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Growth and Pigments of Nitrosoguanidine Treated and Untreated Cells of the Blue-green Alga Anacystis nidulans in Different Nitrogen Sources
Biochem. Physiol. Pflanzen !'is, 82-85 (1978)
Short Communication
Growth and Pigments of Nitrosoguanidine Treated and Untreated Cells of the Blue-green Alga Anacystis nidulans in Different Nitrogen Sources B. D. SINHA and H. D. KUMAR Department of Botany, School of Basic Sciences and Humanities, Udaipur University, Udaipur, India Key Term Index: Nitrosoguanidine, nitrogen sources, culture growth, pigment ratio; Anacystis
nidulans.
Summary NTG-treated cells of the blue-green alga Anacystis nidulans grew well in media supplemented with sodium nitrate and ammonium chloride but not in sodium nitrite in which case a yellowing of the cultures was also noticed. Absorption spectra of acetone extracts showed greater amounts of carotenoids and lesser amount of chlorophyll for the yellow cultures in nitrite medium while the reverse was true for cultures growing in media containing either nitrate or ammonium. No change of this kind occurred in untreated cell suspensions in the presence Of different inorganic nitrogen sources supplied to them.
Because Anacystis nidulans possesses certain desirable attributes in an exerimental organism for genetical, physiological and biochemical studies, it has been used for such studies more frequently than any other strain of blue-green algae. Like some bacteria, many blue-greens are able to fix molecular nitrogen. Several species of the Chroococales, including A. nidulans however do not have this property. Blue-green algae grow best under slightly alkaline conditions (FOGG 1956) and utilize both amonium and nitrate salts as nitrogen sources (KRATZ and MYERS 1955) provided the pH changes are minimal (PROVASOLI 1958; GERLOFF et al. 1952; MACLACHLAN and GORHAM 1962). In general, nitrate is assimilated after its enzymic conversion into ammonia through stepwise reduction processes. Since mutants have been employed for the study of metabolic reactions (SHAFER et al. 1961; STEVANS and VAN BAALEN 1970) it was proposed to study the growth behaviour of .A. nidulans in media containing nitrate, nitrite and ammonium nitrogen after treatment with nitrosoguanidine (NTG) which may reflect the pattern and activities of enzymes concerned in the pathway of nitrate assimilation. For comparison, a parallel experiment was set up with untreated cells of the alga. Organism. Pure cultures of Anacystis nidulans DROUET (MYERS' strain) were grown in a culture chamber in the ALLEN'S (1968) modified HUGHES et al. medium. Its detailed composition, methods of culturing and harvesting the alga have been given in our earlier paper (SINHA and KUMAR 1973). Chemicals. N_methyl-N'nitro-N-nitrosoguanidine (NTG) was a product of Aldrich Chemical co., Milwaukee, Wisconsin. Fresh solutions were prepared in sterile water or culture medium for each experiment and were used without sterilization. Sodium nitrate, sodium nitrite and ammonium chloride were used as the nitrogen sources. Growth estimation. Exponentially growing cells were washed by centrifugation and suspended in glass distilled water. The algal suspension was divided into two sets, one of which served as control
1
F
s s
s
d f f e e n
al or co-
e d
N
o
Nitrosoguanidine-treated Blue-green Algae
while the other was treated with NTG (100 p,g/ml) for one hour. Cells were next inoculated in 0.5 ml aJiquots containing 10 4 cells into liquid media containing sodium nitrate, sodium nitrite and ammonium chloride as nitrogen sources. Untreated cells which served as control were also similarly inocu2 DC and 500-600 lux. Growth was determined by measulated. Cultures were incubated at 37 DC ring optical density at 600 nm in a Bausch and Lomb Spectronic-20 spectrocolorimeter while pigment characteristics were examined in acetone extracts of 10-day old cells.
