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Amino acid incorporation by a natural population of Oscillatoria rubescens. A microautoradiographic study
FEMS MicrobiologyEcology62 (1989) 185-190 Published by Elsevier
185
FEC 00203
Amino acid incorporation by a natural population of Oscillatoria rubescens. A microautoradiographic study Gilles Bourdier, Jacques Bohatier, M a u r i c e t t e Feuillade a n d Jacques Feuillade Universitd Blaise Pascal, Laboratoire de Zoologie Protistologie, Aubidre, and Station d'Hydrobiologie Lacustre, 75 avenue de Corzent, Thonon les Bains, France
Received 25 May 1988 Revision received26 September 1988 Accepted 4 October 1988 Key words: Oscillatoria rubescens; Cyanobacteria; Amino acid incorporation; Microautoradiography
1. SUMMARY The incorporation of a very low concentration (0.015 I~M) of an [3H]amino acid mixture was measured for a natural population of Oscillatoria rubescens DC in samples from a eutrophic lake, Lake Nantua (France). The kinetics of amino acid incorporation in the size fraction >I 12 /~m showed that uptake was fast and that the maximum was reached after 4 h. Microautoradiography demonstrated that Oscillatoria rubescens is able to utilize for protein synthesis an external pool of amino acids whose [3H] label becomes distributed generally throughout the cell.
2. INTRODUCTION Organic nitrogen uptake in the cyanobacterium rubescens DC was studied in a
Oscillatoria
Correspondence to: G. Bourdier, Universit6 Blaise Pascal, Laboratoire de ZoologieProtistologie,UA CNRS 138, B.P. 45, 63170 Aubi6re, France.
eutrophic lake, Lake Nantua (France). It blooms at the beginning of summer in the nitrogen and phosphorus depleted epilimnion and then 'migrates' to the metalimnion where its photosynthetic activity is restricted owing to fight limitation [1]. This observation suggested that the growth of this cyanobacterium may depend in part on utilization of organic matter. It has been previously demonstrated that O. rubescens grown axenically has a high affinity for some amino acids [2] and that it grows equally well on mineral medium and on medium where arginine is the sole source of nitrogen [3]. According to some authors [4-6] uptake of organic compounds in the natural environment is attributable almost entirely to heterotrophic bacteria. Autotrophs, then, would be unable to compete with heterotrophic bacteria for the very low concentrations of organic compounds available in the water. However, heterotrophic nutrition is common among algae [7-11] and some examples of high affinity systems for transport of organic nutrients by algae show them to be competitive with bacteria [12,13]. However, this has seldom been demonstrated under natural conditions [14]. Saunders [7] applied grain autoradiography using [a4C]-glucose,
0168-6496/89/$03.50 © 1989 Federationof European MicrobiologicalSocieties
186
cens utilizes naturally occurring amino acids in low concentrations in freshwater. Using microautoradiography, amino acid uptake could easily be detected at the cellular level after a very low concentration of labeled amino acids had been introduced into the water.
400
3000,
2000.
3. M A T E R I A L S A N D M E T H O D S 1000
RS I
2
3
4
5
Fig. 1. Time curve of [3H]-amino acid fixation for the fraction >/12 ~m (dpm.ml-]).
and both [14C]-acetate and [14C]-glucose to estimate potential heterotrophic assimilation in O. agardhii var. isothrix. However, he did not use labeled amino acids, and without EM autoradiography failed to prove true intracellular incorporation. The aim of this investigation was to test the hypothesis that a natural population of O. rubes-
Samples were collected from the metalimnion of Lake Nantua at a depth of 10 m where the highest population density of O. rubeseens was observed. To 100 ml glass bottles filled with the lake water was added an [3H]-amino acid mixture (Amersham, code T R K 440) to give 0.015 /~M amino acid final concentration and 55 /zCi per bottle. The samples were incubated in the dark [2] at in situ temperature. Initial pre-killed controls (2% formalin) were used to determine whether adsorption of [3H]-amino acids had occurred.
3.1. Heterotrophic uptake Each hour for 5 h of incubation, 100 ml samples were filtered through a Nuclepore membrane
Figs. 2, 3 and 4. Light microscopic autoradiographs of 1/.tm thick sections of Oscillatoria rubescens incubated for 1 h (Fig. 2), 3 h (Fig. 3) and 5 h (Fig. 4) with the [3H]-amino acid mixture; scale bar 10 #m.
