ZupT Is a Zn(II) Uptake System in Escherichia coli

Escherichia coli zupT (ygiE), encoding a ZIP family member,mediated zinc uptake. Growth of cells disrupted in both zupTand the znuABC operon was inhibited by EDTA at a much lowerconcentration than a single mutant or the wild type. Cells expressingZupT from a plasmid exhibited increased uptake of ⁶⁵Zn²⁺.

INTRODUCTION

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Introduction

Zinc is an essential transition metal for all organisms andserves as a cofactor in members of all six major functionalclasses of enzymes (18). It also serves as a structural cofactorfor many proteins. As a result, organisms have developed mechanismsfor maintaining adequate concentrations of intracellular zincwhile preventing metal ion overaccumulation. Cells of Escherichiacoli encounter fluctuating extracellular zinc levels and maintainzinc homeostasis by transporting excess metal out of the celland regulating zinc uptake across the cytoplasmic membrane.Prior to this report, three zinc transport systems had beencharacterized for E. coli. Efflux of zinc is accomplished bythe P-type ATPase ZntA (2, 16) and the cation diffusion facilitatorZitB (7). Under conditions of deficiency, zinc is taken up bythe high-affinity ABC transporter ZnuABC (14). In this reportwe show that ZupT is an additional zinc transporter responsiblefor zinc uptake. ZupT is the first characterized bacterial memberof the ZIP family of proteins, previously only reported to bepresent in eukaryotes. The ZIP family derived its name fromthe first identified members (ZRT, IRT-like protein) (9). Thesetransporters were initially identified as iron or zinc transportersbut were subsequently shown to be able to also transport manganeseand cadmium. However, the specificity and affinity to differentmetals are specific for each ZIP transporter (6, 9).

Either ZupT (YgiE) or ZnuABC is necessary for growth under zinc-limited conditions.

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Either zupt (ygie) or...

ZnuABC had been shown to be responsible for high-affinity zincuptake (14). However, addition of EDTA to mineral salts mediumdid not completely abolish growth of E. coli strain GR352 ${Delta}$ znuABC::cam,indicating the presence of other transporters. Recently, a reviewby Gaither and Eide (6) suggested that the ZIP family is notrestricted to eukaryotes but is also present in bacteria. InE. coli the ygiE gene encodes a putative ZIP protein (6). Toassess the physiological role, ygiE was deleted as describedpreviously (4). Deletion of ygiE in E. coli GR316 ( ${Delta}$ ygiE::cam)only slightly affected growth under the conditions tested (Fig.1A). However, growth of the double mutant E. coli GR354 ( ${Delta}$ znuABC ${Delta}$ ygiE::cam) with both ygiE and znuABC deleted was severely inhibitedby the presence of EDTA. This inhibition was more pronouncedin the double mutant than in the single mutants E. coli GR352( ${Delta}$ znuABC::cam) and GR316 ( ${Delta}$ ygiE::cam) (Fig. 1A). Addition of zincbut not of nickel, copper, or cadmium alleviated this inhibition(Fig. 1B), indicating that ygiE is responsible for zinc uptake.We therefore renamed ygiE as zupT to indicate its role as azinc uptake transporter. ZnuABC appears to have a higher affinityfor zinc than ZupT, since a strain with a deletion of zupT isless inhibited in growth by the addition of high concentrationsof EDTA than a strain with a deletion of znuABC. E. coli GR352( ${Delta}$ znuABC) showed improvement of growth after addition of zincbut also a slight increase after nickel addition (Fig. 1B).This could be caused by slight impurities in the nickel saltused. However, zinc clearly is the growth-limiting factor inthe E. coli GR352 ( ${Delta}$ znuABC) single mutant and the E. coli GR354( ${Delta}$ znuABC ${Delta}$ ygiE::cam) double mutant.