±
Figs. 1 and 2 show the growth responses of untreated and NTG-treated cells in the presence of different nitrogen sources. It appears that the mutagen treated cells grew well in media supplemented with sodium nitrate and ammonium chloride but not in sodium nitrite; the untreated controls exhibited almost identical growth in the presence of different inorganic nitrogen sources supplied to them. Another point of interest was noted in the visually observed pigmentation. A marked colour change from blue-green to yellow was noted on old cultures of NTG-treated cells growing in medium containing nitrite. No change of this kind occurred in untreated cells growing in nitrite medium. The yellow cultures in the nitrite medium as well as those growing in media containing sodium nitrate and ammonium chloride were analysed for acetone-soluble pigments. Fig. 3 shows that the yellow cultures had greater amounts of carotenoids and lesser amount of chlorophyll while the reverse was true for cultures growing in media containing either nitrate of ammonium nitrogen. A selective decrease in photosynthetic pigments of NTG-treated cells in nitrite medium may be attributed to the unmasking of carotenoids leading to the yellowing of the culture. When inocula taken from yellow cultures were transferred from nitrite to media containing either nitrate or ammonium nitrogen, the normal blue-green colour was restored. A similar yellowing of the culture was observed in Plectonema nos to corum by KINGSRURY (1956) who attributes this pigment change to depletion of nitrogen content of the medium. He further observed regreening of cultures upon addition of nitrogen as was true in the present study when the yellow cultures were transferred from nitrite to media containing nitrate or ammonium nitrogen.
ER
'·0
een in,
>I
Z UJ
o 0.5
re ds 3). cal or um
ed rol
83
~
«
u
I-
a..
o
0·0 - t - - - - - , - - - - - - y - - - - - - r - - - - - - , - ' o 8 2 4 6 DAYS
Fig. 1. Growth of Anacystis nidulans (Wild strain) in media containinlJ NaN03 , NH4 Cl and NaN02 • 6*
B. D.
84
SINHA
and H. D.
KUMAR
Since most blue-green algae are obligate photoautotrophs deriving all their carbon and energy source from photosynthesis, chlorophyll and phycocyanin must therefore be two of their critical and determinative constituents. Chlorophyll is the main pigment of photochemical reactions and is made more efficient by phycocyanin. According to this, one should expect impaired growth behaviour in organisms having lesser amounts of photosynthetic pigments. The sluggish growth of mutagen treated cells in nitrite supplemented media may be attributed to its partly toxic nature but this possibility seems ruled out since no such effect was observed in control cultures growing in nitrite medium. An alternative possibility seems related to the problem of phycocyanin synthesis on the one hand, and the mechanism of phycocyanin-chlorophyll energy transfer ,.,T---------------------------------------~
'·0 ~ o·s
~ 0·6 ..J
....
0·4
0..
o
0·2
2
4
8
6
DAYS
Fig. 2. Growth of Anacystis nidulans (NTG-treated) in media containing NaNOs, NH4Cl and NaN0 2
'.0 >-
O.S
....
~ 0.6
o ~ 0·4 ()
....0..
o 0.2 O.O+---_T----~--_T----~--_T----~--_r~
420
460
.500
540 SSO 620 WAVELENGTH lnm)
660
Fig. 3. Absorption spectra of acetone extracts from equal cell volumes growing in media containing nitrate, nitrite and ammonium nitrogen.
700
of A. nidulans
(NTG - treated)
)
Nitrosoguanidine-treated Blue-green Algae
85
on the other. Furthermore, owing to a decrease in chlorophyll pigments, the photochemically generated hydrogen donors may not be availabl.e to the extent of being utilised for the reduction of nitrite nitrogen. These could be some possible causes for the retarded growth of cells in the presence of nitrite. Thus the data presented reveal a remarkable and close intermeshing of nitrite assimilation and photochemical reactions of photosynthesis. Although colour shifts usually allow the organism to adapt itself to a better use of light received, the reverse was true in the pres en t study, where in the pigment changes indicated a relatively lower yield of photosynthetic pigment leading to decreased phototrophic assimilation reflected in the slow and sluggish growth of yellow cultures in nitrite medium. As to the possible causes for this change in pigmentation of cultures, light does not appear to be responsible for it although both the shape of the light intersity curve of photosynthesis and the visually observed pigmentation could be varied considerably by the culture conditions elsewhere (KRATZ and MYERS 1955). However, since nitrite has also been used as a potent mutagen, it may be that mutagenic activities of NGT coupled with that of nitrite may be the cltuse for the change in the visually observable yellowing of cultures leading to a slow and reatarded growth. Acknowledgement The authors thank Dr. H. N. SINGH for helpful suggestions and criticism.
