the zur-regulated zint protein is an auxiliary component of the

JOURNAL OF BACTERIOLOGY, Mar. 2010, p. 1553–1564 Vol. 192, No. 60021-9193/10/$12.00 doi:10.1128/JB.01310-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

The Zur-Regulated ZinT Protein Is an Auxiliary Component of theHigh-Affinity ZnuABC Zinc Transporter That Facilitates Metal

Recruitment during Severe Zinc Shortage�†Patrizia Petrarca,1 Serena Ammendola,1 Paolo Pasquali,2 and Andrea Battistoni1,3*

Dipartimento di Biologia, Universita di Roma Tor Vergata, 00133 Rome, Italy1; Dipartimento di Sanita Alimentare e Animale,Istituto Superiore di Sanita, 00161 Rome, Italy2; and Consorzio Interuniversitario “Istituto Nazionale Biostrutture e Biosistemi,”

Viale delle Medaglie d’Oro 305, 00136 Rome, Italy3

Received 2 October 2009/Accepted 7 January 2010

The pathways ensuring the efficient uptake of zinc are crucial for the ability of bacteria to multiply in theinfected host. To better understand bacterial responses to zinc deficiency, we have investigated the role of theperiplasmic protein ZinT in Salmonella enterica serovar Typhimurium. We have found that zinT expression isregulated by Zur and parallels that of ZnuA, the periplasmic component of the zinc transporter ZnuABC.Despite the fact that ZinT contributes to Salmonella growth in media containing little zinc, disruption of zinTdoes not significantly affect virulence in mice. The role of ZinT became clear using strains expressing a mutatedform of ZnuA lacking a characteristic histidine-rich domain. In fact, Salmonella strains producing thismodified form of ZnuA exhibited a ZinT-dependent capability to import zinc either in vitro or in infected mice,suggesting that ZinT and the histidine-rich region of ZnuA have redundant function. The hypothesis that ZinTand ZnuA cooperate in the process of zinc recruitment is supported by the observation that they form a stablebinary complex in vitro. Although the presence of ZinT is not strictly required to ensure the functionality of theZnuABC transporter, our data suggest that ZinT facilitates metal acquisition during severe zinc shortage.

Transition metals are essential constituents of a huge num-ber of proteins where they play catalytic or structural functions(4, 50). Therefore, all organisms possess complex machineriesto ensure an adequate supply of these elements, while avoidingtheir potentially toxic intracellular accumulation. A large num-ber of studies have documented the relevance of metals formicrobial growth and resistance to a variety of stress conditions(23, 42, 50). In particular, it is well established that the path-ways enabling bacteria to recruit metal ions are key for theability of pathogens to multiply within the host and causedisease (42, 43, 44). The vast majority of studies concerningmetal uptake and bacterial pathogenicity have focused on iron,but strong evidence is emerging that the efficient uptake ofother transition metals plays an important role in the host-pathogen interaction (24, 54). In particular, recent observa-tions suggest that zinc is not freely available within the host (3).After iron, zinc is the second most abundant transition metalion in living organisms and plays catalytic and/or structuralroles in enzymes of all six classes, several of which play func-tions essential for cell viability (12). Investigations initially car-ried out in Escherichia coli and then confirmed in other micro-organisms have established that zinc homeostasis is finelycontrolled by the coordinated activity of import and exportsystems regulated by Zur and ZntR, two metalloproteins ableto regulate gene transcription depending on their metallation

state (36, 40). Zur controls the expression of a few genesinvolved in bacterial response to zinc shortage, whereas ZntRregulates the expression of the zinc efflux pump ZntA. It isworth observing that, while the intracellular zinc concentrationis rather constant and independent of the culture medium(close to 200 �M), both of these regulators are able to respondto femtomolar variations in the intracellular concentration offree zinc (38). These observations give emphasis to the dy-namic nature of metal homeostasis and suggest that very smallalterations in the intracellular zinc concentration may have arelevant influence on cellular physiology.

The metallated form of Zur also influences pathogenicity bythe capability to repress the expression of the high-affinity zincuptake system ZnuABC, a transporter of the ABC family ac-tivated in several bacteria in response to zinc deficiency (40).This transporter is constituted by three proteins: ZnuB, themembrane permease; ZnuC, the ATPase component; andZnuA, a soluble periplasmic protein that captures Zn(II) anddelivers it to ZnuB. Different studies have established that thefunctionality of this transporter is essential to ensure growth ofmicroorganisms in media with little zinc and for bacterial vir-ulence (3, 9, 16, 30, 32, 52). We have recently demonstratedthat the expression of znuABC is repressed in Salmonella en-terica serovar Typhimurium (hereafter referred to as S. Typhi-murium) cultivated in media containing a zinc concentration aslow as 1 �M, whereas its expression is strongly activated inbacteria recovered from the spleens of infected mice or fromcultured epithelial or macrophage cells (3). Since the zincconcentration within eukaryotic cells is close to 0.2 mM, ourstudies indicate that the amount of metal effectively availablefor microorganisms during intracellular infections is very lim-ited and that the ZnuABC transporter is required to ensure theefficient recruitment of zinc within the host.

* Corresponding author. Mailing address: Dipartimento di BiologiaUniversita di Roma “Tor Vergata,” Via della Ricerca Scientifica,00133 Rome, Italy. Phone: 39 0672594372. Fax: 39 0672594311. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

� Published ahead of print on 22 January 2010.

1553

on February 11, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

In addition to the genes encoding the proteins forming theZnuABC transporter, Zur directly regulates one or more genesencoding paralogs of ribosomal proteins (1, 39, 45). The Zur-regulated ribosomal proteins lack a zinc-binding motif that ispresent in their paralogs which are normally produced in zinc-replete conditions. The insertion of these proteins in ribo-somes during zinc starvation likely facilitates growth by reduc-ing the zinc requirements of bacterial cells. An additional gene(zinT) putatively regulated by Zur was identified in E. coliusing a bioinformatics approach (39). zinT, which was formerlyknown as yodA, was originally identified as a member of the E.coli cadmium stress stimulon (21), and it was proposed that itsrole could be to decrease the concentration of cadmium ions inE. coli cells during cadmium stress (41). Subsequent studies ofE. coli have demonstrated that zinT is modulated in bacterialcells exposed to low pH (8, 27), copper ions (29), or thetransition metal chelator N,N,N�,N�-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) (46) or grown in media containingvery low levels of zinc (22). In addition, it has been hypothe-sized that ZinT could play a chaperone function by deliveringzinc to other periplasmic zinc-binding proteins, such as Cu,Znsuperoxide dismutase (Cu,ZnSOD) (24). This possibility, how-ever, appears to be unlikely, as Cu,ZnSOD is strongly down-regulated under conditions of zinc deficiency (2). Although themost recent investigations suggest that ZinT is involved in zinchomeostasis (22, 29, 46), the exact function of this protein has

not been elucidated. Interestingly, ZinT, whose three-dimen-sional structure has been solved in the presence of differentmetal cofactors (cadmium, zinc, and nickel) (14, 15), shows avery high homology to a domain of AdcA, a component of anABC transporter involved in zinc acquisition in Streptococcuspneumoniae (19, 39). As the N-terminal portion of AdcA ishomologous to ZnuA, this observation strongly suggests thatZinT could cooperate with ZnuA in zinc uptake within theperiplasmic space.

To verify this possibility, the role of ZinT has been investi-gated in S. Typhimurium. Our results demonstrate that ZinThas no role in cadmium resistance and that it participates in thezinc uptake process mediated by ZnuABC.

MATERIALS AND METHODS

Salmonella strains and growth conditions. The S. Typhimurium strains used inthis work are listed in Table 1. Cultures were grown aerobically in liquid Luria-Bertani broth (LB) or on LB agar plates at 37°C. For growth under zinc limitingconditions, Vogel-Bonner minimal medium E (MM) (anhydrous MgSO4 [0.04g/liter], citric acid [2 g/liter], anhydrous K2HPO4 [10 g/liter], NaH4PO4 [3.5g/liter[, glucose [2 g/liter]), M9 minimal medium (Na2HPO4 [7.52 g/liter],KH2PO4 [3 g/liter], NH4Cl [1 g/liter], NaCl [5 g/liter], MgSO4 � 7H2O [1.23g/liter], CaCl2 � 2H2O [0.007 g/liter], glucose [0.2%]), or Tris minimal medium(Tris-HCl [120 mM] [pH 7.2], K2HPO4 [0.017 g/liter], MgCl2 [2.03 g/liter],NH4Cl [1.06 g/liter], NaSO4 [0.44 g/liter], CaCl2 [0.06 g/liter], NaCl [4.68 g/liter],KCl [1.48 g/liter], glucose [1.98 g/liter]) was employed. For antibiotic selection,agar plates were supplemented with kanamycin (50 �g/ml) or chloramphenicol(30 �g/ml).

