Abstract

A locus involved in zinc(II) uptake in Escherichia coli K-12 was identified through the generation of a zinc(II)-resistant mutant by transposon (Tn10dCam) mutagenesis. The mutation was located within the pitA gene, which encodes the low-affinity inorganic phosphate transport system (Pit). The pitA mutant accumulated reduced amounts of zinc(II) when exposed to 0.5–2.0 mM ZnSO4 during growth in Luria–Bertani medium.

Introduction

Zinc(II) is an essential trace metal ion for all organisms, but above optimal concentrations it is toxic. The intracellular concentration of zinc(II) must therefore be regulated within very narrow limits. Uptake and intracellular transport systems must ensure an adequate supply of zinc(II) ions to Zn(II)-dependent proteins, but mechanisms must also exist for the sequestration or efflux of zinc(II) when in excess. Recent research has led to a rapid increase in our understanding of both uptake and efflux systems.

Growth of Escherichia coli at high concentrations of zinc(II) results in Zn(II) export through ZntA, a cation transport (P-type) ATPase [1,2]. The expression of zntA is regulated by ZntR, a zinc(II)-responsive homologue of the transcriptional regulator MerR [3]. Homologues of ZntA have been identified in several bacteria such as Synechocystis sp. (ZiaA) [4], Proteus mirabilis (ZntA) [5], Staphylococcus aureus (ZntA) [6], Helicobacter pylori (CadA) [7], Alcaligenes eutrophus (the Czc plasmid-encoded system) [8] and Pseudomonas aeruginosa (Czr) [9].

Uptake systems for metal ions are frequently duplicated in bacteria: constitutive, low-affinity systems may co-exist with inducible high-affinity systems. High-affinity zinc(II) uptake systems have recently been identified in several bacteria. In E. coli, zinc(II) deficiency induces the expression of the ZnuABC Zn(II) uptake system, which belongs to the superfamily of ATP-binding cassette (ABC) transporters [10]. Under conditions of zinc(II) sufficiency, expression of the pump is repressed by the Fur homologue Zur. Genes encoding homologues of ZnuABC have been identified in a number of other bacteria such as Streptococcus pneumoniae[11], Streptococcus pyogenes[12], Haemophilus influenzae[13], Haemophilus ducreyi[14] and Listeria monocytogenes (in which a Zur-like protein was also identified [15]). In Bacillus subtilis, two gene loci have been implicated in zinc(II) uptake [16]: the ycdH-containing operon encodes a putative high-affinity zinc(II)-translocating ABC transporter, whereas yciC encodes an ATP-binding integral membrane protein that may form part of a low-affinity system.

Mutations in genes encoding components of metal uptake systems may confer a metal-resistant or metal-dependent phenotype (e.g. [11,13,14,16–19]). To identify other genetic loci involved in zinc(II) uptake in E. coli, we have sought mutations that confer a zinc(II)-resistant phenotype. We describe here the properties of a mutant that showed enhanced resistance to Zn(II). The mutation is located within pitA, which encodes the inorganic phosphate transport system (Pit) in E. coli[20].

Materials and methods

Bacterial strains and phages

Derivatives of E. coli K-12 were used throughout. Strain GE2515 was used for transposon mutagenesis [21], and strain DH5α[22] was used as a host for cloning. Strains GE2515 and MG1655 (wild-type) were kindly provided by H.D. Williams (Imperial College of Science and Technology, London, UK). Hfr and P1 mapping strains [23] were kindly provided by C.A. Gross (University of California, San Francisco, CA, USA). Bacteriophage P1vir was used for generalised transductions.

Isolation of zinc(II)-resistant mutants

Random insertional mutagenesis of E. coli GE2515 was carried out using the Tn10dCam transposition system as described by Heath et al. [21]. The mutagenised cells were harvested by centrifugation, resuspended in 0.2 ml of Luria–Bertani (LB) medium, and serial dilutions were plated on LB agar containing chloramphenicol and ZnSO4 (at a range of concentrations between 0 and 10 mM).

Analyses of metal sensitivity and intracellular zinc(II) accumulation

Metal sensitivity was assessed by monitoring the turbidity of cultures containing varying concentrations of ZnSO4 or CdCl2. Cultures were grown with shaking at 37°C for 30 h. Sensitivity to these metal ions was also assessed by streaking cells across metal gradient agar plates [1]. Amounts of intracellular Zn(II) were measured after cells were exposed to various concentrations of ZnSO4 during exponential growth as described previously [1].

