The Zur-regulated ZinT protein is an auxiliary component of the high

The Zur-regulated ZinT protein is an auxiliary component of the high affinity ZnuABC zinc 1

transporter that facilitates metal recruitment during severe zinc shortage 2

3

Patrizia Petrarca1, Serena Ammendola

1, Paolo Pasquali

2 and Andrea Battistoni

1,3* 4

5

1Dipartimento di Biologia, Università di Roma Tor Vergata, 00133 Rome, Italy;

2Dipartimento di Sanità 6

Alimentare e Animale, Istituto Superiore di Sanità, 00161 Rome, Italy; 3Consorzio Interuniversitario 7

“Istituto Nazionale Biostrutture e Biosistemi” (I.N.B.B.), Viale delle Medaglie d’Oro 305, 00136 8

Rome, Italy 9

10

11

*Corresponding author. Mailing address: Dipartimento di Biologia Università di Roma “Tor 12

Vergata”, Via della Ricerca Scientifica, 00133 Rome, Italy. Phone: +39 0672594372. Fax: +39 13

0672594311. e-mail: [email protected] 14

15

Running title: ZinT involvement in zinc uptake 16

17

Keywords: zinc homeostasis, zinc transporter, ZnuABC, Salmonella enterica, Zur, cadmium 18

toxicity, ZinT, Histidine-rich region. 19

20

21

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.01310-09 JB Accepts, published online ahead of print on 22 January 2010

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Abstract: The pathways ensuring the efficient uptake of zinc are crucial for bacterial ability to 22

multiply in the infected host. To better understand bacterial responses to zinc deficiency, we have 23

investigated the role of the periplasmic protein ZinT in Salmonella enterica serovar Typhimurium. 24

We have found that zinT expression is regulated by Zur and parallels that of ZnuA, the periplasmic 25

component of the zinc transporter ZnuABC. Despite that ZinT contributes to Salmonella growth in 26

media poor of zinc, disruption of zinT does not significantly affect virulence in mice. The role of 27

ZinT became clear using strains expressing a mutated form of ZnuA lacking a characteristic 28

histidine-rich domain. In fact, Salmonella strains producing this modified form of ZnuA exhibited a 29

ZinT-dependent capability to import zinc either in vitro or in infected mice, suggesting that ZinT 30

and the histidine-rich region of ZnuA have redundant function. The hypothesis that ZinT and ZnuA 31

cooperate in the process of zinc recruitment is supported by the observation that they form a stable 32

binary complex in vitro. Although, the presence of ZinT is not strictly required to ensure the 33

functionality of the ZnuABC transporter, our data suggest that ZinT facilitates metal acquisition 34

during severe zinc shortage. 35

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Introduction 37

Transition metals are essential constituents of a huge number of proteins where they play 38

catalytic or structural functions (4, 50). Therefore, all organisms posses complex machineries to 39

ensure an adequate supply of these elements, while avoiding their potentially toxic intracellular 40

accumulation. A large number of studies have documented the relevance of metals for microbial 41

growth and resistance to a variety of stress conditions (23, 42, 50). In particular, it is well 42

established that the pathways enabling bacteria to recruit metal ions are key for the ability of 43

pathogens to multiply within the host and cause disease (42; 43, 44). The vast majority of the 44

studies concerning metal uptake and bacterial pathogenicity have been focused on iron, but strong 45

evidences are emerging that the efficient uptake of other transition metals plays an important role in 46

the host-pathogen interaction (24, 54). In particular, recent observations suggest that zinc is not 47

freely available within the host (3). Zinc, after iron, is the second most abundant transition metal ion 48

in living organisms and plays catalytic and/or structural roles in enzymes of all six classes, several 49

of which play functions essential for cell viability (12). Investigations initially carried out in 50

Escherichia coli and then confirmed in other microorganisms have established that zinc 51

homeostasis is finely controlled by the coordinated activity of import and export systems regulated 52

by Zur and ZntR, two metalloproteins able to regulate gene transcription depending on their 53

metallation state (36, 40). Zur controls the expression of a few genes involved in bacterial response 54

to zinc shortage, whereas ZntR regulates the expression of the zinc efflux pump ZntA. It is worth 55

observing that, while the intracellular zinc concentration is rather constant and independent of the 56

culture medium (close to 200 µM), both of these regulators are able to respond to femtomolar 57

variations in the intracellular concentration of free zinc (38). These observations give emphasis to 58

the dynamic nature of metal homeostasis and suggest that very small alterations in the intracellular 59

zinc concentration may have a relevant influence on cellular physiology. 60

The metallated form of Zur influences also pathogenicity by the capability to repress the 61

expression of the high affinity zinc uptake system ZnuABC, a transporter of the ABC family 62


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activated in several bacteria in response to zinc deficiency (40). This transporter is constituted by 63

three proteins: ZnuB, the membrane permease, ZnuC, the ATPase component, and ZnuA, a soluble 64

periplasmic protein which captures Zn(II) and delivers it to ZnuB. Different studies have 65

established that the functionality of this transporter is essential to ensure growth of microorganisms 66

in media poor of zinc and for bacterial virulence (3, 9, 16, 30, 32, 52). We have recently 67

demonstrated that the expression of znuABC is repressed in Salmonella enterica serovar 68

Typhimurium (hereafter referred to as S. Typhimurium) cultivated in media containing a zinc 69

concentration as low as 1 µM, whereas its expression is strongly activated in bacteria recovered 70

from the spleens of infected mice or from cultured epithelial or macrophagic cells (3). Since zinc 71

concentration within eukaryotic cells is close to 0.2 mM, our studies indicate that the amount of 72

metal effectively available for microorganisms during intracellular infections is very limited and 73

that the ZnuABC transporter is required to ensure the efficient recruitment of zinc within the host. 74

In addition to the genes encoding for the proteins forming the ZnuABC transporter, Zur 75

directly regulates one or more genes encoding paralogs of ribosomal proteins (1, 39, 45). The Zur-76

regulated ribosomal proteins lack a zinc-binding motif that is present in their paralogs which are 77

normally produced in zinc-replete conditions. The insertion of these proteins in ribosomes during 78

zinc starvation likely facilitates growth by reducing the zinc requirements of bacterial cells. An 79

additional gene (zinT) putatively regulated by Zur was identified in E. coli using a bioinformatics 80

approach (39). zinT, which was formerly known as yodA, was originally identified as a member of 81

the E. coli cadmium-stress stimulon (21) and it was proposed that its role could be to decrease the 82

concentration of cadmium ions in E. coli cells during cadmium stress (41). Subsequent studies on E. 83

coli have demonstrated that zinT is modulated in bacterial cells exposed to low pH (8, 27), to copper 84

ions (29), to the transition metals chelator TPEN (46) or growing in media containing very low 85

levels of zinc (22). In addition, it has been hypothesized that ZinT could play a chaperone function 86

by delivering zinc to other periplasmic zinc-binding proteins, such as Cu,Zn superoxide dismutase 87

(24). This possibility, however, appears to be unlikely as Cu,ZnSOD is strongly down-regulated 88


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under conditions of zinc deficiency (2). Although the most recent investigations suggest that ZinT is 89

involved in zinc homeostasis (22, 29, 46), the exact function of this protein has not been elucidated. 90

Interestingly, ZinT, whose three dimensional structure has been solved in the presence of different 91

metal cofactors (cadmium, zinc or nickel) (14, 15), shows a very high homology to a domain of 92

AdcA, a component of an ABC transporter involved in zinc acquisition in Streptococcus 93

pneumoniae (19, 39). As the N-terminal portion of AdcA is homologous to ZnuA, this observation 94

strongly suggests that ZinT could cooperate with ZnuA in zinc uptake within the periplasmic space. 95

To verify this possibility, the role of ZinT has been investigated in S. Typhimurium. Our 96

results demonstrate that ZinT has no role in cadmium resistance and that it participates to the zinc 97

uptake process mediated by ZnuABC. 98

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Materials and Metods 101

Salmonella strains and growth conditions 102

The S. Typhimurium strains used in this work are listed in Table 1. Cultures were grown 103

aerobically in liquid Luria-Bertani broth (LB) or on LB agar plates at 37°C. For growth under zinc 104

limiting conditions the Vogel-Bonner minimal medium E (MM) (anhydrous MgSO4 0.04 g/l, citric 105

acid 2 g/l, anhydrous K2HPO4 10 g/l, NaH4PO4 3.5 g/l, glucose 2 g/l), the M9 minimal medium 106