TABLE 1. Salmonella enterica serovar Typhimurium strains used in this study

Strain Relevant genotype or description Source, reference, technique(s)a, origin, and/or relevantantibiotic resistance phenotype

MA6926b Wild type L. BossiMA6926(pKD46) Wild-type strain harboring plasmid pKD46 L. BossiSA123 znuA::kan 3SA140 znuA::3�FLAG-kan ilvI::Tn10dTac-cat::3�FLAG-kan 3SA150 znuA::cam Lab collectionSA182 znuABC::kan Lab collectionSA229 yebA::cam Electroporation of fragment �oli167-168 pKD3� in

MA6926(pKD46); verified by PCR (oli169/K1); Camr

SA233 znuA�loop yebA::cam This study (see Materials and Methods)SA287 znuA�loop yebA::scarc Derived from SA233; Cams

SA288 znuA::3�FLAG-scar ilvI::Tn10dTac-cat::3�FLAG-scar Derived from SA140; Kans

PP101 znuB::3�FLAG-kan Electroporation of fragment �oli172-173 pSUB11� inMA6926(pKD46); verified by PCR (oli136/K1); Kanr

PP116 zinT::cam Electroporation of fragment �oli178-179 pKD3� inMA6926(pKD46); verified by PCR (K3/oli181); Camr

PP118 zinT::cam znuA::kan P22-mediated transduction (SA123) on PP116; Kanr

PP120 znuA-3�FLAG-kan zinT::cam P22-mediated transduction (SA140) on PP116; Camr

PP125 zinT-scar Derived from PP116; Cams

PP126 znuA::3�FLAG-scar zinT::scar Derived from PP120; Cams

PP127 zur::kan Electroporation of fragment �oli184-185 pKD4� inMA6926(pKD46); verified by PCR (oli177/K1); Kanr

PP128 znuA::3�FLAG-scar zinT::scar ilvI::Tn10dTac-cat::3�FLAG-kan P22-mediated transduction (SA140) on PP126; Camr

PP130 zinT::cam znuA��loop� yebA::scar P22-mediated transduction (PP116) on SA287; Camr

PP131 znuA::3�FLAG-scar zur::kan ilvI::Tn10dTac-cat::3�FLAG-scar P22-mediated transduction (PP127) on SA288; Kanr

PP132 zinT::3�FLAG-scar zur::kan P22-mediated transduction (PP127) on PP129; Kanr

PP134 zinT::3�FLAG-kan Electroporation of fragment �oli182-195 pSUB11� inMA6926(pKD46); verified by PCR (oli180/K4); Kanr

PP137 zinT::3�FLAG-kan znuA::cam P22-mediated transduction (PP134) on SA150; Kanr

PP138 zinT::3�FLAG-scar Derived from PP134; Kans

PP141 znuA::3�FLAG-kan zinT::3�FLAG-scar P22-mediated transduction (SA140) on PP138; Kanr

a The oligonucleotides are shown in italic type in parentheses or brackets, and the template plasmid is shown underlined in brackets.b MA6926 is S. Typhimurium ATCC 14028.c The term “scar” refers to the DNA sequence remaining after excision of the antibiotic resistance cassette following homologous recombination between two flanking

FLP recombination targets (FRTs) mediated by a recombinase encoded by plasmid pCP20 (13).

1554 PETRARCA ET AL. J. BACTERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

Construction of mutants. All Salmonella knockout mutants and the 3�FLAGepitope-tagged strains were obtained following the one-step inactivation proto-col of Datsenko and Wanner (13) and the epitope tagging method described byUzzau et al. (48), respectively. The oligonucleotides and plasmids used forconstruction of mutants are listed in Table 2. Each new strain was confirmed byPCR with oligonucleotides annealing upstream or downstream of the mutatedallele and an internal primer annealing on the inserted antibiotic resistancecassette. The oligonucleotides used for the construction and verification of eachnew strain are specified in Table 1. The alleles were then transduced into a cleanbackground by generalized transduction with phage P22 HT 105/1 int-201 (47).In some cases, the antibiotic resistance cassette was removed by the FLP recom-binase transiently introduced by electroporation of plasmid pCP20 into thestrain. The znuA�loop mutant was obtained by the Datsenko and Wannermethod (13) modified as follows. First, an antibiotic resistance cassette wasinserted into the Salmonella chromosome downstream of the znuA gene byelectroporating a PCR fragment obtained with oligonucleotides oli167/168 onpKD3 plasmid template. The chromosome of the resulting strain (yebA::cam;strain SA229) was then used as a template for a PCR with primers oli167/163,designed ad hoc to amplify the znuA region with a deletion in the His-rich loop(from nucleotide 411 to nucleotide 480 of the coding sequence) and the down-stream antibiotic cassette. The obtained fragment was then electroporated intostrain MA6926 carrying pKD46, and recombinants were selected on chloram-phenicol selective plates. The deletion of the His-rich loop of znuA was con-firmed by nucleic acid sequencing of the mutant strain.

Western blot analysis. To analyze the accumulation of ZinT, ZnuA, andZnuB, aliquots of bacterial cultures (approximately 5 � 108 cells) were har-vested, lysed by resuspending bacteria in sample buffer containing sodium do-decyl sulfate (SDS) and �-mercaptoethanol, and boiled for 8 min at 100°C.Subsequently, the proteins were run on 12% SDS-polyacrylamide gels and blot-ted onto a nitrocellulose membrane (Hybond ECL; Amersham). The epitope-flagged proteins were revealed by incubating the nitrocellulose membrane withan appropriate dilution of mouse anti-FLAG antibody (anti-FLAG M2; Sigma)and anti-mouse horseradish peroxidase-conjugated antibody (Bio-Rad), fol-lowed by the enhanced chemiluminescence reaction (ECL kit; Amersham).

Growth curves. Each strain was grown overnight in LB broth at 37°C and thendiluted 1:500 in fresh LB broth alone or LB broth supplemented with theappropriate concentration of EDTA and/or of metals. The absorbance at 600 nmwas monitored every hour for 10 h using a Perkin-Elmer Lambda 9 spectropho-tometer.

Mouse infections and competition assays. Overnight cultures of bacteria werediluted in phosphate-buffered saline (PBS) buffer to a final concentration of 104

cells/ml and then mixed in pairs in a 1:1 ratio. Portions (0.2 ml) of each mixturewere used to infect 10-week-old female BALB/c mice intraperitoneally. Theanimals were sacrificed when they exhibited symptoms of terminal septic syn-drome (4 or 5 days postinfection). Bacteria recovered from spleens were platedfor single colonies, and then 200 colonies were picked on selective plates. Thecompetitive index (CI) was calculated by the formula CI � output (strainA/strain B)/inoculum (strain A/strain B). Statistical differences between outputsand inputs were determined by Student’s t test.

Cloning, expression, and purification of ZnuA, ZinT, and ZnuA�loop. The S.Typhimurium znuA and znuA�loop genes were amplified from chromosomalDNA extracted with ZRfungal/bacterial DNA kit (Zymo Research) from thewild-type and SA233 strains, respectively. In both cases, the primers used wereSalZnuAfor (5�-ATAGAATTCCGGGGCTCAATTCAAG-3�) and SalZnuArev(5�-TTTAAGCTTAATCTCCTTTCAGGCAGCT-3�) that amplify the codingsequences plus about 200 bp upstream of the start codon. The purified PCRproducts were digested with EcoRI and HindIII, ligated into the pEMBL-18vector (17), obtaining plasmids p18PznuA and p18PznuA�loop, which wereintroduced into E. coli DH5 cells. The sequences of the cloned DNA fragmentswere confirmed by nucleic acid sequencing.

To study the expression of recombinant proteins, cells were grown overnight inLB medium, harvested by centrifugation for 15 min at 8,000 � g, and resus-pended in 500 ml of isotonic solution, and the periplasmic proteins were releasedby osmotic shock as already described (2). After a 20-min centrifugation at13,000 rpm, the periplasmic proteins contained in the supernatant were appliedto a Ni-nitrilotriacetic acid (Ni-NTA) column (Qiagen) preequilibrated with 50mM sodium phosphate and 250 mM NaCl (pH 7.8) and eluted with a discon-tinuous gradient of 0 to 250 mM imidazole. ZnuA eluted with 20 to 40 mMimidazole. The protein was further purified by anion-exchange chromatographyon a HiLoad Q Sepharose fast-performance liquid chromatography (FPLC)column (Pharmacia Biotech) preequilibrated with 20 mM Tris-HCl (pH 7.0) andeluted using a 0 to 400 mM NaCl linear gradient. The purified protein wasconcentrated to 30 mg/ml in a buffer containing 20 mM HEPES, 10 mM NaCl,and 5% glycerol (pH 7.0) and stored at 20°C.

