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Mutation in the Blue-green Alga Nostoc muscorum II. L-methionine-DL-sulfoximine-resistant Mutants
Biochem. PhysioJ. Pflanzen 176, 631-637 (1981)
Mutation in the Blue-green Alga N ostoc muscorum II. L-methionine-DL-sulfoximine-resistant Mutants!) A. VAISHAMPAYAN 2) and H. N. SINGH3) Department of Botany, Banaras Hindu Universi ty, Varanasi, India Key Term Index: L~methionine-DL-su]foximine, mutation, glutamine synthetase, heterocyst, nitrogenase; Nosloc muscorurn.
Summary The blue-green alga N ostoe museorum and its Em-R het- nif2 - mutant are completely sensitive to 15 ppm concentration of L-methionine-DL-sulfoximille (MSO). Growth-toxicity created by MSO is independent of the inorganic nitrogen sources, i.e., NO a-, N0 2 - and NH" +. The pa.rent N. museorum does not form heterocysts in inorganic nitrogen media lacking MSO, but does form them in all these media (except glutamine) containing MSO. MSO-R strain does not form heterocysts in NO a-, N0 2 -, NH4 + or glutamine medium containing or lacking MSO.
Introduction
There are evidences that under aerobic conditions, heterocysts are major sites for nitrogenase biosynthesis and activity in blue-green algae possessing these specialized cells (FAY et a1. 1968; WOLK and WOJCIUCH 1971 a, b; TEL-OR and STEWART 1977; FLEMING and HASELKORN 1973). The fate of newly produced ammonia as a result of N2 fixation is of much current interest. The analogue L-methionine-DL-sulfoximine (MSO) (TATE and MEISTER 1973) is an inhibitor of glutamine synthetase (GS) activity in blue-green algae (STEWART and RoWELL 1975; ROWELL et al. 1977), and it has been shown based on the use of MSO (STEWART and ROWELL 1975; WOLK et al. 1976; SINGH et a1. 1977) that under N2fixing conditions the primary route for NHa assimilation involves GS and that the inhibition of GS is accompanied by nitrogenase synthesis and heterocysts production even in the presence of exogenous ammonia. It is thus highly desirable to find out the ways in which GS is regulated and the mechanism by which GS affects heterocysts and nitrogenase regulation. In order to trace these it was intended to isolate a mutant of the blue-green alga N ostoc muscorum, resis .. tant to MSO, with a view to examining the GS-regulated control of heterocyst and nitrogenase. The results suggest that factors other than GS or glutamine may be involved in the control of heterocyst and nitrogenase in N ostoe muscorum. 1) Part of Ph.D. thesis submitted by the senior author at the Banaras Hindu University, Varanasi ~ 221005, India.
2) Universtiy Department of Botany, University of Bihar, Muzaffarpur - 81l200l, India. 3) School of Life-sciences, Central University of Hyderabad, Hyderabad - 500134, India. 42 Biochem. Physiol. Pflanzen, Bd.176
'632
A. VAISHAMP AY AN and H. N. SINGH
Materials and Methods The source and characteristics of parent N. muscorum and its Em-R het- nif2 - mutant strain as well as their culture conditions have already been described in the preceding paper. The het+ nif+ parent and the Em-R het- nif2 - mutant strain were tested for their MSO-sensitivity and 15 ppm MSO/ml was found to be completely toxic and growth-inhibitory in both these strains. MSO was thus used in a concentration of 100 ppm for mutagenesis experiments. The procedure for the isolation of MSO-R mutant was similar to that applied for the isolation of MA-R mutant (Part I). MSO-R mutants were scored on 100 ppm MSO/ml supplemented NO a- agar plates. The viable population size was determined on MSO-unsupplemented NOa- agar plates and the mutation frequency was established by determining the number of resistant colonies present in the popUlation in proportion to the total number of colony-forming units present in that population. Two morphologically different types of mutant colonies were