Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13.

Area of Science:

• Microbiology
• Biochemistry
• Environmental Science

Background:

• Nitrate assimilation is crucial for bacterial growth and nitrogen cycling.
• The specific mechanisms of nitrate uptake and assimilation in prokaryotes, particularly soil bacteria like Pseudomonas fluorescens, remain largely uncharacterized.

Purpose of the Study:

• To elucidate the mechanism of nitrate (NO3-) uptake and assimilation in Pseudomonas fluorescens.
• To investigate the role of ammonium (NH4+) in regulating nitrate assimilation pathways.

Main Methods:

• Utilized the radioactive isotope 13N-labeled nitrate (13NO3-) to trace uptake and assimilation.
• Cultured Pseudomonas fluorescens under various conditions (ammonium sulfate, ammonium nitrate, nitrite, ammonium limitation, tungstate treatment).
• Assessed nitrate reductase activity, internal 13N forms, and kinetics of nitrate transport and assimilation.

Main Results:

• Ammonium repressed the synthesis of assimilatory enzymes and inhibited nitrate uptake, suggesting transport is the primary inhibition site.
• Nitrate assimilation proceeded via glutamine synthetase and glutamate synthase, with 13N incorporated into ammonium and amino acids.
• Nitrate transport and assimilation followed Michaelis-Menten kinetics (Km ≈ 7 μM) and were inhibited by azide and cyanide.
• Tungstate treatment to inactivate nitrate reductase led to significant NO3- concentration within cells.

Conclusions:

• Pseudomonas fluorescens employs active transport for nitrate uptake.
• Ammonium plays a dual role, repressing enzyme synthesis and inhibiting transport, thereby controlling nitrate assimilation.
• These findings provide critical insights into nitrogen metabolism regulation in soil bacteria.