187 (12/~m pore size) using gentle suction ( < 100 m m Hg). The filters were rinsed with filter-sterilized (0.2 /~m) lake water, dried on filter paper, placed in a vial containing a toluene-based scintillation cocktail and counted in a Packard SL 400 liquid scintillation counter. Incorporation of [3H] was expressed in d p m corrected for formalin treated control counts. All counts were corrected for quench by the external standards method.
3.2. Microautoradiography After 1, 3 and 5 h incubation with [3H]-amino acids, filaments of O. rubescens were collected on a 12 /~m Nuclepore membrane and fixed for one hour with 2% glutaraldehyde in a 0.1 M sodium cacodylate buffer, p H 7.0. After washing, the cells
63
were post-fixed with 1% OsO4 in the same buffer and then embedded in Epon 812 resin [15].
3.2.1. Light microscopy autoradiography: 1 i~m thick sections were cut and placed on glass slides previously coated with Ulrich adhesive [16]. The slides were then coated with a layer of Ilford K5 emulsion by dipping [17]. After 72 hours exposure in a dark box, the autoradiographs were developed for 4 minutes at 1 8 ° C with K o d a k D19B developer diluted with an equal volume of distilled water. After washing, they were mounted in a neutral synthetic medium and observed under a phase contrast microscope. Silver grains were enumerated on autoradiographic micrographs using arbitrary surface units [18]. Some slides were dipped in 5% T C A aqueous solution at 6 0 ° C prior to the emulsion layering. 3.2.2. Electron microscopy autoradiography: Ultra-thin sections were collected on 200 mesh Nickel grids. The carbon coated grids were placed on magnetic slides and covered with Ilford L4 emulsion using the bubble technique [19]. After four weeks exposure they were developed with K o d a k Microdol X, fixed and washed by immersion in suitable baths. The grids were removed from the supports, stained with uranyl acetate and lead citrate and examined under a Jeol 1200 EX transmission electron microscope. 4. R E S U L T S
ii
.....
1
2
a
,
s
~
~
~
6
1"o
Fig. 5. Histogram representing the silver grain distribution in 1 /~m-thick sections of Oscillatoria rubescens incubated in the presence of the [3H]-arnino acid mixture: (a), for 1 h (n = 82); (b) for 3 h (n = 86); (c) for 5 h (n = 94). Silver grains were counted on autoradiographic micrographs using an arbitrary surface unit.
The amino-acid uptake bioassay was carried out on water samples in which O. rubescens constituted 97% of the total cell biomass >i 12 #m. Bacteria attached to filaments of O. rubescens represented only 0.065% of the total biomass as calculated from cell volumes (Dufour, personal communication). Free bacteria were not considered in the analysis. The kinetics of labeled amino-acid incorporation in the size fraction >~ 12 t~m (Fig. 1) showed that m a x i m u m incorporation was reached after 4 h incubation with [3H]-amino acids and then decreased between 4 and 5 h incubation. Total 3H-incorporation represented approximately 0.4% of the added radioactivity, equivalent to 60 pmol amino acid. Extremely low adsorption was detected on pre-killed controls ( < 100 dpm).
188
,.3
,
7
Figs. 6, 7, 8 and 9. Electron microscope autoradiographs of ultra-thin sections of Oscillatoria rubescens incubated for 5 h with the [3H]-amino acid mixture, scale bar 1 gin. CY-cyanophycin granules; Th = Thylakoids; Gv = gas vacuoles; Lip = lipid inclusions.
189 Autoradiographic examination of 1 /~m thick sections (Figs. 2-4) showed a labeling localized above the sections of Oscillatoria and a very low background. Labelling of cells as determined by silver grain counts was lower after 1 h incubation (Fig. 2) than it was after 3 h (Fig. 3) or 5 h (Fig. 4). Dipping slides in TCA did not significantly modify the silver grain count. In addition, the silver grain count for samples incubated for 3 h and for those incubated for 5 h was different because model classes were (2-3) and (1-2), respectively (Fig. 5). This change corresponds to the decrease in radioactivity incorporated by this fraction of the phytoplankton after 4 h incubation. Nevertheless, autoradiographic observation of ultrathin sections of Oscillatoria revealed significant intracellular labelling after 5 h of incubation (Figs. 6-9). The labelling was distributed generally throughout the cell.