obtained. Out of these two types, one was a pure colonial form and it was thus, designated phenotypically as MSO-c, whereas the other was obtained in a diffused form and it was thus, designated phenotypically as MSO-d. The mutants thus scored, showed to have developed resistance to only 50 ppm MSO/ml of the medium. Clonal culture of this 50 ppm MSO/ml-resistant mutant was raised and the same was again subjected to a spontaneous mutation for resistance to 100 ppm MSO/ml. Quantitative enumeration of all these sets were separately done. It was found that MSO-c was non-heterocystous and non nitrogen-fixing (Em-R MSO-R hetnif-), and that MSO-d was heterocystous and nitrogen fixing (Em-R MSO-R het+ nif+). These two strains, i.e. MSO-c and MSO-d were characterized with regard to their growth behaviour, heterocyst development nad nitrogenase activity (heterocyst and nitrogenase were seen only in MSO-d strain) in N2, 5 mM NO a-, 5 mM N0 2- or 1 mM NH t + medium containing or lacking MSO, and the same were compared with the behaviour of the parent strain. Nitrogenase activity was assessed by the method of STEWAR'f et al. (1967) as n moles ethylene,ug chlorophyll a- 1 h- 1 . L-methionine-DL-sulfoximine (MSO) was a Sigma grade chemical and other chemicals used were of BDH grade. The experiments in general were done in five replicates. The results were analyzed statistically using the method of standard error (FISHER and YATES 1957).
Results
Em-R het- nif2 - strain was found to mutate spontaneously to MSO-resistance with a frequency of 1.7 + 3.4 X 10-7 out of which 78.31 + 3.46 % population belonged to the category of MSO-c and 21.69 + 3.46 0/ 0 population belonged to the category of MSO-~. The mutants thus scored were having a resistance to only 50 ppm of MSO. When this 50 ppm MBO-R culture was subjected again for a mutation leading to a resistance to 100 ppm MSO/ml, the same were scored with a frequency of 3.2 + 1.3 x 10-4, out of which 87.63 + 4.73 % population belonged to the category of MSO-c and 12.37 ±,4.73 % to that of M SO-d. A clonal culture of both these MBO-R strains (MSO-c and MBO-d) was raised and the detailed characterization was made. Fig. 1 shows the effect of 15 ppm MSO on the growth of parent and Em-R het- nif2strains in different inorganic nitrogen media. Both these strains died at an MSO concentra tion of 15 ppm and the inorganic nitrogen sources, i.e., N0 3 -, NO2- and NH4+ could not protect these strains from MSO-toxicity. However, the culture of MBO-S het+ nif+ parental strain showed a heterocyst differentiation with a toxic concentra-
633
Mutation in Nosloc muscorU1n. II.
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TIME (DAYS)
Fig. 1. Growth of parental and Em-R het- nif2- strains in N2 (x), 5 mM N0 3 - (0),5 mM N0 2 (~), or .1 mM NH4 + (0) medium. Parent unsupplemented ( ) or supplemented with 15 ppm M80 (_.-.-.-.-); Em-R het- nif2strain unsupplemented (---------) or supplemented with 15 ppm M80 (-.. -.. -.. -).
Table 1. Heterocyst frequency of parental and MSO-d (Em-R MSO-R hct+ nif+) strains of Nostoc muscorum in different inorganic m:trogen-supplernented medl:a with or without MSO (.15 ppm). Heterocyst frequency was determined by counting the number of heterocysts per hundred vegetative cells per filament. Five independent readings were taken each based on the observation of a random sample of 12 filaments Medium
N2 NO s - (5 mM)
N0 2 - (5 mM) NH4+ (1 mM)
Glutamine (100 ppm) 42*
MSO-d (Em-R MSO-R het+ nif+)
Parent -M80
+ M80
- M80
+MSO
5.0-6.0 0.0 0.0 0.0 0.0
5.0-6.5 2.0- 3.0 2.5-3.5 3.5-5.5 0.0
12.0-13.0 0.0 0.0 0.0 0.0
12.0-13.0 0.0 0.0 0.0 0.0
634
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V,USHAMPAYAN
and H. N.
SINGH
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Fig. 2. Growth of MSO-c ( ) and MSO-d NO z- (0) or 1 mM NH4+ medium (6.).