Periplasmic extracts containing the ZnuA�loop protein were initially purifiedby anion-exchange chromatography on a column equilibrated with 20 mM Tris-HCl (pH 7.0) and eluted using a 0 to 400 mM NaCl linear gradient. Subse-quently, the protein was loaded on a cation-exchange HiLoad SP Sepharose

TABLE 2. Oligonucleotides and plasmids used to construct mutants

Oligonucleotide orplasmid Oligonucleotide sequence or plasmid feature (reference)

OligonucleotidesK1............................................................CAGTCATAGCCGAATAGCCTK3............................................................AGCTCACCGTCTTTCATTGCK4............................................................CACTGCAAGCTACCTGCTTToli136.......................................................GTACGCGTGGTTTTAGGACToli163.......................................................GCAACTTGCCGATGTAAAACCGTTACTCATGAAGGGCGCGGGCGAATATAACATGCATCToli167.......................................................AGACCTTCCGTGCGCGGCAATTTTGCTGTCAGAGGGTTAATGTAGGCTGGAGCTGCTTCGoli168.......................................................GCCCTATGTTTACCACCCAGAATCCGCGCCAATCGTTAAACATATGAATATCCTCCTTAGoli169.......................................................TTTCGTCGTTACGACGCATColi172.......................................................GCTGCTGTTTATCTTCAGTATGATGAAAAAGCAGGCAAGCGACTACAAAGACCATGACGGoli173.......................................................TTGAGGATGTGCTGGAGCCGTATCTGATTCAGCAAGGCTTCATATGAATATCCTCCTTAGoli177.......................................................TTCATGCCATTCGAGGTGCToli178.......................................................TTCTGGGAATGCTGTTGGTAAATAGTCCTGCCTTCGCGCATGTAGGCTGGAGCTGCTTCGoli179.......................................................CCAGTTTTCCATCTCTTGCAACAATGCCTGCTGCGAGGTACATATGAATATCCTCCTTAGoli180.......................................................CCGGATAGAGCATAGAGCTToli181.......................................................TGACACGAGTAATCAGGCGAoli182.......................................................GTTAAAGGCGAATGAGGTCGTTGACGAAATGCTACATCATGACTACAAAGACCATGACGGoli184.......................................................GACCACAACGCAAGAGTTACTGGCGCAAGCTGAAAAACTCTGTAGGCTGGAGCTGCTTCGoli185.......................................................TTTTCTTCACCAGCACCGAGTGATCGTGACCACAGTTTCCCATATGAATATCCTCCTTAGoli195.......................................................TTGTCACCAGCAGATCAATGTCGCTGTTTGGCTTCAGACCCATATG

PlasmidspKD46 ....................................................Lambda red recombinase function (13)pKD4 ......................................................Kanamycin resistance cassette template (13)pKD3 ......................................................Chloramphenicol resistance cassette template (13)pCP20 .....................................................FLP recombinase function (13)pSUB11 ..................................................3�FLAG-kanamycin resistance cassette template (48)

VOL. 192, 2010 ZinT INVOLVEMENT IN ZINC UPTAKE 1555


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

column equilibrated with 20 mM sodium phosphate (pH 7.0) and eluted with a0 to 400 mM NaCl linear gradient. A final chromatographic step was carried outon a HiLoad 26/10 Phenyl Sepharose HP column equilibrated with 30 mMTris-HCl and 1.5 M (NH4)SO4 (pH 7.0). Proteins were eluted with a lineargradient of 1.5 to 0 M (NH4)SO4. The fractions containing the ZnuA�loopprotein were pooled, and the resulting protein was more than 98% pure, asjudged by SDS-PAGE analysis. The purified protein was concentrated to 30mg/ml in a buffer containing 20 mM HEPES, 10 mM NaCl, and 5% glycerol withpH 7.0 and stored at 20°C.

The S. Typhimurium zinT gene was amplified from chromosomal DNA ex-tracted from the wild-type strain utilizing primers zinT5 (5�-TCCATGGATATTCATTTAAAAAAACTGACAATG-3�) and zinT2 (5�-ATCAAGCTTAATCAGACTTAATGATGTAGCAT-3�). The PCR product was digested with NcoIand HindIII, ligated into the pSE420 vector (Invitrogen), yielding plasmidpSEzinT, and then transformed into E. coli DH5 cells. The sequence of the wholecloned DNA fragment was verified by nucleic acid sequencing.

Cells harboring plasmid pSEzinT were grown in LB medium supplementedwith 100 �g/ml ampicillin, and when the absorbance at 600 nm of the culturereached 0.5, protein expression was induced in the culture overnight with 0.1 mMisopropyl-�-D-thiogalactopyranoside (IPTG). Cells were harvested by centrifu-gation for 15 min at 5,000 rpm, and periplasmic proteins were extracted bylysozyme treatment (2). Spheroplasts were separated from periplasmic proteinsby centrifugation, and the supernatant was applied to a Ni-NTA column pre-equilibrated with 50 mM sodium phosphate and 250 mM NaCl (pH 7.8) andeluted with a linear gradient of 0 to 500 mM imidazole. ZinT eluted at 250 mMimidazole. Fractions containing ZinT (�98% pure) were pooled, dialyzedagainst a solution of 20 mM HEPES, 10 mM NaCl, and 5% glycerol (pH 7.0),concentrated to 30 mg/ml, and stored at 20°C.

To analyze the possible formation of a complex between ZnuA and ZinT, thetwo proteins were mixed in a 2:1 (ZnuA-ZinT) (wt/wt) ratio, corresponding to a1.4 molar ratio, to favor the formation of complexes. After a 90-min incubationat room temperature, proteins were injected onto a HiLoad 16/60 Superdex 75gel filtration FPLC column (Amersham Biosciences) equilibrated with 20 mMHEPES and 100 mM NaCl (pH. 7.0). Elution was carried out at room temper-ature.

Metal-free ZnuA and ZinT were prepared by extensive dialysis against 50 mMsodium acetate buffer and 2 mM EDTA (pH 5.5). The proteins were subse-quently dialyzed twice against 50 mM sodium acetate and 0.1 M NaCl (pH 5.5)to remove excess EDTA. Finally, the proteins were dialyzed against 20 mMHEPES and 100 mM NaCl (pH 7.0) to carry out gel filtration experiments. Themetal content of the apoproteins was evaluated by atomic absorption using aPerkin-Elmer spectrometer AAnalyst 300 equipped with the graphite furnaceHGA-800. The zinc content of the demetallated proteins was below 5%.

RESULTS

zinT distribution in eubacteria. A previous study has shownthat ZinT is present in some bacterial species as an isolatedprotein, whereas in other bacteria it is a domain of the AdcAprotein involved in zinc uptake (39). The amino acid alignmentshown in Fig. 1 shows that the C-terminal portion of AdcAfrom different bacteria displays high homology with ZinT(53.4% amino acid identity between the overlap region of S.pneumoniae AdcA and mature Salmonella ZinT), whereas itsN-terminal portion is similar to ZnuA (26% amino acid iden-tity between the overlap region of the S. pneumoniae proteinand mature Salmonella ZnuA). The functional and structuralhomology between ZnuA and AdcA is confirmed by two ad-ditional features: the conservation of the three histidine resi-dues which coordinate the zinc ion in the crystal structures ofE. coli (10, 31, 53) and Synechocystis 6803 (5, 51) ZnuA and thepresence in the same sequence position of a charged loop richin acidic and histidine residues (His-rich loop), typical of pro-teins involved in the transport of zinc (5, 11). Interestingly, thethree residues involved in zinc binding in ZinT are strictlyconserved in the AdcA proteins, as well as a C-terminal histi-dine residue, which is likely involved in metal binding (15).However, it should be noted that ZinT differs from the C-

terminal domain of AdcA in the presence of a N-terminalhistidine-rich domain. The observations that in some bacterialspecies ZinT is fused to a ZnuA-like protein involved in zinctransport and that zinT and znuA are likely coregulated by Zur(22, 39) strongly suggest that ZinT could participate in theZnuABC-mediated zinc uptake process. This possibility hasbeen further corroborated by an analysis of the distribution ofZinT, ZnuA (as well as ZnuB and ZnuC), and AdcA in avail-able bacterial genomes (see Table S1 in the supplementalmaterial). This analysis shows that several bacteria possessingthe ZnuABC system do not contain ZinT. However, bacterialspecies lacking a ZnuA homologue do not have a ZinT protein,whereas the occurrence of ZinT is always associated with thepresence of ZnuA. These observations support the hypothesisthat the role of ZinT is related to the role of ZnuA.