5. DISCUSSION This study shows that the Lake Nantua phytoplanktonic fraction >/12 /xm, largely dominated by O. rubescens was able to incorporate [3HIamino acids at naturally occurring concentrations. Dissolved free amino acid concentrations were previously determined in Lake Nantua, and were between 0.1 and 1.1 /~M (Feuillade, unpublished results). The amount of labeled amino acid added in our experiment was much lower than the natural concentration. It has long been surmised that autotrophs in natural conditions are unable to compete effectively with heterotrophic bacteria for organic compounds present at very low concentrations. However, evidence existed that heterotrophy may be very important among eutrophic cyanobacteria [20]. Our microautoradiographic study confirmed that O. rubescens is capable of assimilating amino acids in lake water in bottles in the dark. Radioactive labeling of Oscillatoria could be demonstrated in 1 ~m sections after 1 h incubation of the organisms with a low concentration of 3H-amino acids and the labeling increased with increasing incubation time. This agrees with observations from axenic cultures of O. rubescens [2] and shows t h a t this species is able to transport
amino acids across the cell membrane and to utilise them. Washing with hot TCA would remove unincorporated [3H]-amino acids or those converted into macromolecules other than proteins. Because the silver grain count was not affected by the TCA treatment, it can be concluded that the absorbed amino acids were incorporated into proteins. The decrease in the silver grain count after 3 h of incubation correlates with a decrease in radioactivity for the fraction >~ 12/~m. The reasons for this decrease are unclear. Perhaps cell physiology was modified during the experiment. It is possible that instead of decreasing uptake rates, the rates at which [3H] amino-acids were degraded or excreted (as secondary metabolites) changed between 3 and 5 h of incubation. Furthermore, isotopic equilibrium may not have been fully established during the entire 5 h incubation period [21,22]. In addition, cell autolysis could have taken place ('bottle effect') during incubation. The electron microscope autoradiography of ultra-thin sections demonstrated that the labeling was intracellular, thus indicating that an external pool of amino acids has been utilized for cellular biosynthesis. The localization of labeling above cyanophycin granules suggests that synthesis of cyanophycin was not inhibited during the experiment. Cyanophycin is a polypeptide copolymer of aspartic acid and arginine [23] and its formation can be induced by a variety of treatments [24]. Variation in the number and size of granules results from altering growth conditions [25]. The ability to utilize amino-acids present in the water at very low concentrations does not imply that Oscillatoria rubescens can maintain a high rate of growth and division in light- and nutrientlimited conditions. However its ability to metabolize external amino acids may be one factor that could explain its persistence and dominance in certain environments.
REFERENCES [1] Feuillade, J. (ed.), Balvay, G., Barroin, G., Blanc, P., Feuillade, M., Orand, A., Pelletier, J. and Chahuneau, F.
190 (1985) Caractrrisation et essais de restauration d'un 6cosytrme drgradr: le lac de Nantua. I.N.R.A., Versailles. [2] Feuillade, M. and Krupka, H.M. (1986) Assimilation des acides aminrs par Oscillatoria rubescens D.C. (Cyanophycre). Arch. Hydrobiol. 107, 441-463. [3] Krupka, H.M. and Feuillade, M. (1988) Amino acids as a nitrogen source for growth of Oscillatoria rubescens D.C. Ecological significance. Arch. Hydrobiol. 112, 125-142. [4] Allen, H.L. (1969) Chemo-organotrophic utilization of dissolved organic compounds by planktonic algae and bacteria in a pond. Int. Rev. Ges. Hydrobiol. 54, 1-33. [5] Hobbie, J.E. and Wright, R.T. (1965) Competition between planktonic bacteria and algae for organic solutes, in Primary productivity in aquatic environments, (C.R. Goldman ed.), pp. 175-185, University of California Press, Berkeley. [6] Sepers, A.B.J. (1977) The utilization of dissolved organic compounds in aquatic environments. Hydrobiologia 51, 39-54. [7] Saunders, G.W. (1972) Potential heterotrophy in a natural population of Oscillatoria agardhii var. isothrix Skuja. Limnol. Oceanogr. 17, 704-711. [8] Antia, N.J., Berland, B.R., Bonin, D.J. and Maestrini, S.Y. (1978) Utilisation de la mati&e organique dissoute en tant que substrat par les algues unicellulaires marines. Actual. Biochimie Marine. GABIM, 147-178. [9] Vincent, W.F. (1980) The physiological ecology of a Scenedesmus population in the hypolimnion of a hypertrophic pond. II. Heterotrophy. Br. Phycol. J. 15, 35-41. [10] Vincent, W.F. and Goldman, C.R. (1980) Evidence for algal heterotrophy in Lake Tahoe, California--Nevada. Limnol. Oceanogr. 25, 89-99. [11] Flynn, K.J. and Butler, I. (1986) Nitrogen sources for the growth of marine microalgae: role of dissolved free amino-acids. Mar. Ecol. Prog. Ser. 34, 281-304. [12] Hellebust, J.A. and Lewin, J. (1977) Heterotrophic nutrition, in The Biology of Diatoms. (D. Werner, ed.), Blackwell Scientific Publications, Oxford. [13] Bollman, R.C. and Robinson, G.G.C. (1985) Heterotrophic potential of the green alga Ankistrodesmus braunii (Naeg.). Can. J. Microbiol. 31, 549-554.