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Fig. 3. Growth of parent:(O-~O) and MSO-d ( X - - x ) strains in N2 medium.
Mutation in Nomoc muscorum. II.
635
tion of MSO in NOa-, N0 2- or NH4+ medium, while its MSO-unsupplemented cultures never formed heterocyst in these inorganic nitrogen media (Table 1). Both MSO-c and MSO-d strains grew well in NOa-, N0 2- or NH4+ medium with or without MSO (Fig. 2). The MSO-d strain grew well in N2 medium but its growth was slower in comparison to the MSO-S het+ nif+ parent (Fig. 3). It showed an average heterocyst frequency of 12-130/0 (Table 1). It did not show heterocysts in combined inorganic nitrogen medium containing MSO, while the parent N. museorum did so under similar conditions (Table 1). This appears to indicate that MSO-resistance is not accompanied by relief of heterocyst formation from inorganic nitrogen media. Like parent, the MSO-d strain did not form heterocysts in glutamine medium whether or not supplemented with MSO (Table 1). Nitrogenase activity of the parental strain was 6.2 n moles ethylene pg chlorophyll a-I h-1, whereas that of the MSO-d strain was of the order of 4.8 n moles ethylene I'g chlorophyll a-I h-t, e.i., approximately 3j4th of the activity of the parental strain. Discussion
The MSO-d produced heterocysts during its growth with molecular nitrogen. However, unlike the parental strain, it did not form heterocysts in MSO-containing inorganic nitrogen media. In the light of reputed MSO-sensitivity of GS activity leading to a relief of heterocyst and nitrogenase inhibition from NH4 +, it is difficult to visualize at the moment how the product of common genetic determinant could be involved in GS controlled regulation of heterocyst and nitrogenase. It could also be that the MSO-d strain is a result of multiple mutation. In that case inability of MSO to relieve heterocyst and nitrogenase from NH4+ in MSO-d strain could be due to permeability mutation. The alternative reason could be that GS in MSO-d strain has become resistant to MSO so much that MSO could no longer be able to bind to and inactivate GS. Mutation to MSO-resistance in Em-R het- nif2 - strain, showing diffuse growth and hence called MSO-d was stimultaneously accompanied by mutation to prototrophic het+ nif+ character. This is certainly the case of a pleiotropic mutation. The simplest explanation of observed pleiotropy could be due to a common genetic determinant controlling both MSO-sensitivity and inhibition of heterocyst and nitrogenase formation. The loss of this common determinant by mutation would thus confer resistance to MSO as well as would release the inhibition of heterocyst and nitrogenase formation. The findings of MSO action on het+ nif+ parent, appears no doubt to correspond to those Anabaena cylindrica (STACEY et a1. 1979) and N ostoe linekia (SINGH et a1. 1977). MSO-containing cultures of both Anabaena and N ostoe do form heterocysts in NO a- or NH,+ medium but not in glutamine medium. Similar is the case here with the parent as well as the MSO-d mutant. This appears to suggest glutamine as an inhibitor of heterocyst and nitrogenase formation both. Recently, SINGH and SINGH (1978) isolated an azideresistant mutant of N. muscorum derepressed for heterocyst and nitrogenase, and based on the results of glutamine effect on this as well as the parent N. muscorum, they suggested the operation of two levels of glutamine-sensitive regulation - one which oper-
636
A. VAISHAMP.\YAN and H. N. SINGH
ates through the common genetic determinant of heterocyst and nitrogenase regulation, and the other exclusively to nitrogenase regulation, thereby suggesting that glutamine or a close metabolic product of it is the real in vivo common inhibitor of the production of heterocyst and nitrogenaso in N. ·muscorum (SINGH and SINGH 1978). Recently, HASELKORN (1978) reported that the cyanobacterial nit operon is not likely to be operated by the adenylylation state of GS or even by GS at all. He found that Anabaena GS is not affected in vitro by levels of MSO, 1,000 or at least 100-fold higher than that needed to inhibit E. coli GS by 95 % and thus suggested that possibly MSO is metabolized in vivo to another compound which is tho actual inhibitor of GS. Glutamine itself is a good candidate for a co-ropresor. A more clear understanding of the actual role of GS or glutamine or any derivative product of it in controlling heterocyst and nitrogenase may be made if a glutamine-requiring mutant is isolated in blue-green algae. Acknowledgements The authors wish to thank Professor PETER J. DART and his technical assistant Mr. T. BAGCHI, Microbiology Division, ICRISAT, Hyderabad - 28 for assessing the nitrogenase activity of the mutant on gas chromatograph. The senior author gratefully acknowledges the financial assistance received in the form of a research fellowship granted by the Indian Atomic Energy Commission, Bhabha Atomic Research Centre, Bombay - 400085.
