OxyR-Dependent Transcription Response of Sinorhizobium meliloti to Oxidative Stress

Abstract

Reactive oxygen species such as peroxides play an important role in plant development, cell wall maturation, and defense responses. During nodulation with the host plant Medicago sativa, Sinorhizobium meliloti cells are exposed to H₂O₂ in infection threads and developing nodules (R. Santos, D. Hérouart, S. Sigaud, D. Touati, and A. Puppo, Mol Plant Microbe Interact 14:86-89, 2001, https://doi.org/10.1094/MPMI.2001.14.1.86). S. meliloti cells likely also experience oxidative stress, from both internal and external sources, during life in the soil. Here, we present microarray transcription data for S. meliloti wild-type cells compared to a mutant deficient in the key oxidative regulatory protein OxyR, each in response to H₂O₂ treatment. Several alternative sigma factor genes are upregulated in the response to H₂O₂; the stress sigma gene rpoE2 shows OxyR-dependent induction by H₂O₂, while rpoH1 expression is induced by H₂O₂ irrespective of the oxyR genotype. The activity of the RpoE2 sigma factor in turn causes increased expression of two more sigma factor genes, rpoE5 and rpoH2 Strains with deletions of rpoH1 showed improved survival in H₂O₂ as well as increased levels of oxyR and total catalase expression. These results imply that ΔrpoH1 strains are primed to deal with oxidative stress. This work presents a global view of S. meliloti gene expression changes, and of regulation of those changes, in response to H₂O₂IMPORTANCE Like all aerobic organisms, the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti experiences oxidative stress throughout its complex life cycle. This report describes the global transcriptional changes that S. meliloti makes in response to H₂O₂ and the roles of the OxyR transcriptional regulator and the RpoH1 sigma factor in regulating those changes. By understanding the complex regulatory response of S. meliloti to oxidative stress, we may further understand the role that reactive oxygen species play as both stressors and potential signals during symbiosis.

Keywords: OxyR; RpoH; Sinorhizobium meliloti; catalase; oxidative stress; sigma factors; transcriptome.

FIG 1

Comparison qPCR and chip expression data. Data represent fold changes in gene expression of selected genes differentially regulated by the use of 1 mM H₂O₂ as determined in three replicates each using Affymetrix GeneChip and qPCR. Raw data and statistical analysis are shown in Table S4.

FIG 2

Overlap of genes differentially regulated in response to two concentrations of H₂O₂ and in the oxyR mutant. (A) Genes upregulated in response to H₂O₂ in wild-type CL150 (treated with 1 mM or 0.5 mM H₂O₂) and in the oxyR mutant (treated with 0.5 mM H₂O₂). (B) Genes downregulated in response to H₂O₂ in wild-type CL150 (treated with 1 mM or 0.5 mM H₂O₂) and in the oxyR mutant (treated with 0.5 mM H₂O₂).

FIG 3

Effect of H₂O₂ on S. meliloti mutants with complete deletions of alternative sigma factors. Survival rates were determined after 30 min of exposure to H₂O₂ of S. meliloti strains deleted for alternative sigma factors implicated in the response to H₂O₂. Survival was normalized to wild-type CL150 survival (10.3% ± 1.6%). Error bars indicate standard deviations; *, P ≤ 0.05. Experiments were performed in triplicate; two other independent experiments were performed with comparable results.

FIG 4

Transcription of oxyR in various S. meliloti mutants deleted for alternative sigma factors. Data represent expression of a oxyR-uidA transcriptional fusion with and without 30 min of treatment with 1 mM H₂O₂ in mutants lacking various S. meliloti alternative sigma factors. Error bars indicate standard deviations. “a” indicates a P value of ≤0.05 compared to 0 mM H₂O₂. “b” indicates a P value of ≤0.05 comparing the activity of the mutant strain at 0 mM H₂O₂ to the activity of the wild-type strain at 0 mM H₂O₂. Experiments were performed in triplicate.

FIG 5

Catalase activity in S. meliloti stains deleted for alternative sigma factors. (A) Total catalase activity of various alternative sigma factor deletions with and without 30 min of treatment with 1 mM H₂O₂. Catalase activity was normalized to OD₆₀₀. Error bars indicate standard deviations. “a” indicates a P value of ≤0.05 compared to 0 mM H₂O₂. “b” indicates a P value of ≤0.05 comparing the activity of the mutant strain at 0 mM H₂O₂ to the activity of the wild-type strain at 0 mM H₂O₂. Experiments were performed in triplicate. (B) Activity of katA-uidA transcriptional fusion with and without 30 min of treatment with 1 mM H₂O₂. Error bars indicate standard deviations. *, P ≤ 0.05 (comparing the activity of the mutant strain at 0 mM H₂O₂ to the activity of the wild-type strain at 0 mM H₂O₂). Experiments were performed in triplicate.