ZinT is not involved in cadmium resistance. To verifywhether ZinT is involved in cadmium resistance, we have con-structed an epitope-tagged mutant by introducing a 3�FLAGsequence at the 3� end of the chromosomal copy of the zinTcoding sequence. The PP134 strain (zinT::3�FLAG) shows agrowth rate similar to that of the wild-type strain (data notshown).

In line with previous investigations carried out with E. coli,ZinT accumulates in S. Typhimurium grown in LB mediumsupplemented with 0.5 mM cadmium acetate (Fig. 2A). How-ever, ZnuA and ZnuB, the periplasmic and transmembranecomponent of the ZnuABC zinc transporter, respectively, alsoshow comparable increases in protein accumulation in bacteriacultivated in the presence of cadmium (Fig. 2B and C, respec-tively). This finding suggests that cadmium induces the expres-sion of all Zur-regulated proteins participating in zinc ho-meostasis.

To better evaluate whether the induction of zinT plays a rolein cadmium resistance, we have analyzed the ability of a zinTmutant strain to grow in LB plates containing variable amountsof cadmium. As shown in Table 3, the zinT mutant strain(PP116) exhibited a cadmium susceptibility comparable to thatof the wild-type strain, thus confirming recent observationsshowing that ZinT does not contribute to bacterial growth/survival in the presence of this toxic metal (22, 29). The re-duced ability of a znuA mutant strain to grow in the presenceof cadmium confirmed that this metal interferes with zinc ho-meostasis.

zinT is induced under zinc starvation and belongs to the Zurregulon. Previous studies have shown that ZnuA accumulationis induced by metal-chelating agents (EDTA and TPEN) andrepressed by the addition of zinc (3) (Fig. 3A). To verifywhether ZinT accumulation is also modulated by zinc avail-ability, we have grown strain PP134 in the presence of 0.5 mMEDTA with or without equimolar amounts of zinc. As shown inFig. 3, ZinT accumulation is strongly induced by EDTA but isrepressed by zinc (Fig. 3A). The inhibition of ZinT accumula-tion is specific for zinc, as manganese, iron, and copper have noeffect (Fig. 3B). To verify that zinT and znuA are coregulatedby the transcriptional factor Zur, the znuA::3�FLAG andzinT::3�FLAG alleles have been transduced in a strain inwhich zur had been deleted, obtaining PP131 and PP132strains, respectively. The accumulation of ZinT and ZnuA inthese strains was completely deregulated and insensitive eitherto EDTA, zinc, or cadmium supplementation (Fig. 3A). This



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

observation confirms that in Salmonella enterica transcriptionof zinT is under the control of Zur, as recently observed in E.coli (22, 29).

To better analyze the relationships between ZinT/ZnuA ac-cumulation and zinc availability, we have constructed a doubleepitope-tagged strain (znuA::3�FLAG zinT::3�FLAG; strainPP141). When this strain was grown in a zinc-depleted medium(MM), both ZnuA and ZinT accumulated at high levels (Fig.4A). Similar results were obtained when strain PP141 wasgrown in defined media of different formulation, i.e., M9 andTris minimal media (data not shown). However, the two pro-teins exhibit a slightly different response to zinc availability. Infact, the results shown in the figure show that the addition of0.5 �M ZnSO4 to the culture medium causes the completeabrogation of ZinT accumulation, but not that of ZnuA (Fig.4A), which also shows a low level of accumulation at higher

zinc concentrations. In agreement with this observation, ZinTand ZnuA are maximally expressed in bacteria cultivated in LBmedium supplemented with 0.5 mM EDTA, but accumulationof ZnuA is observed at EDTA concentrations lower than thoserequired to induce accumulation of ZinT (Fig. 4B). Moreover,ZinT accumulation in a strain lacking the znuA gene (PP137)is not inhibited by the addition of zinc (Fig. 4C). In contrast,ZnuA accumulation in a strain lacking the zinT gene (PP128)is comparable to that observed in the wild-type strain (Fig. 4C).

These experiments show that znuA is induced at higher zincconcentrations than those required to activate zinT transcrip-tion, indicating that ZnuA is a protein involved in the frontlineresponse to zinc deficiency, whereas ZinT participated in thebacterial response to more severe zinc deficiency. Moreover,the observation that ZinT accumulation cannot be repressedby the external supply of zinc suggests that ZnuA plays a role

FIG. 1. Sequence alignment of AdcA, ZnuA, and ZinT. Sequence alignment of the mature (without the signal peptide) AdcA protein fromStreptococcus pneumoniae, Streptococcus suis, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus haemolyticus; ZnuA from S.Typhimurium; and ZinT from S. Typhimurium, E. coli, and B. subtilis. Residues involved in zinc binding in ZnuA and ZinT are shown on a graybackground. A strictly conserved histidine residue present at the C terminus of ZinT is shown in boldface type. The protein sequencescorresponding to the His-rich loop of ZnuA and AdcA and the N-terminal His-rich region of ZinT are in bold and underlined.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

in zinc import within the cell that cannot be substituted byZinT.

Consequences of zinT deletion on Salmonella growth. Inagreement with the above reported expression studies, Table 3shows that growth of a zinT mutant strain on LB agar plates isinhibited by divalent metal chelators, such as EDTA andTPEN. The growth impairment due to zinT deletion is lowerthan that observed for a znuA mutant strain, confirming theprominent role of ZnuA in zinc homeostasis.

This observation was confirmed by the results of analysis ofthe growth curves of the wild-type strain and znuA and zinTmutant strains in the presence of EDTA (Fig. 5). In fact,whereas the growth of all the strains tested is nearly identicalin standard LB medium, in the presence of 2 mM EDTA, thegrowth of the zinT mutant strain is impaired compared to thewild-type strain. The growth defect, however, is lower than thatexhibited by the strain lacking znuA. Zinc, but not iron ormanganese, supplementation to the growth medium com-pletely abolished this phenotype (data not shown), indicatingthat it is specifically due to the zinc sequestration ability ofEDTA and not to EDTA-induced shortage of other metals.Interestingly, the growth of the zinT znuA double mutant iscomparable to that of the znuA mutant strain, confirming thatZinT cannot substitute for ZnuA in the process of zinc importwithin the cytoplasm.

Role of ZinT in Salmonella pathogenicity. Previous studieshave established that disruption of znuA dramatically de-creases Salmonella pathogenicity (3). In this work we havecompared the contribution of zinT and znuA to the ability ofSalmonella to colonize host tissues by carrying out competitionexperiments in BALB/c mice. This approach confirmed therelevance of ZnuA in Salmonella infections (Table 4). In con-trast, the ability of the strain lacking zinT (strain PP116) tocolonize the spleens of infected mice was comparable to that ofthe wild-type strain, whereas the zinT mutant (PP116) outcom-

peted the zinT znuA double mutant (PP118). In addition, wedid not observe differences in spleen colonization between theznuA (SA123) and znuABC (SA182) mutant strains. Quitesurprisingly, a zinT znuABC mutant strain (PP119) was favoredcompared to the znuABC mutant strain (SA182). This obser-vation could be suggestive of a detrimental role of ZinT in theabsence of ZnuABC, although more studies are required toverify this hypothesis. These experimental results suggest thatZinT facilitates zinc transport through the ZnuABC system butthat it has a dispensable role during mouse infections. More-over, the observation that the disruption of zinT does notattenuate mutant strains lacking a functional ZnuABC trans-porter (strain SA123 or SA182) provides further support to thehypothesis that ZinT is not involved in a ZnuABC-indepen-dent mechanism of zinc transport.

