Planta (1990)182:194-198
P l a n t a 9 Springer-Verlag1990
Extracellular deamination of L-amino acids by Chlamydomonas reinhardtii cells J. Mufioz-Blanco, J. Hidalgo-Martinez, and J. Cfirdenas* Departamento de Bioquimica y Biologia Molecular y Fisiologia, Facultad de Ciencias, Universidad de C6rdoba, Avda. S. Alberto Magno, s/n, E-14071 C6rdoba, Spain Received 14 December 1989; accepted 2 April 1990
Abstract. When grown in the light and in a Tris-acetate phosphate medium, cells o f Chlamydomonas reinhardtii Dang. can use the following L-amino acids as a sole nitrogen source: asparagine, glutamine, arginine, lysine, alanine, valine, leucine, isoleucine, serine, methionine, histidine, and phenylalanine, whereas, in the absence of acetate, the cells only used L-arginine. The utilization system in the acetate m e d i u m consisted of an extracellular deaminating activity induced by L-amino acids; it t o o k between 10 to 30 h before the system appeared in cells previously grown with a m m o n i u m . This deaminase activity was nonspecific, required an organic carbon source for its de-novo synthesis, and was sensitive to high a m m o n i u m concentration and light deprivation.
(Paul and Cooksey 1979), whereas L-arginine is taken up through an active transport system induced by nitrogen starvation (Kirk and Kirk 1978a). In the present paper, we demonstrate that the unicellular green alga Chlamydomonas reinhardtii can grow in the presence of acetate, at the expense of a m m o n i u m supplied by twelve L-amino acids (asparagine, glutamine, arginine, lysine, alanine, valine, leucine, isoleucine, serine, methionine, histidine, and phenylalanine), which are first deaminated in a reaction mediated by a nonspecific de-novo inducible deaminase sensitive to high amm o n i u m concentration and light deprivation. It is the first time that such a general deaminating system for L-amino acids has been described in green algae.
Key words: L-amino acids (utilization) - Chlamydomona s - Deaminase (extracellular)
Material and methods
Introduction Photosynthetic organisms are able to use L-amino acids as nitrogen sources for growth (Liu and Hellebust 1974; Wheeler et al. 1974; Baden and Mende 1979). This takes place either by means of energy-dependent transport systems (Kirk and K i r k 1978a, b; Cho et al. 1981; Cho and K o m o r 1983; Flynn and Syrett 1985; Sauer and Tanner 1985) or by deamination mechanisms occurring at the cell surface with the subsequent uptake and assimilation o f the a m m o n i u m produced (Paul and Cooksey 1979, 1981a, b). In Chlamydomonas, it has been reported that L-asparagine and L-arginine are the only amino acids that can be used as nitrogen sources to support algal growth. L-Asparagine provides a m m o n i u m after deamination * To whom correspondence should be addressed Abbreviations: HPLC = high-performance liquid chromatography; TAP = Tris-acetate-phosphate
Cell growth conditions. Cells of Chlamydomonas reinhardtii Dang. 6145c (a gift of Dr. R. Sager, Cancer Faber Center, New York, USA) were cultured at pH 7.0 in the minimal liquid medium of Sueoka et al. (1967) or in liquid media containing acetate (TAP, Tris-acetate-phosphate media; Gorman and Levine 1965), with ammonium or L-amino acids as sole nitrogen sources. Solid TAP media contained, in addition, 1.5% Difco Bacto-Agar (Difco Laboratories, Detroit, Mich., USA). Cells were maintained under saturating light conditions (15 W.m-z) until the mid-logarithmic phase of growth, then harvested by centrifugation at 10000-g (5 min), washed twice with deionised water, and resuspended, as required, either in TAP-ammonium or TAP-amino acid media. Growth was followed by measuring the turbidity of cell suspensions at 660 nm. The chlorophyll content of cells ranged between 1.2 to 1.4 mg-(g FW)- 1. Analytical determinations. Nitrate was measured spectrophotometrically according to Cawse (1967). Ammonium was estimated enzymatically with bovine-liver glutamate dehydrogenase as described by Schmidt and Schmidt (1983). L-Amino acids were determined either by high-performance liquid chromatography (HPLC) according to Mfirquez et al. (1986) or colorimetrically by the ninhydrin-ascorbate method (Robinson 1978). Oxo acids were determined either colorimetrically by measuring the 2,4-dinitrophenylhydrazones formed (Borchers 1977) or by HPLC (Mfirquez et al. 1986). Chlorophyll was estimated by the Arnon method (1949).