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FIG. 1. Effect of zinc depletion on growth of E. coli W3110, GR352 ( ${Delta}$ znuABC::cam), and GR354 ( ${Delta}$ zupT::cam ${Delta}$ znuABC). Growth yields with different EDTA concentrations are shown. (A) Overnight cultures grown in LB medium were diluted 1:400 into fresh mineral salts medium (12) with the indicated concentrations of EDTA. Cell growth was monitored by measuring the OD₆₀₀ after 6 h of incubation at 37°C with shaking and converted to dry weight. Results are shown for E. coli W3110 ( ${blacklozenge}$ ), E. coli GR316 ( ${Delta}$ zupT::cam) (•), E. coli GR352 ( ${Delta}$ znuABC::cam) ( ${blacksquare}$ ), and E. coli GR354 ( ${Delta}$ zupT::cam ${Delta}$ znuABC) ( ${blacktriangleup}$ ). Experiments were performed in triplicate, and the average was calculated. (B) Cells were treated as for panel A except that equimolar amounts of EDTA and metals were added (both at 250 µM). Results are shown for E. coli W3110 (light grey bars), E. coli GR352 ( ${Delta}$ znuABC::cam) (dark grey bars), and E. coli GR354 ( ${Delta}$ zupT::cam ${Delta}$ znuABC) (black bars).

Expression of ZupT makes cells zinc hypersensitive.

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Expression of zupt makes...

To further characterize ZupT-mediated metal transport, ZupTwas cloned into the inducible expression vector pASK-IBA3 (IBA,Göttingen, Germany), creating pZUPT. Expression of zupTwas induced by the addition of 200 ng of anhydrotetracycline(AHT) (Sigma-Genosys)/ml, which made cells hypersensitive tozinc (Fig. 2A). This effect was most pronounced in strain E.coli GG48, in which the zntA and zitB genes, encoding zinc effluxpumps, had been disrupted. Expression of zupT in E. coli GG48(zntA::kan ${Delta}$ zitB::cam) even led to reduced viability in Luria-Bertani(LB) broth without added zinc due to residual zinc present inthe medium (Fig. 2A). However, addition of EDTA alleviated thetoxic effects of expression of zupT (Fig. 2B). This indicatedthat zinc hypersensitivity is caused by overaccumulation ofcytosolic zinc and is not due to possible toxic effects of proteinoverproduction. Furthermore, a slight increase in copper sensitivityin wild-type cells expressing zupT was observed, indicatingthat ZupT transports copper in addition to zinc (Fig. 2C). Incontrast, addition of cadmium to cells expressing zupT did notlead to a pronounced increase in sensitivity as observed withzinc (Fig. 2D). On the contrary, the addition of 5 µMCd(II) led to an increase in viability over that for cells inLB broth without added metal, indicating that Cd(II) might inhibituptake of zinc by ZupT. This is in agreement with previouslyreported metal specificities of ZIP proteins from Arabidopsisthaliana (8).

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FIG. 2. Growth of E. coli strains expressing zupT in the presence of metals or EDTA. Overnight cultures grown in LB medium were diluted 1:400 into fresh LB medium with the indicated concentrations of zinc (A), EDTA (B), copper (C), or cadmium (D), and expression of zupT was induced with 200 ng of AHT/ml. Growth was monitored after 6 h by measuring the OD₆₀₀ and converted to dry weight (mg/ml). (A, B, D) ${blacksquare}$ , E. coli GG48 ( ${Delta}$ zntA::kan ${Delta}$ zitB::cam)(pZUPT); ${blacktriangleup}$ , E. coli GG48 ( ${Delta}$ zntA::kan ${Delta}$ zitB::cam) vector control. (C) ${blacksquare}$ , E. coli W3110(pZUPT); ${blacktriangleup}$ , E. coli W3110 vector control. Results of representative experiments are shown.

ZupT mediates uptake of ⁶⁵Zn.

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Zupt mediates uptake of...

To measure uptake of ⁶⁵Zn²⁺ by ZupT, all known zinc transportsystems in E. coli were disrupted (4). The zntA and zitB genesboth encode efflux systems (7, 16), whereas znuABC and zupTare responsible for zinc uptake (14; this report). In addition,the zntB gene, encoding a putative zinc efflux system, was deleted(data not shown). This strain, E. coli GR362 (zntA::kan ${Delta}$ zitB ${Delta}$ zupT ${Delta}$ znuABC ${Delta}$ zntB::cam), showed normal growth in LB medium andwas transformed with the plasmid pZUPT. Expression of zupT wasinduced with different concentrations of AHT for 10 min. Cellswith the largest amount of inducer added (200 ng/ml) showedthe largest increase in zinc uptake compared to the vector control,E. coli GR362(pASK-IBA3) (Fig. 3). Addition of smaller amountsof inducer still led to a significantly higher level of accumulationof ⁶⁵Zn²⁺ than was found with control cells (Fig. 3).

