reductase activity even in the presence of ammonium. The enzyme activity in the MSX-treated cells was also remarkably high in the presence of nitrate and nitrite (Table 1). These results indicate that ammonium metabolism through GS is required for ammonium repression of nitrite reductase activity in Nostoc ANTH. Thus, the repression of nitrite reductase in Nostoc ANTH is not exerted by ammonium itself or, at least not by ammonium alone, but it requires the operation of GS and probably involves the participation of some organic-nitrogen containing metabolites. The data with MSX reaffirmed the positive effect of nitrate or nitrite on nitrite reductase activity, even when ammonium was simultaneously available to the cells. To further assess the possibilities whether the development of nitrite reductase activity could either be exerted by nitrate itself or by nitrite resulting from its reduction, cells devoid of functional nitrate reductase have been used. Cells of Nostoc ANTH devoid of functional nitrate reductase, but with full activity of other components of nitrate assimilating systems were obtained by treatment with tungstate (HERRERO and GUERRERO 1986). Molybdenum is a prosthetic group of cyanobacterial nitrate reductase with an essential role in catalysis (MIKAMI and IDA 1984). As with nitrate reductase from other sources (GUERRERO et al. 1981), tungsten, a structural analogue of molybdenum, can substitute for molybdenum in Nostoc ANTH enzyme, leading to the formation of inactive molecules (SINGH 1992). Nostoc ANTH cells, grown on ammonium when transferred to nitrate medium without molybdate but with tungstate, showed negligible nitrate reductase activity (Fig. 2A). The tungstate-treated cells with non functional nitrate reductase did not show the nitrate stimulation of nitrite reductase activity, indicating that nitrate itself is 244
BPP 188 (1992) 4
Fig. 2. Effect of tungstate on the derepression of nitrate reductase (A) and nitrate reductase (B) activities of Nostoc ANTH when ammonium-grown cells were transferred to nitrate-containing medium. Nostoc ANTH cells grown on ammonium medium (without molybdate) were incubated in nitrate-medium lacking molybdate, but with 300 ~M sodium tungstate, for 12 h. The medium was then supplemented with ammonium chloride (5 mM). After 24 h the cells were harvested and resuspended in nitrate-medium with 4 ~M sodium molybdate (0); or + 300 ~M sodium tungstate (e).
not the inducer of nitrite reductase but the reduction of nitrate to nitrite through nitrate reductase is required for positive effect. This is in contrasts with the findings obtained with A. nidulans (HERRERO and GUERRERO 1986) in which nitrate induces the nitrite reductase without being reduced to nitrite. Thus, the differential responses of nitrite reductase to the nitrogen sources may represent a characteristic feature of the regulation of nitrate assimilation in cyanobacteria.
Acknowledgements This work was supported by DSTP, Council of Scientific & Industrial Research (Govt. of ' India), New Delhi. BPP 188 (1992) 4
245
References FLORES , E., RAMOS , J. L. , HERRERO , A. , and GUERRERO, M. G .: Nitrate assimilation by cyanobacteria. Photosynthetic Prokaryotes : Cell Differentiation and Function (Eds.): PAPAGEORGIOU, G. c., and PACKER , K .) pp. 363-387. Elsevier, New York 1983. FOGG, G. E., STEWART, W. D. P., FAY , P., and WALSBY , A. E.: The Blue-green Algae. Academic Press, London 1973. GUERRERO, M. G., VEGA, J. M. , and LOSADA, M.: The assimilatory nitrate reducing system and its regulation. Annu. Rev. Plant Physiol. 32, 169-204 (1981) . HERRERO , A., FLORES, E., and GUERRERO , M. G.: Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. strain 7119, and Nostoc sp. strain 6719. J. Gen. Microbiol. 145, 175-180 (1981) . HERRERO , A., FLORES , E. , and GUERRERO , M. G.: Regulation of nitrate reductase level in Anacystis nidulans: activity decay under nitrogen stress . Arch . Biochem. Biophys. 234 , 454-459 (1984) . HERRERO , A., FLORES, E. , and GUERRERO, M. G.: Regulation of nitrate reductase cellular levels in the cyanobacteria Anabaena variabilis and Synechocystis sp. FEMS Microbial. Lett. 26, 21-25 (1985). HERRERO, A., and GUERRERO , M . G.: Regulation of nitrite reductase in the cyanobacterium Anacystis nidulans. J. Gen. Microbiol. 132, 2463- 2468 (1986). LOWRY , D . H., ROSEBROUGH, N. J. , FARR , A. L., and RANDALL, R . J.: Protein measurement with the falin-phenol reagent. J. BioI. Chern. 193,265-275 (1951). MANZANO, c. , CANDAV , P. , GOMEZ-MoRENO, c. , RELIMPIO, A . M., and LOSADA, M.: Ferredoxin-dependent photosynthetic reduction of nitrate and nitrite by particles of Anacystis nidulans. Mol. Cell. Biochem . 10, 161-169 (1976). MENDEZ , 1. M. , and VEGA, J . M.: Purification and molecular properties of nitrite reductase from Anabaena sp. 7119 . Physiol. Plant. 52, 7-14 (1981). MIKAMI, B., and IDA, S.: Purification and properties of ferredoxin-nitrate reductase from the cyanobacterium Plectonema boryanum. Biochim. Biophys . Acta 791,294-304 (1984). OHMORI, K., and HATTORI, A.: Induction of nitrate and nitrite reductases in Anabaena cylindrica. Plant Cell Physiol. 11,873- 878 (1970). RAI, A . K ., and SINGH, S.: Regulation of nitrate uptake in Nostoc muscorum by glutamine synthetase. FEMS Microbial. Lett . 14,303-306 (1982). RIPPKA, R., DERUELLES, J., WATERBURY, J. B., HERDMAN , M., and STANIER, R. Y.: Generic assignment, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111 , 1-61 (1979) . SINGH , S.: Regulation of nitrite uptake in the cyanobiont Nostoc ANTH. Indian J. Exp. BioI. 30, 288-291 (1992). SNELL , F. D., and SNELL , C. T .: Colorimetric Methods of Analysis , vol. 3, pp . 804-805. Van Nostrand, New York 1949. STEWART, W. D. P., and ROWELL , P.: Effect of L-methionine-DL-sulphoximine on the assimilation of newly fixed NH), acetylene reduction and heterocyst production in Anabaena cylindrica. Biochem. Biophys. Res. Commun. 65 , 846-856 (1975).
Received June 3,1991; revised form accepted April 8, 1992 Author's address: Dr. SVRENDRA SINGH, Department of Biochemistry , School of LifeSciences, North-Eastern Hill University , Shillong-793014 , India.
246
BPP 188 (1992) 4