195
J. Mufioz-Blanco et al. : Amino-acid deamination in Chlamydomonas
Amino-acid-utilization experiments. Amino-acid utilization was measured in TAP-amino acids media at cell densities corresponding tO A660 = 1.5. Cells were induced in the presence of the indicated L-amino acid, then harvested by centrifugation at 10000-g (5 min), washed twice with deionised water, resuspended in fresh TAP medium and bubbled with air (1% in CO2) for 30 min. Experiments were started by addition of L-amino acids to cell suspensions to a final amino-acid concentration of 2 mM. In radioactive experiments, cold L-amino acids (1 mM) and H3-amino acids DuPont, Boston, Mass., USA, labeled in the side chains (6.4.109 Bq.mol-1), were added to culture media. When indicated, 200-~tl samples were withdrawn and placed in vials containing a mixture of 80 ~tl of 15% (v/v) perchloric acid and 20 Ixl of a solution consisting of a phenylmethylsilicone (Silicon oil DC550 from Fluka, Buchs, Switzerland) and Bis (3,5,5-trimethylhexyl) phthalate (Fluka; 60:40; v/v). Samples were centrifuged at 10000' g (30 s) and the supernatant (culture medium) and the centrifugate (whole cells and cell debris) were removed, mixed with 2 ml of scintillation cocktail, and their radioactivity measured in a Beckman/LS-4700 counter (Beckman, Mfinchen, FRG).
arginine, L-lysine, L-alanine, L-valine, L-leucine, L-isoleucine, L-serine, L - m e t h i o n i n e , L-histidine, a n d L-phenylala n i n e as sole n i t r o g e n sources. F i g u r e 1 shows L - m e t h i o nine a n d L-alanine u t i l i z a t i o n . G r o w t h a n d a m i n o - a c i d u t i l i z a t i o n p a t t e r n s were v e r y similar in all cases studied. G e n e r a t i o n times a n d rates o f a m i n o - a c i d u t i l i z a t i o n for cells p r e v i o u s l y g r o w n o n a m m o n i u m r a n g e d b e t w e e n 9 - 1 8 h a n d 10-17 g m o l . h - l . ( m g Chl) - 1 , respectively, a n d it t o o k b e t w e e n 10-30 h b e f o r e the u t i l i z a t i o n s y s t e m was i n d u c e d (Fig. 1). A c c u m u l a t i o n o f a m m o n i u m in the e x t r a c e l l u l a r m e d i u m o f T A P - a m i n o a c i d - g r o w i n g cells was n e v e r d e t e c t e d , a n d , in the a b s e n c e o f a c e t a t e , cells o n l y used L-arginine as n i t r o g e n s o u r c e for g r o w t h by an uptake process induced under nitrogen-starvation c o n d i t i o n s (results n o t shown). A s i m i l a r selectivity o f a r g i n i n e u p t a k e in m i n i m a l m e d i a has b e e n p r e v i o u s l y r e p o r t e d (even in n i t r o g e n - s t a r v e d cultures) in s o m e spe-
13
Results and discussion L- serine
In the light a n d in T A P - a c e t a t e m e d i u m , Chlamydomonas cells were a b l e to use L - a s p a r a g i n e , L-glutamine, L-
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P l a n t a 9 Springer-Verlag1990
Extracellular deamination of L-amino acids by Chlamydomonas reinhardtii cells J. Mufioz-Blanco, J. Hidalgo-Martinez, and J. Cfirdenas* Departamento de Bioquimica y Biologia Molecular y Fisiologia, Facultad de Ciencias, Universidad de C6rdoba, Avda. S. Alberto Magno, s/n, E-14071 C6rdoba, Spain Received 14 December 1989; accepted 2 April 1990
Abstract. When grown in the light and in a Tris-acetate phosphate medium, cells o f Chlamydomonas reinhardtii Dang. can use the following L-amino acids as a sole nitrogen source: asparagine, glutamine, arginine, lysine, alanine, valine, leucine, isoleucine, serine, methionine, histidine, and phenylalanine, whereas, in the absence of acetate, the cells only used L-arginine. The utilization system in the acetate m e d i u m consisted of an extracellular deaminating activity induced by L-amino acids; it t o o k between 10 to 30 h before the system appeared in cells previously grown with a m m o n i u m . This deaminase activity was nonspecific, required an organic carbon source for its de-novo synthesis, and was sensitive to high a m m o n i u m concentration and light deprivation.
(Paul and Cooksey 1979), whereas L-arginine is taken up through an active transport system induced by nitrogen starvation (Kirk and Kirk 1978a). In the present paper, we demonstrate that the unicellular green alga Chlamydomonas reinhardtii can grow in the presence of acetate, at the expense of a m m o n i u m supplied by twelve L-amino acids (asparagine, glutamine, arginine, lysine, alanine, valine, leucine, isoleucine, serine, methionine, histidine, and phenylalanine), which are first deaminated in a reaction mediated by a nonspecific de-novo inducible deaminase sensitive to high amm o n i u m concentration and light deprivation. It is the first time that such a general deaminating system for L-amino acids has been described in green algae.
