JOURNAL OF BACTERIOLOGY, Nov. 1976, p. 683-688 Copyright C 1976 American Society for Microbiology
Vol. 128, No. 2 Printed in U.S.A.
Inorganic Nitrogen Assimilation by the Photosynthetic Bacterium Rhodopseudomonas capsulata BO C. JOHANSSON AND HOWARD GEST* Department of Microbiology, Indiana University, Bloomington, Indiana 47401
Received for publication 2 July 1976
The photosynthetic bacterium Rhodopseudomonas capsulata lacks glutamate dehydrogenase and normally uses the glutamine synthetase/glutamate synthase sequence of reactions for assimilation of N2 and ammonia. The glutamine synthetase in cell-free extracts of the organism is completely sedimented by centrifugation at 140,000 x g for 2 h, is inhibited by L-alanine but not by adenosine 5'-monophosphate, and exhibits two apparent Km values for ammonia (ca. 13 ,uM and 1 mM).
Purple bacteria typically can use N2 or ammonium salts as sole nitrogen sources for photosynthetic growth (6), and preliminary experiments by Nagatani et al. (12) suggested that certain species may use the "glutamine synthetase/glutamate synthase" reaction sequence (19) as a major pathway for the assimilation of inorganic nitrogen. In this communication we report more detailed experiments in this connection with the nonsulfur purple bacterium Rhodopseudomonas capsulata. This organism can use, in addition to N2 and ammonia, a wide variety of compounds as nitrogen sources (e.g., glutamate, glutamine, aspartate, alanine, proline, ornithine, thymine, urea) and has been recently used to demonstrate (21) genetic transfer of nitrogenase-hydrogenase activity by means of a "gene transfer agent," a phagelike vector of still unknown nature. One aim of the present work was to examine the possible role of glutamate dehydrogenase in the nitrogen metabolism of R. capsulata, since in various microorganisms this enzyme activity provides a mechanism for assimilation of ammonia when the latter is available at relatively high concentration (2). Our attempts to demonstrate glutamate dehydrogenase activity in extracts of R. capsulata cells grown under various conditions have given negative results; thus, significant activity could not be detected in extracts from cells I grown with ammonia or glutamate as nitrogen sources, or on glutamate as both nitrogen and carbon source. It is noteworthy that certain blue-green algae (13) and many Bacillus species (7) also appear to lack glutamate dehydrogenase. Such organisms usually manifest alanine dehydrogenase activity, and it has been suggested that this could account for ammonia assimilation under certain conditions (7, 13).
Both aminating and deaminating reactions of alanine dehydrogenase are observed in crude cell-free extracts of R. capsulata (Table 1). The aminating reaction (optimal pH, 7.3) is specifically dependent on nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), whereas the deaminating reaction (optimal pH > 9.5) requires NAD+. Ammonia inhibits the deaminating reaction (50% inhibition at 0.8 mM) and NAD+ is a potent inhibitor of the aminating reaction (50% inhibition at 30 ,AM). Alanine dehydrogenase activity in R. capsulata is observed at significant levels only under certain growth conditions (Table 1). With malate (30 mM) as carbon source and excess ammonia (15 mM) providing nitrogen, the activity is low in the mid-exponential phase of growth and reaches a maximum in the early stationary phase. Activity in mid-exponential-phase cells grown on pyruvate plus ammonia is somewhat elevated, but the highest activities are observed when alanine provides both carbon and nitrogen for growth. It is particularly significant that with N2 as the nitrogen source alanine dehydrogenase activity is virtually absent. These results indicate that alanine dehydrogenase is not primarily involved in N2 assimilation by R. capsulata and that its role in ammonia assimilation is also limited. Consistent with this conclusion is the finding that the Km for ammonia of the aminating activity is 8.3 mM (at pH 7.6); the corresponding Km of the enzyme from various other microorganisms has similarly been reported (10, 15, 24) to be relatively high. Thus, alanine dehydrogenase may be active in an ammonia assimilation capacity only when ammonia is present at high concentration. Formation of glutamate under such growth conditions could occur, in part at least, by coupling of the aminating alanine dehydro-
683
684
J. BACTERIOL.
NOTES
TABLE 1. Levels of alanine dehydrogenase, L-alanine:2-oxoglutarate aminotransferase, glutamate synthase, and glutamine synthetase activities in extracts ofR. capsulatagrown on various carbon and nitrogen sourcesa Sp act (nmol/min per mg of protein&) Carbon source
Nitrogen source
Doubling
time (min)
ADH Aminating
Deaminating
AOAT
GOGAT
GS
Malate (NH4)2SO4 140 8.1 _C 68.0 52.4 280.3 Pyruvate (NH4)2SO4 95 36.9 141.2 39.2 Malate/alanine Alanine 165 71.1 39.6 134.9 18.3 Alanine Alanine 160 264.3 109.8 3.5 Malate/glutamate Glutamate 180 3.4 118.4 21.0 Malate N2 220
Vol. 128, No. 2 Printed in U.S.A.