(Na2HPO4 7.52g/l, KH2PO4 3g/l, NH4Cl 1g/l, NaCl 5g/l, MgSO4 7H2O 1.23g/l, CaCl2 2H2O 107

0.007g/l, glucose 0.2%) or Tris Minimal Medium (Tris HCl 120mM pH7.2, K2HPO4 0.017g/l, 108

MgCl2 2.03g/l, NH4Cl 1.06g/l, NaSO4 0.44g/l, CaCl2 0.06g/l, NaCl 4.68g/l, KCl 1.48 g/l, glucose 109

1.98g/l) were employed. For antibiotic selection agar plates were supplemented with kanamycin 110

(50µg/ml) or chloramphenicol (30µg/ml). 111

112

Mutants construction 113

All Salmonella knockout mutants and the 3xFLAG strains were obtained following the one-114

step inactivation protocol described by Datsenko (13) and the epitope tagging method described by 115

Uzzau (48), respectively. The oligonucleotides and plasmids used for mutants construction are 116

listed in Table 2. Each new strain was confirmed by PCR with oligonucleotides annealing upstream 117

or downstream the mutated allele and an internal primer annealing on the inserted antibiotic 118

resistance cassette. The oligonucleotides used for the construction and the verification of each new 119

strain are specified in Table 1. Alleles were then transduced into a clean background by generalized 120

transduction with phage P22 HT 105/1 int-201 (Uzzau et al, 2002). In some cases the antibiotic 121

resistance cassette was removed by the FLP recombinase transiently introduced by electroporation 122

of plasmid pCP20 into the strain. The znuA∆loop mutant was obtained by the Datsenko and Wanner 123

method modified as follows. First, an antibiotic resistance cassette was inserted into the Salmonella 124

chromosome downstream the znuA gene by electroporating a PCR fragment obtained with 125

oligonucleotides oli167/168 on pKD3 plasmid template. The chromosome of the resulting strain 126


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(yebA::cam, SA229) was then used as a template for a PCR with primers oli167/163, designed ad 127

hoc to amplify the znuA region with a deletion in the His-rich loop (from nucleotide 411 to 128

nucleotide 480 of the coding sequence) and the downstream antibiotic cassette . The obtained 129

fragment was then electroporated into strain MA6926 pKD46 and recombinants were selected on 130

chloramphenicol selective plates. The deletion of the His-rich loop of znuA was confirmed by 131

nucleic acid sequencing of the mutant strain. 132

133

Western blot analysis 134

To analyze the accumulation of ZinT, ZnuA and ZnuB, aliquots of bacterial cultures 135

(approximately 5x108

cells) were harvested, lysed by resuspending bacteria in sample buffer 136

containing sodium dodecyl sulphate (SDS) and β-mercaptoethanol, and boiled for 8 min at 100 °C. 137

Subsequently, proteins were run on 12% SDS-page gels and blotted onto a nitrocellulose membrane 138

(Hybond ECL, Amersham). The epitope–flagged proteins were revealed by incubation of 139

nitrocellulose membrane with an appropriate dilution of mouse anti-FLAG antibody (anti-FLAG 140

M2, Sigma) and anti-mouse horseradish peroxidase-conjugated antibody (Bio-Rad), followed by the 141

enhanced chemiluminescence reaction (ECL, Amersham). 142

143

Growth curves 144

Each strain was grown overnight in LB broth at 37 °C and then diluted 1:500 in fresh LB 145

broth supplemented or not with appropriate concentration of EDTA and/or of metals. The 146

absorbance at 600 nm was monitored every hour for 10 hours using a Perkin Elmer Lambda 9 147

spectrophotometer. 148

149

Mouse infections and competition assays 150

Overnight cultures of bacteria were diluted in PBS buffer to a final concentration of 104

151

cells/ml and then mixed in pairs in a 1:1 ratio. 0.2 ml of each mixture was used to infect 152


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intraperitoneally female balb/c mice of 10 weeks of age. Animals were sacrificed when exhibiting 153

symptoms of terminal septic syndrome (4-5 days post infection). Bacteria recovered from spleens 154

were plated for single colonies and then 200 colonies were picked on selective plates. The 155

competitive index (CI) was calculated by the formula CI =output (Strain A/Strain B)/inoculum 156

(Strain A/StrainB). Statistical differences between outputs and inputs were determined by the 157

Student’s t-test. 158

159

Cloning, expression and purification of ZnuA, ZinT and ZnuA∆loop 160

The S. Typhimurium znuA and znuA∆loop genes were amplified from chromosomal DNA 161

extracted with ZRfungal/bacterial DNA Kit TM (Zymo Research) from wild type and SA233 162

strains respectively. In both cases the primers used were SalZnuAfor (5’-ATAGAATTCCGG-163

GGCTCAATTCAAG-3’) and SalZnuArev (5’-TTTAAGCTTAATCTCCTTTCAGGCAGCT-3’) 164

that amplify the coding sequences plus about 200 base pairs upstream the start codon. The purified 165

PCR products were digested with EcoRI and HindIII, ligated into the pEMBL-18 vector (Dente et 166

al., 1983) obtaining plasmids p18PznuA and p18PznuA∆loop, which were introduced into E. coli 167

DH5α cells. The sequences of the cloned DNA fragments were confirmed by nucleic acid 168

sequencing. 169

For the expression of recombinant proteins, cells were grown overnight in LB medium, 170

harvested by centrifugation for 15 min at 8000 g, resuspended in 500 ml of isotonic solution and 171

periplasmic proteins were released by osmotic shock, as already described (2). After a 20 min 172

centrifugation at 13000 rpm, the periplasmic proteins contained in the supernatant were applied to a 173

Ni-NTA column (Qiagen) pre-equilibrated with 50 mM Na-phosphate, 250 mM NaCl, pH 7.8 and 174

eluted with a discontinuous gradient of 0-250 mM imidazole. ZnuA eluted with 20-40 mM 175

imidazole. The protein was further purified by anionic exchange chromatography on a HiLoadTm

Q 176

Sepharose FPLC column (Pharmacia Biotech) pre-equilibrated with 20 mM Tris-HCl, pH 7.0 and 177


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eluted using a 0-400 mM NaCl linear gradient. The purified protein was concentrated to 30 mg/ml 178

in a buffer containing 20 mM Hepes, 10 mM NaCl, 5% glycerol pH 7.0 and stored at -20°C. 179

Periplasmic extracts containing the ZnuA∆loop protein were initially purified by anionic 180

exchange chromatography on a column equilibrated with 20 mM Tris-HCl, pH 7.0 and eluted using 181

a 0-400 mM NaCl linear gradient. Subsequently, the protein was loaded on a cationic exchange 182

HiLoad SP Sepharose column equilibrated with 20 mM Na-phosphate, pH 7.0 and eluted with a 0-183

400 mM NaCl linear gradient. A final chromatographic step was carried out on a HiLoad 26/10 184

Phenyl Sepharose HP column equilibrated with 30 mM Tris-HCl, 1.5 M (NH4)SO4, pH 7.0. Proteins 185

were eluted with a 1.5-0 M (NH4)SO4 0-400 mM NaCl linear gradient. The fractions containing the 186

ZnuA∆loop protein were pooled and the protein resulted more than 98% pure, as judged by SDS-187

PAGE analysis. The purified protein was concentrated to 30 mg/ml in a buffer containing 20 mM 188