References FAY, P., STEWART, W. D. P., WALSBY, A. E., and FOGG, G. E.: Is the heterocyst the site of nitrogen fixation in blue-green algae? Nature (Lond.) 220, 810-812 (1968). FISHER, S. R. A., and YAIES, F. A.: Statistical Tables. Oliver and Boyd, Edinburgh 1957. FLEMING, H., and HASELKORN, R.: Differentiation in Nosloc muscorum: Nitrogenase is synthesized in heterocysts. Proc. Nat. Acad. Sci. USA 70, 2727-2731 (1973). ROWELL, P., ENTICOTT, S., and STEWART,W. D. P.: Glutamine synthetase and nitrogenase activity in the blue-green alga Anaebaena cylindrica. New Phytol. 79, 41-54 (1977). SINGH, H. N., LAHDA, J. K., and KUMAR, H. D.: Genetic control of heterocyst formation in the blue-green algae Nostoc muscorum and Nostoc linckia. Arch. Microbiol. 114:, 155-159 (1977). - and SINGH, H. N. Jr.: An azide-resistant mutant of the blue-green alga Nostoc muscorum producing heterocyst and nitrogenase in the presence of fixed inorganic nitrogen source. Arch. Microbiol. 119, 197- 201 (1978). STACEY, G., BOTTOMLEY, P. J., VAN BAALEN,C., and TABITA, F. R.: Control of heterocyst and nitrogenase synthesis in cyanobacteria. J. BioI. Chem; 137, 321-326 (1979). STEWART, W. D. P., FITZGERALD, G. P., and BURRIS, R. H.: In situ studies on N2 fixation using the acetylene reduction technique. Proc. Nat. Acad. Sci. USA 68, 2071-2078 (1967). - and ROWELL, P.: Effect of L-methionine-DL-sulfoximine on the assimilation of newly fixed NH a, acetylene reduction and heterocyst production in Anabaena cylindrica. Biochem. Biophys. Res. Commun. 65, 846- 856 (1975). TATE, S. S., and MEISTER, A.: Glutamine synthetase of mamalian liver and brain. In: "The enzymes of glutamine metabolism" (Eds. S. PRUSINER and E. R. STADTMAN) pp. 77-128. Academic Press, London-New York 1973. TEL-OR, E., and STEWART, W. D. P.: Photosynthetic components and activities of nitrogen-fixing isolated heterocysts of Anabaena cylindrica. Proc. Roy. Soc. London 198, 61-86 (1977).
Mutation in Nostoc muscorum. II.
637
WOLK, C. P., THOMAS, J., and SHAFFER, P. W.: Pathway of nitrogen metabolism after fixation of l3N-Iabeled nitrogen gas by the cyanobacterium Anabaena cylindrica. J. BioI. Chern. 251, 5027 to 5034 (1976). - and WOJCIUCn, E.: Biphasic time course of solubilization of nitrogenase during cavitation of aerobically grown Anabaena cylindrica. J. Phycol. 7, 339-344 (1971 a). - - Photoreduction of acetylene by heterocyst. Planta 97, 126-134 (1971 b). Received September 26, 1980; in revised form January 23, 1981.
Authors' addersses: AKHOURI VAISHAMPAYAN, University Department of Botany, University of Bihar, Muzaffarpur ~ 842001 (India); HRIDAYA NARAYAN SINGH, School of Life~sciences, Central University of Hyderabad, Hyderabad - 500134 (India).
