Fine-Tuning Modulation of Oxidation-Mediated Posttranslational Control of Bradyrhizobium diazoefficiens FixK₂...

FixK₂ is a CRP/FNR-type transcription factor that plays a central role in a sophisticated regulatory network for the anoxic, microoxic and symbiotic lifestyles of the soybean endosymbiont Bradyrhizobium diazoefficiens. Aside from the balanced expression of the fixK gene under microoxic conditions (induced by the two-component regulatory system FixLJ and negatively auto-repressed), FixK₂ activity is posttranslationally controlled by proteolysis, and by the oxidation of a singular cysteine residue (C183) near its DNA-binding domain. To simulate the permanent oxidation of FixK₂, we replaced C183 for aspartic acid. Purified C183D FixK₂ protein showed both low DNA binding and in vitro transcriptional activation from the promoter of the fixNOQP operon, required for respiration under symbiosis. However, in a B. diazoefficiens strain coding for C183D FixK₂, expression of a fixNOQP'-'lacZ fusion was similar to that in the wild type, when both strains were grown microoxically. The C183D FixK₂ encoding strain also showed a wild-type phenotype in symbiosis with soybeans, and increased fixK gene expression levels and FixK₂ protein abundance in cells. These two latter observations, together with the global transcriptional profile of the microoxically cultured C183D FixK₂ encoding strain, suggest the existence of a finely tuned regulatory strategy to counterbalance the oxidation-mediated inactivation of FixK₂ in vivo.