Hepes, 10 mM NaCl, 5% glycerol, pH 7.0 and stored at -20°C. 189

The S. Typhimurium zinT gene was amplified from chromosomal DNA extracted from wild 190

type strain utilizing primers zinT5 (5’-TCCATGGATATTCATTTAAAAAAACTGACAATG-3’) 191

and zinT2 (5’-ATCAAGCTTAATCAGACTTAATGATGTAGCAT-3’). The PCR product was 192

digested with NcoI and HindIII, ligated into the pSE420 vector (Invitrogen) obtaining plasmid 193

pSEzinT and then transformed into E. coli DH5α cells. The sequence of the whole cloned DNA 194

fragment was verified by nucleic acid sequencing. 195

Cells harbouring plasmid pSEzinT were grown in LB medium supplemented with 100 µg/ml 196

ampicillin, and when the absorbance at 600 nm of the culture reached the value of 0.5, protein 197

expression was induced overnight with 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG). Cells 198

were harvested by centrifugation for 15 min at 5000 rpm and periplasmic proteins were extracted by 199

lysozyme treatment (2). Spheroplasts were separated from periplasmic proteins by centrifugation 200

and the supernatant was applied to Ni-NTA column preequilibrated with 50 mM Na-phosphate, 250 201

mM NaCl pH 7.8 and eluted with a linear gradient of 0-500 mM imidazole. ZinT eluted at 250 mM 202


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imidazole. Fractions containing ZinT (>98% pure) were pooled, dialyzed against 20 mM Hepes, 10 203

mM NaCl, 5% glycerol, pH 7.0, concentrated to 30 mg/ml and stored at -20 °C. 204

To analyze the possible formation of a complex between ZnuA and ZinT, the two proteins 205

were mixed in a 2:1 (ZnuA:ZinT) w/w ratio, corresponding to a 1.4 molar ratio, to favour complex 206

formation. After a 90 min incubation at room temperature, proteins were injected onto a High Load 207

16/60 Superdex 75 gel filtration FPLC column (Amersham Biosciences) equilibrated with 20 mM 208

Hepes, 100 mM NaCl, pH. 7.0. Elution was carried out at room temperature. 209

Metal-free ZnuA and ZinT were prepared by extensive dialysis against 50 mM sodium 210

acetate buffer, 2 mM EDTA, pH 5.5. The proteins were subsequently dialyzed twice against 50 mM 211

sodium acetate, 0.1 M NaCl, pH 5.5 to remove excess EDTA. Finally, the proteins were dialyzed 212

against 20 mM Hepes, 100 mM NaCl, pH. 7.0 to carry out gel filtration experiments. The metal 213

content of the apo-proteins was evaluated by atomic absorption using a Perkin Elmer spectrometer 214

AAnalyst 300 equipped with the graphite furnace HGA-800. The zinc content of the demetallated 215

proteins was below the 5 %. 216

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Results 218

zinT distribution in eubacteria. 219

A previous study has shown that ZinT is present in some bacterial species as an isolated 220

protein, whereas in other bacteria it is a domain of the AdcA protein involved in zinc uptake (39). 221

The amino acids alignment reported in Figure 1 shows that the C-terminal portion of AdcA from 222

different bacteria displays high homology with ZinT (53,4% amino acids identity between the 223

overlap region of S. pneumoniae AdcA and mature Salmonella ZinT), whereas its N-terminal 224

portion is similar to ZnuA (26% identity amino acids identity between the overlap region of the S. 225

pneumoniae protein and mature Salmonella ZnuA). The functional and structural homology 226

between ZnuA and AdcA is confirmed by two additional features: the conservation of the three 227

histidine residues which coordinate the zinc ion in the crystal structures of E. coli (10, 31, 53) and 228

Synechocystis 6803 (5, 51) ZnuA and the presence in the same sequence position of a charged loop 229

rich in acidic and histidine residues (His-rich loop), typical of proteins involved in the transport of 230

zinc (5, 11). Interestingly, the three residues involved in zinc binding in ZinT are strictly conserved 231

in the AdcA proteins, as well as a C-terminal histidine residue which is likely involved in metal 232

binding (15). However, it should be noted that ZinT differs from the C-terminal domain of AdcA 233

for the presence of a N-terminal histidine-rich domain. The observations that in some bacterial 234

species ZinT is fused to a ZnuA-like protein involved in zinc transport and that zinT and znuA are 235

likely coregulated by Zur (22, 39), strongly suggest that ZinT could participate in the ZnuABC 236

mediated zinc uptake process. This possibility has been further corroborated by an analysis of the 237

distribution of ZinT, ZnuA (as well as ZnuB and ZnuC) and AdcA in available bacterial genomes 238

(Supplementary Table 1). This analysis shows that several bacteria possessing the ZnuABC system 239

do not contain ZinT. However, bacterial species lacking a ZnuA homologue do not have a ZinT 240

protein, whereas occurrence of ZinT is always associated to the presence of ZnuA. These 241

observations support the hypothesis that the role of ZinT is related to that of the ZnuA. 242

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ZinT is not involved in cadmium resistance. 244

To verify whether ZinT is involved in cadmium resistance, we have constructed an epitope 245

tagged mutant by introducing a 3xFLAG sequence at the 3’ end of the chromosomal copy of the 246

zinT coding sequence. The PP134 strain (zinT::3xFLAG) shows a growth rate similar to that of the 247

wild type strain (data not show). 248

In line with previous investigations carried out with E. coli, ZinT accumulates in S. 249

Typhimurium grown in LB medium supplemented with 0.5 mM cadmium acetate (Fig. 2A). 250

However, also ZnuA and ZnuB, the periplasmic and the transmembrane component of the ZnuABC 251

zinc transporter, respectively, show a comparable increase in protein accumulation in bacteria 252

cultivated in presence of cadmium (Fig. 2B and 2C, respectively). This finding suggests that 253

cadmium induces the expression of all Zur-regulated proteins participating to zinc homeostasis. 254

To better evaluate whether the induction of zinT plays a role in cadmium resistance, we have 255

analyzed the ability of a zinT mutant strain to grow in LB plates containing variable amounts of 256

cadmium. As shown in Table 3, the zinT mutant strain exhibited a cadmium susceptibility 257

comparable to that of the wild type strain, thus confirming recent observations showing that ZinT 258

does not contribute to bacterial growth/survival in the presence of this toxic metal (22, 29). The 259

reduced ability of a znuA mutant strain to grow in presence of cadmium confirmed that this metal 260

interferes with zinc homeostasis. 261

262

zinT is induced under zinc starvation and belongs to the Zur-regulon. 263

Previous studies have shown that ZnuA accumulation is induced by metal chelating agents 264

(EDTA or TPEN) and repressed by the addition of zinc (3, and Fig. 3A). To verify if also ZinT 265

accumulation is modulated by zinc availability, we have grown strain PP134 in presence of 0.5 mM 266

EDTA with or without equimolar amounts of zinc. As shown in figure 3, ZinT accumulation is 267

strongly induced by EDTA, but is repressed by zinc (Fig. 3A). The inhibition of ZinT accumulation 268

is specific for zinc, as manganese, iron and copper have no effect (Fig. 3B). To verify that zinT and 269


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znuA are co-regulated by the transcriptional factor Zur, the znuA::3xFLAG and the zinT::3xFLAG 270

alleles have been transduced in a zur deleted strain, obtaining the PP131 and PP132 strains, 271

respectively. The accumulation of ZinT and ZnuA in these strains was completely deregulated and 272

insensitive either to EDTA, to zinc or cadmium supplementation (Fig. 3A). This observation 273

confirms that in Salmonella enterica transcription of zinT is under control of Zur, as recently 274

observed in E. coli (22, 29). 275

To better analyze the relationships between ZinT/ZnuA accumulation and zinc availability, 276

we have constructed a double epitope tagged strain (znuA::3xFLAG zinT::3xFLAG, PP141). When 277

this strain was grown in a zinc depleted medium (MM) both ZnuA and ZinT accumulated at high 278

levels (Fig. 4A). Similar results were obtained when strain PP141 was grown in defined media of 279

different formulation, i.e. M9 and Tris Minimal Medium (data not shown). However, the two 280

proteins exhibit a slightly different response to zinc availability. In fact, the results reported in the 281

figure show that the addition of 0.5 µM ZnSO4 to the culture medium causes the complete 282

abrogation of ZinT accumulation, but not that of ZnuA (Fig. 4A), which show a low level of 283

accumulation also at higher zinc concentrations. In agreement with this observation, ZinT and 284

ZnuA are maximally expressed in bacteria cultivated in LB medium supplemented with 0.5 mM 285

EDTA, but accumulation of ZnuA is observed at EDTA concentrations lower than those required to 286

induce accumulation of ZinT (Fig. 4B). Moreover, ZinT accumulation in a strain lacking the znuA 287

gene (PP137) is not inhibited by the addition of zinc (Fig. 4C). In contrast, ZnuA accumulation in a 288

strain lacking the zinT gene (PP128) is comparable to that observed in the wild type strain (Fig. 4C). 289

