AMINO ACID NUTRITION IN THE BLUE-GREEN ALGA NOSTOC MUSCORUM

Transcription

1 New Phytol. (1982) 90, AMINO ACID NUTRITION IN THE BLUE-GREEN ALGA NOSTOC MUSCORUM BY A. VAISHAMPAYAN* Department of Botany, Banaras Hindu University, Varanasi , India (Accepted 20 August 1981) SUMMARY The L-isomers of 21 amino acids have been screened for their ability to serve as carbon or nitrogen sources for growth of the het^ nif^^ heterocystous and non-nitrogen fixing mutant strain of the blue-green alga Nostoc muscorum by a simple test system combining the use of 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU) (an inhibitor of photosynthetic assimilation of CO2) and the auxotrophic (non-nitrogen fixing) characteristic of the mutant. The amino acids glutamate, alanine, tyrosine and cysteine were toxic; glutamine, histidine, asparagine, tryptophan and serine were utilized as nitrogen sources; arginine, proline and phenylalanine were utilized as carbon sources; leucine, isoleucine, lysine, methionine, valine and citrulline were utilized as sources of both carbon and nitrogen; aspartate, threonine and glycine served neither as carbon nor nitrogen sources. INTRODUCTION Amino acids in general are inhibitory to the growth of methylotrophs (Eccleston and Kelly, 1972; Smith and Hoare, 1977) and poor sources of nitrogen for blue-green algae (Neilson and Doudoroff, 1973). However, systematic studies on the nutritive role of amino acids in blue-green algae are lacking. In the present investigation I have attempted to see, the help of 3-(3,4-dichlorophenyl)- 1,1-dimethylurea (DCMU), whether amino acids can serve as a carbon or nitrogen source for the growth of the hef^ nif{^ (heterocystous and non-nitrogen fixing) mutant strain of N. muscorum. DCMU inhibits heterocyst formation in N. muscorum but this inhibitory effect is reversed in the presence of an exogenous usable carbon source (Vaishampayan, Singh and Singh, 1978; Singh and Vaishampayan, 1978). Thus, the hef^ nif{^ strain always requires combined nitrogen for growth (because it is a non-nitrogen-fixing strain) but, when treated DCMU, it requires both combined nitrogen and utilizable organic carbon for growth. This simple technique has been employed to investigate which of the 21 amino acids tested served as nitrogen and/or carbon sources for the hef^ nifr. mutant of the blue-green alga Nostoc muscorum. MATERIALS AND METHODS Organism The axenic clonal culture of the heterocystous, non-nitrogen-fixing mutant of A^. muscorum (phenotypically designated as het"^ nif^^ and isolated by Stewart, Rowell and Tel-Or, 1975) was supplied by Professor H. N. Singh of the Central University of Hyderabad, India. This mutant develops heterocysts in minimal (nitrogen-free) a frequency of 55 to 6-5 % but does not fix nitrogen. Present address: Mutagenesis and Cytogenetics Laboratory, University Department of Botany, University of Bihar, Muzaffarpur , India X/82/ ho5 $02.00/ The New Phytologist

2 546 A. VAISHAMPAYAN When grown on a combined nitrogen source, e.g. nitrate, nitrite or ammonium it grows well out forming heterocysts, but when transferred to fresh minimal it starts producing heterocysts in 20 to 24 h. Methods The hef^ nif{^ mutant strain was grown routinely in Chu 10 as modified by Gerloff, Fitzgerald and Skoog (1950) combined nitrogen (5 mivi potassium nitrate or 1 mm ammonium chloride) at a temperature of C and at a continuous fiuorescent light flux of about 10 W m~^ (400 to 700 nm). Growth was determined by optical density measurements at 663 nm, and heterocyst frequency was determined from microscopic observations and expressed as number of heterocysts per 100 vegetative cells. A nitrate-grown non-heterocystous log phase culture of the organism was harvested, washed sterile glass-distilled water and inoculated into minimal (nitrogen-free) containing 3 mm glucose and/or 1 mm ammonium chloride; or 50 p.p.m. each (in different sets) of different amino acids, unsupplemented or supplemented 1 x 10~^ M (an inhibitory concentration) of DCMU, a well-known inhibitor of photosynthetic COg fixation (Bishop, 1958; Rippka, 1972; Stanier, 1973). Growth and heterocyst frequency were recorded 9 days after inoculation. The amino acids which reversed the inhibitory action of DCMU on heterocyst formation (in a manner similar to glucose) but did not support growth, were categorized as carbon sources only; those which supported the growth of DCMU-untreated cultures (in a manner similar to ammonium chloride) but not the cultures DCMU, were categorized as nitrogen sources only; those which supported the growth of DCMU-treated cultures (in a manner similar to glucose and ammonium chloride in combination) were categorized as both carbon and nitrogen sources. DCMU and all amino acids were from Sigma Chemical Company and were filter sterilized and added aseptically to the already sterile. All other chemicals used were from British Drug Houses, Poole. RESULTS Growth and heterocyst frequency ofthe hef^ nif^^ mutant glucose, ammonium chloride or different amino acids added to minimal + DCMU media, are shown in Tables 1 and 2. Under control conditions (i.e. in minimal unsupplemented glucose, ammonium chloride, amino acids or DCMU) the mutant formed heterocysts a frequency of 5*9 % but, as expected, failed to grow. Addition of DCMU blocked the formation of heterocysts in minimal. Ammonium chloride, being a nitrogen source, allowed the growth of DCMUuntreated cultures but failed to do so after addition of DCMU. Glucose, being a readily assimilable carbon source, reversed the inhibitory action of DCMU on heterocyst formation, and, in a containing ammonium chloride, it supported good growth of the DCMU-treated cultures. The amino acids glutamate, alanine, tyrosine and cysteine supported neither growth nor heterocyst formation in the presence of DCMU; indeed these amino acids inhibited the formation of heterocysts in the control (DCMU-free) cultures. In addition these amino acids the cultures underwent massive fragmentation and chlorosis followed by vacuolation of cells and, ultimately, cell lysis. The amino acids aspartate, threonine or glycine supported neither growth ofthe

