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

Research in Microbiology 162 (2011) 214e222 www.elsevier.com/locate/resmic

Response regulator ArcA of Salmonella enterica serovar Typhimurium downregulates expression of OmpD, a porin facilitating uptake of hydrogen peroxide Iva´n L. Caldero´n, Eduardo Morales, Nelson J. Caro, Catalina A. Chahu´an, Bernardo Collao, Fernando Gil, Jose´ M. Villarreal, Francisco Ipinza, Guido C. Mora, Claudia P. Saavedra* Laboratorio de Microbiologı´a Molecular, Facultad de Ciencias Biolo´gicas, Universidad Andre´s Bello, Repu´blica 217, Santiago, Chile Received 29 July 2010; accepted 1 October 2010 Available online 7 December 2010

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. Ó 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: OmpD porin; ArcA; Hydrogen peroxide

1. Introduction The outer membrane (OM) of Gram-negative bacteria comprises a lipid bilayer that allows the passage of small hydrophilic solutes (