These experiments show that znuA is induced at higher zinc concentration than those 290

required to activate zinT transcription, indicating that ZnuA is a protein involved in the frontline 291

response to zinc deficiency, whereas ZinT participated in the bacterial response to more severe zinc 292

deficiency. Moreover, the observation that ZinT accumulation can not be repressed by the external 293

supply of zinc suggests that ZnuA plays a role in zinc import within the cell that can not be 294

substituted by ZinT. 295


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Consequences of zinT deletion on Salmonella growth 296

In agreement with the above reported expression studies, Table 3 shows that growth on LB 297

agar plates of a zinT mutant strain is inhibited by divalent metals chelators such as EDTA and 298

TPEN. The growth impairment due to zinT deletion is lower than that observed for a znuA mutant 299

strain, confirming the prominent role of ZnuA in zinc homeostasis. 300

This observation was confirmed by the analysis of the growth curves of wild type and znuA 301

and zinT mutant strains in presence of EDTA (Fig. 5). In fact, whereas the growth of all the strains 302

tested is nearly identical in standard LB medium, in presence of 2 mM EDTA the growth of the 303

zinT mutant strain is impaired compared to the wild type strain. The growth defect, however, is 304

lower than that exhibited by the strain lacking znuA. Zinc, but not iron or manganese, 305

supplementation to the growth medium completely abolished this phenotype (data not shown), 306

indicating that it is specifically due to the zinc sequestration ability of EDTA and not to EDTA-307

induced shortage of other metals. Interestingly, the growth of the double mutant zinT-znuA is 308

comparable to that of the znuA mutant strain, confirming that ZinT can not substitute ZnuA in the 309

process of zinc import within the cytoplasm. 310

311

ZinT role in Salmonella pathogenicity. 312

Previous studies have established that disruption of znuA dramatically decreases Salmonella 313

pathogenicity (3). In this work we have compared the contribution of zinT and znuA to Salmonella 314

ability to colonize host tissues by carrying out competition experiments in balb/c mice. This 315

approach confirmed the relevance of ZnuA in Salmonella infections (Table 4). In contrast, the 316

ability of the strain lacking zinT (PP116) to colonize the spleen of infected mice was comparable to 317

that of the wild type strain, whereas the zinT mutant (PP116) outcompeted the double mutant 318

zinTznuA (PP118). In addition, we did not observe differences in spleen colonization between znuA 319

(SA123) and the znuABC (SA182) mutant strains. Quite surprisingly a zinTznuABC (PP119) mutant 320

strain was favoured with respect to the znuABC (SA182) mutant strain. This observation could be 321


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suggestive of a detrimental role of ZinT in the absence of ZnuABC, although more studies are 322

required to verify this hypothesis. These experiments suggest that ZinT facilitates zinc transport 323

through the ZnuABC system, but that it has a dispensable role during mice infections. Moreover, 324

the observation that the disruption of zinT does not attenuate mutant strains lacking a functional 325

ZnuABC transporter (SA123 or SA182) provides further support to the hypothesis that ZinT is not 326

involved in a ZnuABC-independent mechanism of zinc transport. 327

Additional experiments, however, shed more light on the role of ZinT in the mechanism of 328

zinc uptake. The ZnuA protein possesses a charged flexible loop rich in histidines and acidic 329

residues, whose function has not yet been clarified (His-rich loop). It has been hypothesized that the 330

role of this loop could be to increase the zinc sequestering ability of ZnuA in environments poor of 331

this metal (6, 18), whereas other Authors have suggested that it could be a sensor of periplasmic 332

zinc concentration (51). To investigate the role of this His-rich region, we have constructed a znuA 333

mutant strain producing a ZnuA protein devoid of this loop (SA233). As shown in Figure 6, the 334

growth of this mutant strain is not impaired in LB medium containing EDTA, indicating that ZnuA 335

can mediate zinc transport also in the absence of the His-rich region. However, a severe growth 336

defect was observed in a mutant strain expressing such mutated form of ZnuA and unable to 337

produce ZinT. The growth of this mutant, in fact, was comparable to that of the strain lacking znuA. 338

Similar results were obtained in competition experiments. The data reported in Table 4 show that 339

deletion of yebA, which was required for the construction of the znuA∆loop mutant strain has no 340

effect on Salmonella virulence. Similarly, the znuA∆loop mutant strain was not significantly 341

attenuated in comparison to the wild type strain, although a slightly higher number of wild type 342

bacteria were recovered from the spleens of infected mice. In agreement, there was not a significant 343

difference in the spleen colonization ability of a zinT mutant strain (PP125) and of the znuA∆loop 344

strain (SA233). However, the Salmonella strain lacking zinT and expressing the mutated form of 345

znuA (PP130) was attenuated with respect to the strain unable to produce ZinT (PP125). This strain 346


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maintained some ability to transport zinc through the ZnuB channel, as it was advantaged over 347

strain PP118, which does not express znuA. 348

These experiments suggest that ZinT and the His-rich domain of ZnuA are two independent 349

elements which participate to the ZnuABC-mediated zinc transport by playing an overlapping role 350

in facilitating zinc recruitment by ZnuA. Although the simultaneous presence of ZinT and of the 351

His-rich domain is apparently not indispensable to ensure the functionality of the transporter under 352

the conditions investigated in this work, disruption of each one of the two elements disclose a role 353

for the other one in enhancing the efficiency of zinc uptake. 354

355

ZinT and ZnuA form a binary complex in vitro 356

The above experiments showing that ZnuA and ZinT functionally interact in the mechanism 357

of ZnuABC-mediated zinc import suggest that ZinT might physically interact with ZnuA. To prove 358

this possibility we have analyzed the ability of these two proteins to form a complex in vitro. ZinT 359

and ZnuA were cloned, expressed and purified as described in Experimental Procedures. The two 360

proteins were mixed together, incubated for 90 min at room temperature and then loaded on a gel 361

filtration column. Figure 7shows a comparison of the elution of the individual proteins and of the 362

ZnuA:ZinT mixture. ZinT eluted from the column with an apparent molecular weight of 22.3 kDa 363

(peak centred at 75 ml, Fig. 7A), in excellent agreement with the ZinT molecular weight deduced 364

from the amino acids sequence (22,2 kDa). ZnuA eluted with an apparent molecular weigth of 34.9 365

kDa (peak centred at 67 ml, Fig. 7B), a value which is slightly higher than the expected molecular 366

weight (31.5 kDa). When the mixture of ZinT and ZnuA was applied to the column, the elution 367

profiles of the two proteins significantly changed. In fact, in this case maximal ZinT and ZnuA 368

concentration was found in fraction 63 (Fig. 7C), corresponding to a protein of approximately 48 369

kDa. The observation that the apparent molecular weight of the two proteins was shifted to values 370

significantly higher than those of the single proteins strongly supports the hypothesis of formation 371

of a binary complex between the two proteins. The elution profile of the complex was rather broad, 372


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possibly due to incomplete involvement of the two proteins in complex formation and/or to partial 373

complex dissociation during the elution. However, when the proteins recovered from fraction 63 374

were concentrated and subjected to a new gel filtration chromatography, the largest part of ZnuA 375

and ZinT still coeluted with a high molecular weight, indicating that the complex between the two 376

proteins is stable (data not shown). The observation that the ZnuA/ZinT complex has an apparent 377

molecular weight lower than that expected (53,7 kDa) is indicative of changes in the hydrodynamic 378

properties of the two proteins following their interaction. 379

Interestingly, the two apo-proteins were not able to form a stable complex (Fig 7D). 380

However, the ability of ZnuA and ZinT to stably interact was restored when the two proteins were 381

individually reconstituted with an equimolar amount of zinc before protein incubation (Fig. 7E). 382