References ALLEN, M. M.: Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4, 1-4 (1968). FOGG, G. E.: Comparative physiology and biochemistry of the blue-green algae. Bacteriol. Rev. 20, 148-165 (1956). GERLOFF, G. C. FITZGERALD, G. P., and SKOOG, F.: The mineral nutrition of Microcystis aeruginosa Am. J. Botany 39, 26-32 (1952). KINGSBURY, J. M.: On pigment changes and growth in the blue-green alga, Plectonema nostocorum Bornet Ex Gomont. BioI. Bull. 10,310-319 (1956). KRATZ, W. A., and MYERS, J.: Nutrition and growth of several blue-green algae. Am.- J. Botany 42, 282-287 (1955). MCLACHLAN, J., and GORHAM, P. R.: Effects of pH and Nitrogen sources on growth of Microcystis aeruginosa Kutz. Can. J. Microbiol. 8, 1-11 (1962). PROVASOLI, L.: Nutrition and ecology of protozoa and algae. Ann. Rev. Microbiol. 12, 279-308 (1958). SHAFER, J. Jr., BAKER, J. E., and THOMSON, J. F.: A Chlorella mutant lacking nitrate reductase, Am. J. Botany 48,896-897 (1961). SINHA, B. D., and KUMAR, H. D.: Spontaneous and induced mutation frequencies in the blue-green alga Anacystis nidulans. Proc. Ind. Nat. Science Acad. 39, 143-149 (1973). STEVENS, S. E. Jr., and VAN BAALEN, C.: Growth characteristics of selected mutants of a coccoid blue-green alga. Arch. Mikrobiol. 72, 1-8 (1970). Received February 21, 1978.
Authors' addresses: B. D. SINHA, Department of Botany, University of Bihar, Muzaffarpur842001, India, and H. D. KUMAR Banaras Hindu University, Varanasi, 221005, India.
Short Communication
Growth and Pigments of Nitrosoguanidine Treated and Untreated Cells of the Blue-green Alga Anacystis nidulans in Different Nitrogen Sources B. D. SINHA and H. D. KUMAR Department of Botany, School of Basic Sciences and Humanities, Udaipur University, Udaipur, India Key Term Index: Nitrosoguanidine, nitrogen sources, culture growth, pigment ratio; Anacystis
nidulans.
Summary NTG-treated cells of the blue-green alga Anacystis nidulans grew well in media supplemented with sodium nitrate and ammonium chloride but not in sodium nitrite in which case a yellowing of the cultures was also noticed. Absorption spectra of acetone extracts showed greater amounts of carotenoids and lesser amount of chlorophyll for the yellow cultures in nitrite medium while the reverse was true for cultures growing in media containing either nitrate or ammonium. No change of this kind occurred in untreated cell suspensions in the presence Of different inorganic nitrogen sources supplied to them.