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FIG. 3. Uptake of ⁶⁵Zn(II) by cells of E. coli GR362 ( ${Delta}$ znuABC ${Delta}$ zupT ${Delta}$ zntB::cam ${Delta}$ zitB zntA::kan) expressing zupT. Cells were grown overnight in LB medium, diluted 50-fold into fresh prewarmed LB medium, and grown to an OD₆₀₀ of 0.5, and expression of zupT was induced with 200 or 20 ng of AHT/ml for 10 min or not induced. The cells were washed with buffer A (10 mM Tris-HCl [pH 7.0], 2 g of glucose/liter, 10 mM Na₂HPO₄) and resuspended in the same buffer. ⁶⁵ZnSO₄ was added to a final concentration of 10 µM. The cells were incubated at 37°C, and 0.1-ml aliquots were filtered through nitrocellulose membranes (pore size, 0.45 µm) at various times and immediately washed with 10 ml of buffer B (10 mM Tris-HCl [pH 7.0], 10 mM MgCl₂). The membranes were dried, and radioactivity was measured using a liquid scintillation counter. The protein concentration was determined using the method of Lowry et al. (11), and the amount of Zn(II) per milligram of protein was calculated. •, E. coli GR362 ( ${Delta}$ znuABC ${Delta}$ zupT ${Delta}$ zntB::cam ${Delta}$ zitB zntA::kan)(pZUPT) (no inducer); ( ${circ}$ ), E. coli GR362 ( ${Delta}$ znuABC ${Delta}$ zupT ${Delta}$ zntB::cam ${Delta}$ zitB zntA::kan)(pZUPT) (20-ng/ml AHT); ${blacktriangledown}$ , E. coli GR362 ( ${Delta}$ znuABC ${Delta}$ zupT ${Delta}$ zntB::cam ${Delta}$ zitB zntA::kan)(pZUPT) (200 ng/ml); ${triangledown}$ , E. coli GR362 ( ${Delta}$ znuABC ${Delta}$ zupT ${Delta}$ zntB::cam ${Delta}$ zitB zntA::kan)(pASK-IBA3).

Conclusions.

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Conclusions.

In this report we show that ZupT is responsible for zinc uptakein E. coli. ZupT represents the first bacterial member of theZIP family (5, 6, 9). Therefore, the transporters involved inzinc homeostasis in E. coli constitute a mixture of uniquelybacterial systems, such as ZnuABC and transporters that belongto ubiquitously distributed protein families, such as the CDFmember ZitB (13, 15) and the ZIP protein ZupT. P1-type ATPasessuch as ZntA have also been identified but not yet characterizedin plants (1), indicating that this family is also present inall three kingdoms. In addition to these well-characterizedmetal transporters, zinc might also be able to enter as a metalphosphate via the Pit phosphate uptake system (3), but thisappears to be adventitious. Since zinc is such an importantnutrient, it is not surprising to observe redundancy in transportsystems. However, the number and families of putative zinc transportersare not conserved in different bacteria and might reflect differentphysiological needs. In addition, metal transporters sometimesseem to be acquired by horizontal gene transfer, since the sequencesof transporters often do not follow the overall phylogeneticpatterns of the particular microbes as determined by sequencecomparison of small-subunit rRNAs (10, 17, 17a).

Finally, it appears likely that ZupT is a broad-range metalion transporter in E. coli, perhaps mediating transport of otherdivalent cations including Cd(II) and Cu(II). It demonstrablytransports Zn(II); Cd(II) antagonizes the effect of Zn(II) ina zupT-overexpressing strain; and cells expressing ZupT exhibita slight increase in Cu(II) sensitivity.

ACKNOWLEDGMENTS

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This work was supported by Hatch project 136713 and NIEHS grantESO4940 with funds from the EPA to C.R. and U.S. Public HealthGrant GM 55425 to B.P.R.

FOOTNOTES

* Corresponding author. Mailing address: Department of Soil, Water, and Environmental Science, University of Arizona, Shantz Bldg. no. 38, Rm. 429, Tucson, AZ 85721. Phone: (520) 626-8482. Fax: (520) 621-1647. E-mail: rensingc{at}ag.arizona.edu.

REFERENCES

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