[14] Moll, R. (1984) Heterotrophy by phytoplankton and bacteria in Lake Michigan. Verh. Int. Ver. Limnol. 22, 431-434. [15] Bourdier, G. and Bohatier, J. (1986-1987) Illustration de l'activit6 photosynth&ique du phytoplancton par des techniques autoradiographiques. Ann. Sci. Nat., Zoologie. 2, 75-79. [16] Brock, M.L. and Brock, T.D. (1968) The application of microautoradiographic techniques to ecological studies. Mitt. Int. Ver. Limnol. 15, 1-29. [17] Larra, F. and Droz, B. (1970) Techniques radioautoradiographiques et leur application au renouvellement de constituants cellulaires. J. Microsc 9, 845-880. [18] Pelc, S.R. (1972) Theory of autoradiography, in Autoradiography for Biologists, (Gahan, P.B., ed.), pp. 1-17, Academic Press, London and New York. [19] Hubert, J. and Bohatier, J. (1975) Magnetic slides for support of grids in electron microscopic autoradiography: use with the bubble technique of emulsion application. Stain Technol. 50, 60-61. [20] Wetzel, R.G. (1983) Planktonic communities: Algae in Limnology. pp. 342-407, Saunders College Publishing, Philadelphia. [21] Storch, T.A. and Saunders, G.W. (1978) Phytoplankton extracellular release and its relation to the seasonal cycle of dissolved organic carbon in a eutrophic lake. Limnol. Oceanogr. 23, 112-119. [22] Jensen, L.M., Jorgensen, N.O.G. and Sondergaard, M. (1985) Specific activity. Significance in estimating release rates of extracellular dissolved organic carbon (EOC) by algae. Verh. Int. Ver. Limnol. 22, 2893-2897. [23] Simon, R.D. (1971) Cyanophycin granules from the bluegreen alga Anabaena cylindrica: a reserve material consisting of copolymers of aspartic acid and arginine. Proc. Natl. Acad. Sci. U.S.A. 68, 265-267. [24] Lawry, N.H. and Simon, R.D. (1982) The normal and induced occurrence of cyanophycin inclusion bodies in several blue-green algae. J. Phycol. 18, 391-399. [25] Allen, M.M. (1984) Cyanobacterial cell inclusions. Ann. Rev. Microbiol. 38, 1-25.
185
FEC 00203
Amino acid incorporation by a natural population of Oscillatoria rubescens. A microautoradiographic study Gilles Bourdier, Jacques Bohatier, M a u r i c e t t e Feuillade a n d Jacques Feuillade Universitd Blaise Pascal, Laboratoire de Zoologie Protistologie, Aubidre, and Station d'Hydrobiologie Lacustre, 75 avenue de Corzent, Thonon les Bains, France
Received 25 May 1988 Revision received26 September 1988 Accepted 4 October 1988 Key words: Oscillatoria rubescens; Cyanobacteria; Amino acid incorporation; Microautoradiography
1. SUMMARY The incorporation of a very low concentration (0.015 I~M) of an [3H]amino acid mixture was measured for a natural population of Oscillatoria rubescens DC in samples from a eutrophic lake, Lake Nantua (France). The kinetics of amino acid incorporation in the size fraction >I 12 /~m showed that uptake was fast and that the maximum was reached after 4 h. Microautoradiography demonstrated that Oscillatoria rubescens is able to utilize for protein synthesis an external pool of amino acids whose [3H] label becomes distributed generally throughout the cell.