Molecular genetic techniques

Generalised transductions were carried out using bacteriophage P1vir as described by Silhavy et al. [24]. Hfr matings were performed as described by Singer et al. [23]. Methods for the manipulation and analysis of DNA were performed as described in Sambrook et al. [25]. Nucleotide sequencing was performed using an ABI Prism 377 automated sequencer.

Cloning and sequencing of a DNA fragment containing Tn10dCam

Genomic DNA from SJB201 was digested with SalI and ligated to SalI-cut pBR322. This mixture was introduced into E. coli DH5α and chloramphenicol-resistant transformants were selected. A plasmid (pRKP1080) isolated from one CmR transformant contained a 4.6-kb insert, which was shown to contain Tn10dCam by restriction analysis. To construct a plasmid containing only one end of Tn10dCam for sequencing, plasmid pRKP1080 was digested with EcoRI (which cuts once within Tn10dCam), religated and introduced into strain DH5α. A plasmid (pRKP1081) was isolated from an ApR, CmS transformant, and it was found to contain a 1.6-kb insert. This was sequenced using an oligonucleotide primer RP57 (5′-GTATTTTACCAAAATCATTAGGGG-3′) which is complementary to a region at the end of Tn10dCam.

Results and discussion

Isolation of a zinc(II)-resistant mutant

Following Tn10dCam mutagenesis, zinc(II)-resistant mutants were identified as colonies growing on LB agar containing 2.5 mM added ZnSO4; no colonies appeared after cells from a non-mutagenised culture were plated onto the same medium. The disrupted allele in one mutant was co-transduced with the CmR marker into the wild-type strain MG1655. The resulting transductant (designated SJB201) appeared to exhibit increased resistance to both Zn(II) and Cd(II) ions when growth was assessed across metal gradient plates (Fig. 1). The zinc(II)-resistant phenotype of SJB201 was confirmed by comparing its growth with that of MG1655 in batch cultures containing added ZnSO4 (Fig. 2). At the lowest Zn(II) concentration (1.5 mM), the wild-type strain began to grow after a considerably prolonged lag phase (>8 h), by which time the pitA mutant had reached stationary phase. We were unable to confirm the Cd(II)-resistant phenotype of SJB201 in batch culture.

1

Growth of SJB201 (pitA) and GE2515 (pitA+) across concentration gradients of Zn(II) and Cd(II). Freshly grown biomass was streaked across solidified glycerol-glycerophosphate minimal medium [1] containing chloramphenicol and concentration gradients of (A) ZnSO4 (0.60 mM maximum) and (B) CdCl2 (0.20 mM maximum). The plates were incubated at 37°C for 3 days.

1

Growth of SJB201 (pitA) and GE2515 (pitA+) across concentration gradients of Zn(II) and Cd(II). Freshly grown biomass was streaked across solidified glycerol-glycerophosphate minimal medium [1] containing chloramphenicol and concentration gradients of (A) ZnSO4 (0.60 mM maximum) and (B) CdCl2 (0.20 mM maximum). The plates were incubated at 37°C for 3 days.

2

Growth of strains SJB201 (closed symbols) and MG1655 (open symbols) in LB in the absence (●) or presence of 1.5 mM (▪), 1.75 mM (♦) or 2.0 mM (▲) ZnSO4. Each experimental point is a single measurement, but the entire experiment was repeated three times with similar results.

2

Growth of strains SJB201 (closed symbols) and MG1655 (open symbols) in LB in the absence (●) or presence of 1.5 mM (▪), 1.75 mM (♦) or 2.0 mM (▲) ZnSO4. Each experimental point is a single measurement, but the entire experiment was repeated three times with similar results.

Genetic mapping of the Tn10dCam insertion

Using a set of Hfr mapping strains that contain Tn10[23], the Tn10dCam element was estimated to lie between 67 and 81.75 min on the chromosome of SJB201. Transduction with a P1vir lysate prepared from one (CAG18450) of a set of P1 mapping strains [23] that cover this region generated chloramphenicol-sensitive transductants (with concomitant loss of the zinc(II)-resistant phenotype) at a frequency of approximately 10%. The mutation in SJB201 was estimated to map between 74.5 and 75.5 min on the E. coli genetic map [26].