Because Anacystis nidulans possesses certain desirable attributes in an exerimental organism for genetical, physiological and biochemical studies, it has been used for such studies more frequently than any other strain of blue-green algae. Like some bacteria, many blue-greens are able to fix molecular nitrogen. Several species of the Chroococales, including A. nidulans however do not have this property. Blue-green algae grow best under slightly alkaline conditions (FOGG 1956) and utilize both amonium and nitrate salts as nitrogen sources (KRATZ and MYERS 1955) provided the pH changes are minimal (PROVASOLI 1958; GERLOFF et al. 1952; MACLACHLAN and GORHAM 1962). In general, nitrate is assimilated after its enzymic conversion into ammonia through stepwise reduction processes. Since mutants have been employed for the study of metabolic reactions (SHAFER et al. 1961; STEVANS and VAN BAALEN 1970) it was proposed to study the growth behaviour of .A. nidulans in media containing nitrate, nitrite and ammonium nitrogen after treatment with nitrosoguanidine (NTG) which may reflect the pattern and activities of enzymes concerned in the pathway of nitrate assimilation. For comparison, a parallel experiment was set up with untreated cells of the alga. Organism. Pure cultures of Anacystis nidulans DROUET (MYERS' strain) were grown in a culture chamber in the ALLEN'S (1968) modified HUGHES et al. medium. Its detailed composition, methods of culturing and harvesting the alga have been given in our earlier paper (SINHA and KUMAR 1973). Chemicals. N_methyl-N'nitro-N-nitrosoguanidine (NTG) was a product of Aldrich Chemical co., Milwaukee, Wisconsin. Fresh solutions were prepared in sterile water or culture medium for each experiment and were used without sterilization. Sodium nitrate, sodium nitrite and ammonium chloride were used as the nitrogen sources. Growth estimation. Exponentially growing cells were washed by centrifugation and suspended in glass distilled water. The algal suspension was divided into two sets, one of which served as control
1
F
s s
s
d f f e e n
al or co-
e d
N
o
Nitrosoguanidine-treated Blue-green Algae
while the other was treated with NTG (100 p,g/ml) for one hour. Cells were next inoculated in 0.5 ml aJiquots containing 10 4 cells into liquid media containing sodium nitrate, sodium nitrite and ammonium chloride as nitrogen sources. Untreated cells which served as control were also similarly inocu2 DC and 500-600 lux. Growth was determined by measulated. Cultures were incubated at 37 DC ring optical density at 600 nm in a Bausch and Lomb Spectronic-20 spectrocolorimeter while pigment characteristics were examined in acetone extracts of 10-day old cells.
±
Figs. 1 and 2 show the growth responses of untreated and NTG-treated cells in the presence of different nitrogen sources. It appears that the mutagen treated cells grew well in media supplemented with sodium nitrate and ammonium chloride but not in sodium nitrite; the untreated controls exhibited almost identical growth in the presence of different inorganic nitrogen sources supplied to them. Another point of interest was noted in the visually observed pigmentation. A marked colour change from blue-green to yellow was noted on old cultures of NTG-treated cells growing in medium containing nitrite. No change of this kind occurred in untreated cells growing in nitrite medium. The yellow cultures in the nitrite medium as well as those growing in media containing sodium nitrate and ammonium chloride were analysed for acetone-soluble pigments. Fig. 3 shows that the yellow cultures had greater amounts of carotenoids and lesser amount of chlorophyll while the reverse was true for cultures growing in media containing either nitrate of ammonium nitrogen. A selective decrease in photosynthetic pigments of NTG-treated cells in nitrite medium may be attributed to the unmasking of carotenoids leading to the yellowing of the culture. When inocula taken from yellow cultures were transferred from nitrite to media containing either nitrate or ammonium nitrogen, the normal blue-green colour was restored. A similar yellowing of the culture was observed in Plectonema nos to corum by KINGSRURY (1956) who attributes this pigment change to depletion of nitrogen content of the medium. He further observed regreening of cultures upon addition of nitrogen as was true in the present study when the yellow cultures were transferred from nitrite to media containing nitrate or ammonium nitrogen.
ER
'·0
een in,
>I
Z UJ
o 0.5
re ds 3). cal or um
ed rol
83
~
«
u
I-
a..
o
0·0 - t - - - - - , - - - - - - y - - - - - - r - - - - - - , - ' o 8 2 4 6 DAYS
Fig. 1. Growth of Anacystis nidulans (Wild strain) in media containinlJ NaN03 , NH4 Cl and NaN02 • 6*
B. D.
84
SINHA
and H. D.
KUMAR
Since most blue-green algae are obligate photoautotrophs deriving all their carbon and energy source from photosynthesis, chlorophyll and phycocyanin must therefore be two of their critical and determinative constituents. Chlorophyll is the main pigment of photochemical reactions and is made more efficient by phycocyanin. According to this, one should expect impaired growth behaviour in organisms having lesser amounts of photosynthetic pigments. The sluggish growth of mutagen treated cells in nitrite supplemented media may be attributed to its partly toxic nature but this possibility seems ruled out since no such effect was observed in control cultures growing in nitrite medium. An alternative possibility seems related to the problem of phycocyanin synthesis on the one hand, and the mechanism of phycocyanin-chlorophyll energy transfer ,.,T---------------------------------------~
'·0 ~ o·s
~ 0·6 ..J
....