Mutation in the Blue-green Alga N ostoc muscorum II. L-methionine-DL-sulfoximine-resistant Mutants!) A. VAISHAMPAYAN 2) and H. N. SINGH3) Department of Botany, Banaras Hindu Universi ty, Varanasi, India Key Term Index: L~methionine-DL-su]foximine, mutation, glutamine synthetase, heterocyst, nitrogenase; Nosloc muscorurn.
Summary The blue-green alga N ostoe museorum and its Em-R het- nif2 - mutant are completely sensitive to 15 ppm concentration of L-methionine-DL-sulfoximille (MSO). Growth-toxicity created by MSO is independent of the inorganic nitrogen sources, i.e., NO a-, N0 2 - and NH" +. The pa.rent N. museorum does not form heterocysts in inorganic nitrogen media lacking MSO, but does form them in all these media (except glutamine) containing MSO. MSO-R strain does not form heterocysts in NO a-, N0 2 -, NH4 + or glutamine medium containing or lacking MSO.
Introduction
There are evidences that under aerobic conditions, heterocysts are major sites for nitrogenase biosynthesis and activity in blue-green algae possessing these specialized cells (FAY et a1. 1968; WOLK and WOJCIUCH 1971 a, b; TEL-OR and STEWART 1977; FLEMING and HASELKORN 1973). The fate of newly produced ammonia as a result of N2 fixation is of much current interest. The analogue L-methionine-DL-sulfoximine (MSO) (TATE and MEISTER 1973) is an inhibitor of glutamine synthetase (GS) activity in blue-green algae (STEWART and RoWELL 1975; ROWELL et al. 1977), and it has been shown based on the use of MSO (STEWART and ROWELL 1975; WOLK et al. 1976; SINGH et a1. 1977) that under N2fixing conditions the primary route for NHa assimilation involves GS and that the inhibition of GS is accompanied by nitrogenase synthesis and heterocysts production even in the presence of exogenous ammonia. It is thus highly desirable to find out the ways in which GS is regulated and the mechanism by which GS affects heterocysts and nitrogenase regulation. In order to trace these it was intended to isolate a mutant of the blue-green alga N ostoc muscorum, resis .. tant to MSO, with a view to examining the GS-regulated control of heterocyst and nitrogenase. The results suggest that factors other than GS or glutamine may be involved in the control of heterocyst and nitrogenase in N ostoe muscorum. 1) Part of Ph.D. thesis submitted by the senior author at the Banaras Hindu University, Varanasi ~ 221005, India.
2) Universtiy Department of Botany, University of Bihar, Muzaffarpur - 81l200l, India. 3) School of Life-sciences, Central University of Hyderabad, Hyderabad - 500134, India. 42 Biochem. Physiol. Pflanzen, Bd.176
'632
A. VAISHAMP AY AN and H. N. SINGH
Materials and Methods The source and characteristics of parent N. muscorum and its Em-R het- nif2 - mutant strain as well as their culture conditions have already been described in the preceding paper. The het+ nif+ parent and the Em-R het- nif2 - mutant strain were tested for their MSO-sensitivity and 15 ppm MSO/ml was found to be completely toxic and growth-inhibitory in both these strains. MSO was thus used in a concentration of 100 ppm for mutagenesis experiments. The procedure for the isolation of MSO-R mutant was similar to that applied for the isolation of MA-R mutant (Part I). MSO-R mutants were scored on 100 ppm MSO/ml supplemented NO a- agar plates. The viable population size was determined on MSO-unsupplemented NOa- agar plates and the mutation frequency was established by determining the number of resistant colonies present in the popUlation in proportion to the total number of colony-forming units present in that population. Two morphologically different types of mutant colonies were obtained. Out of these two types, one was a pure colonial form and it was thus, designated phenotypically as MSO-c, whereas the other was obtained in a diffused form and it was thus, designated phenotypically as MSO-d. The mutants thus scored, showed to have developed resistance to only 50 ppm MSO/ml of the medium. Clonal culture of this 50 ppm MSO/ml-resistant mutant was raised and the same was again subjected to a spontaneous mutation for resistance to 100 ppm MSO/ml. Quantitative enumeration of all these sets were separately done. It was found that MSO-c was non-heterocystous and non nitrogen-fixing (Em-R MSO-R hetnif-), and that MSO-d was heterocystous and nitrogen fixing (Em-R MSO-R het+ nif+). These two strains, i.e. MSO-c and MSO-d were characterized with regard to their growth behaviour, heterocyst development nad nitrogenase activity (heterocyst and nitrogenase were seen only in MSO-d strain) in N2, 5 mM NO a-, 5 mM N0 2- or 1 mM NH t + medium containing or lacking MSO, and the same were compared with the behaviour of the parent strain. Nitrogenase activity was assessed by the method of STEWAR'f et al. (1967) as n moles ethylene,ug chlorophyll a- 1 h- 1 . L-methionine-DL-sulfoximine (MSO) was a Sigma grade chemical and other chemicals used were of BDH grade. The experiments in general were done in five replicates. The results were analyzed statistically using the method of standard error (FISHER and YATES 1957).
