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Journal of Bacteriology, August 2002, p . 4369-4373, Vol . 184, No . 16

ZntB Is a Novel Zn2+ Transporter in Salmonella enterica Serovar Typhimurium

Ashley J . Worlock and Ronald L . Smith*

Department of Biology, The University of Texas at Arlington, Arlington, Texas 76019

Received 23 April 2002/ Accepted 31 May 2002


   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 .


   INTRODUCTION

 
Zinc is the second most abundant transition metal in biological systems (6, 11, 24) . It is an essential element that is employed in a wide range of biochemical and biophysical roles . It is required to maintain the structural stability of macromolecules and to serve as a cofactor for more than 300 metabolic enzymes . It also plays a prominent role in gene expression as a structural component in a large number of Zn2+-dependent transcription factors (4, 19) . While the cellular requirement for zinc is absolute, excess concentrations of the cation are highly toxic . Consequently, the ability to maintain the intracellular Zn2+ concentration within very narrow limits is a fundamental property of all living cells . Enteric bacteria are currently thought to maintain Zn2+ homeostasis through the activities of at least four Zn2+-specific transport systems . Zinc uptake is facilitated by the combined activities of the ZnuABC and ZupT transport systems (10, 13) . The ZnuABC system is the primary Zn2+ influx pathway and is induced in response to zinc deprivation (16) . Bacteria respond to high levels of exogenous Zn2+ by increased expression of the ZntA and ZitB efflux systems (2, 5, 9, 17) . The previously uncharacterized open reading frame b1342 of Escherichia coli encodes a protein that is homologous to the CorA family of cation transporters (3, 20) . In Salmonella enterica serovar Typhimurium and E . coli, CorA functions as the primary influx pathway for Mg2+ and is distinguished by its unique ability to mediate both the influx and efflux of Mg2+ (8, 22, 23) . In an effort to ascertain the function of the peptide encoded by b1342, our laboratory identified the corresponding allele in S . enterica serovar Typhimurium and disrupted it by the insertion of an antibiotic resistance cassette . In this report, we show that the serovar Typhimurium homolog of b1342 encodes an additional zinc transport system . Mutations at this locus resulted in an increased sensitivity to cytotoxic levels of Zn2+ and Cd2+ and a reduced capacity for zinc efflux . Consequently, the mutated gene and its protein product were designated ZntB .


   MATERIALS AND METHODS

 
Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1 . Plasmids were maintained in E . coli Top10F' unless indicated otherwise .


TABLE 1 . Bacterial strains and plasmids

 
Culture media and reagents. Bacteria were grown at 37°C with aeration in Luria-Bertani (LB) medium or on LB agar plates (14), except when otherwise stated . Minimal medium was based on the N medium of Nelson and Kennedy (15), supplemented with 0.25% glucose, 0.02% Casamino Acids, and 1 mM MgSO4 . Antibiotics were added to complex and minimal culture media, respectively, at the following concentrations: sodium ampicillin salt (100 and 30 µg/ml), kanamycin sulfate (50 and 100 µg/ml), chloramphenicol (25 and 10 µg/ml) . Restriction endonucleases, T4 DNA ligase, and shrimp alkaline phosphatase were obtained from Promega Corporation . Sequencing enzymes and associated biochemicals were obtained from Epicentre Technologies . 65ZnCl2 was purchased from Perkin Elmer Life Sciences, Inc . Additional chemicals were obtained from standard suppliers .

DNA manipulations and zntB mutant construction. Construction of the serovar Typhimurium zntB mutant strain was performed as described previously by Datsenko and Wanner (7) with the following modifications: pKD46 was first moved into serovar Typhimurium 14028s by electroporation . The zntB1::{Omega}Cm mutagenic cassette was gel purified and transformed into 14028s/pKD46 . Chloramphenicol-resistant transformants were subsequently cured of pKD46 by growth at 40°C . Chromosomal integration of the mutagenic cassette was confirmed by PCR and sequenced using oligonucleotides external to the integrated cassette . Plasmid DNA was prepared from 5-ml cultures using the Qiaprep Spin Miniprep kit obtained from Qiagen . Genomic DNA was isolated using a PureGene purification kit obtained from Gentra Systems . Restriction endonuclease digestion, DNA ligation, and transformation of linear and plasmid DNA were performed as described previously (1) .

Quantitation of cation sensitivity. Susceptibility profiles were generated by disk diffusion and growth curve analyses . Bacterial lawns were spread on N-minimal agar plates, and 8-mm sterile filter disks were placed in the center . Lethal levels of ZnSO4, CoCl2, NiCl2, MnCl2, and CdCl2 (100 mM, 100 mM, 1 M, 1 M, and 100 mM, respectively) were added to the disks . Zones of inhibition were measured after 24 h of growth at 37°C . In addition, growth was monitored over time in N-minimal media containing various levels of ZnSO4 . Overnight cultures were diluted 1:100 into appropriate growth assay media and readings of the optical density at 600 nm (OD600) were made every hour until stationary phase was reached .