2. INTRODUCTION Organic nitrogen uptake in the cyanobacterium rubescens DC was studied in a
Oscillatoria
Correspondence to: G. Bourdier, Universit6 Blaise Pascal, Laboratoire de ZoologieProtistologie,UA CNRS 138, B.P. 45, 63170 Aubi6re, France.
eutrophic lake, Lake Nantua (France). It blooms at the beginning of summer in the nitrogen and phosphorus depleted epilimnion and then 'migrates' to the metalimnion where its photosynthetic activity is restricted owing to fight limitation [1]. This observation suggested that the growth of this cyanobacterium may depend in part on utilization of organic matter. It has been previously demonstrated that O. rubescens grown axenically has a high affinity for some amino acids [2] and that it grows equally well on mineral medium and on medium where arginine is the sole source of nitrogen [3]. According to some authors [4-6] uptake of organic compounds in the natural environment is attributable almost entirely to heterotrophic bacteria. Autotrophs, then, would be unable to compete with heterotrophic bacteria for the very low concentrations of organic compounds available in the water. However, heterotrophic nutrition is common among algae [7-11] and some examples of high affinity systems for transport of organic nutrients by algae show them to be competitive with bacteria [12,13]. However, this has seldom been demonstrated under natural conditions [14]. Saunders [7] applied grain autoradiography using [a4C]-glucose,
0168-6496/89/$03.50 © 1989 Federationof European MicrobiologicalSocieties
186
cens utilizes naturally occurring amino acids in low concentrations in freshwater. Using microautoradiography, amino acid uptake could easily be detected at the cellular level after a very low concentration of labeled amino acids had been introduced into the water.
400
3000,
2000.
3. M A T E R I A L S A N D M E T H O D S 1000
RS I
2
3
4
5
Fig. 1. Time curve of [3H]-amino acid fixation for the fraction >/12 ~m (dpm.ml-]).
and both [14C]-acetate and [14C]-glucose to estimate potential heterotrophic assimilation in O. agardhii var. isothrix. However, he did not use labeled amino acids, and without EM autoradiography failed to prove true intracellular incorporation. The aim of this investigation was to test the hypothesis that a natural population of O. rubes-
Samples were collected from the metalimnion of Lake Nantua at a depth of 10 m where the highest population density of O. rubeseens was observed. To 100 ml glass bottles filled with the lake water was added an [3H]-amino acid mixture (Amersham, code T R K 440) to give 0.015 /~M amino acid final concentration and 55 /zCi per bottle. The samples were incubated in the dark [2] at in situ temperature. Initial pre-killed controls (2% formalin) were used to determine whether adsorption of [3H]-amino acids had occurred.
3.1. Heterotrophic uptake Each hour for 5 h of incubation, 100 ml samples were filtered through a Nuclepore membrane
Figs. 2, 3 and 4. Light microscopic autoradiographs of 1/.tm thick sections of Oscillatoria rubescens incubated for 1 h (Fig. 2), 3 h (Fig. 3) and 5 h (Fig. 4) with the [3H]-amino acid mixture; scale bar 10 #m.
187 (12/~m pore size) using gentle suction ( < 100 m m Hg). The filters were rinsed with filter-sterilized (0.2 /~m) lake water, dried on filter paper, placed in a vial containing a toluene-based scintillation cocktail and counted in a Packard SL 400 liquid scintillation counter. Incorporation of [3H] was expressed in d p m corrected for formalin treated control counts. All counts were corrected for quench by the external standards method.
3.2. Microautoradiography After 1, 3 and 5 h incubation with [3H]-amino acids, filaments of O. rubescens were collected on a 12 /~m Nuclepore membrane and fixed for one hour with 2% glutaraldehyde in a 0.1 M sodium cacodylate buffer, p H 7.0. After washing, the cells
63
were post-fixed with 1% OsO4 in the same buffer and then embedded in Epon 812 resin [15].