The addition of zinc to a single protein (ZnuA or ZinT) was not sufficient to observe the coelution 383

of the two proteins from the gel filtration column (data not shown). These results suggest that the 384

binding of zinc induces structural rearrangements in ZnuA and ZinT which are necessary for the 385

formation of a stable complex between the two proteins. 386

To verify if the histidine rich region of ZnuA is involved in the formation of the complex 387

between ZinT and ZnuA, similar experiments were carried out using the ZnuA∆loop mutant 388

protein. As show in figure 7G, when these proteins were coincubated they eluted in the same 389

fractions with apparent molecular weights higher than those of isolated ZnuA∆loop and ZinT. In 390

line with functional studies, this observation demonstrates that the His-rich region of ZnuA is not 391

required for the formation of a ZinT /ZnuA complex in vitro. 392

The elution profile of ZinT was not altered when the protein was preincubated with bovine 393

serum albumin or Cu,Zn superoxide dismutase (Fig. 7H and 7I, respectively), thus indicating that 394

the interaction of ZinT and ZnuA is highly specific. 395

396


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Discussion 397

Zinc serves important functions in a wide range of cellular processes and, therefore, the 398

mechanisms ensuring a highly efficient zinc recruitment are critical for bacterial growth and 399

survival in all the environments where this metal is not abundant. Recent studies have established 400

that zinc is not freely available within the host tissues and that the ability of several pathogens to 401

multiply in the infected host is strictly dependent on the zinc transporter ZnuABC. As this is the 402

only high affinity zinc transporter identified in many bacterial species, these observations suggest 403

that ZnuABC could be an interesting target for novel antimicrobial strategies. To deepen our 404

understanding of the mechanisms governing zinc homeostasis in bacteria, we have investigated the 405

role of ZinT, a poorly characterized protein which has been proposed to be involved in the 406

mechanisms of resistance to toxic metals or to zinc deficiency. 407

In agreement with recent observations (22, 29) this investigation demonstrates that ZinT has 408

no role in bacterial resistance to cadmium toxicity. In fact, although zinT is strongly induced by this 409

metal, deletion of the gene does not impair bacterial growth in cadmium-containing media. 410

Moreover, cadmium induces the accumulation of ZinT as well as of other proteins involved in zinc 411

transport, i.e. ZnuA and ZnuB. The mechanisms responsible for cadmium toxicity are not 412

completely understood, but can be largely explained by the ability of cadmium to deplete cells from 413

intracellular glutathione, to react with the sulphydryl groups of proteins and to compete with other 414

metals for the binding to metalloproteins (7, 49). These results provide novel suggestions to 415

understand the complex molecular basis of cadmium toxicity in bacteria. In fact, cadmium induces 416

the expression of the Zur-regulated genes in bacteria growing in a zinc replete medium (LB), 417

suggesting that cadmium alters zinc homeostasis in bacteria. Additional studies are required to 418

understand whether this is due to a general ability of cadmium to substitute the proper metal 419

cofactor in zinc-containing proteins or to a specific effect on Zur (which, upon cadmium binding, 420

might adopt an altered conformation unable to bind to DNA) or on the functionality of the ZnuABC 421

transporter. However, it is worth nothing that cadmium binds with high affinity to metal sites 422


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characterized by cysteine ligands, such as metallothioneins (26) or zinc-dependent transcription 423

factors (25) and that X-ray absorption studies have shown that zinc binding to Zur involves 424

different cysteine residues (38). This last observation suggests that Zur could be a privileged target 425

for cadmium ions. 426

All the results described in this work converge in demonstrating that ZinT participates to the 427

process of zinc uptake mediated by ZnuABC. This conclusion is supported by different evidences. 428

First of all, ZinT can be found, either as an independent protein or as a domain of the AdcA 429

proteins, in bacteria containing ZnuA or homologous periplasmic ligand binding proteins involved 430

in zinc uptake. In contrast, ZinT is not present in bacteria lacking ZnuA (see supplementary Table 431

1). Moreover, in line with recent studies carried out in E. coli (22, 29), we have confirmed the 432

original proposal of Panina and coworkers (39) that zinT is a member of the Zur regulon. We have 433

also demonstrated that zinT is induced under conditions of zinc deficiency (Fig. 3 and 4) and that it 434

contributes to bacterial growth in media poor of this metal (Fig. 5 and 6). Interestingly, either the 435

analysis of zinT role in bacterial growth in vitro (Fig. 6) or competition experiments in mice (Table 436

4), failed to identify a major contribution of ZinT to zinc transport in bacteria lacking znuA or the 437

whole znuABC operon, indicating that the role of ZinT in zinc uptake is dependent on the presence 438

of ZnuA. Noteworthy, a mutant strain expressing zinT but deleted of the znuABC operon was 439

disadvantaged with respect to the zinT-znuABC mutant strain (Tab. 4), suggesting that in the 440

absence of the ZnuABC transporter the expression of ZinT is harmful, possibly due to the zinc 441

sequestration ability of this protein which might decrease zinc import trough low affinity zinc 442

transporters. This possibility is supported by the observation that ZinT accumulates constitutively in 443

bacteria lacking znuA (Fig. 4C). However, further work is required to verify this hypothesis because 444

the same effect was not observed in the competition between the znuA (SA123) and the znuAzinT 445

(PP118) mutant strains. All these observations suggest that ZinT is an additional component of the 446

ZnuABC transporter facilitating zinc recruitment in the periplasmic space. The results reported in 447

Figure 7 demonstrate that ZinT is also able to form a stable complex with ZnuA in vitro, which we 448


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suppose could resemble the structural organization of AdcA proteins. Interestingly, the stability of 449

the complex is dependent on the presence of zinc, suggesting that in vivo the interaction between 450

ZnuA and ZinT could be destabilized upon zinc exchange within the ZnuA/ZinT complex or 451

following zinc release from the complex to ZnuB.The scattered distribution of zinT in eubacteria 452

expressing znuA, the observation that ZnuABC is critical for successful infection in several bacteria 453

lacking zinT, including H. ducreyi (30), B. abortus (52) and C. jejuni (16) and the results showing 454

that deletion of zinT does not attenuate S. Typhimurium and only marginally decreases bacterial 455

growth in presence of very high concentrations of chelating agents, all together indicate that ZinT 456

has a role in zinc transport which is significantly less important than that of ZnuA. In support of this 457

view, we have observed slight differences in the regulation of zinT and znuA, suggesting that 458

transcription of znuA occurs at zinc concentrations higher than those required to activate zinT 459

expression. In fact, ZinT accumulation is completely repressed in bacteria growing in a minimal 460

medium containing 0.5 µM ZnSO4 or in LB medium supplemented with 0.05 mM EDTA, whereas 461

under the same conditions ZnuA is partially induced (Fig. 4). Such a flexible response to zinc 462

deficiency, likely justified by small differences in the Zur-binding region (data not shown), may 463

provide an explanation for the production of two separate proteins, instead of a single one (AdcA) 464

as in some Gram-positive bacteria. It should be noted that this finding is in apparent contrast with 465

previous transcriptomics studies showing a greater increase in mRNA levels of zinT with respect to 466

znuA in responce to TPEN (46) or to zinc deficiency (22). However, both the studies were carried 467

out using defined media containing low levels of zinc and that Graham and coworkers (22) used a 468

medium containing high EDTA concentrations. We believe that the media used in these works 469

provided conditions sufficient to induce a significant basal expression of znuA, thus explaining the 470

much greater mRNA induction of zinT upon further depletion of zinc. 471

Although dispensable for the functionality of ZnuABC, ZinT enhances bacterial capability 472

to grow under severe zinc shortage. The presence of another periplasmic protein distinct from ZnuA 473

may be useful to facilitate metal sequestration in this cellular compartment. We have observed that 474


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ZinT has a very high affinity for immobilized nickel (see Experimental Procedures) possibly due to 475

its N-terminal His-rich domain (Fig. 1). This region of ZinT differs from the ZnuA His-rich loop as 476

this last motif is characterized by the presence of a very high number of acidic residues, which 477

likely affects the protonation state of the neighbouring histidine residues. We suggest that His-rich 478

domain of ZinT could play a role in facilitating zinc displacement from other proteins and the 479

subsequent entry of the metal into the ZnuABC-mediated process of zinc transport from the 480

periplasm to the cytoplasm. 481

Some experiments reported in this study also shed new light on the role of His-rich region of 482