65Zn2+ uptake. Overnight cultures were diluted 100-fold into supplemented N-minimal medium and grown at 37°C with aeration to an OD600 of 0.4 . Cells were then washed twice with ice-cold N-minimal medium without MgSO4, Casamino Acids, and glucose supplementation . The pellet was resuspended in wash buffer to a final OD600 of between 1.0 and 2.0 . Uptake was measured at 37°C in N-minimal medium containing 10 mM MgSO4, 0.25% glucose, and 6 µM 65ZnCl2 . The reaction was initiated by addition of prewarmed cells . At timed intervals, 100-µl samples were removed and filtered through nitrocellulose filters (0.45-µm pore size; Whatman) . Filters were washed with 8 ml of cold wash buffer containing 0.5 mM EDTA . Radioactivity was analyzed by gamma counting . A background value, obtained from 1 ml of assay mixture without the addition of cells, was subtracted from all readings .

65Zn2+ efflux. Cells were grown and washed as stated above with the following changes . Washed cells were resuspended in N-minimal medium supplemented with glucose, MgSO4, and Casamino Acids and containing 100 µM MnCl2 . Cells were incubated at 37°C for 1 h to allow Mn2+ to bind intracellular sites and allow efflux . 65ZnCl2 was added to 600-µl cell suspension aliquots to a final concentration of 5 µM and incubated for 20 min . Cells were pelleted at 12,000 x g for 3 min, the supernatant was removed, and the cells were resuspended in 600 µl of assay buffer (N-minimal medium with nutrient supplements and 10 µM ZnSO4) . At timed intervals, 100-µl aliquots of the cell suspension were removed and collected on nitrocellulose filters as described above . Radioactivity was analyzed by gamma counting .

DNA sequencing and PCR. DNA sequencing was performed by the dideoxy chain termination method of Sanger et al . (18) as modified by Tabor and Richardson (25), using T7 DNA polymerase obtained from Epicentre Technologies . Initial reactions were performed using M13 -20 promoter primers obtained from LI-COR Biosciences . Subsequent reactions employed synthetic oligodeoxynucleotide primers complementary or identical to segments within a previously sequenced segment . PCRs were performed using the Opti-Prime PCR optimization kit obtained from Stratagene and oligodeoxynucleotide primers obtained from Integrated DNA Technologies .

Nucleotide sequence accession number. The nucleotide sequence of the S . enterica serovar Typhimurium zntB locus was deposited in the nucleotide database of the National Center for Biotechnology Information with the accession number AF308568 .


   RESULTS

 
Disruption of zntB increases sensitivity to zinc. Allelic replacement techniques were used to disrupt the S . enterica serovar Typhimurium homolog of b1342 by the insertion of a chloramphenicol resistance cassette into the coding sequence, producing strain RS1100 (7) . Bacteriophage P22-mediated cotransduction techniques were used to determine the position of the zntB locus on the serovar Typhimurium chromosome . The insertion mutation mapped to centisome 36 and was 100% linked to oxrA(fnr), which is in agreement with the recently completed genome sequence . The effects of the insertion mutation on metal sensitivity were determined by disk diffusion assay . Disruption of this locus did not significantly alter the sensitivities to cobalt, nickel, or manganese compared to those of the wild-type strain RS404 . However, the loss of this allele had a marked effect on resistance levels to zinc and cadmium (Fig . 1) . The increased zones of sensitivity displayed by the mutant suggest that the product of b1342 might participate in the extrusion of Zn2+ and possibly Cd2+ . Consequently, b1342 and its gene product were designated ZntB (for Zn2+ transport) . Expression of a wild-type zntB allele in the mutant background partially restored resistance to zinc but had no measurable effect on resistance to cadmium . Growth of the zntB mutant (RS1100) and wild-type (RS404) strains was measured in minimal growth media containing a range of inhibitory zinc concentrations (Fig . 2) . The wild type displayed half-maximal growth in medium containing 60 µM Zn2+ . By comparison, the zntB mutant displayed half-maximal growth at 20 µM Zn2+ . The zinc-sensitive phenotype displayed by RS1100 could be partially rescued through the introduction of a low-copy-number plasmid encoding the wild-type zntB allele (pAJW54) . This strain (RS1100/pAJW54) attained half-maximal growth at 45 µM Zn2+ .


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 NiCl2. 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 ({square}), RS404/pAJW54 ({blacksquare}), RS1100 ({circ}), 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.

