Antioxidant Ability of Glutaredoxins and Their Role in Symbiotic Nitrogen Fixation in Rhizobium leguminosarum bv. viciae 3841

Glutaredoxins (Grx) are redoxin family proteins that reduce disulfides and mixed disulfides between glutathione and proteins. Rhizobium leguminosarum by. viciae 3841 contains three genes coding for glutaredoxins as follows: RL4289 (grxA) codes for a dithiol glutaredoxin, RL2615 (grx8) codes for a monothiol glutaredoxin, and RL4261 (grxC) codes for a glutaredoxin-like NrdH protein. We generated mutants interrupted in one, two, or three glutaredoxin genes. These mutants had no obvious differences in growth phenotypes from the wild-type RL3841. However, while a mutant of grxC did not affect the antioxidant or symbiotic capacities of R. leguminosarum, grxA-derived or grx8 mutants decreased antioxidant and nitrogen fixation capacities. Furthermore, grxA mutants were severely impaired in rhizosphere colonization and formed smaller nodules with defects of bacteroid differentiation, whereas nodules induced by grx8 mutants contained abnormally thick cortices and prematurely senescent bacteroids. The grx triple mutant had the greatest defect in antioxidant and symbiotic capacities of R. leguminosarum, and quantitative proteomics revealed it had 56 upregulated and 81 downregulated proteins relative to wild type. Of these proteins, 28 are involved in transporter activity, 20 are related to stress response and virulence, and 16 are involved in amino acid metabolism. Overall, R. leguminosarum glutaredoxins behave as antioxidant proteins mediating root nodule symbiosis. IMPORTANCE Glutaredoxin catalyzes glutathionylation/deglutathionylation reactions, protects mercapto groups from oxidation, and restores functionally active thiols. Three glutaredoxins exist in R. leguminosarum, and their properties were investigated in free-living bacteria and during nitrogen-fixing symbiosis. All of the glutaredoxins were necessary for oxidative stress defense. Dithiol GrxA affects nodulation and nitrogen fixation of bacteroids by altering deglutathionylation reactions, monothiol GrxB is involved in symbiotic nitrogen fixation by regulating Fe-S cluster biogenesis, and GrxC may participate in symbiosis by an unknown mechanism. Proteome analysis provides clues to explain the differences between the grx triple mutant and wild-type nodules.

Automated summary

Glutaredoxins in R. leguminosarum play essential roles in antioxidation and symbiosis, with different mutants affecting nodulation, nodule structure, and nitrogen fixation capacities. Proteome analysis provides insights into the mechanisms underlying these effects.