3 Amino acid nutrition of Nostoc 547 Table 1. Growth* of the het"*" nifj^ strain 0/Nostoc muscorum in minimal ; minimal containing 3 mm glucose and/or 1 mm ammonium chloride; minimal containing 50 p.p.m. concentration of different amino acids, unsupplemented or supplemented 1 x 10"^ M DCMU supplemented Growth -I supplemented Growth + DCMU Nil Glucose NH4CI Glucose-I-NH4CI Glutamate Alanine Tyrosine Cysteine Aspartate Threonine Glycine Histidine Glutamine 0-45 ± ± ± Asparagine Tryptophan Serine Arginine Proline Phenylalanine Leucine Isoleucine Methionine Valine Citrulline Lysine 0-41 ± ± ± ± ± ± ± ± ± * Difference of initial and final optical density (663 nm) recorded on 9th day of inoculation. The values are means of five independent readings their respective standard errors. Table 2. Heterocyst frequency* of the het"*" niff^ strain of Nostoc muscorum in minimal ; minimal containing 3 mm glucose and/or 1 mm ammonium chloride; minimal containing 50 p.p.m. concentration of different amino acids, unsupplemented or supplemented 1 x 10~^ M DCMU supplemented Heterocyst frequency + DCMU supplemented Heterocyst frequency + DCMU Nil Glucose NH4CI Glucose + NH4CI Glutamate Alanine Tyrosine Cysteine Aspartate Threonine Glycine Histidine Glutamine 5-90 ± ± ± ± ± Asparagine Tryptophan Serine Arginine Proline Phenylalanine Leucine Isoleucine Methionine Valine Citrulline Lysine 6-42 ± ± ± ±O ± ± ± ±O ± ± ±O ±O ± ± ±0-15 * Number of heterocysts per 100 vegetative cells. The values are means of five independent readings their respective standard errors. mutant (in the presence or absence of DCMU) nor reversed the inhibitory effect of DCMU on heterocyst formation but they did not interfere normal heterocyst formation in DCMU-free. The organism differentiated heterocysts similar frequencies in DCMU-free in the presence of 19 ANP90

4 54^ A. VAISHAMPAYAN aspartate, threonine or glycine as it did in the absence of added amino acids, i.e. these amino acids had no observable effect on the organism. The amino acids glutamine, histidine, asparagine, tryptophan and serine each supported significant growth of the mutant in DCMU-free but they supported neither growth nor formation of heterocysts in containing DCMU. Thus these amino acids, like ammonium chloride, suppressed the formation of heterocysts in DCMU-free (control) media and provided nitrogen for growth. The amino acids arginine, proline and phenylalanine (in a manner similar to glucose) completely reversed the inhibitory effect of DCMU on heterocyst formation and enhanced heterocyst frequency of DCMU-free cultures; they did not support the growth of the mutant in DCMU-free or DCMU-containing. The amino acids leucine, isoleucine, methionine, valine, citruuine and lysine were remarkable in that each partially reversed the inhibition of heterocyst formation by DCMU and supported considerable growth of the cultures in either DCMU-containing or DCMU-free media. Consequently in the presence of these amino acids the organism grew and formed heterocysts equally well in the presence and absence of DCMU. However, growth was slower than that supported by ammonium chloride, glutamine, histidine, asparagine, tryptophan or serine, and heterocyst frequency was lower than in cultures supplied glucose, arginine, proline or phenylalanine. DISCUSSION DCMU inhibition of both heterocyst differentiation and growth in Nostoc muscorum is readily reversed by glucose (see also Singh and Vaishampayan, 1978) thus suggesting that in N. muscorum growth and heterocyst differentiation require either photosynthetically fixed carbon or a utilizable organic carbon source. It can be assumed, therefore, that all those amino acids which successfully reverse the heterocyst-inhibitory action of DCMU are metabolizable sources of organic carbon for the hef^ i^ifn niutant strain of N. muscorum. Accordingly, the amino acids arginine, proline, phenylalanine, leucine, isoleucine, methionine, valine, citrulline and lysine can be regarded as good organic carbon sources in this alga. Of these, the first three allowed more heterocyst formation but they did not support growth. The hef^ nif^^ mutant strain does not grow in minimal as it does not fix nitrogen (Stewart et al., 1975). Any substrate which supports the growth of this strain in minimal must do so by acting as a nitrogen source. Moreover, combined nitrogen sources like ammonium chloride are inhibitory to heterocyst formation in N. muscorum (Stewart and Singh, 1975). Thus, all amino acids which supported the growth of this organism in minimal and at the same time suppressed the formation of heterocysts, can be regarded as metabolizable sources of organic nitrogen; the amino acids were glutamine, histidine, asparagine, tryptophan, serine, leucine, isoleucine, methionine, valine, citrulline and lysine. Of these, the first five were the best sources of nitrogen because they supported better growth of the mutant and at the same time completely suppressed the formation of heterocysts. It is remarkable that the organism apparently could utilize carbon from the amino acids arginine, proline and phenylalanine out also utilizing nitrogen which should have been released in the form of ammonium.

