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Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13

Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13 203 129 129 2 2 Michael R. Betlach James M. Tiedje Richard B. Firestone Department of Microbiology and Public Health Michigan State University 48824 East Lansing Michigan USA Department of Microbiology and Public Health Michigan State University 48824 East Lansing Michigan USA Department of Crop and Soil Sciences Michigan State University 48824 East Lansing Michigan USA Cyclotron Laboratory Michigan State University 48824 East Lansing Michigan USA Extraterrestrial Research Div. NASA Ames Research Center 94035 Moffett Field California Lawrence Berkeley Laboratory 94720 Berkeley California USA Abstract The mechanism of nitrate uptake for assimilation in procaryotes is not known. We used the radioactive isotope, 13 N as NO 3 - , to study this process in a prevalent soil bacterium, Pseudomonas fluorescens . Cultures grown on ammonium sulfate or ammonium nitrate failed to take up labeled nitrate, indicating ammonium repressed synthesis of the assimilatory enzymes. Cultures grown on nitrite or under ammonium limitation had measurable nitrate reductase activity, indicating that the assimilatory enzymes need not be induced by nitrate. In cultures with an active nitrate reductase, the form of 13 N internally was ammonium and amino acids; the amino acid labeling pattern indicated that 13 NO 3 - was assimilated via glutamine synthetase and glutamate synthase. Cultures grown on tungstate to inactivate the reductase concentrated NO 3 - at least sixfold. Chlorate had no effect on nitrate transport or assimilation, nor on reduction in cell-free extracts. Ammonium inhibited nitrate uptake in cells with and without active nitrate reductases, but had no effect on cell-free nitrate reduction, indicating the site of inhibition was nitrate transport into the cytoplasm. Nitrate assimilation in cells grown on nitrate and nitrate uptake into cells grown with tungstate on nitrite both followed Michaelis-Menten kinetics with similar K m values, 7 μM. Both azide and cyanide inhibited nitrate assimilation. Our findings suggest that Pseudomonas fluorescens can take up nitrate via active transport and that nitrate assimilation is both inhibited and repressed by ammonium. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Microbiology Springer Journals

Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13

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References (30)

Publisher
Springer Journals
Copyright
Copyright © 1981 by Springer-Verlag GmbH & Co. KG
Subject
Life Sciences; Biotechnology; Biochemistry, general; Cell Biology; Ecology; Microbial Ecology; Microbiology
ISSN
0302-8933
eISSN
1432-072X
DOI
10.1007/BF00455349
Publisher site
See Article on Publisher Site

Abstract

203 129 129 2 2 Michael R. Betlach James M. Tiedje Richard B. Firestone Department of Microbiology and Public Health Michigan State University 48824 East Lansing Michigan USA Department of Microbiology and Public Health Michigan State University 48824 East Lansing Michigan USA Department of Crop and Soil Sciences Michigan State University 48824 East Lansing Michigan USA Cyclotron Laboratory Michigan State University 48824 East Lansing Michigan USA Extraterrestrial Research Div. NASA Ames Research Center 94035 Moffett Field California Lawrence Berkeley Laboratory 94720 Berkeley California USA Abstract The mechanism of nitrate uptake for assimilation in procaryotes is not known. We used the radioactive isotope, 13 N as NO 3 - , to study this process in a prevalent soil bacterium, Pseudomonas fluorescens . Cultures grown on ammonium sulfate or ammonium nitrate failed to take up labeled nitrate, indicating ammonium repressed synthesis of the assimilatory enzymes. Cultures grown on nitrite or under ammonium limitation had measurable nitrate reductase activity, indicating that the assimilatory enzymes need not be induced by nitrate. In cultures with an active nitrate reductase, the form of 13 N internally was ammonium and amino acids; the amino acid labeling pattern indicated that 13 NO 3 - was assimilated via glutamine synthetase and glutamate synthase. Cultures grown on tungstate to inactivate the reductase concentrated NO 3 - at least sixfold. Chlorate had no effect on nitrate transport or assimilation, nor on reduction in cell-free extracts. Ammonium inhibited nitrate uptake in cells with and without active nitrate reductases, but had no effect on cell-free nitrate reduction, indicating the site of inhibition was nitrate transport into the cytoplasm. Nitrate assimilation in cells grown on nitrate and nitrate uptake into cells grown with tungstate on nitrite both followed Michaelis-Menten kinetics with similar K m values, 7 μM. Both azide and cyanide inhibited nitrate assimilation. Our findings suggest that Pseudomonas fluorescens can take up nitrate via active transport and that nitrate assimilation is both inhibited and repressed by ammonium.

Journal

Archives of MicrobiologySpringer Journals

Published: Apr 1, 1981

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