ZnuA. ZnuA possesses a central domain of variable length in different bacterial species which is 483

characterized by the presence of a high number of histidine and acidic residues whose function is 484

not yet explained. A few studies have suggested that this loop could enhance zinc binding ability 485

and its subsequent transfer to the primary binding site of ZnuA (6, 18). Alternatively it has been 486

suggested that it could be a sensor of periplasmic zinc concentration, able to inhibit zinc transfer 487

from ZnuA to ZnuB at high zinc concentrations (51). Interestingly, similar domains are present also 488

in a vast number of eukaryotic zinc transporters (20). Different studies have explored the role of 489

these histidine-rich regions by producing mutant proteins devoid of this protein domain. The results 490

obtained have failed to provide an univocal answer to the role of such His-rich loops, as it has been 491

proposed that they can contribute to protein stability (33), to the process of metal transfer (34), to 492

metal selectivity (35) or to the modulation of protein activity (28). We have observed that a S. 493

Typhimurium strain expressing a ZnuA variant devoid of the His-rich region is not attenuated in 494

infection studies and grows as the wild type strain in LB medium containing 2 mM EDTA. This 495

observation apparently argues against a role of this domain in metal recruitment. However, when 496

this mutation was inserted in a strain deleted of zinT, mutant ZnuA proved not to be able to work as 497

well as native ZnuA either in vitro (Fig. 6) or in the infected animal (Table 4). Although we can not 498

exclude that the His-rich region could play additional roles (for example in facilitating the selection 499

of the correct metal ion), our findings suggest that ZinT and the His-rich region play an overlapping 500


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role in increasing the metal binding ability of ZnuA. Under the experimental conditions we have 501

explored in this work, this function can be disclosed only in bacteria lacking both ZinT and the His-502

rich region of ZnuA, suggesting that they play similar roles. It is likely that the simultaneous 503

presence of two structurally distinct elements with apparently redundant roles could enhance metal 504

recruitment during conditions of severe zinc shortage and maximize zinc import under variable 505

environmental conditions. 506

This study, which shows that ZinT participates in the ZnuABC-mediated process of zinc 507

transport, provides additional insights into the mechanisms employed by some bacteria to obtain 508

zinc. ZinT is dispensable for the functionality of the transporter, thus explaining its absence in 509

several bacteria, but plays a role auxiliary to that of ZnuA in the recruitment of zinc within the 510

periplasmic space. 511

512

Acknowledgments 513

This work was partially supported by an ISS grant to A.B. and P.P. and by a grant of 514

Fondazione Roma to A.B. 515

516


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663

Legends to figures 664

665

Figure 1. Sequence alignment of AdcA, ZnuA and ZinT: Sequence alignment of the mature 666

(without the signal peptide) AdcA from S. pneumoniae, S. suis, E. faecalis, S. aureus, S. 667

haemoliticus; ZnuA from S. Typhimurium and ZinT from S. Typhimurium, E. coli, B. subtilis. 668

Residues involved in zinc binding in ZnuA and ZinT are highlighted in gray. A strictly conserved 669

histidine residue present at the C-terminus of ZinT is shown in bold. The protein sequences 670

corresponding to the His-rich loop of ZnuA and AdcA and the N-terminal His-rich region of ZinT 671

have been put in evidence. 672

673

Figure 2. Effect of cadmium on ZinT, ZnuA and ZnuB accumulation: Western Blots of 674

bacterial lysates from strains PP134 (zinT::3xFLAG-kan) (Panel A), SA140 (znuA::3xFLAG-kan 675

ilvI::Tn10dTac-cat::3xFLAG-kan) (Panel B) and PP101 (znuB::3xFLAG-kan ) (Panel C) grown in 676

LB medium (lane 1) and LB supplemented with 0.5 mM cadmium acetate (lane 2). In Panel A, the 677

accumulation of ZnuA is shown in comparison with an internal standard (Cat::3xFLAG). It was not 678

possible to use this protein as an internal standard to follow ZinT and ZnuB accumulation, as 679

migration of these proteins overlapped with that of Cat. 680

681

Figure 3. ZinT and ZnuA accumulation in wild type and in Zur deleted strains. Western Blots 682

of bacterial lysates In panel A strains SA140 (znuA::3xFLAG-kan ilvI::Tn10dTac-cat::3xFLAG-683

kan), PP134 (zinT::3xFLAG-kan), PP131 (znuA::3xFLAG-scar zur::kan ilvI::Tn10dTac-684

cat::3xFLAG-scar) and PP132 (zinT::3xFLAG-scar zur::kan) were grown in LB medium alone or 685

supplemented with cadmium acetate, EDTA or ,ZnSO4 as indicated. Panel B shows ZinT 686

accumulation in strain PP134 (zinT::3xFLAG-kan) grown in minimal medium supplemented with 5 687

µM ZnSO4, 5 µM MnCl2, 5 µM CuSO4 or 5 µM FeSO4. 688

689

Figure 4. Differential accumulation of ZinT and ZnuA in zinc repleted and zinc deficient 690

conditions. Western Blots of bacterial lysates. Panel A: strain PP141 (znuA::3xFLAG-kan 691

zinT::3xFLAG-scar) was grown in minimal medium alone or supplemented with increasing 692

amounts of ZnSO4 as indicated. Panel B: the same strain was grown in LB medium alone or 693

supplemented with increasing amounts of EDTA as indicated. Panel C shows zinc dependent 694

accumulation of ZinT in a wild type background (strain PP134) and in a znuA deleted strain PP137 695

(zinT::3xFLAG-kan znuA::cam) and accumulation of ZnuA in a wild type background (strain 696


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PP140) and in a zinT deleted strain PP128 (znuA::3xFLAG-scar zinT::scar ilvI::Tn10dTac-697

cat::3xFLAG-kan). 698

699

Figure 5: Growth curves of S. Typhimurium mutants: Growth curves of the wild-type 700

(triangles), zinT::cam (squares), znuA::kan (circles) and zinT::cam-znuA::kan (diamonds) strains. 701

Each strain was grown in LB medium (closed symbols) and LB medium supplemented with 2 mM 702

EDTA (open symbols). 703

704

Figure 6: Growth curves of znuA∆loop mutant strain: Growth curves of the wild-type 705

(triangles), zinT::cam (squares), znuA::kan (circles), znuA∆loop-yebA::cam (diamonds) and 706

znuA∆loop-yebA::cam-zinT::scar (inverted triangles). Each strain was grown in LB medium 707

supplemented with 2 mM EDTA. 708

709

Figure 7: Analysis of ZinT and ZnuA interaction by gel filtration chromatography: SDS-710

PAGE analysis of Salmonella ZinT and ZnuA eluted from a HiLoadTM 16/60 SuperdexTM 75 gel 711

filtration FPLC column, calibrated with bovine serum albumin (67000 Da), ovoalbumin (43000 712

Da), chymotrypsinogen A (25000 Da) and ribonuclease A (13700 Da). The samples loaded on the 713

column were collected in 1 ml fractions. The gel shows the proteins contained in the 61-78 ml 714

fractions, as indicated at the bottom of the figure. Panels correspond to: A) isolated ZinT; B) 715

isolated ZnuA; C) mixture of ZnuA and ZinT; D) mixture of apo-ZnuA and apo-ZinT; E) mixture 716

of apo-ZnuA and apo-ZinT reconstituted with an equimolar amount of zinc; F) isolated 717

ZnuA∆loop; G) mixture of ZnuA∆loop and ZinT; H) mixture of Cu,ZnSOD and ZinT; I) mixture of 718

BSA and ZinT. 719

720


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Table 1 Salmonella strains

*MA6926 is S.Typhimurium ATCC14028

†The term “scar” refers to the DNA sequence remaining after excision of antibiotic-resistance cassette following

homologous recombination between two flanking FRT mediated by a recombinase encoded by plasmid pCP20 (13).

