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

Abstract

Glutaredoxins (Grx) are redoxin family proteins that reduce disulfides and mixed disulfides between glutathione and proteins. <i>Rhizobium leguminosarum</i> bv. <i>Viciae</i> 3841 contains three genes coding for glutaredoxins: <i>RL4289</i> (<i>grxA</i>) codes for a dithiolic glutaredoxin, <i>RL2615</i> (<i>grxB</i>) codes for a monothiol glutaredoxin, while <i>RL4261</i> (<i>grxC</i>) 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 <i>grxC</i> did not affect the antioxidant or symbiotic capacities of <i>R. leguminosarum</i>, <i>grxA-</i>derived or <i>grxB</i> mutants decreased antioxidant and nitrogen fixation capacities. Furthermore, <i>grxA</i> mutants were severely impaired in rhizosphere colonization, and formed smaller nodules with defects of bacteroid differentiation, whereas nodules induced by <i>grxB</i> mutants contained abnormally thick cortices and prematurely senescent bacteroids. The <i>grx</i> triple mutant had the greatest defect in antioxidant and symbiotic capacities of <i>R. leguminosarum</i> and quantitative proteomics revealed it had 56 up-regulated and 81 down-regulated proteins relative to wildtype. Of these proteins, twenty-eight are involved in transporter activity, twenty are related to stress response and virulence, and sixteen are involved in amino acid metabolism. Overall, <i>R. leguminosarum</i> glutaredoxins behave as antioxidant proteins mediating root nodule symbiosis.<b>IMPORTANCE</b> Glutaredoxin catalyzes glutathionylation/deglutathionylation reactions, protects SH-groups from oxidation and restores functionally active thiols. Three glutaredoxins exist in <i>R. leguminosarum</i> and their properties were investigated in free-living bacteria and during nitrogen-fixing symbiosis. All 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 <i>grx</i> triple mutant and wild-type nodules.