3.2.1. Light microscopy autoradiography: 1 i~m thick sections were cut and placed on glass slides previously coated with Ulrich adhesive [16]. The slides were then coated with a layer of Ilford K5 emulsion by dipping [17]. After 72 hours exposure in a dark box, the autoradiographs were developed for 4 minutes at 1 8 ° C with K o d a k D19B developer diluted with an equal volume of distilled water. After washing, they were mounted in a neutral synthetic medium and observed under a phase contrast microscope. Silver grains were enumerated on autoradiographic micrographs using arbitrary surface units [18]. Some slides were dipped in 5% T C A aqueous solution at 6 0 ° C prior to the emulsion layering. 3.2.2. Electron microscopy autoradiography: Ultra-thin sections were collected on 200 mesh Nickel grids. The carbon coated grids were placed on magnetic slides and covered with Ilford L4 emulsion using the bubble technique [19]. After four weeks exposure they were developed with K o d a k Microdol X, fixed and washed by immersion in suitable baths. The grids were removed from the supports, stained with uranyl acetate and lead citrate and examined under a Jeol 1200 EX transmission electron microscope. 4. R E S U L T S
ii
.....
1
2
a
,
s
~
~
~
6
1"o
Fig. 5. Histogram representing the silver grain distribution in 1 /~m-thick sections of Oscillatoria rubescens incubated in the presence of the [3H]-arnino acid mixture: (a), for 1 h (n = 82); (b) for 3 h (n = 86); (c) for 5 h (n = 94). Silver grains were counted on autoradiographic micrographs using an arbitrary surface unit.
The amino-acid uptake bioassay was carried out on water samples in which O. rubescens constituted 97% of the total cell biomass >i 12 #m. Bacteria attached to filaments of O. rubescens represented only 0.065% of the total biomass as calculated from cell volumes (Dufour, personal communication). Free bacteria were not considered in the analysis. The kinetics of labeled amino-acid incorporation in the size fraction >~ 12 t~m (Fig. 1) showed that m a x i m u m incorporation was reached after 4 h incubation with [3H]-amino acids and then decreased between 4 and 5 h incubation. Total 3H-incorporation represented approximately 0.4% of the added radioactivity, equivalent to 60 pmol amino acid. Extremely low adsorption was detected on pre-killed controls ( < 100 dpm).
188
,.3
,
7
Figs. 6, 7, 8 and 9. Electron microscope autoradiographs of ultra-thin sections of Oscillatoria rubescens incubated for 5 h with the [3H]-amino acid mixture, scale bar 1 gin. CY-cyanophycin granules; Th = Thylakoids; Gv = gas vacuoles; Lip = lipid inclusions.
189 Autoradiographic examination of 1 /~m thick sections (Figs. 2-4) showed a labeling localized above the sections of Oscillatoria and a very low background. Labelling of cells as determined by silver grain counts was lower after 1 h incubation (Fig. 2) than it was after 3 h (Fig. 3) or 5 h (Fig. 4). Dipping slides in TCA did not significantly modify the silver grain count. In addition, the silver grain count for samples incubated for 3 h and for those incubated for 5 h was different because model classes were (2-3) and (1-2), respectively (Fig. 5). This change corresponds to the decrease in radioactivity incorporated by this fraction of the phytoplankton after 4 h incubation. Nevertheless, autoradiographic observation of ultrathin sections of Oscillatoria revealed significant intracellular labelling after 5 h of incubation (Figs. 6-9). The labelling was distributed generally throughout the cell.