Additional experiments, however, shed more light on therole of ZinT in the mechanism of zinc uptake. The ZnuAprotein possesses a charged flexible loop rich in histidines andacidic residues (His-rich loop), whose function has not yetbeen clarified. It has been hypothesized that the role of thisloop could be to increase the ability of ZnuA to sequester zincin environments with low concentrations of this metal (6, 18),whereas other authors have suggested that it could be a sensorof periplasmic zinc concentration (51). To investigate the roleof this His-rich region, we have constructed a znuA mutantstrain producing a ZnuA protein devoid of this loop (SA233).As shown in Fig. 6, the growth of this mutant strain is notimpaired in LB medium containing EDTA, indicating thatZnuA can also mediate zinc transport in the absence of the

TABLE 3. Salmonella growth on LB plates

Chemicalagent added

Growth of S. enterica serovarTyphimurium straina:

WT SA123 PP116

Cadmium0 � � �0.06 mM � � �0.1 mM � � �0.5 mM � �/ �0.6 mM �/ �/0.7 mM

EDTA0 � � �0.02 mM � � �0.06 mM � �/ �0.1 mM � �0.5 mM � �1 mM � �/1.2 mM � 1.4 mM �

TPEN0 � � �0.005 mM � � �0.025 mM � �0.05 mM � 0.5 mM

a Three strains of bacteria were grown overnight in LB medium and thenstreaked on LB plates containing the indicated amounts of EDTA, TPEN, andcadmium acetate. The three strains were the wild-type strain (WT) (ATCC14028), znuA mutant strain SA123, and zinT mutant strain PP116. Symbols: �,growth; , no growth; �/, weak growth of very small colonies.

FIG. 2. Effects of cadmium on the accumulation of ZinT, ZnuA, andZnuB proteins. (A to C) Western blots of bacterial lysates from strainsPP134 (zinT::3�FLAG-kan) (A), SA140 (znuA::3�FLAG-kan ilvI::Tn10dTac-cat::3�FLAG-kan) (B), and PP101 (znuB::3�FLAG-kan)(C) grown in LB medium (lane 1) and LB medium supplemented with0.5 mM cadmium acetate (lane 2). In panel A, the accumulation ofZnuA is shown in comparison with an internal standard (cat::3�FLAG). It was not possible to use this protein as an internal standardto monitor the accumulation of ZinT and ZnuB, as migration of theseproteins overlapped with that of chloramphenicol acetyltransferase(CAT).



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

His-rich region. However, a severe growth defect was observedin a mutant strain expressing a mutated form of ZnuA andunable to produce ZinT. The growth of this mutant, in fact,was comparable to that of the strain lacking znuA. Similarresults were obtained in competition experiments. The datareported in Table 4 show that deletion of yebA, which wasrequired for the construction of the znuA�loop mutant strainhas no effect on Salmonella virulence. Similarly, the znuA�loop

FIG. 3. ZinT and ZnuA accumulation in the wild type and in Zur-deleted strains. (A) Western blots of bacterial lysates of strainsSA140 (znuA::3�FLAG-kan ilvI::Tn10dTac-cat::3�FLAG-kan), PP134 (zinT::3�FLAG-kan), PP131 (znuA::3�FLAG-scar zur::kanilvI::Tn10dTac-cat::3�FLAG-scar [scar explained in Table 1, footnote c), and PP132 (zinT::3�FLAG-scar zur::kan) were grown in LB mediumalone or supplemented with cadmium acetate, EDTA, or ZnSO4 as indicated. Cat, chloramphenicol acetyltransferase. (B) ZinT accumulation instrain PP134 (zinT::3�FLAG-kan) grown in minimal medium alone () or supplemented with 5 �M ZnSO4, 5 �M MnCl2, 5 �M CuSO4, or 5 �MFeSO4.

FIG. 4. Differential accumulation of ZinT and ZnuA in zinc-replete and zinc-deficient conditions. (A to C) Western blots ofbacterial lysates of strains. (A) Strain PP141 (znuA::3�FLAG-kanzinT::3�FLAG-scar) was grown in minimal medium alone () orsupplemented with increasing amounts of ZnSO4 as indicated. (B) Thesame strain (PP141) was grown in LB medium alone () or sup-plemented with increasing amounts of EDTA as indicated. (C) Zinc-dependent accumulation of ZinT in a wild-type background (strainPP134) and in a znuA-deleted strain (PP137) (zinT::3�FLAG-kanznuA::cam) and accumulation of ZnuA in a wild-type back-ground (strain PP140) and in a zinT-deleted strain (PP128)(znuA::3�FLAG-scar zinT::scar ilvI::Tn10dTac-cat::3�FLAG-kan).

FIG. 5. Growth curves of S. Typhimurium mutants. Growth curvesof the wild type (triangles), zinT::cam (squares), znuA::kan (circles),and zinT::cam-znuA::kan (diamonds) strains. Each strain was grown inLB medium alone (closed symbols) and LB medium supplementedwith 2 mM EDTA (open symbols). OD600, optical density at 600 nm.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

mutant strain was not significantly attenuated in comparison tothe wild-type strain, although a slightly higher number of wild-type bacteria were recovered from the spleens of infected mice.In agreement, there was not a significant difference in thespleen colonization ability of a zinT mutant strain (PP125) andof the znuA�loop strain (SA233). However, the Salmonellastrain lacking zinT and expressing the mutated form of znuA(PP130) was attenuated with respect to the strain unable toproduce ZinT (PP125). This strain maintained some ability totransport zinc through the ZnuB channel, so it had an advan-tage over strain PP118, which does not express znuA.

These experiments suggest that ZinT and the His-rich do-main of ZnuA are two independent elements which participatein ZnuABC-mediated zinc transport by playing an overlappingrole in facilitating zinc recruitment by ZnuA. Although thesimultaneous presence of ZinT and the His-rich domain isapparently not indispensable to ensure the functionality of thetransporter under the conditions investigated in this work,

disruption of each one of the two elements disclose a role forthe other element in enhancing the efficiency of zinc uptake.

ZinT and ZnuA form a binary complex in vitro. The aboveexperiments showing that ZnuA and ZinT functionally interactin the mechanism of ZnuABC-mediated zinc import suggestthat ZinT might physically interact with ZnuA. To prove thispossibility, we have analyzed the abilities of these two proteinsto form a complex in vitro. ZinT and ZnuA were cloned,expressed, and purified as described in Materials and Methods.The two proteins were mixed together, incubated for 90 min atroom temperature, and then loaded on a gel filtration column.Figure 7 shows a comparison of elution of the individual pro-teins and of the ZnuA-ZinT mixture. ZinT eluted from thecolumn with an apparent molecular mass of 22.3 kDa (peakcentered at 75 ml [Fig. 7A]), in excellent agreement with theZinT molecular mass deduced from the amino acid sequence(22.2 kDa). ZnuA eluted with an apparent molecular mass of34.9 kDa (peak centered at 67 ml [Fig. 7B]), a value which isslightly higher than the expected molecular mass (31.5 kDa).When the mixture of ZinT and ZnuA was applied to thecolumn, the elution profiles of the two proteins changed sig-nificantly. In fact, in this case maximal ZinT and ZnuA con-centration was found in fraction 63 (Fig. 7C), corresponding toa protein of approximately 48 kDa. The observation that theapparent molecular mass of the two proteins was shifted tovalues significantly higher than those of the single proteinsstrongly supports the hypothesis of formation of a binary com-plex between the two proteins. The elution profile of the com-plex was rather broad, possibly due to incomplete involvementof the two proteins in complex formation and/or to partialcomplex dissociation during the elution. However, when theproteins recovered from fraction 63 were concentrated andsubjected to a new gel filtration chromatography, the largestpart of ZnuA and ZinT still coeluted with a high molecularmass, indicating that the complex between the two proteins isstable (data not shown). The observation that the ZnuA/ZinTcomplex has an apparent molecular mass lower than that ex-pected (53.7 kDa) is indicative of changes in the hydrodynamicproperties of the two proteins following their interaction.

Interestingly, the two apoproteins were not able to form a

TABLE 4. Competition assays in BALB/c micea

Strain A (relevant genotype) Strain B (relevant genotype) No. of mice Median CIb Pc

MA6926 (wild type) SA123 (znuA::kan) 5 �200d 0.001MA6926 (wild type) PP116 (zinT::cam) 4 1.202 NSSA123 (znuA::kan) SA182 (znuABC::kan) 10 1.021 NSPP116 (zinT::cam) PP118 (zinT::cam znuA::kan) 5 �200d 0.001SA123 (znuA::kan) PP118 (zinT::cam znuA::kan) 5 1.041 NSSA182 (znuABC::kan) PP119 (zinT::cam znuABC::kan) 5 0.394 0.037SA229 (yebA::cam) MA6926 (wild type) 5 0.85 NSPP125 (zinT-scar) SA233 (znuA�loop yebA::cam) 5 1.522 NSSA229 (yebA::cam) SA233 (znuA�loop yebA::cam) 4 1.062 NSPP125 (zinT-scar) PP130 (zinT::cam znuA�loop yebA::cam) 10 1.820 0.006PP118 (zinT::cam znuA::kan) PP130 (zinT::cam znuA�loop yebA::cam) 5 0.006 0.001

a Experiments were performed with BALB/c mice inoculated by the intraperitoneal route.b Competitive index (CI) � output (strain A/strain B)/inoculum (strain A/strain B).c Statistical differences between output and inocula (P values) were determined by Student’s t test. NS, not significant.d Only colonies of one of the two strains were isolated during the selection procedure. However, a small number of bacteria of the outcompeted strains were present

in the spleens of infected mice, as demonstrated by plating the concentrated recovered bacteria on appropriate selective plates. No attempts were carried out to evaluatethe exact ratio between the two strains.