Identity of the mutated gene conferring resistance to Zn(II)

To identify the precise location of the Tn10dCam insertion in the genome of SJB201, a DNA fragment containing the transposon was cloned and sequenced. The first 279 nucleotides adjacent to one end of Tn10dCam showed 100% identity to the pitA gene from E. coli[27], which encodes Pit [20]. The Tn10dCam element was located at nucleotide 1169 in the 1497-bp pitA open reading frame (ORF). The downstream ORF yhiO is transcribed towards pitA, so it is unlikely that the Tn10dCam insertion affected its function. The predicted amino acid sequence of PitA indicates that it contains 10 potential transmembrane domains, and that it lacks any of the typical zinc(II)-binding motifs.

Intracellular accumulation of Zn(II) by strains SJB201 and MG1655

The amounts of intracellular Zn(II) accumulated by strains SJB201 (pitA) and MG1655 (pitA+) were measured after cells were exposed to various concentrations of ZnSO4 during exponential growth, as described previously [1] (Fig. 3). In these experiments, the minimal inhibitory concentrations (MICs) of ZnSO4 were 1.5 mM for MG1655 and 2.0 mM for SJB201. The amounts of intracellular Zn(II) accumulated by SJB201 were significantly lower than the amounts accumulated by MG1655 when ZnSO4 was added in the concentration range 0.5–2.0 mM (Fig. 3). At higher, growth inhibitory Zn(II) concentrations (2.5–9.5 mM), however, SJB201 was found to accumulate slightly higher levels of Zn(II) than the wild-type (data not shown). The amounts of Zn(II) accumulated by MG1655 were found repeatedly to be about one order of magnitude greater than the amounts accumulated by its RecA derivative, strain SJB124 (MG1655 recA::Tn10), as reported previously [1]. A possible explanation for this discrepancy may be that SJB124 exhibits enhanced Zn(II) efflux, since the Tn10-encoded TetA protein has been shown to transport divalent cation–tetracycline complexes [28].

3

Intracellular accumulation of Zn(II) by strains SJB201 (pitA) (▪) and MG1655 (pitA+) (◻) after the addition of ZnSO4 (up to 2 mM) to cultures in exponential phase [1]. Means of triplicate experiments are plotted; S.D.s are smaller than the symbol size.

3

Intracellular accumulation of Zn(II) by strains SJB201 (pitA) (▪) and MG1655 (pitA+) (◻) after the addition of ZnSO4 (up to 2 mM) to cultures in exponential phase [1]. Means of triplicate experiments are plotted; S.D.s are smaller than the symbol size.

Conclusions

We have demonstrated that insertional mutagenesis of the pitA gene in E. coli confers increased resistance to zinc(II), and that this is associated with reduced accumulation of Zn(II) when cells are exposed to concentrations of ZnSO4 below the MIC. This indicates that the Pit system represents a second, low-affinity uptake system for zinc(II) in E. coli, in contrast to the ZnuABC high-affinity uptake system that has been described recently [10]. The Pit system is expressed constitutively in E. coli[29], whereas expression of ZnuABC is repressed at high concentrations of Zn(II) [10].

Previous studies (e.g. [17,30–32]) have shown that Pit of prokaryotes mediates the uptake of Pi and divalent cations, although transport of zinc(II) ions via Pit has not been reported before. For example, Mg2+, Ca2+, Mn2+ and Co2+ were shown to be transported as neutral metal–phosphate (MeHPO4) complexes via the Pit systems in E. coli[30] and Acinetobacter johnsonii[31]. In B. subtilis, uptake of these metal ions was stimulated by inorganic phosphate [17], and a Pit mutant exhibited reduced transport of Ca2+ and Co2+. Pit mutants of B. subtilis and E. coli also exhibit increased resistance to arsenate [17,18], a competitive inhibitor of Pi transport. Although cobalt(II) was shown to be transported via the Pit system in E. coli[30], the pitA mutant SJB201 did not exhibit a Co(II)-resistant phenotype: a possible explanation for this is that the mutant accumulated normal levels of Co(II) as a result of uptake via the constitutively expressed CorA system [19]. We have performed experiments to examine the effects on Zn(II) resistance of altering the concentration of inorganic phosphate in the growth medium: these were hampered, however, by the formation of a precipitate in the glycerol-glycerophosphate minimal medium [1] containing Pi (instead of glycerophosphate) and ZnSO4 (results not shown).

