Pyruvate is synthesized by two pathways in pea bacteroids with different efficiencies for nitrogen fixation

Area of Science:

• Microbiology
• Plant Science
• Biochemistry

Background:

• Nitrogen fixation in legume bacteroids relies on dicarboxylic acid metabolism, producing oxaloacetate and pyruvate.
• Alfalfa bacteroids primarily use NAD+ malic enzyme (Dme) for pyruvate production, unlike Rhizobium leguminosarum which has dual pathways.

Purpose of the Study:

• To investigate the roles of Dme, phosphoenolpyruvate carboxykinase (PckA), and pyruvate kinase (PykA) in pyruvate formation and nitrogen fixation in Rhizobium leguminosarum.
• To determine the necessity of these pathways for bacteroid growth on dicarboxylates and for nitrogen (N2) fixation in legumes.

Main Methods:

• Construction and analysis of single and double mutants of Rhizobium leguminosarum lacking key enzymes (Dme, PckA, PykA).
• Assessing nitrogen fixation efficiency using acetylene reduction assays in wild-type and mutant strains in alfalfa and pea plants.
• Investigating the impact of expressing PckA in alfalfa dme mutants and analyzing the effect of deleting the phosphotransacetylase (Pta) domain of Dme.

Main Results:

• Rhizobium leguminosarum utilizes both Dme and PckA/PykA pathways for pyruvate synthesis from dicarboxylates, with Dme supporting higher N2 fixation rates.
• Double mutants lacking both pathways showed no N2 fixation and impaired growth on dicarboxylates, confirming their essentiality.
• Alfalfa bacteroids strictly require Dme for pyruvate synthesis and N2 fixation, as PckA is not expressed. Deleting the Pta domain of Dme enhanced N2 fixation in pea plants but did not improve overall plant growth.

Conclusions:

• Dme is indispensable for pyruvate formation and nitrogen fixation in alfalfa bacteroids.
• While dual pathways exist in Rhizobium leguminosarum, the Dme pathway is more efficient for N2 fixation.
• Modulating pyruvate synthesis pathways, particularly Dme, offers potential for enhancing nitrogen fixation, though sustained plant growth benefits require further investigation.