FIG. 6. Growth curves of znuA�loop mutant strains. Growth curvesof the wild type (Œ), zinT::cam (f), znuA::kan (F), znuA�loop-yebA::cam([diaf2]), and znuA�loop-yebA::cam-zinT::scar (ƒ) strains. Each strain wasgrown in LB medium supplemented with 2 mM EDTA.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

stable complex (Fig. 7D). However, the ability of ZnuA andZinT to stably interact was restored when the two proteinswere individually reconstituted with an equimolar amount ofzinc before protein incubation (Fig. 7E). The addition of zincto a single protein (ZnuA or ZinT) was not sufficient to ob-serve the coelution of the two proteins from the gel filtrationcolumn (data not shown). These results suggest that the bind-ing of zinc induces structural rearrangements in ZnuA andZinT which are necessary for the formation of a stable complexbetween the two proteins.

To determine whether the histidine-rich region of ZnuA isinvolved in the formation of a complex between ZinT andZnuA, similar experiments were carried out using the

ZnuA�loop mutant protein. As shown in Fig. 7G, when theseproteins were coincubated, they eluted in the same fractionswith apparent molecular masses higher than those of isolatedZnuA�loop and ZinT proteins. In line with functional studies,this observation demonstrates that the His-rich region ofZnuA is not required for the formation of a ZinT/ZnuA com-plex in vitro.

The elution profile of ZinT was not altered when the proteinwas preincubated with bovine serum albumin or Cu,Zn super-oxide dismutase (Fig. 7H and I, respectively), thus indicatingthat the interaction of ZinT and ZnuA is highly specific.

DISCUSSION

Zinc serves important functions in a wide range of cellularprocesses; therefore, the mechanisms ensuring highly efficientzinc recruitment are critical for bacterial growth and survival inall the environments where this metal is not abundant. Recentstudies have established that zinc is not freely available withinhost tissues and that the ability of several pathogens to multiplyin the infected host is strictly dependent on the zinc transporterZnuABC. As this is the only high-affinity zinc transporter iden-tified in many bacterial species, these observations suggest thatZnuABC could be an interesting target for novel antimicrobialstrategies. To deepen our understanding of the mechanismsgoverning zinc homeostasis in bacteria, we have investigatedthe role of ZinT, a poorly characterized protein which has beenproposed to be involved in the mechanisms of resistance totoxic metals or to zinc deficiency.

In agreement with recent observations (22, 29), this investi-gation demonstrates that ZinT has no role in bacterial resis-tance to cadmium toxicity. In fact, although zinT is stronglyinduced by this metal, deletion of the gene does not impairbacterial growth in cadmium-containing media. Moreover,cadmium induces the accumulation of ZinT as well as of otherproteins involved in zinc transport, i.e., ZnuA and ZnuB. Themechanisms responsible for cadmium toxicity are not com-pletely understood, but they can be largely explained by theability of cadmium to deplete cells of intracellular glutathione,to react with the sulfhydryl groups of proteins, and to competewith other metals for binding to metalloproteins (7, 49). Theseresults provide novel suggestions to understand the complexmolecular basis of cadmium toxicity in bacteria. In fact, cad-mium induces the expression of the Zur-regulated genes inbacteria growing in a zinc-replete medium (LB), suggestingthat cadmium alters zinc homeostasis in bacteria. Additionalstudies are required to understand whether this is due to ageneral ability of cadmium to substitute for the proper metalcofactor in zinc-containing proteins or to a specific effect onZur (which, upon cadmium binding, might adopt an alteredconformation unable to bind to DNA) or on the functionalityof the ZnuABC transporter. However, it is worth noting thatcadmium binds with high affinity to metal sites characterized bycysteine ligands, such as metallothioneins (26) and zinc-depen-dent transcription factors (25) and that X-ray absorption stud-ies have shown that zinc binding to Zur involves differentcysteine residues (37). This last observation suggests that Zurcould be a privileged target for cadmium ions.

All the results described in this work converge in demon-strating that ZinT participates in the process of zinc uptake

FIG. 7. Analysis of ZinT and ZnuA interaction by gel filtrationchromatography. SDS-PAGE analysis of Salmonella ZinT and ZnuAeluted from a HiLoad 16/60 Superdex 75 gel filtration FPLC column,calibrated with bovine serum albumin (67,000 Da), ovalbumin (43,000Da), chymotrypsinogen A (25,000 Da), and RNase A (13,700 Da). Thesamples loaded on the column were collected in 1-ml fractions. The gelshows the proteins contained in the 61- to 78-ml fractions, as indicatedat the bottom of the figure. (A) Isolated ZinT; (B) isolated ZnuA;(C) mixture of ZnuA and ZinT; (D) mixture of apo-ZnuA and apo-ZinT; (E) mixture of apo-ZnuA and apo-ZinT reconstituted with anequimolar amount of zinc; (F) isolated ZnuA�loop; (G) mixture ofZnuA�loop and ZinT; (H) mixture of Cu,ZnSOD and ZinT; (I) mix-ture of bovine serum albumin (BSA) and ZinT.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

mediated by ZnuABC. This conclusion is supported by differ-ent pieces of evidence. First of all, ZinT can be found, either asan independent protein or as a domain of the AdcA proteins,in bacteria containing ZnuA or homologous periplasmic li-gand-binding proteins involved in zinc uptake. In contrast,ZinT is not present in bacteria lacking ZnuA (see Table S1 inthe supplemental material). Moreover, in line with recent stud-ies carried out in E. coli (22, 29), we have confirmed theoriginal proposal of Panina and coworkers (39) that zinT is amember of the Zur regulon. We have also demonstrated thatzinT is induced under conditions of zinc deficiency (Fig. 3 and4) and that it contributes to bacterial growth in media with lowconcentrations of this metal (Fig. 5 and 6). Interestingly, boththe analysis of the role of zinT in bacterial growth in vitro (Fig.6) and the competition experiments in mice (Table 4) failed toidentify a major contribution of ZinT to zinc transport inbacteria lacking znuA or the entire znuABC operon, indicatingthat the role of ZinT in zinc uptake is dependent on thepresence of ZnuA. It is noteworthy that a mutant strain ex-pressing zinT but with the znuABC operon deleted was disad-vantaged compared to the zinT znuABC mutant strain (Table4), suggesting that in the absence of the ZnuABC transporter,the expression of ZinT is harmful, possibly due to the zincsequestration ability of this protein, which might decrease zincimport through low-affinity zinc transporters. This possibility issupported by the observation that ZinT accumulates constitu-tively in bacteria lacking znuA (Fig. 4C). However, furtherwork is required to verify this hypothesis because the sameeffect was not observed in the competition between the znuA(SA123) and znuA zinT (PP118) mutant strains. All these ob-servations suggest that ZinT is an additional component of theZnuABC transporter facilitating zinc recruitment in theperiplasmic space. The results reported in Fig. 7 demonstratethat ZinT is also able to form a stable complex with ZnuA invitro, which we suppose could resemble the structural organi-zation of AdcA proteins. Interestingly, the stability of the com-plex is dependent on the presence of zinc, suggesting that invivo the interaction between ZnuA and ZinT could be desta-bilized upon zinc exchange within the ZnuA/ZinT complex orfollowing zinc release from the complex to ZnuB. The scat-tered distribution of zinT in eubacteria expressing znuA, theobservation that ZnuABC is critical for successful infection inseveral bacteria lacking zinT, including Haemophilus ducreyi(30), Brucella abortus (52), and Campylobacter jejuni (16), andthe results showing that deletion of zinT does not attenuate S.Typhimurium and only marginally decreases bacterial growthin the presence of very high concentrations of chelating agentsall together indicate that ZinT has a role in zinc transportwhich is significantly less important than that of ZnuA. Insupport of this view, we have observed slight differences in theregulation of zinT and znuA, suggesting that transcription ofznuA occurs at zinc concentrations higher than those requiredto activate zinT expression. In fact, ZinT accumulation is com-pletely repressed in bacteria growing in a minimal mediumcontaining 0.5 �M ZnSO4 or in LB medium supplementedwith 0.05 mM EDTA, whereas under the same conditionsZnuA is partially induced (Fig. 4). Such a flexible response tozinc deficiency, likely justified by small differences in the Zur-binding region (data not shown), may provide an explanationfor the production of two separate proteins, instead of a single

one (AdcA), as in some Gram-positive bacteria. It should benoted that this finding is in apparent contrast with previoustranscriptomic studies showing a greater increase in zinTmRNA levels than in znuA mRNA levels in response to TPEN(46) or zinc deficiency (22). However, both these studies werecarried out using defined media containing low levels of zincand Graham and coworkers (22) used a medium containinghigh concentrations of EDTA. We believe that the media usedin these works provided conditions sufficient to induce a signifi-cant basal expression of znuA, thus explaining the much greatermRNA induction of zinT upon further depletion of zinc.