0·4
0..
o
0·2
2
4
8
6
DAYS
Fig. 2. Growth of Anacystis nidulans (NTG-treated) in media containing NaNOs, NH4Cl and NaN0 2
'.0 >-
O.S
....
~ 0.6
o ~ 0·4 ()
....0..
o 0.2 O.O+---_T----~--_T----~--_T----~--_r~
420
460
.500
540 SSO 620 WAVELENGTH lnm)
660
Fig. 3. Absorption spectra of acetone extracts from equal cell volumes growing in media containing nitrate, nitrite and ammonium nitrogen.
700
of A. nidulans
(NTG - treated)
)
Nitrosoguanidine-treated Blue-green Algae
85
on the other. Furthermore, owing to a decrease in chlorophyll pigments, the photochemically generated hydrogen donors may not be availabl.e to the extent of being utilised for the reduction of nitrite nitrogen. These could be some possible causes for the retarded growth of cells in the presence of nitrite. Thus the data presented reveal a remarkable and close intermeshing of nitrite assimilation and photochemical reactions of photosynthesis. Although colour shifts usually allow the organism to adapt itself to a better use of light received, the reverse was true in the pres en t study, where in the pigment changes indicated a relatively lower yield of photosynthetic pigment leading to decreased phototrophic assimilation reflected in the slow and sluggish growth of yellow cultures in nitrite medium. As to the possible causes for this change in pigmentation of cultures, light does not appear to be responsible for it although both the shape of the light intersity curve of photosynthesis and the visually observed pigmentation could be varied considerably by the culture conditions elsewhere (KRATZ and MYERS 1955). However, since nitrite has also been used as a potent mutagen, it may be that mutagenic activities of NGT coupled with that of nitrite may be the cltuse for the change in the visually observable yellowing of cultures leading to a slow and reatarded growth. Acknowledgement The authors thank Dr. H. N. SINGH for helpful suggestions and criticism.
References ALLEN, M. M.: Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4, 1-4 (1968). FOGG, G. E.: Comparative physiology and biochemistry of the blue-green algae. Bacteriol. Rev. 20, 148-165 (1956). GERLOFF, G. C. FITZGERALD, G. P., and SKOOG, F.: The mineral nutrition of Microcystis aeruginosa Am. J. Botany 39, 26-32 (1952). KINGSBURY, J. M.: On pigment changes and growth in the blue-green alga, Plectonema nostocorum Bornet Ex Gomont. BioI. Bull. 10,310-319 (1956). KRATZ, W. A., and MYERS, J.: Nutrition and growth of several blue-green algae. Am.- J. Botany 42, 282-287 (1955). MCLACHLAN, J., and GORHAM, P. R.: Effects of pH and Nitrogen sources on growth of Microcystis aeruginosa Kutz. Can. J. Microbiol. 8, 1-11 (1962). PROVASOLI, L.: Nutrition and ecology of protozoa and algae. Ann. Rev. Microbiol. 12, 279-308 (1958). SHAFER, J. Jr., BAKER, J. E., and THOMSON, J. F.: A Chlorella mutant lacking nitrate reductase, Am. J. Botany 48,896-897 (1961). SINHA, B. D., and KUMAR, H. D.: Spontaneous and induced mutation frequencies in the blue-green alga Anacystis nidulans. Proc. Ind. Nat. Science Acad. 39, 143-149 (1973). STEVENS, S. E. Jr., and VAN BAALEN, C.: Growth characteristics of selected mutants of a coccoid blue-green alga. Arch. Mikrobiol. 72, 1-8 (1970). Received February 21, 1978.
Authors' addresses: B. D. SINHA, Department of Botany, University of Bihar, Muzaffarpur842001, India, and H. D. KUMAR Banaras Hindu University, Varanasi, 221005, India.