Inorganic Nitrogen Assimilation by the Photosynthetic Bacterium Rhodopseudomonas capsulata BO C. JOHANSSON AND HOWARD GEST* Department of Microbiology, Indiana University, Bloomington, Indiana 47401
Received for publication 2 July 1976
The photosynthetic bacterium Rhodopseudomonas capsulata lacks glutamate dehydrogenase and normally uses the glutamine synthetase/glutamate synthase sequence of reactions for assimilation of N2 and ammonia. The glutamine synthetase in cell-free extracts of the organism is completely sedimented by centrifugation at 140,000 x g for 2 h, is inhibited by L-alanine but not by adenosine 5'-monophosphate, and exhibits two apparent Km values for ammonia (ca. 13 ,uM and 1 mM).
Purple bacteria typically can use N2 or ammonium salts as sole nitrogen sources for photosynthetic growth (6), and preliminary experiments by Nagatani et al. (12) suggested that certain species may use the "glutamine synthetase/glutamate synthase" reaction sequence (19) as a major pathway for the assimilation of inorganic nitrogen. In this communication we report more detailed experiments in this connection with the nonsulfur purple bacterium Rhodopseudomonas capsulata. This organism can use, in addition to N2 and ammonia, a wide variety of compounds as nitrogen sources (e.g., glutamate, glutamine, aspartate, alanine, proline, ornithine, thymine, urea) and has been recently used to demonstrate (21) genetic transfer of nitrogenase-hydrogenase activity by means of a "gene transfer agent," a phagelike vector of still unknown nature. One aim of the present work was to examine the possible role of glutamate dehydrogenase in the nitrogen metabolism of R. capsulata, since in various microorganisms this enzyme activity provides a mechanism for assimilation of ammonia when the latter is available at relatively high concentration (2). Our attempts to demonstrate glutamate dehydrogenase activity in extracts of R. capsulata cells grown under various conditions have given negative results; thus, significant activity could not be detected in extracts from cells I grown with ammonia or glutamate as nitrogen sources, or on glutamate as both nitrogen and carbon source. It is noteworthy that certain blue-green algae (13) and many Bacillus species (7) also appear to lack glutamate dehydrogenase. Such organisms usually manifest alanine dehydrogenase activity, and it has been suggested that this could account for ammonia assimilation under certain conditions (7, 13).
Both aminating and deaminating reactions of alanine dehydrogenase are observed in crude cell-free extracts of R. capsulata (Table 1). The aminating reaction (optimal pH, 7.3) is specifically dependent on nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), whereas the deaminating reaction (optimal pH > 9.5) requires NAD+. Ammonia inhibits the deaminating reaction (50% inhibition at 0.8 mM) and NAD+ is a potent inhibitor of the aminating reaction (50% inhibition at 30 ,AM). Alanine dehydrogenase activity in R. capsulata is observed at significant levels only under certain growth conditions (Table 1). With malate (30 mM) as carbon source and excess ammonia (15 mM) providing nitrogen, the activity is low in the mid-exponential phase of growth and reaches a maximum in the early stationary phase. Activity in mid-exponential-phase cells grown on pyruvate plus ammonia is somewhat elevated, but the highest activities are observed when alanine provides both carbon and nitrogen for growth. It is particularly significant that with N2 as the nitrogen source alanine dehydrogenase activity is virtually absent. These results indicate that alanine dehydrogenase is not primarily involved in N2 assimilation by R. capsulata and that its role in ammonia assimilation is also limited. Consistent with this conclusion is the finding that the Km for ammonia of the aminating activity is 8.3 mM (at pH 7.6); the corresponding Km of the enzyme from various other microorganisms has similarly been reported (10, 15, 24) to be relatively high. Thus, alanine dehydrogenase may be active in an ammonia assimilation capacity only when ammonia is present at high concentration. Formation of glutamate under such growth conditions could occur, in part at least, by coupling of the aminating alanine dehydro-
683
684
J. BACTERIOL.
NOTES
TABLE 1. Levels of alanine dehydrogenase, L-alanine:2-oxoglutarate aminotransferase, glutamate synthase, and glutamine synthetase activities in extracts ofR. capsulatagrown on various carbon and nitrogen sourcesa Sp act (nmol/min per mg of protein&) Carbon source
Nitrogen source
Doubling
time (min)
ADH Aminating
Deaminating
AOAT
GOGAT
GS
Malate (NH4)2SO4 140 8.1 _C 68.0 52.4 280.3 Pyruvate (NH4)2SO4 95 36.9 141.2 39.2 Malate/alanine Alanine 165 71.1 39.6 134.9 18.3 Alanine Alanine 160 264.3 109.8 3.5 Malate/glutamate Glutamate 180 3.4 118.4 21.0 Malate N2 220
