ZntB is a novel Zn2+ transporter in Salmonella enterica serovar Typhimurium

Abstract

A Zn2+ transport system encoded by the zntB locus of Salmonella enterica serovar Typhimurium has been identified. The protein encoded by this locus is homologous to the CorA family of Mg2+ transport proteins and is widely distributed among the eubacteria. Mutations at zntB confer an increased sensitivity to the cytotoxic effects of Zn2+ and Cd2+, a phenotype that suggests that the encoded protein mediates the efflux of both cations. A direct analysis of transport activity identified a capacity for Zn2+ efflux. These data identify ZntB as a zinc efflux pathway in the enteric bacteria and assign a new function to the CorA family of cation transporters.

FIG. 1.

Cation sensitivity profiles of serovar Typhimurium strains RS404 (white), RS1100 (black), and RS1100/pAJW54 (gray). Cells were grown overnight in supplemented N-minimal medium, and 100 μl of the culture was spread onto N-minimal agar plates. An 8-mm filter disk was placed in the center and saturated with 17 μl of a 100 mM cation solution of ZnSO₄, CdCl₂, and CoCl₂ and 1 M solutions of MnCl₂ and NiCl_2. The areas of the resulting inhibition zones were determined after 18 h of incubation at 37°C. Values presented are averages of three independent trials, with standard deviations of the means indicated.

FIG. 2.

Effects of zinc on growth of serovar Typhimurium strains RS404 (□), RS404/pAJW54 (▪), RS1100 (○), and RS1100/pAJW54 (•). Overnight cultures were prepared in supplemented N-minimal medium and diluted 1:500 into fresh N-minimal medium with the indicated concentrations of ZnCl₂. Cell growth was measured as the OD₆₀₀ as the cultures entered late log phase after 6 h of aerobic incubation at 37°C. The data are the averages of six independent trials, with standard deviations of the means indicated.

FIG. 3.

Magnesium-dependent growth of serovar Typhimurium strains MM281 (♦), MM281/pRS310 (•), MM281/pRS319 (□), and MM281/pAJW54 (▪). Overnight cultures were prepared in LB growth medium supplemented with 100 mM Mg²⁺. Prior to the assay, the cell cultures were washed twice with medium containing no supplemental Mg²⁺ and diluted 1:500 into LB medium containing the indicated concentration of MgSO₄. Cell growth was measured as the OD₆₀₀ after 8 h of aerobic incubation at 37°C. The data presented are the averages of five independent trials, with standard deviations of the means indicated.

FIG. 4.

Effects of zntB expression on Zn²⁺ uptake. Bulk uptake of ⁶⁵Zn²⁺ was measured in serovar Typhimurium strains RS404 (wild type □), RS1100 (zntB1 ▪), and RS1100/pAJW54 (•). Cation uptake was measured for 20 min at 37°C as described in Materials and Methods. The data shown are from a single representative experiment.

FIG. 5.

Effects of zntB expression on Zn²⁺ efflux. Efflux was measured at 37°C in E. coli strains GR480 (□), RS1220 (▪), and RS1220/pAJW54 (•) as described in Materials and Methods. GR480 carries chromosomal deletions in each of the known zinc transport loci (ΔzntA, ΔzitB, ΔzupD, ΔznuABC, and ΔyiiP) but retains a functional zntB allele. RS1220 was derived from GR480 by introducing an additional mutation in zntB. Plasmid pAJW54 encodes a wild-type zntB allele from serovar Typhimurium. The data shown are the averages of three independent experiments. Variability was less than ±5% at each point. Correlation coefficients derived from linear regression analysis were >0.95 for each individual experiment.