Regulation of CorA Mg ²⁺ Channel Function Affects the Virulence of Salmonella enterica Serovar Typhimurium

Abstract

Mg2ϩ is implicated in several stages of Salmonella enterica serovar Typhimurium infection. For example, exposure to low extracellular Mg 2ϩ concentrations results in activation of the PhoP/PhoQ two-component system and consequently markedly alters the expression of many genes necessary for virulence, including but not limited to those that encode Salmonella pathogenicity island 1, Salmonella pathogenicity island 2, host antimicrobial peptide resistance, bile resistance, and biofilm formation (1,5,18). mgtA and mgtB, which encode two inducible Mg 2ϩ transport systems, are also PhoP/PhoQ regulated (5,32,34).The primary source of intracellular Mg 2ϩ in S. enterica serovar Typhimurium, about 50% of other bacteria, and about 20% of archaea is the CorA Mg 2ϩ channel (12)(13)(14)21). A strain lacking corA shows no Mg 2ϩ growth phenotype or other growth deficit. Despite this lack of an overt growth phenotype, in another study we found that a strain lacking corA (MM2242) was attenuated in mice after infection by both the oral and intraperitoneal routes and was defective for invasion and replication within Caco-2 epithelial cells (22). Microarray data indicated that multiple metabolic pathways are affected by corA mutation. The simplest explanation for these defects is that, in the absence of CorA, the organism cannot obtain sufficient intracellular Mg 2ϩ for optimal virulence despite having two other Mg 2ϩ transporters. However, here we show that intracellular Mg 2ϩ is relatively unaffected by loss of corA. The virulence phenotype and changes in gene expression are related to the presence of a functional CorA protein and most likely regulation of CorA function rather than the actual transport process. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains used in this study are listed in Table 1. Unless otherwise indicated, all are derived from S. enterica serovar Typhimurium SL1344, referred to as our wild-type strain. Cloning of Methanococcus jannaschii corA into the pUC18 plasmid (pMJcorA) has been previously reported (27). This plasmid carrying M. jannaschii corA was isolated from MM1556 and transformed into the corA mutant strain (MM2242), creating MM3203. The Escherichia coli corA and Bacillus subtilis mgtE genes were cloned from E. coli DH5␣ cells (MM3097) and B. subtilis genomic DNA (ATCC 23857D), respectively, into a pBADMycHis(a) vector (Invitrogen), creating pBADECcorA and pBADBSmgtE. The restriction sites used for insertion into this vector were XhoI and EcoRI. An additional stop codon was added to prevent the MycHis tag from being added. Expression from the pBAD vectors was dependent on the arabinose-inducible araBAD promoter. These plasmids were shuttled into the restrictionless MM1242 strain and then into MM2242, creating strains MM3218 and MM3217, respectively. MM3217 and MM3218 were grown in Luria-Bertani (LB) broth with 1 mM arabinose overnight for induction prior to experiments. pCorAF266A and pCorAP269A are low-copy pAlter plasmids carrying the corA gene with site-directed substitu...

The Pseudomonas aeruginosa Magnesium Transporter MgtE Inhibits Transcription of the Type III Secretion System

“…Our results with this binding site mutant suggest that inhibition of cytotoxicity is effected by some part of the MgtE structure rather than the act of transporting magnesium or switching between open and closed conformations. Recent studies of the CorA magnesium transporter in S. Typhimurium (strain SL1344) have reached a similar conclusion; CorA affected virulence despite inhibition of magnesium transport through the protein (37). The data presented in this study suggest the hypothesis that P. aeruginosa has evolved dual functions for MgtE: magnesium transport and virulence regulation.…”

Comparative community genomics in the Dead Sea: an increasingly extreme environment

“…The enrichment in putative archaeal (see phylogenetic tree in Figure 7b) CorA magnesium channels (up to 11-fold for COG 0598; see Figure 7a) in the highly magnesium-enriched Dead Sea environment points to a potential, yet unknown, resistance mechanism used by Dead Sea halophiles. Although CorA (COG 0598) is usually associated with Mg 2 þ influx activity, when exposed to high extracellular Mg 2 þ concentrations, CorA can mediate Mg 2 þ efflux (Gibson et al, 1991;Papp-Wallace and Maguire, 2008). Interestingly, the Mg 2 þ /Co 2 þ /Ni 2 þ channel MgtE (COG 2239; Figure 7a) was completely absent from the Dead Sea libraries.…”

Mg 2+ regulates transcription of mgtA in Salmonella Typhimurium via translation of proline codons during synthesis of the MgtL peptide

“…Salmonella has three uptake systems for Mg 2+ : MgtA, MgtB, and CorA. The transcription of mgtA and mgtB, which is flanked by mgtC and mgtR in an operon, is inducible over a hundred-fold by Mg 2+ limitation, whereas the transcription of corA is not regulated by Mg 2+ (3).…”