Name Relevant genotype Source/Tecnique

(oligonucleotides and template plasmid)

MA6926* Wild type L. Bossi

MA6926(pKD46) wild type harbouring plasmid pKD46 L. Bossi

SA123 znuA::kan (3)

SA140 znuA::3xFLAG-kan ilvI::Tn10dTac-

cat::3xFLAG-kan

(3)

SA150 znuA::cam Lab collection

SA182 znuABC::kan Lab collection

SA229 yebA::cam electroporation of fragment [oli167-168 pKD3] in

MA6926 pKD46; verified by PCR (oli169/K1)

camR

SA233 znuA∆loop yebA::cam see Materials and Methods section

SA287 znuA∆loop yebA::scar† derived from SA233; camS

SA288 znuA::3xXFLAG-scar† ilvI::Tn10dTac-

cat::3xFLAG-scar†

derived from SA140; kanS

PP101 znuB::3xFLAG-kan electroporation of fragment [oli172-173 pSUB11] in

MA6926 pKD46; verified by PCR (oli136/K1);

kanR

PP116 zinT::cam electroporation of fragment [oli178-179 pKD3] in

MA6926 pKD46; verified by PCR (K3/oli181)

camR

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

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

camR

PP125 zinT-scar† derived from PP116; camS

PP126 znuA::3xFLAG-scar† zinT::scar

† derived from PP120; camS

PP127 zur::kan electroporation of fragment [oli184-185 pKD4] in

MA6926 pKD46; verified by PCR (oli177/K1);

kanR

PP128 znuA::3xFLAG-scar† zinT::scar†

ilvI::Tn10dTac-cat::3xFLAG-kan

P22-mediated transduction (SA140) on PP126;

camR

PP130 zinT::cam znuA[∆ loop] yebA::scar† P22-mediated transduction (PP116) on SA287;

camR

PP131 znuA::3xFLAG-scar† zur::kan

ilvI::Tn10dTac-cat::3xFLAG-scar†

P22-mediated transduction (PP127) on SA288; kanR

PP132 zinT::3xFLAG-scar† zur::kan P22-mediated transduction (PP127) on PP129; kanR

PP134 zinT::3xFLAG-kan electroporation of fragment [oli182-195 pSUB11] in

MA6926 pKD46; verified by PCR (oli180/K4) kanR

PP137 zinT::3xFLAG-kan znuA::cam P22-mediated transduction (PP134) on SA150; kanR

PP138 zinT::3xFLAG-scar† derived from PP134; kanS

PP141 znuA::3xFLAG-kan zinT::3xFLAG-scar† P22-mediated transduction (SA140) on PP138; kanR


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Table 2.

Oligonucleotides and plasmid used for mutant construction

Oligonucleotides

Name Sequence

K1 CAGTCATAGCCGAATAGCCT

K3 AGCTCACCGTCTTTCATTGC

K4 CACTGCAAGCTACCTGCTTT

oli136 GTACGCGTGGTTTTAGGACT

oli163 GCAACTTGCCGATGTAAAACCGTTACTCATGAAGGGCGCGGGCGAATATAACATGCATCT

oli167 AGACCTTCCGTGCGCGGCAATTTTGCTGTCAGAGGGTTAATGTAGGCTGGAGCTGCTTCG

oli168 GCCCTATGTTTACCACCCAGAATCCGCGCCAATCGTTAAACATATGAATATCCTCCTTAG

oli169 TTTCGTCGTTACGACGCATC

oli172 GCTGCTGTTTATCTTCAGTATGATGAAAAAGCAGGCAAGCGACTACAAAGACCATGACGG

oli173 TTGAGGATGTGCTGGAGCCGTATCTGATTCAGCAAGGCTTCATATGAATATCCTCCTTAG

oli177 TTCATGCCATTCGAGGTGCT

oli178 TTCTGGGAATGCTGTTGGTAAATAGTCCTGCCTTCGCGCATGTAGGCTGGAGCTGCTTCG

oli179 CCAGTTTTCCATCTCTTGCAACAATGCCTGCTGCGAGGTACATATGAATATCCTCCTTAG

oli180 CCGGATAGAGCATAGAGCTT

oli181 TGACACGAGTAATCAGGCGA

oli182 GTTAAAGGCGAATGAGGTCGTTGACGAAATGCTACATCATGACTACAAAGACCATGACGG

oli184 GACCACAACGCAAGAGTTACTGGCGCAAGCTGAAAAACTCTGTAGGCTGGAGCTGCTTCG

oli185 TTTTCTTCACCAGCACCGAGTGATCGTGACCACAGTTTCCCATATGAATATCCTCCTTAG

oli195 TTGTCACCAGCAGATCAATGTCGCTGTTTGGCTTCAGACCCATATG

Plasmids

Name Features (reference) pKD46 lambda red recombinase function (13)

pKD4 kanamycin resistance cassette template (13)

pKD3 chloramphenicol resistance cassette template (13)

pCP20 FLP recombinase function (13)

pSUB11 3xFLAG-kanamycin resistance cassette template (48)


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Table 3 Salmonella growth on LB plates

Chemical agent added

Growth of S. enterica serovar Typhimurium

ATCC14028 strains a

WT SA123 PP116

Cadmium

0 + + +

0.06 mM + + +

0.1 mM + + +

0.5 mM + +/- +

0.6 mM +/- - +/-

0.7 mM - - -

EDTA

0 + + +

0.02 mM + + +

0.06 mM + +/- +

0.1 mM + - +

0.5 mM + - +

1 mM + - +/-

1.2 mM + - -

1.4 mM + - -

TPEN

0 + + +

0.005 mM + + +

0.025 mM + - +

0.05 mM + - -

0.5 mM - - -

a. Bacteria were grown overnight in LB medium and then streaked on LB plates containing the indicated amounts of

EDTA, TPEN and Cadmium acetate. WT, wild type. Symbols: +, growth; -, no growth; +/-, weak growth of very small

colonies.


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Table 4 Competition assays in balb/c mice

Strain A (relevant genotype) Strain B (relevant genotype) Median

CI a

No. of

mice b P c

MA6926 (wild type) SA123 (znuA::kan) >200d 5 <0.001

MA6926 (wild type) PP116 (zinT::cam) 1.202 4 NS

SA123 (znuA::kan) SA182 (znuABC::kan) 1.021 10 NS

PP116 (zinT::cam) PP118 (zinT::cam znuA::kan ) >200d 5 <0.001

SA123 (znuA::kan) PP118 (zinT::cam znuA::kan ) 1.041 5 NS

SA182(znuABC::kan) PP119(zinT::cam znuABC::kan) 0.394 5 0.037

SA229 (yebA::cam) MA6926 (wild type) 0.85 5 NS

PP125 (zinT-scar) SA233 (znuA∆loop yebA::cam) 1.522 5 NS

SA229 (yebA::cam) SA233 (znuA∆loop yebA::cam) 1.062 4 NS

PP125 (zinT-scar) PP130 (zinT::cam znuA∆loop yebA::cam) 1.820 10 0.006

PP118 (zinT::cam znuA::kan) PP130 (zinT::cam znuA∆loop yebA::cam) 0.006 5 <0.001

a. Competitive index= output (Strain A/Strain B)/inoculum (Strain A/Strain B).

b. Experiments were performed whit BALB/c mice inoculated by the intraperitoneal route.

c. Statistical differences between output and inocula (the P-values) were determined by the Student 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 evaluate

the exact ratio between the two strains.


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S.pneumoniae ..........CSNQKQA.DGKLNIVTTFYPVYEFTKQVAGDTANVELLIGAGTEPHEYEPSAKAVAKIQDADTFVYENENMETWVPKLLDTL..DKKKVKTI

S.suis ..........CGNSTASEDGKLNIVTTFYPVYEFTKQVAGDEANVDLLVKAGTEVHGYEPSAKDIARIQEADAFVYENENMETWVHDVEKSL..DTTKVNVI

E.faecalis .....CGKTNTSDKTADGKEKLSIVTTFYPMYDFTKNIVGDEGDVKLLIPAGSEPHDYEPSAKDMATIHDADVFVYHNENMESWVPKAAKGW..KKGAPNVI

S.aureus .....CGNDDGKDK...DG.KVTIKTTVYPLQSFAEQIGGKHVKVSSIYPAGTDLHSYEPTQKDILSASKSDLFMYTGDNLDPVAKKVASTIKDKDKKLSLE

S.haemolyticus CVLVACGKEDSSNKGSKDGDKINVSTTVYPLQSFIKQIGGNHVNVSSIYPAGSDLHDYEPTQKDMLKVNKSDLFVYTGDDLDPVAKKVAATIKDDKKKVSLQ

S.Typhimurium ZnuA ......................AVVASLKPLGFIASAIADGVTDTQVLLPDGASEHDYSLRPSDVKRLQGADLVVWVGPEMEAFMEKSVRNIPDNKQVTIAQ

S.Typhimurium ZinT ......................................................................................................

