Structural genes for the vanadium nitrogenase from Azotobacter chroococcum.

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Nitrogen fixation is crucial for life, with molybdenum and vanadium nitrogenases being key enzymes.
• Azotobacter chroococcum possesses both molybdenum and vanadium nitrogenase systems.
• Understanding the genetic basis of vanadium nitrogenase is essential for its biotechnological applications.

Purpose of the Study:

• To clone and sequence the structural genes of the vanadium nitrogenase (Ac1V) VFe-protein from Azotobacter chroococcum.
• To investigate the genetic organization and regulation of these genes.
• To determine if alternative nitrogen fixation systems are present in this organism.

Main Methods:

• Gene cloning and DNA sequencing.
• Sequence homology analysis comparing VFe-protein subunits to MoFe-protein subunits.
• Gene deletion analysis to assess the role of the vnfD, G, and K gene cluster.
• Phenotypic analysis of mutant strains lacking nitrogenase components.

Main Results:

• The structural genes vnfD (alpha), vnfG (delta), and vnfK (beta) for the VFe-protein were identified and found to be contiguous, forming an operon.
• The VFe-protein subunits show limited sequence identity to the MoFe-protein subunits.
• Transcription of the vnf operon is repressed by ammonia.
• The Fe-protein component (Ac2V) is encoded by nifH*, located upstream of the vnf operon.
• Deletions in the vnfD, G, K cluster abolish V-dependent nitrogen fixation.
• A strain lacking both V-nitrogenase and Mo-nitrogenase showed no nitrogen fixation, indicating no third system.

Conclusions:

• The genetic organization and components of the vanadium nitrogenase system in Azotobacter chroococcum have been elucidated.
• The identified genes are essential for V-dependent nitrogen fixation.
• Azotobacter chroococcum does not possess a functional third nitrogen fixation system.