Sinorhizobium meliloti Glutathione Reductase Is Required for both Redox Homeostasis and Symbiosis

Abstract

Glutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH), one of the key antioxidants in Sinorhizobium meliloti, is required for the development of alfalfa (Medicago sativa) nitrogen-fixing nodules. Glutathione exists as either reduced glutathione (GSH) or oxidized glutathione (GSSG), and its content is regulated by two pathways in S. meliloti The first pathway is the de novo synthesis of glutathione from its constituent amino acids, namely, Glu, Cys, and Gly, catalyzed by γ-glutamylcysteine synthetase (GshA) and glutathione synthetase (GshB). The second pathway is the recycling of GSSG via glutathione reductase (GR). However, whether the S. meliloti GR functions similarly to GshA and GshB1 during symbiotic interactions with alfalfa remains unknown. In this study, a plasmid insertion mutation of the S. melilotigor gene, which encodes GR, was constructed, and the mutant exhibited delayed alfalfa nodulation, with 75% reduction in nitrogen-fixing capacity. The gor mutant demonstrated increased accumulation of GSSG and a decreased GSH/GSSG ratio in cells. The mutant also showed defective growth in rich broth and minimal broth and was more sensitive to the oxidants H₂O₂ and sodium nitroprusside. Interestingly, the expression of gshA, gshB1, katA, and katB was induced in the mutant. These findings reveal that the recycling of glutathione is important for S. meliloti to maintain redox homeostasis and to interact symbiotically with alfalfa.IMPORTANCE The antioxidant glutathione is regulated by its synthetase and reductase in cells. In the symbiotic bacterium S. meliloti, the de novo synthesis of glutathione is essential for alfalfa nodulation and nitrogen fixation. In this study, we observed that the recycling of glutathione from GSSG not only was required for redox homeostasis and oxidative stress protection in S. meliloti cells but also contributed to alfalfa nodule development and competition capacity. Our findings demonstrate that the recycling of glutathione plays a key role in nitrogen fixation symbiosis.

Keywords: Sinorhizobium meliloti; glutathione reductase; reactive oxygen species; redox homeostasis; symbiosis.

FIG 1

Strategies for plasmid insertion and characterization of the gor19 mutant. (A) Schematic representation of the location of the genomic locus before and after integration of the plasmid into the genome in the WT, gor19, and cpgor19 strains. WT, S. meliloti 1021; gor19, mutant with a plasmid insertion in gor; cpgor19, strain containing pSRK19 in the gor19 mutant. (B) Agarose gel with a DNA size marker and DNA fragments amplified with different genomic DNA. Lane M, DNA marker 2K Plus; lane 1, amplified gor PCR fragment from the S. meliloti 1021 (WT) strain; lane 2, amplified gor PCR fragment from the gor19 mutant; lane 3, amplified gor PCR fragment from the cpgor19 strain. (C) Real-time PCR analysis of gor expression levels in the WT, gor19, and cpgor19 strains. (D) GR activity of total protein in the WT, gor19, and cpgor19 strains. The data shown are the means of three experiments, with the error bars indicating the standard errors.

FIG 2

Growth curves of the S. meliloti strains. At time zero, rhizobial cells were diluted with LB/MC medium or M9 medium to an OD₆₀₀ of 0.01. The growth of the WT, gor19, and cpgor19 strains in LB/MC and M9 media was monitored by measuring the optical density at 600 nm. (A) Growth curves of free-living S. meliloti strains grown in LB/MC medium. (B) Growth curves of free-living S. meliloti strains grown in M9 medium. The data points represent the averages of triplicate samples, with the error bars indicating the standard errors.

FIG 3

Accumulation of glutathione in S. meliloti. (A and B) High-performance liquid chromatography (HPLC) analysis of the GSH (A) and GSSG (B) contents in the WT, gor19, and cpgor19 strains. (C) GSH/GSSG ratios in the WT, gor19, and cpgor19 strains. The data represent the means ± standard errors for triplicate samples. Significant differences were identified by one-way analysis of variance (ANOVA), followed by Tukey's post hoc test, and are noted as different letters (P < 0.05).

FIG 4

qRT-PCR analysis of the expression levels of various genes in the WT, gor19, and cpgor19 strains. The relative amount of mRNA for each gene was calculated using the threshold cycle (C_T) method and was normalized to that of the S. meliloti rpsF gene. Error bars indicate standard deviations. This experiment was performed in triplicate. Significant differences were identified by one-way ANOVA, followed by Tukey's post hoc test, and are noted as different letters (P < 0.05). Vertical bars indicate the standard errors for three biological replicates. nd, not detected.

FIG 5

Sensitivity of the WT, gor19, and cpgor19 strains to oxidants. (A) H₂O₂ sensitivity of the WT, gor19, and cpgor19 strains grown in LB/MC medium. Cell cultures were adjusted to an OD₆₀₀ of 0.5, treated for 30 min at 28°C with the indicated amount of H₂O₂, washed twice, resuspended in fresh LB/MC medium, and spotted onto LB/MC agar plates. The 10-fold serial dilutions are indicated above each column. The plates were incubated at 28°C for 4 days. (B) SNP sensitivity of the WT, gor19, and cpgor19 strains grown in LB/MC medium. Cell cultures were adjusted to an OD₆₀₀ of 0.5 and spotted onto LB/MC agar in the absence or presence of 1 or 3 mM SNP. The 10-fold serial dilutions are indicated above each column. The plates were incubated at 28°C for 4 days.

FIG 6

Deficiency of the gor19 strain in symbiosis with alfalfa. (A) Symbiotic phenotypes of the WT, gor19, and cpgor19 strains. (B) Dry weights of shoots inoculated with the WT, gor19, and cpgor19 strains. (C) Nitrogenase activity of alfalfa nodules per plant, as detected by C₂H₂ reduction, in plants inoculated with the WT, gor19, and cpgor19 strains. (D) Numbers of nodules per plant, assessed 5, 9, and 14 dpi. In panels B to D, the data represent means ± standard deviations (n = 32). The experiments were repeated four times, and a representative experiment is shown. Significant differences (P < 0.05) were identified by one-way ANOVA, followed by Tukey's post hoc test, and are noted as different letters (P < 0.05).