Role of Salmonella Typhimurium small RNAs RyhB-1 and RyhB-2 in the oxidative stress response.

As part of the response to specific stress conditions, bacteria express small molecules of non-coding RNA which maintain cellular homeostasis by regulating gene expression, commonly at the post-transcriptional level. Among these, in Salmonella enterica sv. Typhimurium, the paralog small non-coding RNAs RyhB-1 and RyhB-2 play an important role in iron homeostasis. In addition, in the present work, we show that RyhB-1 and RyhB-2 also participate in the response to hydrogen peroxide (H2O2). Deletion of RyhB-1 and/or RyhB-2 resulted in increased levels of intracellular reactive oxygen species, protein carbonylation and an altered NADH/NAD(+) ratio. Analyses of the transcriptional profiles of ryhB-1 and ryhB-2 by northern blot and qRT-PCR showed that they are induced in response to H2O2 in an OxyR-dependent manner. By using lacZ-fusions and electrophoretic mobility shift assays, we confirmed the requirement of OxyR for inducing expression of both ryhB-1 and ryhB-2. Taken together, our results support a model in which, in response to peroxide treatment, ryhB-1 and ryhB-2 are upregulated by OxyR through direct interaction with their promoter region.

Response regulator ArcA of Salmonella enterica serovar Typhimurium downregulates expression of OmpD, a porin facilitating uptake of hydrogen peroxide.

Here we demonstrate that OmpD, the most abundant porin in Salmonella enterica serovar Typhimurium, facilitates uptake of hydrogen peroxide (H2O2) and that its expression is negatively regulated by ArcA upon peroxide exposure. When exposed to sublethal concentrations of H2O2, a S. Typhimurium ompD mutant showed decreased peroxide levels compared to those observed in the wild type strain, suggesting that H2O2 could be channeled inside the cell through OmpD. Further evidence came from in vitro studies using OmpD-containing reconstituted proteoliposomes, which showed enhanced H2O2 uptake compared to control liposomes with no porins. RT-PCR and western blot analyses were consistent with a negative regulation mechanism of ompD expression in wild type S. Typhimurium exposed to H2O2. In silico analysis aimed at detecting putative transcriptional regulator binding regions led to identification of an ArcA global regulator motif in the ompD promoter region. The interaction of ArcA with its putative binding site was confirmed in vitro by electrophoretic mobility shift assays. In addition, RT-PCR and western blot experiments demonstrated that the ompD downregulation, observed when the wild type strain was grown in the presence of H2O2, was not retained in arcA mutants, suggesting that ArcA could act as an ompD transcriptional repressor.