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

Abstract

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.