5 Amino acid nutrition of Nostoc 549 It is difficult to explain how this could be so and further study of the mechanisms of utilization of these amino acids is necessary. The amino acids aspartate, threonine and glycine neither supported grovv^th nor heterocyst formation by the mutant in DCMU-containing. Also, they did not suppress the formation of heterocysts in DCMU-free. These amino acids therefore served neither as carbon nor as nitrogen sources. The amino acids glutamate, alanine, tyrosine and cysteine were toxic in this system and no conclusions about their metabolism can be drawn. From these results it can be inferred that for the blue-green alga, Nostoc muscorum, several amino acids can act as good carbon and/or nitrogen sources. However, the growth supported by a few amino acids was very poor. This may be due to the incorporation of relatively small amounts into the algal cells. The reason for this has been attributed, for methylotrophs, to a limited capacity of the cells to utilize the compounds which are for the most part incorporated unchanged into protein (Eccleston and Kelly, 1972). It is thus essential to characterize the reactions that make the amino acids so specific in their nutritive behaviour. Experiments in this direction are in progress. ACKNOWLEDGEMENTS Thanks are due to Professor H. N. Singh for his suggestion to use his hef^ ^ifn mutant strain for this investigation. The receipt of financial assistance for this work in the form of a Research Fellowship from the Indian Atomic Energy Commission, Bhabha Atomic Research Centre, Trombay, Bombay , is gratefully acknowledged. REFERENCES BISHOP, N. 1.(1958). The influence ofthe herbicide DCMU on the oxygen-evolving system of photosynthesis. Bioehimica et Biophysica Acta, 27, ECCLESTON, M. & KELLY, D. P. (1972). Assimilation and toxicity of exogenous amino acids in the methane-oxidizing bacterium Methylococcus capsulatus. Journal of General Microbiology, 71, GERLOFF, G. C, FITZGERALD, G. P. & SKOOG, F. (1950). The isolation, purification and culture of blue-green algae. American Journal of Botany, 37, NEILSON, A. H. & DouDOROFF, M. (1973). Ammonia assimilation in blue-green algae. Archives of Microbiology, 89, RiPPKA, R. (1972). Photoheterotrophy and chemoheterotrophy among unicellular blue-green algae. Archives of Microbiology, 87, SINGH, H. N. & VAISHAMPAYAN, A. (1978). Biological effects ofthe rice-field herbicide machete on various strains of the nitrogen-fixing blue-green alga Nostoc muscorum. Environmental and Experimental Botany, 18, SMITH, A. J. & HOARE, D. S. (1977). Specialist phototrophs, lithotrophs, and methylotrophs: a unity among a diversity of prokaryotes? Bacteriological Reviews, 41, STANIER, R. Y. (1973). Autotrophy and heterotrophy in unicellular blue-green algae. In: The Biology of Blue-green Algae (Ed. by N. G. Carr & B. A. Whitton), pp University of California Press, Berkeley and Los Angeles. STEWART, W. D. P., ROWELL, P. & TEL-OR, E. (1975). Nitrogen fixation and the heterocysts in blue-green algae. Biochemical Society Transactions, 3, STEWART, W. D. P. & SINGH, H. N. (1975). Transfer of nitrogen-fixing {Nif) genes in the blue-green alga Nostoc muscorum. Biochemical and Biophysical Research Communications, 62, VAISHAMPAYAN, A., SINGH, H. R. & SINGH, H. N. (1978). Biological effects of rice-field herbicide stamf-34 on various strains of the nitrogen-fixing blue-green alga Nostoc muscorum. Biochemie und Physiologie der Pflanzen, 173,

6