Results
Em-R het- nif2 - strain was found to mutate spontaneously to MSO-resistance with a frequency of 1.7 + 3.4 X 10-7 out of which 78.31 + 3.46 % population belonged to the category of MSO-c and 21.69 + 3.46 0/ 0 population belonged to the category of MSO-~. The mutants thus scored were having a resistance to only 50 ppm of MSO. When this 50 ppm MBO-R culture was subjected again for a mutation leading to a resistance to 100 ppm MSO/ml, the same were scored with a frequency of 3.2 + 1.3 x 10-4, out of which 87.63 + 4.73 % population belonged to the category of MSO-c and 12.37 ±,4.73 % to that of M SO-d. A clonal culture of both these MBO-R strains (MSO-c and MBO-d) was raised and the detailed characterization was made. Fig. 1 shows the effect of 15 ppm MSO on the growth of parent and Em-R het- nif2strains in different inorganic nitrogen media. Both these strains died at an MSO concentra tion of 15 ppm and the inorganic nitrogen sources, i.e., N0 3 -, NO2- and NH4+ could not protect these strains from MSO-toxicity. However, the culture of MBO-S het+ nif+ parental strain showed a heterocyst differentiation with a toxic concentra-
633
Mutation in Nosloc muscorU1n. II.
>-
..... If)
z UJ o 0'3 --1 « u ..... e..
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-- "-. '- ----- ''-.
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4
'-.
.
--=-__--=-__
0'0 1..--_ _-'--_ _- ' -_ _
o
.
6
8
----oL..--..I
10
TIME (DAYS)
Fig. 1. Growth of parental and Em-R het- nif2- strains in N2 (x), 5 mM N0 3 - (0),5 mM N0 2 (~), or .1 mM NH4 + (0) medium. Parent unsupplemented ( ) or supplemented with 15 ppm M80 (_.-.-.-.-); Em-R het- nif2strain unsupplemented (---------) or supplemented with 15 ppm M80 (-.. -.. -.. -).
Table 1. Heterocyst frequency of parental and MSO-d (Em-R MSO-R hct+ nif+) strains of Nostoc muscorum in different inorganic m:trogen-supplernented medl:a with or without MSO (.15 ppm). Heterocyst frequency was determined by counting the number of heterocysts per hundred vegetative cells per filament. Five independent readings were taken each based on the observation of a random sample of 12 filaments Medium
N2 NO s - (5 mM)
N0 2 - (5 mM) NH4+ (1 mM)
Glutamine (100 ppm) 42*
MSO-d (Em-R MSO-R het+ nif+)
Parent -M80
+ M80
- M80
+MSO
5.0-6.0 0.0 0.0 0.0 0.0
5.0-6.5 2.0- 3.0 2.5-3.5 3.5-5.5 0.0
12.0-13.0 0.0 0.0 0.0 0.0
12.0-13.0 0.0 0.0 0.0 0.0
634
A.
V,USHAMPAYAN
and H. N.
SINGH
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2
4
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TIME (DAYS>
Fig. 2. Growth of MSO-c ( ) and MSO-d NO z- (0) or 1 mM NH4+ medium (6.).
