the zur-regulated zint protein is an auxiliary component of the high
TRANSCRIPT
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
36
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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
99
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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
243
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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
S.Typhimurium ZinT ......................................................................................................
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
S.Typhimurium ZinT ......................................................................................................
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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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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