Key words: L-amino acids (utilization) - Chlamydomona s - Deaminase (extracellular)
Material and methods
Introduction Photosynthetic organisms are able to use L-amino acids as nitrogen sources for growth (Liu and Hellebust 1974; Wheeler et al. 1974; Baden and Mende 1979). This takes place either by means of energy-dependent transport systems (Kirk and K i r k 1978a, b; Cho et al. 1981; Cho and K o m o r 1983; Flynn and Syrett 1985; Sauer and Tanner 1985) or by deamination mechanisms occurring at the cell surface with the subsequent uptake and assimilation o f the a m m o n i u m produced (Paul and Cooksey 1979, 1981a, b). In Chlamydomonas, it has been reported that L-asparagine and L-arginine are the only amino acids that can be used as nitrogen sources to support algal growth. L-Asparagine provides a m m o n i u m after deamination * To whom correspondence should be addressed Abbreviations: HPLC = high-performance liquid chromatography; TAP = Tris-acetate-phosphate
Cell growth conditions. Cells of Chlamydomonas reinhardtii Dang. 6145c (a gift of Dr. R. Sager, Cancer Faber Center, New York, USA) were cultured at pH 7.0 in the minimal liquid medium of Sueoka et al. (1967) or in liquid media containing acetate (TAP, Tris-acetate-phosphate media; Gorman and Levine 1965), with ammonium or L-amino acids as sole nitrogen sources. Solid TAP media contained, in addition, 1.5% Difco Bacto-Agar (Difco Laboratories, Detroit, Mich., USA). Cells were maintained under saturating light conditions (15 W.m-z) until the mid-logarithmic phase of growth, then harvested by centrifugation at 10000-g (5 min), washed twice with deionised water, and resuspended, as required, either in TAP-ammonium or TAP-amino acid media. Growth was followed by measuring the turbidity of cell suspensions at 660 nm. The chlorophyll content of cells ranged between 1.2 to 1.4 mg-(g FW)- 1. Analytical determinations. Nitrate was measured spectrophotometrically according to Cawse (1967). Ammonium was estimated enzymatically with bovine-liver glutamate dehydrogenase as described by Schmidt and Schmidt (1983). L-Amino acids were determined either by high-performance liquid chromatography (HPLC) according to Mfirquez et al. (1986) or colorimetrically by the ninhydrin-ascorbate method (Robinson 1978). Oxo acids were determined either colorimetrically by measuring the 2,4-dinitrophenylhydrazones formed (Borchers 1977) or by HPLC (Mfirquez et al. 1986). Chlorophyll was estimated by the Arnon method (1949).
195
J. Mufioz-Blanco et al. : Amino-acid deamination in Chlamydomonas
Amino-acid-utilization experiments. Amino-acid utilization was measured in TAP-amino acids media at cell densities corresponding tO A660 = 1.5. Cells were induced in the presence of the indicated L-amino acid, then harvested by centrifugation at 10000-g (5 min), washed twice with deionised water, resuspended in fresh TAP medium and bubbled with air (1% in CO2) for 30 min. Experiments were started by addition of L-amino acids to cell suspensions to a final amino-acid concentration of 2 mM. In radioactive experiments, cold L-amino acids (1 mM) and H3-amino acids DuPont, Boston, Mass., USA, labeled in the side chains (6.4.109 Bq.mol-1), were added to culture media. When indicated, 200-~tl samples were withdrawn and placed in vials containing a mixture of 80 ~tl of 15% (v/v) perchloric acid and 20 Ixl of a solution consisting of a phenylmethylsilicone (Silicon oil DC550 from Fluka, Buchs, Switzerland) and Bis (3,5,5-trimethylhexyl) phthalate (Fluka; 60:40; v/v). Samples were centrifuged at 10000' g (30 s) and the supernatant (culture medium) and the centrifugate (whole cells and cell debris) were removed, mixed with 2 ml of scintillation cocktail, and their radioactivity measured in a Beckman/LS-4700 counter (Beckman, Mfinchen, FRG).
arginine, L-lysine, L-alanine, L-valine, L-leucine, L-isoleucine, L-serine, L - m e t h i o n i n e , L-histidine, a n d L-phenylala n i n e as sole n i t r o g e n sources. F i g u r e 1 shows L - m e t h i o nine a n d L-alanine u t i l i z a t i o n . G r o w t h a n d a m i n o - a c i d u t i l i z a t i o n p a t t e r n s were v e r y similar in all cases studied. G e n e r a t i o n times a n d rates o f a m i n o - a c i d u t i l i z a t i o n for cells p r e v i o u s l y g r o w n o n a m m o n i u m r a n g e d b e t w e e n 9 - 1 8 h a n d 10-17 g m o l . h - l . ( m g Chl) - 1 , respectively, a n d it t o o k b e t w e e n 10-30 h b e f o r e the u t i l i z a t i o n s y s t e m was i n d u c e d (Fig. 1). A c c u m u l a t i o n o f a m m o n i u m in the e x t r a c e l l u l a r m e d i u m o f T A P - a m i n o a c i d - g r o w i n g cells was n e v e r d e t e c t e d , a n d , in the a b s e n c e o f a c e t a t e , cells o n l y used L-arginine as n i t r o g e n s o u r c e for g r o w t h by an uptake process induced under nitrogen-starvation c o n d i t i o n s (results n o t shown). A s i m i l a r selectivity o f a r g i n i n e u p t a k e in m i n i m a l m e d i a has b e e n p r e v i o u s l y r e p o r t e d (even in n i t r o g e n - s t a r v e d cultures) in s o m e spe-
13
Results and discussion L- serine
In the light a n d in T A P - a c e t a t e m e d i u m , Chlamydomonas cells were a b l e to use L - a s p a r a g i n e , L-glutamine, L-
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