Magnesium uptake by CorA is essential for viability of the gastric pathogen Helicobacter pylori

Abstract

We show here that Mg(2+) acquisition by CorA is essential for Helicobacter pylori in vitro, as corA mutants did not grow in media without Mg(2+) supplementation. Complementation analysis performed with an Escherichia coli corA mutant revealed that H. pylori CorA transports nickel and cobalt in addition to Mg(2+). However, Mg(2+) is the dominant CorA substrate, as the corA mutation affected neither cobalt and nickel resistance nor nickel induction of urease in H. pylori. The drastic Mg(2+) requirement (20 mM) of H. pylori corA mutants indicates that CorA plays a key role in the adaptation to the low-Mg(2+) conditions predominant in the gastric environment.

FIG. 1.

Genetic organization and mutagenesis of H. pylori corA with overview of the corA region in H. pylori strain 26695. Genes and primer binding sites are marked by gray and black arrows, respectively. The insertion site of the cat cassette (white arrow) is indicated. Genes are numbered according to the annotated genome sequence (24). The orientation of the DNA region cloned in plasmids pCORA+ and pCORA− with respect to the lacZ promoter (P_lacZ) present in the cloning vector pZERO-1 is indicated.

FIG. 2.

Determination of the Mg²⁺ requirement of H. pylori corA mutants. (A) Mg²⁺ requirement caused by the corA mutation in H. pylori strain 1061. The growth of H. pylori strain 1061 (black bars) and of the corA mutant 1061-CORA1 (gray bars) in BBF was determined by measuring the OD₆₀₀. Bacteria were precultured to an OD₆₀₀ of 1.0 in BBF medium with 20 mM Mg²⁺ and then subcultured in BBF supplemented with Mg²⁺ at increasing concentrations, indicated on the x axis. (B) Mg²⁺ requirement caused by the corA mutation in H. pylori strain 26695. The growth of H. pylori strains 26695 (black bars), 26695-CORA1 (corA::cat; gray bars) and 26695-CORA2 (corA::Pcat; white bars) was determined by measuring the OD₆₀₀. Bacteria were precultured to an OD₆₀₀ of 1.0 in BBF medium with 20 mM Mg²⁺ and then subcultured in BBF supplemented with Mg²⁺ at increasing concentrations, indicated on the x axis. The data shown represent mean values from three independent determinations. Standard deviations are indicated.

FIG. 3.

Analysis of H. pylori corA transport functions in the E. coli corA mutant H5324. (A) Sensitivity to Mg²⁺ and to various metals mediated by H. pylori corA in E. coli. The growth of E. coli strains MC4100 (black bars), H5324 (gray bars), and H5324 with plasmid pCORA+ carrying H. pylori corA (white bars) in the presence of Mg²⁺, nickel (Ni), cobalt (Co), iron (Fe), and copper (Cu) was determined by measuring the OD₅₅₀. The Mg²⁺ and metal concentrations which were toxic for the parental strain but not for the corA mutant are indicated on the x axis. The data represent mean values from three independent determinations. Standard deviations are indicated. (B) Determination of Mg²⁺ uptake mediated by H. pylori CorA. The E. coli strains MC4100 (black bars), H5324 (gray bars), and H5324(pCORA+) carrying the H. pylori corA gene (white bars) grown for 24 h in LB medium were removed by centrifugation and the Mg²⁺ concentrations in the supernatants were determined. Uptake of Mg²⁺ was calculated from the Mg²⁺ concentration in the LB medium prior to inoculation. The data represent mean values from two independent determinations. Standard deviations are indicated. (C) The influence of Ni-HA onCorA-mediated nickel transport. The E. coli strains MC4100, H5324(pCORA+), and H5324 were grown in LB medium supplemented with 3.5 mM nickel in the absence (black bars) or presence (shaded bars) of 2 mM Ni-HA. Growth was monitored by measuring the OD₅₅₀. The growth of the E. coli corA mutant, which was not affected by nickel or by the hexaamine, is shown as a control. The data represent mean values from three independent determinations. Standard deviations are indicated.