5. DISCUSSION This study shows that the Lake Nantua phytoplanktonic fraction >/12 /xm, largely dominated by O. rubescens was able to incorporate [3HIamino acids at naturally occurring concentrations. Dissolved free amino acid concentrations were previously determined in Lake Nantua, and were between 0.1 and 1.1 /~M (Feuillade, unpublished results). The amount of labeled amino acid added in our experiment was much lower than the natural concentration. It has long been surmised that autotrophs in natural conditions are unable to compete effectively with heterotrophic bacteria for organic compounds present at very low concentrations. However, evidence existed that heterotrophy may be very important among eutrophic cyanobacteria [20]. Our microautoradiographic study confirmed that O. rubescens is capable of assimilating amino acids in lake water in bottles in the dark. Radioactive labeling of Oscillatoria could be demonstrated in 1 ~m sections after 1 h incubation of the organisms with a low concentration of 3H-amino acids and the labeling increased with increasing incubation time. This agrees with observations from axenic cultures of O. rubescens [2] and shows t h a t this species is able to transport
amino acids across the cell membrane and to utilise them. Washing with hot TCA would remove unincorporated [3H]-amino acids or those converted into macromolecules other than proteins. Because the silver grain count was not affected by the TCA treatment, it can be concluded that the absorbed amino acids were incorporated into proteins. The decrease in the silver grain count after 3 h of incubation correlates with a decrease in radioactivity for the fraction >~ 12/~m. The reasons for this decrease are unclear. Perhaps cell physiology was modified during the experiment. It is possible that instead of decreasing uptake rates, the rates at which [3H] amino-acids were degraded or excreted (as secondary metabolites) changed between 3 and 5 h of incubation. Furthermore, isotopic equilibrium may not have been fully established during the entire 5 h incubation period [21,22]. In addition, cell autolysis could have taken place ('bottle effect') during incubation. The electron microscope autoradiography of ultra-thin sections demonstrated that the labeling was intracellular, thus indicating that an external pool of amino acids has been utilized for cellular biosynthesis. The localization of labeling above cyanophycin granules suggests that synthesis of cyanophycin was not inhibited during the experiment. Cyanophycin is a polypeptide copolymer of aspartic acid and arginine [23] and its formation can be induced by a variety of treatments [24]. Variation in the number and size of granules results from altering growth conditions [25]. The ability to utilize amino-acids present in the water at very low concentrations does not imply that Oscillatoria rubescens can maintain a high rate of growth and division in light- and nutrientlimited conditions. However its ability to metabolize external amino acids may be one factor that could explain its persistence and dominance in certain environments.
REFERENCES [1] Feuillade, J. (ed.), Balvay, G., Barroin, G., Blanc, P., Feuillade, M., Orand, A., Pelletier, J. and Chahuneau, F.
190 (1985) Caractrrisation et essais de restauration d'un 6cosytrme drgradr: le lac de Nantua. I.N.R.A., Versailles. [2] Feuillade, M. and Krupka, H.M. (1986) Assimilation des acides aminrs par Oscillatoria rubescens D.C. (Cyanophycre). Arch. Hydrobiol. 107, 441-463. [3] Krupka, H.M. and Feuillade, M. (1988) Amino acids as a nitrogen source for growth of Oscillatoria rubescens D.C. Ecological significance. Arch. Hydrobiol. 112, 125-142. [4] Allen, H.L. (1969) Chemo-organotrophic utilization of dissolved organic compounds by planktonic algae and bacteria in a pond. Int. Rev. Ges. Hydrobiol. 54, 1-33. [5] Hobbie, J.E. and Wright, R.T. (1965) Competition between planktonic bacteria and algae for organic solutes, in Primary productivity in aquatic environments, (C.R. Goldman ed.), pp. 175-185, University of California Press, Berkeley. [6] Sepers, A.B.J. (1977) The utilization of dissolved organic compounds in aquatic environments. Hydrobiologia 51, 39-54. [7] Saunders, G.W. (1972) Potential heterotrophy in a natural population of Oscillatoria agardhii var. isothrix Skuja. Limnol. Oceanogr. 17, 704-711. [8] Antia, N.J., Berland, B.R., Bonin, D.J. and Maestrini, S.Y. (1978) Utilisation de la mati&e organique dissoute en tant que substrat par les algues unicellulaires marines. Actual. Biochimie Marine. GABIM, 147-178. [9] Vincent, W.F. (1980) The physiological ecology of a Scenedesmus population in the hypolimnion of a hypertrophic pond. II. Heterotrophy. Br. Phycol. J. 15, 35-41. [10] Vincent, W.F. and Goldman, C.R. (1980) Evidence for algal heterotrophy in Lake Tahoe, California--Nevada. Limnol. Oceanogr. 25, 89-99. [11] Flynn, K.J. and Butler, I. (1986) Nitrogen sources for the growth of marine microalgae: role of dissolved free amino-acids. Mar. Ecol. Prog. Ser. 34, 281-304. [12] Hellebust, J.A. and Lewin, J. (1977) Heterotrophic nutrition, in The Biology of Diatoms. (D. Werner, ed.), Blackwell Scientific Publications, Oxford. [13] Bollman, R.C. and Robinson, G.G.C. (1985) Heterotrophic potential of the green alga Ankistrodesmus braunii (Naeg.). Can. J. Microbiol. 31, 549-554.
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