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mutant strains in 5 mM NO a- (x), 5 mM
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Fig. 3. Growth of parent:(O-~O) and MSO-d ( X - - x ) strains in N2 medium.
Mutation in Nomoc muscorum. II.
635
tion of MSO in NOa-, N0 2- or NH4+ medium, while its MSO-unsupplemented cultures never formed heterocyst in these inorganic nitrogen media (Table 1). Both MSO-c and MSO-d strains grew well in NOa-, N0 2- or NH4+ medium with or without MSO (Fig. 2). The MSO-d strain grew well in N2 medium but its growth was slower in comparison to the MSO-S het+ nif+ parent (Fig. 3). It showed an average heterocyst frequency of 12-130/0 (Table 1). It did not show heterocysts in combined inorganic nitrogen medium containing MSO, while the parent N. museorum did so under similar conditions (Table 1). This appears to indicate that MSO-resistance is not accompanied by relief of heterocyst formation from inorganic nitrogen media. Like parent, the MSO-d strain did not form heterocysts in glutamine medium whether or not supplemented with MSO (Table 1). Nitrogenase activity of the parental strain was 6.2 n moles ethylene pg chlorophyll a-I h-1, whereas that of the MSO-d strain was of the order of 4.8 n moles ethylene I'g chlorophyll a-I h-t, e.i., approximately 3j4th of the activity of the parental strain. Discussion
The MSO-d produced heterocysts during its growth with molecular nitrogen. However, unlike the parental strain, it did not form heterocysts in MSO-containing inorganic nitrogen media. In the light of reputed MSO-sensitivity of GS activity leading to a relief of heterocyst and nitrogenase inhibition from NH4 +, it is difficult to visualize at the moment how the product of common genetic determinant could be involved in GS controlled regulation of heterocyst and nitrogenase. It could also be that the MSO-d strain is a result of multiple mutation. In that case inability of MSO to relieve heterocyst and nitrogenase from NH4+ in MSO-d strain could be due to permeability mutation. The alternative reason could be that GS in MSO-d strain has become resistant to MSO so much that MSO could no longer be able to bind to and inactivate GS. Mutation to MSO-resistance in Em-R het- nif2 - strain, showing diffuse growth and hence called MSO-d was stimultaneously accompanied by mutation to prototrophic het+ nif+ character. This is certainly the case of a pleiotropic mutation. The simplest explanation of observed pleiotropy could be due to a common genetic determinant controlling both MSO-sensitivity and inhibition of heterocyst and nitrogenase formation. The loss of this common determinant by mutation would thus confer resistance to MSO as well as would release the inhibition of heterocyst and nitrogenase formation. The findings of MSO action on het+ nif+ parent, appears no doubt to correspond to those Anabaena cylindrica (STACEY et a1. 1979) and N ostoe linekia (SINGH et a1. 1977). MSO-containing cultures of both Anabaena and N ostoe do form heterocysts in NO a- or NH,+ medium but not in glutamine medium. Similar is the case here with the parent as well as the MSO-d mutant. This appears to suggest glutamine as an inhibitor of heterocyst and nitrogenase formation both. Recently, SINGH and SINGH (1978) isolated an azideresistant mutant of N. muscorum derepressed for heterocyst and nitrogenase, and based on the results of glutamine effect on this as well as the parent N. muscorum, they suggested the operation of two levels of glutamine-sensitive regulation - one which oper-
636
A. VAISHAMP.\YAN and H. N. SINGH
ates through the common genetic determinant of heterocyst and nitrogenase regulation, and the other exclusively to nitrogenase regulation, thereby suggesting that glutamine or a close metabolic product of it is the real in vivo common inhibitor of the production of heterocyst and nitrogenaso in N. ·muscorum (SINGH and SINGH 1978). Recently, HASELKORN (1978) reported that the cyanobacterial nit operon is not likely to be operated by the adenylylation state of GS or even by GS at all. He found that Anabaena GS is not affected in vitro by levels of MSO, 1,000 or at least 100-fold higher than that needed to inhibit E. coli GS by 95 % and thus suggested that possibly MSO is metabolized in vivo to another compound which is tho actual inhibitor of GS. Glutamine itself is a good candidate for a co-ropresor. A more clear understanding of the actual role of GS or glutamine or any derivative product of it in controlling heterocyst and nitrogenase may be made if a glutamine-requiring mutant is isolated in blue-green algae. Acknowledgements The authors wish to thank Professor PETER J. DART and his technical assistant Mr. T. BAGCHI, Microbiology Division, ICRISAT, Hyderabad - 28 for assessing the nitrogenase activity of the mutant on gas chromatograph. The senior author gratefully acknowledges the financial assistance received in the form of a research fellowship granted by the Indian Atomic Energy Commission, Bhabha Atomic Research Centre, Bombay - 400085.