Although dispensable for the functionality of ZnuABC,ZinT enhances the ability of bacteria to grow under severe zincshortage. The presence of another periplasmic protein distinctfrom ZnuA may be useful to facilitate metal sequestration inthis cellular compartment. We have observed that ZinT has avery high affinity for immobilized nickel (see Materials andMethods) possibly due to its N-terminal His-rich domain (Fig.1). This region of ZinT differs from the ZnuA His-rich loop, asthe last motif is characterized by the presence of a very highnumber of acidic residues, which likely affects the protonationstate of the neighboring histidine residues. We suggest that theHis-rich domain of ZinT could play a role in facilitating zincdisplacement from other proteins and the subsequent entry ofthe metal into the ZnuABC-mediated process of zinc transportfrom the periplasm to the cytoplasm.

Some experiments reported in this study also shed new lighton the role of the His-rich region of ZnuA. ZnuA possesses acentral domain of variable length in different bacterial specieswhich is characterized by the presence of a high number ofhistidine and acidic residues whose function is not yet known.A few studies have suggested that this loop could enhance zincbinding ability and its subsequent transfer to the primary bind-ing site of ZnuA (6, 18). Alternatively, it has been suggestedthat it could be a sensor of periplasmic zinc concentration, ableto inhibit zinc transfer from ZnuA to ZnuB at high zinc con-centrations (51). Interestingly, similar domains are also presentin a vast number of eukaryotic zinc transporters (20). Differentstudies have explored the role of these histidine-rich regions byproducing mutant proteins devoid of this protein domain. Theresults obtained have failed to provide an unequivocal answerto the role of His-rich loops, as it has been proposed that theycan contribute to protein stability (33), the process of metaltransfer (34), metal selectivity (35), and modulation of proteinactivity (28). We have observed that an S. Typhimurium strainexpressing a ZnuA variant devoid of the His-rich region is notattenuated in infection studies and grows as the wild-typestrain does in LB medium containing 2 mM EDTA. This ob-servation apparently argues against a role of this domain inmetal recruitment. However, when this mutation was insertedin a strain in which zinT had been deleted, mutant ZnuAproved not to be able to work as well as native ZnuA either invitro (Fig. 6) or in the infected animal (Table 4). Although wecannot exclude the possibility that the His-rich region couldplay additional roles (for example, in facilitating the selectionof the correct metal ion), our findings suggest that ZinT andthe His-rich region play overlapping roles in increasing themetal binding ability of ZnuA. Under the experimental condi-tions we have explored in this work, this function can be dis-closed only in bacteria lacking both ZinT and the His-rich



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

region of ZnuA, suggesting that they play similar roles. It islikely that the simultaneous presence of two structurally distinctelements with apparently redundant roles could enhance metalrecruitment during conditions of severe zinc shortage and maxi-mize zinc import under variable environmental conditions.

This study, which shows that ZinT participates in the Znu-ABC-mediated process of zinc transport, provides additionalinsights into the mechanisms employed by some bacteria toobtain zinc. ZinT is dispensable for the functionality of thetransporter, thus explaining its absence in several bacteria, butplays a role auxiliary to that of ZnuA in the recruitment of zincwithin the periplasmic space.

ACKNOWLEDGMENTS

This work was partially supported by an Istituto Superiore di Sanita(ISS) grant to A.B. and P.P. and by a grant from the Fondazione Romato A.B.

REFERENCES

1. Akanuma, G., H. Nanamiya, Y. Natori, N. Nomura, and F. Kawamura. 2006.Liberation of zinc-containing L31 (RpmE) from ribosomes by its paralogousgene product, YtiA, in Bacillus subtilis. J. Bacteriol. 188:2715–2720.

2. Ammendola, S., P. Pasquali, F. Pacello, G. Rotilio, M. Castor, S. J. Libby, N.Figueroa-Bossi, L. Bossi, F. C. Fang, and A. Battistoni. 2008. Regulatory andstructural differences in the Cu,Zn-superoxide dismutases of Salmonellaenterica and their significance for virulence. J. Biol. Chem. 283:13688–13699.

3. Ammendola, S., P. Pasquali, C. Pistoia, P. Petrucci, P. Petrarca, G. Rotilio,and A. Battistoni. 2007. The high-affinity Zn2� uptake system ZnuABC isrequired for bacterial zinc homeostasis in intracellular environments andcontributes to virulence of Salmonella enterica. Infect. Immun. 75:5867–5876.

4. Andreini, C., I. Bertini, and A. Rosato. 2004. A hint to search for metallo-proteins in gene banks. Bioinformatics 20:1373–1380.

5. Banerjee, S., B. Wei, M. B. Pakrasi, H. B. Pakrasi, and T. J. Smith. 2003.Structural determinants of metal specificity in the zinc transport proteinsZnuA from Synechocystis 6803. J. Mol. Biol. 333:1061–1069.

6. Berducci, G., A. P. Mazzetti, G. Rotilio, and A. Battistoni. 2004. Periplasmiccompetition for zinc uptake between the metallochaperone ZnuA andCu,Zn superoxide dismutase. FEBS Lett. 569:289–292.

7. Binet, M. R., R. Ma, C. W. McLeod, and R. K. Poole. 2003. Detection andcharacterization of zinc- and cadmium-binding proteins in Escherichia coli bygel electrophoresis and laser ablation-inductively coupled plasma-mass spec-trometry. Anal. Biochem. 318:30–38.

8. Birch, R. M., C. O’Byrne, I. R. Booth, and P. Cash. 2003. Enrichment ofEscherichia coli proteins by column chromatography on reactive dye col-umns. Proteomics 3:764–776.

9. Campoy, S., M. Jara, N. Busquets, A. M. Perez de Rozas, I. Badiola, and J.Barbe. 2002. Role of the high-affinity zinc uptake znuABC system in Salmo-nella enterica serovar Typhimurium virulence. Infect. Immun. 70:4721–4725.

10. Chandra, B. R., M. Yogavel, and A. Sharma. 2007. Structural analysis ofABC-family periplasmic zinc binding protein provides new insights intomechanism of ligand uptake and release. J. Mol. Biol. 367:970–987.

11. Claverys, J. P. 2001. A new family of high-affinity ABC manganese and zincpermeases. Res. Microbiol. 152:231–243.

12. Coleman, J. E. 1998. Zinc enzymes. Curr. Opin. Chem. Biol. 2:222–234.13. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromo-

somal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad.Sci. U. S. A. 97:6640–6645.

14. David, G., K. Blondeau, M. Renouard, and A. Lewit-Bentley. 2002. Crystal-lization and preliminary analysis of Escherichia coli YodA. Acta Crystallogr.D Biol. Crystallogr. 58:1243–1245.

15. David, G., K. Blondeau, M. Schiltz, S. Penel, and A. Lewit-Bentley. 2003.YodA from Escherichia coli is a metal-binding, lipocalin-like protein. J. Biol.Chem. 278:43728–43735.

16. Davis, L. M., T. Kakuda, and V. J. DiRita. 2009. A Campylobacter jejuni znuAorthologue is essential for growth in low-zinc environments and chick colo-nization. J. Bacteriol. 191:1631–1640.

17. Dente, L., G. Cesareni, and R. Cortese. 1983. pEMBL: a new family of singlestranded plasmids. Nucleic Acids Res. 11:1645–1655.

18. Desrosiers, D. C., Y. C. Sun, A. A. Zaidi, C. H. Eggers, D. L. Cox, and J. D.Radolf. 2007. The general transition metal (Tro) and Zn2� (Znu) trans-porters in Treponema pallidum: analysis of metal specificities and expressionprofiles. Mol. Microbiol. 65:137–152.

