The development of legume nitrogen-fixing nodules is regulated by reactive oxygen species (ROS) produced by symbionts. Several regulators from Rhizobium are involved in ROS sensing. In a previous study, we found that Sinorhizobium meliloti LsrB regulates lipopolysaccharide production and is associated with H O accumulation in alfalfa (Medicago sativa) nodules. However, its underlying regulatory mechanism remains unclear. Here, we report that the cysteine residues in LsrB are required for adaptation to oxidative stress, gene expression, alfalfa nodulation and nitrogen fixation. Moreover, LsrB directly activated the transcription of lrp3 and gshA (encoding γ-glutamylcysteine synthetase, responsible for glutathione synthesis) and this regulation required the cysteine (Cys) residues in the LsrB substrate-binding domain. The Cys residues could sense oxidative stress via the formation of intermolecular disulfide bonds, generating LsrB dimers and LsrB-DNA complexes. Among the Cys residues, C238 is a positive regulatory site for the induction of downstream genes, whereas C146 and C275 play negative roles in the process. The lsrB mutants with Cys-to-Ser substitutions displayed altered phenotypes in respect to their adaptation to oxidative stress, nodulation and nitrogen fixation-related plant growth. Our findings demonstrate that S. meliloti LsrB modulates alfalfa nodule development by directly regulating downstream gene expression via a post-translational strategy.

The two-component system ActS/ActR plays important roles in bacterial adaptation to abiotic stress, including acid tolerance and oxidant resistance. However, the underlying regulatory mechanism is not clear. In this study, we found that the ActS/ActR system is required for adaptation to oxidative stress by regulating the transcription of the genes actR, katB, gshA and gshB1. The actS and actR mutants were sensitive to low pH and oxidants such as HO, oxidized glutathione (GSSG) and sodium nitroprusside (SNP). The expression of actR by using a plasmid rescued the defect of SNP sensitivity for all actS and actR mutants. The expression of actS and actR were suppressed by treatment with HO. The expression of actS, actR, oxyR, katA and katB was required for ActS and ActR under normal conditions. The induction of katB, gshA and gshB1 depended on ActS and ActR during treatment with HO and SNP. Our findings revealed that the ActS/ActR system is a key redox regulator in S. meliltoi and provides a new cue to understanding Rhizobium-legume symbiosis.

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