 
ZntB does not function as a Mg2+ uptake system. ZntB is homologous to the CorA family of cation transporters (12, 20) . In serovar Typhimurium and E . coli, CorA functions as the primary influx and efflux pathway for Mg2+ (22, 23) . The ability of ZntB to function as a Mg2+ transport system was tested . Strain MM281 carries insertion mutations in each of the three known Mg2+ transport loci (mgtA, mgtCB, and corA) which render the cell unable to grow unless supplemented with 100 mM Mg2+ . The Mg2+-dependent growth of MM281 can be rescued by introducing a plasmid encoding a functional Mg2+ transport system (21) . Strain RS1225 (MM281/pAJW54) displayed a Mg2+ growth dependence that was indistinguishable from that exhibited by MM281 (Fig . 3) . In stark contrast, strains expressing CorAs from serovar Typhimurium (MM281/pRS310) and the archaeon Methanococcus jannaschii (MM281/pRS319) displayed a pronounced leftward shift in Mg2+ dependence for growth . Thus, at the level of growth, ZntB does not function as a Mg2+ uptake system .


FIG . 3 . Magnesium-dependent growth of serovar Typhimurium strains MM281 ({diamondsuit}), MM281/pRS310 (•), MM281/pRS319 ({square}), and MM281/pAJW54 ({blacksquare}) . 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.

 
65Zn2+ transport assays. To characterize the transport activity of ZntB, uptake of 65Zn2+ was measured in RS404 (wild-type serovar Typhimurium), RS1100 (zntB mutant), and the complementing strain RS1100/pAJW54 . All three strains exhibited similar rates of uptake . The total amount of 65Zn2+ accumulated by the zntB mutant was 1.2-fold greater than the wild-type control accumulation . Expression of zntB from pAJW54 reduced the level of zinc accumulation to 1.1-fold of the levels demonstrated by the wild type (Fig . 4) . These data indicate that ZntB does not facilitate zinc uptake, but rather suggest a possible role in the efflux of zinc . This was directly tested by introducing pAJW54 into a Zn2+ transport-deficient strain of E . coli and measuring the rate of 65Zn2+ efflux . GR480 contains mutations in each of the known Zn2+ transport loci (zntA, zitB, zupD, znuABC, and yiiP) but retains a functional zntB allele (9, 10) . This gene was disrupted by the insertion of a chloramphenicol resistance cassette producing strain RS1220 (7) . The presence of the single chromosomal zntB allele carried in strain GR480 resulted in an efflux rate that was 5-fold greater than the rate for the transport-deficient strain (RS1220) . Expression of ZntB from pAJW54 further increased the rate of 65Zn2+ efflux 8.8-fold (Fig . 5) . Thus, ZntB is able to facilitate the efflux of zinc .


FIG . 4 . Effects of zntB expression on Zn2+ uptake . Bulk uptake of 65Zn2+ was measured in serovar Typhimurium strains RS404 (wild type {square}), RS1100 (zntB1 {blacksquare}), 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 ({square}), RS1220 ({blacksquare}), and RS1220/pAJW54 (•) as described in Materials and Methods . GR480 carries chromosomal deletions in each of the known zinc transport loci ({Delta}zntA, {Delta}zitB, {Delta}zupD, {Delta}znuABC, and {Delta}yiiP) 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.

 

   DISCUSSION

 
In this report, we demonstrate that the zntB locus of S . enterica serovar Typhimurium encodes a protein that is involved in the transmembrane flux of zinc . Mutations at zntB render the cell hypersensitive to the cytotoxic effects of zinc, as indicated by disk diffusion analysis and growth characterization . This phenotype suggests that ZntB plays a role in the efflux of this cation . A direct examination of transport activity revealed that zntB mutations diminish the capacity to extrude Zn2+ without significantly affecting the uptake activity . Moreover, this transport deficiency could be complemented by a plasmid encoding a wild-type zntB allele . The ZntB transporter is homologous to the CorA Mg2+ transport protein, which is the defining member of a large class of unusual proteins that appear to be widely distributed throughout the bacteria and the archaea (20, 22) . In S . enterica serovar Typhimurium and E . coli, CorA functions as the primary influx pathway for magnesium and is responsible for more than 95% of the total magnesium accumulated under normal growth conditions . The ZntB transporter, however, is not able to function as a Mg2+ uptake pathway . Our results identify ZntB as a novel transport system for Zn2+ in the enteric bacteria and, as such, assign a new function to the ubiquitous CorA family of cation transporters .

 


   ACKNOWLEDGMENTS

 
This work was supported by a grant from The Welch Foundation (Y1485) to R.L.S .


   FOOTNOTES

 
* Corresponding author . Mailing address: Department of Biology, The University of Texas at Arlington, Arlington, TX 76019 . Phone: (817) 272-2411 . Fax: (817) 272-2855 . E-mail: rlsmith{at}airmail.net .


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