E.coli ......................................................................................................

B.subtilis ......................................................................................................

ZnuA His-rich region

S.pneumoniae KATGDMLLLP.....GGEEEEGDHDH..GE.EGHHHEFDPHVWLSPVRAIKLVEHIRDSLSADYPDKKETFEKNAAAYIEKLQALDKAYAEGLSQAKQKSFV

S.suis SATEGMLLLP.....GGEEEHEGHDH..SE.EGHSHAYDPHVWLSPERAITLVENIRDSLVAKYPEKKDAFETNAAAYIEKLDALDAKYSETLSAAKQKYFV

E.faecalis KGTENMVLLP.....GSDEDGHDHDHEHGE.EGHHHELDPHTWVSPHRAIQEVTNIKEQLVKLYPKKAKTFETNAEKYLTKLTALDKEFQTALKDAKQKSFV

S.aureus DKLDKAKLLTDQHEHGEEHEHEGHDHEKEE.HHHHGGYDPHVWLDPKINQTFAKEIKDELVKKDPKHKDDYEKNYKKLNDDLKKIDNDMKQVTKDKQGNAVF

S.haemolyticus DKLDRSTLLTDQHEHGDEEHADSHEHH....HHHHGGYDPHVWLDPEKNKVFAIEIKDHLVAKDPKHKNEYEKNFKKLEHALDDIDNQLKEITKDKQGNAVF

S.Typhimurium ZnuA LADVKPLLMKGADDDEDEHAHTGADEEKGDVHHHHGEYNMHLWLSPEIARATAVAIHEKLVELMPQSRAKLDANLKDFEAQLAATDKQVGNELAPLKGKGYF


E.coli ......................................................................................................

B.subtilis ......................................................................................................

S.pneumoniae TQHAAFNYLALDYGLKQVAISGLSPDAEPSAARLAELTEYVKKNKIAYIYFEENASQALANTLSKEAGVKTDVLNPLESLTEEDTKAGE.NYISIMEKNLKA

S.suis TQHTAFAYLALDYGLKQVSITGVAADEDPTPSRLAELTEYINKYGIKYIYFEENASKSVAETLAKETGVQLDVLNPLESLTDEDMKNGK.DYISVMEDNLIA

E.faecalis TQHAAFGYLALDYGLKQVPIAGLTPEQEPTAGRLAELKKYVTDNQIRYIYFEKNANDKIAKTLADEANVQLEVLNPLESLTQKQMDNGE.DYLSVMKENLTA

S.aureus ISHESIGYLADRYGFVQKGIQNMNAE.DPSQKELTKIVKEIRDSNAKYILYEDNVANKVTETIRKETDAKPLKFYNMESLNKEQQKKDNITYQSLMKSNIEN

S.haemolyticus ISHESLGYLADRYGFVQKGIQNMNAE.DPSQKALTQLVKEINEKNVKYILYEDNVANKVTETIRKETNAKPLKFYNMESLNKEQSKDESMDYQTLMNKNIEA

S.Typhimurium ZnuA VFHDAYGYYEKHYGLTPLGHFTVNPEIQPGAQRLHEIRTQLVEQKATCVFAEPQFRPAVVEAVARGTSVRMGTLDPLGTNIKLGKTSYSAFLSQLANQYASC


E.coli ......................................................................................................

B.subtilis ........................................................................CQTSGSSAGESNQTTSSSAVEEDSSKTQEQ

S.pneumoniae LKQTTDQEGPAIEPEK.AEDTKTVQNGYFEDAAVKDRTLSDYAGNWQSVYPFLEDGTFDQVFDYKAKLTGKMTQAEYKAYYTKGYQTDVTKINITDNTMEFV

S.suis LEKTTSQEGSEILPEEGAETAQTVYNGYFEDSAVKDRTLSDYAGEWQSVYPYLLDGTLDQVWDYKAKIKGGMTAEEYKTYYDTGYKTDVDQINITDNTMEFV

E.faecalis LKKTTDTAGKEVQPETSEKTEKTVANGYFKDSEVAERTLTDYAGNWQSVYPLLKDGTLDQVFDYKAKLKKDKTPAEYKTYYDAGYQTDVDHINITDSTIEFL

S.aureus IGKALDSGVKVKDDKAESKHDKAISDGYFKDEQVKDRELSDYAGEWQSVYPYLKDGTLDEVMEHKAENDPKKSAKDLKAYYDKGYKTDITNIDIKGNEITFT

S.haemolyticus LDKALDSNIKVEDEKAEHKHDKAISDGYFKDNQVKDRALSDYEGKWQSVYPYLKNGDLDDVMKHKSEEDSAMTEKEYKAYYEKGYKTDISSINIKGDNITFE

S.Typhimurium ZnuA LKGD..................................................................................................

S.Typhimurium ZinT .......HGHHAHGAPMTEVEQKAAAGVFDDANVRDRALTDWDGMWQSVYPYLVSGELDPVFRQKAKKDPEKTFEDIKAYYRKGYVTNVETIGIENGVIEFH

E.coli .......HGHHSHGKPLTEVEQKAANGVFDDANVQNRTLSDWDGVWQSVYPLLQSGKLDPVFQKKADADKTKTFAEIKDYYHKGYATDIEMIGIEDGIVEFH

B.subtilis TSDSHTHEHSHDHSHAHDEETEKIYEGYFKNSQVKDRLLSDWEGDWQSVYPYLQDGTLDEVFSYKSEHEGGKTAEEYKEYYKKGYQTDVDRIVIQKDTVTFF

ZinT His-rich region

S.pneumoniae QGGQSKKYTYKYVGKKILTYKKGNRGVRFLFEATDADAGQF.KYVQFSDHNIAPVKAEHFHIFFGGTSQEALFEEMDNWPTYYPDNLSGQEIAQEMLAH

S.suis VGDKKEKFTYKYVGYKILTYKKGNRGVRFLFEATDANAGNY.KYVQFSDHNIAPVKTGHFHIYFGGESQEKLLEELENWPTYYPVGLTGLEIGQEMLAH

E.faecalis VNGKPQKFTYKAAGYKILNYAKGNRGVRFLFETDDANAGRF.KYVQFSDHNIAPTKAAHFHIFFGGDSQESLFNEMDNWPTYYPSDLSKQEIAQEMIAH

S.aureus KDGKKHTGKYEYNGKKTLKYPKGNRGVRFMFKLVDGNDKDLPKFIQFSDHNIAPKKAEHFHIFMGNDN.DALLKEMDNWPTYYPSKLNKDQIKEEMLAH

S.haemolyticus KNGKKVTGTYKYVGKKILDYKKGNRGVRFIFKLTNDDTQQLPKYVQFSDHNIAPKKAEHFHIFMGDNN.DSLLKEMDHWPTYYPASLDKDDIKEEMLAH

S.Typhimurium ZnuA ...................................................................................................

S.typhimurium ZINT RDNNVASCKYNYAGYKILTYASGKKGVRYLFECKDANSKA.PKYVQFSDHIIAPRKSAHFHIFMGNTSQQALLQEMENWPTYYPYQLKANEVVDEMLHH

E.coli RNNETTSCKYDYDGYKILTYKSGKKGVRYLFECKDPESKA.PKYIQFSDHIIAPRKSSHFHIFMGNDSQQSLLNEMENWPTYYPYQLSSEEVVEEMMSH

B.subtilis KNGKEYSGKYTYDGYEILTYDAGNRGVRYIFKLAK.KAEGLPQYIQFSDHSIYPTKASHYHLYWGDDR.EALLDEVKNWPTYYPSKMDGHDIAHEMMAH

Figure 1


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Figure 2


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Figure 3


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Figure 4


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Fig.5

0 1 2 3 4 5 6 7 8 9 100.0

0.5

1.0

1.5

2.0

2.5

time (hours)

OD

60

0


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Figure 6

0 1 2 3 4 5 6 7 8 9 10 11 120.00

0.25

0.50

0.75

1.00

1.25

1.50

Time (h)

OD

60

0


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Figure 7


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Documents

The Zur-regulated ZinT protein is an auxiliary component of the high