19. Dintilhac, A., G. Alloing, C. Granadel, and J. P. Claverys. 1997. Competenceand virulence of Streptococcus pneumoniae: Adc and PsaA mutants exhibit arequirement for Zn and Mn resulting from inactivation of putative ABCmetal permeases. Mol. Microbiol. 25:727–739.

20. Eide, D. J. 2006. Zinc transporters and the cellular trafficking of zinc. Bio-chim. Biophys. Acta 1763:711–722.

21. Ferianc, P., A. Farewell, and T. Nystrom. 1998. The cadmium-stress stimulonof Escherichia coli K-12. Microbiology 144:1045–1050.

22. Graham, A. I., S. Hunt, S. L. Stokes, N. Bramall, J. Bunch, A. G. Cox, C. W.McLeod, and R. K. Poole. 2009. Severe zinc depletion of Escherichia coli:roles for high affinity zinc binding by ZinT, zinc transport and zinc-indepen-dent proteins. J. Biol. Chem. 284:18377–18389.

23. Hantke, K. 2001. Iron and metal regulation in bacteria. Curr. Opin. Micro-biol. 4:172–177.

24. Hantke, K. 2005. Bacterial zinc uptake and regulators. Curr. Opin. Micro-biol. 8:196–202.

25. Hartwig, A. 2001. Zinc finger proteins as potential targets for toxic metalions: differential effects on structure and function. Antioxid. Redox Signal.3:625–634.

26. Henkel, G., and B. Krebs. 2004. Metallothioneins: zinc, cadmium, mercury,and copper thiolates and selenolates mimicking protein active site features–structural aspects and biological implications. Chem. Rev. 104:801–824.

27. Kannan, G., J. C. Wilks, D. M. Fitzgerald, B. D. Jones, S. S. Bondurant, andJ. L. Slonczewski. 2008. Rapid acid treatment of Escherichia coli: transcrip-tomic response and recovery. BMC Microbiol. 8:37.

28. Kawachi, M., Y. Kobae, T. Mimura, and M. Maeshima. 2008. Deletion of ahistidine-rich loop of AtMTP1, a vacuolar Zn(2�)/H(�) antiporter ofArabidopsis thaliana, stimulates the transport activity. J. Biol. Chem. 283:8374–8383.

29. Kershaw, C. J., N. L. Brown, and J. L. Hobman. 2007. Zinc dependence ofzinT (yodA) mutants and binding of zinc, cadmium and mercury by ZinT.Biochem. Biophys. Res. Commun. 364:66–71.

30. Lewis, D. A., J. Klesney-Tait, S. R. Lumbley, C. K. Ward, J. L. Latimer, C. A.Ison, and E. J. Hansen. 1999. Identification of the znuA-encoded periplasmiczinc transport protein of Haemophilus ducreyi. Infect. Immun. 67:5060–5068.

31. Li, H., and G. Jogl. 2007. Crystal structure of the zinc-binding transportprotein ZnuA from Escherichia coli reveals an unexpected variation in metalcoordination. J. Mol. Biol. 368:1358–1366.

32. Lu, D., B. Boyd, and C. A. Lingwood. 1997. Identification of the key proteinfor zinc uptake in Haemophilus influenzae. J. Biol. Chem. 272:29033–29038.

33. Mao, X., B. E. Kim, F. Wang, D. J. Eide, and M. J. Petris. 2007. A histidine-rich cluster mediates the ubiquitination and degradation of the human zinctransporter, hZIP4, and protects against zinc cytotoxicity. J. Biol. Chem.282:6992–7000.

34. Milon, B., Q. Wu, J. Zou, L. C. Costello, and R. B. Franklin. 2006. Histidineresidues in the region between transmembrane domains III and IV of hZip1are required for zinc transport across the plasma membrane in PC-3 cells.Biochim. Biophys. Acta 1758:1696–1701.

35. Nishida, S., T. Mizuno, and H. Obata. 2008. Involvement of histidine-richdomain of ZIP family transporter TjZNT1 in metal ion specificity. PlantPhysiol. Biochem. 46:601–606.

36. Outten, C. E., F. W. Outten, and T. V. O’Halloran. 1999. DNA distortionmechanism for transcriptional activation by ZntR, a Zn(II)-responsive MerRhomologue in Escherichia coli. J. Biol. Chem. 274:37517–37524.

37. Outten, C. E., D. A. Tobin, J. E. Penner-Hahn, and T. V. O’Halloran. 2001.Characterization of the metal receptor sites in Escherichia coli Zur, anultrasensitive zinc(II) metalloregulatory protein. Biochemistry 40:10417–10423.

38. Outten, J. E., and T. V. O’Halloran. 2001. Femtomolar sensitivity of metal-loregulatory proteins controlling zinc homeostasis. Science 292:2488–2492.

39. Panina, E. M., A. A. Mironov, and M. S. Gelfand. 2003. Comparative genom-ics of bacterial zinc regulons: enhanced ion transport, pathogenesis, andrearrangement of ribosomal proteins. Proc. Natl. Acad. Sci. U. S. A. 100:9912–9917.

40. Patzer, S., and K. Hantke. 1998. The ZnuABC high-affinity zinc-uptakesystem and its regulator Zur in Escherichia coli. Mol. Microbiol. 28:1199–1210.

41. Puskarova, A., P. Ferianc, J. Kormanec, D. Homerova, A. Farewell, and T.Nystrom. 2002. Regulation of yodA encoding a novel cadmium-protein inEscherichia coli. Microbiology 148:3801–3811.

42. Ratledge, C., and L. G. Dover. 2000. Iron metabolism in pathogenic bacteria.Annu. Rev. Microbiol. 54:881–941.

43. Schaible, U. E., and S. H. Kaufmann. 2004. Iron and microbial infection.Nat. Rev. Microbiol. 2:946–953.

44. Schaible, U. E., and S. H. Kaufmann. 2005. Nutritive view on the host-pathogen interplay. Trends Microbiol. 13:373–380.

45. Shin, J. H., S. Y. Oh, S. J. Kim, and J. H. Roe. 2007. The zinc-responsiveregulator Zur controls a zinc uptake system and some ribosomal proteins inStreptomyces coelicolor A3(2). J. Bacteriol. 189:4070–4077.

46. Sigdel, T. K., J. A. Easton, and M. W. Crowder. 2006. Transcriptionalresponse of Escherichia coli to TPEN. J. Bacteriol. 188:6709–6713.

47. Uzzau, S., L. Bossi, and N. Figueroa-Bossi. 2002. Differential accumulationof Salmonella [Cu, Zn] superoxide dismutases SodCI and SodCII in intra-cellular bacteria: correlation with their relative contribution to pathogenicity.Mol. Microbiol. 46:147–156.

48. Uzzau, S., N. Figueroa-Bossi, S. Rubino, and L. Bossi. 2001. Epitope tagging



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

of chromosomal genes in Salmonella. Proc. Natl. Acad. Sci. U. S. A. 98:15264–15269.

49. Valko, M., H. Morris, and M. T. Cronin. 2005. Metals, toxicity and oxidativestress. Curr. Med. Chem. 12:1161–1208.

50. Waldron, K. J., and N. J. Robinson. 2009. How do bacterial cells ensure thatmetalloproteins get the correct metal? Nat. Rev. Microbiol. 7:25–35.

51. Wei, B., A. M. Randich, M. Bhattacharyya-Pakrasi, H. B Pakrasi, and T. J.Smith. 2007. Possible regulatory role for the histidine-rich loop in the zinctransport protein, ZnuA. Biochemistry 46:8734–8743.

52. Yang, X., T. Becker, N. Walters, and D. W. Pascual. 2006. Deletion of znuAvirulence factor attenuates Brucella abortus and confers protection againstwild-type challenge. Infect. Immun. 74:3874–3879.

53. Yatsunyk, L. A., J. A. Easton, L. R. Kim, S. A Sugarbaker, B. Bennett, R. M.Breece, I. I. Vorontsov, D. L. Tierney, M. W. Crowder, and A. C. Rosenzweig.2008. Structure and metal binding properties of ZnuA, a periplasmic zinctransporter from Escherichia coli. J. Biol. Inorg. Chem. 13:271–288.

54. Zaharik, M. L., and B. B. Finlay. 2004. Mn2� and bacterial pathogenesis.Front. Biosci. 9:1035–1042.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

the zur-regulated zint protein is an auxiliary component of the

Documents