References FAY, P., STEWART, W. D. P., WALSBY, A. E., and FOGG, G. E.: Is the heterocyst the site of nitrogen fixation in blue-green algae? Nature (Lond.) 220, 810-812 (1968). FISHER, S. R. A., and YAIES, F. A.: Statistical Tables. Oliver and Boyd, Edinburgh 1957. FLEMING, H., and HASELKORN, R.: Differentiation in Nosloc muscorum: Nitrogenase is synthesized in heterocysts. Proc. Nat. Acad. Sci. USA 70, 2727-2731 (1973). ROWELL, P., ENTICOTT, S., and STEWART,W. D. P.: Glutamine synthetase and nitrogenase activity in the blue-green alga Anaebaena cylindrica. New Phytol. 79, 41-54 (1977). SINGH, H. N., LAHDA, J. K., and KUMAR, H. D.: Genetic control of heterocyst formation in the blue-green algae Nostoc muscorum and Nostoc linckia. Arch. Microbiol. 114:, 155-159 (1977). - and SINGH, H. N. Jr.: An azide-resistant mutant of the blue-green alga Nostoc muscorum producing heterocyst and nitrogenase in the presence of fixed inorganic nitrogen source. Arch. Microbiol. 119, 197- 201 (1978). STACEY, G., BOTTOMLEY, P. J., VAN BAALEN,C., and TABITA, F. R.: Control of heterocyst and nitrogenase synthesis in cyanobacteria. J. BioI. Chem; 137, 321-326 (1979). STEWART, W. D. P., FITZGERALD, G. P., and BURRIS, R. H.: In situ studies on N2 fixation using the acetylene reduction technique. Proc. Nat. Acad. Sci. USA 68, 2071-2078 (1967). - and ROWELL, P.: Effect of L-methionine-DL-sulfoximine on the assimilation of newly fixed NH a, acetylene reduction and heterocyst production in Anabaena cylindrica. Biochem. Biophys. Res. Commun. 65, 846- 856 (1975). TATE, S. S., and MEISTER, A.: Glutamine synthetase of mamalian liver and brain. In: "The enzymes of glutamine metabolism" (Eds. S. PRUSINER and E. R. STADTMAN) pp. 77-128. Academic Press, London-New York 1973. TEL-OR, E., and STEWART, W. D. P.: Photosynthetic components and activities of nitrogen-fixing isolated heterocysts of Anabaena cylindrica. Proc. Roy. Soc. London 198, 61-86 (1977).
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WOLK, C. P., THOMAS, J., and SHAFFER, P. W.: Pathway of nitrogen metabolism after fixation of l3N-Iabeled nitrogen gas by the cyanobacterium Anabaena cylindrica. J. BioI. Chern. 251, 5027 to 5034 (1976). - and WOJCIUCn, E.: Biphasic time course of solubilization of nitrogenase during cavitation of aerobically grown Anabaena cylindrica. J. Phycol. 7, 339-344 (1971 a). - - Photoreduction of acetylene by heterocyst. Planta 97, 126-134 (1971 b). Received September 26, 1980; in revised form January 23, 1981.
Authors' addersses: AKHOURI VAISHAMPAYAN, University Department of Botany, University of Bihar, Muzaffarpur ~ 842001 (India); HRIDAYA NARAYAN SINGH, School of Life~sciences, Central University of Hyderabad, Hyderabad - 500134 (India).