The observation that the pitA mutant accumulated slightly more zinc(II) than the wild-type at high, toxic, external Zn(II) concentrations (>2 mM) is unexplained, since the phenotype of the pitA mutant, the metal analyses of cells challenged with lower zinc concentrations (Fig. 3) and earlier reports of divalent metal transport by PitA (reviewed in [32]) all demonstrate that this system is an importer. One possible explanation is that high Zn concentrations lead to decline in intracellular polyphosphate pools, as demonstrated for Klebsiella aerogenes exposed to cadmium (reviewed in [33]). Keasling [33] proposes that the phosphate released from polyphosphate (by increased polyphosphatase activity) is transported via Pit out of the cell accompanied by cations, which may be toxic metal ions. Thus the Pit system may under certain conditions mediate Zn(II) efflux, or metal exchange [32] as demonstrated for Mg2+ in E. coli[30].

Acknowledgements

We thank Gareth Owen for his assistance in isolating the zinc(II)-resistant mutant. We are grateful to C.A. Gross and H.D. Williams for providing E. coli strains. S.J. Beard was supported by a BBSRC studentship. R. Hashim was supported by SIRIM Bhd., Government of Malaysia.

References

[1]
Beard
S.J.
Hashim
R.
Membrillo-Hernández
J.
Hughes
M.N.
Poole
R.K.
(
1997
)
Zinc(II) tolerance in Escherichia coli K-12: evidence that the zntA gene (o732) encodes a cation transport ATPase
.
Mol. Microbiol.
 
25
,
883
891
.
[2]
Rensing
C.
Mitra
B.
Rosen
B.P.
(
1997
)
The zntA gene of Escherichia coli encodes a Zn(II)-translocating P-type ATPase
.
Proc. Natl. Acad. Sci. USA
 
94
,
14326
14331
.
[3]
Brocklehurst
K.R.
Hobman
J.L.
Lawley
B.
Blank
L.
Marshall
S.J.
Brown
N.L.
Morby
A.P.
(
1999
)
ZntR is a Zn(II)-responsive MerR-like transcriptional regulator of zntA in Escherichia coli
.
Mol. Microbiol.
 
31
,
893
902
.
[4]
Thelwell
C.
Robinson
N.J.
Turner-Cavet
J.S.
(
1998
)
An SmtB-like repressor from Synechocystis PCC 6803 regulates a zinc exporter
.
Proc. Natl. Acad. Sci. USA
 
95
,
10728
10733
.
[5]
Rensing
C.
Mitra
B.
Rosen
B.P.
(
1998
)
A Zn(II)-translocating P-type ATPase from Proteus mirabilis
.
Biochem. Cell Biol.
 
76
,
787
790
.
[6]
Xiong
A.
Jayaswal
R.K.
(
1998
)
Molecular characterization of a chromosomal determinant conferring resistance to zinc and cobalt ions in Staphylococcus aureus
.
J. Bacteriol.
 
180
,
4024
4029
.
[7]
Herrmann
L.
Schwan
D.
Garner
R.
Mobley
H.L.T.
Haas
R.
Schafer
K.P.
Melchers
K.
(
1999
)
Helicobacter pylori cadA encodes an essential Cd(II)-Zn(II)-Co(II) resistance factor influencing urease activity
.
Mol. Microbiol.
 
33
,
524
536
.
[8]
Nies
D.H.
Silver
S.
(
1989
)
Plasmid-determined inducible efflux is responsible for resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus
.
J. Bacteriol.
 
171
,
896
900
.
[9]
Hassan
M.E.T.
van der Lelie
D.
Springael
D.
Romling
U.
Ahmed
N.
Mergeay
M.
(
1999
)
Identification of a gene cluster, czr, involved in cadmium and zinc resistance in Pseudomonas aeruginosa
.
Gene
 
238
,
417
425
.
[10]
Patzer
S.I.
Hantke
K.
(
1998
)
The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli
.
Mol. Microbiol.
 
28
,
1199
1210
.
[11]
Dinthilhac
A.
Alloing
G.
Granadel
C.
Claverys
J.-P.
(
1997
)
Competence and virulence of Streptococcus pneumoniae: AdcA and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases
.
Mol. Microbiol.
 
25
,
727
739
.
[12]
Janulczyk
R.
Pallon
J.
Björck
L.
(
1999
)
Identification and characterization of a Streptococcus pyogenes ABC transporter with multiple specificity for metal cations
.
Mol. Microbiol.
 
34
,
596
606
.
[13]
Lu
D.S.
Boyd
B.
Lingwood
B.
(
1997
)
Identification of the key protein for zinc uptake in Hemophilus influenzae
.
J. Biol. Chem.
 
272
,
29033
29038
.
[14]
Lewis
D.A.
Klesney-Tait
J.
Lumbley
S.R.
Ward
C.K.
Latimer
J.L.
Ison
C.A.
Hansen
E.J.
(
1999
)
Identification of the znuA-encoded periplasmic zinc transport protein of Haemophilus ducreyi
.
Infect. Immun.
 
67
,
5060
5068
.
[15]
Dalet
K.
Gouin
E.
Cenatiemo
Y.
Cossart
P.
Hechard
Y.
(
1999
)
Characterisation of a new operon encoding a Zur-like protein and an associated ABC zinc permease in Listeria monocytogenes
.
FEMS Microbiol. Lett.
 
174
,
111
116
.
[16]
Gaballa
A.
Helmann
J.D.
(
1998
)
Identification of a zinc-specific metalloregulatory protein, Zur, controlling zinc transport operons in Bacillus subtilis
.
J. Bacteriol.
 
180
,
5815
5821
.
[17]
Kay
W.W.
Ghei
O.K.
(
1981
)
Inorganic cation transport and the effects on C4 dicarboxylate transport in Bacillus subtilis
.
Can. J. Microbiol.
 
27
,
1194
1201
.
[18]
Bennett
R.L.
Malamy
M.H.
(
1970
)
Arsenate resistant mutants of Escherichia coli and phosphate transport
.
Biochem. Biophys. Res. Commun.
 
40
,
496
503
.
[19]
Park
M.H.
Wong
B.B.
Lusk
J.E.
(
1976
)
Mutants in three genes affecting transport of magnesium in Escherichia coli: genetics and physiology
.
J. Bacteriol.
 
126
,
1096
1103
.
[20]
Elvin
C.M.
Dixon
N.E.
Rosenberg
H.
(
1986
)
Molecular cloning of the phosphate (inorganic) transport (pit) gene of Escherichia coli K12
.
Mol. Gen. Genet.
 
204
,
477
484
.
[21]
Heath
J.D.
Perkins
J.D.
Sharma
B.
Weinstock
G.M.
(
1992
)
NotI genomic cleavage map of Escherichia coli K-12 strain MG1655
.
J. Bacteriol.
 
174
,
558
567
.
[22]
Hanahan
D.
(
1983
)
Studies on transformation of Escherichia coli with plasmids
.
J. Mol. Biol.
 
166
,
557
580
.
[23]
Singer
M.
Baker
T.A.
Schnitzer
G.
Dieschel
S.M.
Goel
M.
Dove
W.
Jaacks
K.J.
Grossman
A.D.
Erickson
J.W.
Gross
C.A.
(
1989
)
A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli
.
Microbiol. Rev.
 
53
,
1
24
.
[24]
Silhavy
T.J.
Berman
M.L.
Enquist
L.W.
(
1984
)
Experiments with Gene Fusions
 .
Cold Spring Harbor Laboratory Press
,
Cold Spring Harbor, NY
.
[25]
Sambrook
J.
Fritsch
E.F.
Maniatis
T.
(
1989
)
Molecular Cloning: A Laboratory Manual
 ,
2nd
edn.
Cold Spring Harbor Laboratory Press
,
Cold Spring Harbor, NY
.
[26]
Berlyn
M.K.B.
Low
K.B.
Rudd
K.E.
(
1996
)
Linkage map of Escherichia coli K-12
. In:
Escherichia coli and Salmonella, Cellular and Molecular Biology
 ,
2nd
edn. (
Neidhardt
F.C.
Curtiss
R.
III
Ingraham
J.L.
Lin
E.C.C.
Low
K.B.
Magasanik
B.
Reznikoff
W.S.
Riley
M.
Schaechter
M.
Umbarger
H.E.
, Eds.), pp.
1715
1902
.
American Society for Microbiology Press
,
Washington, DC
.
[27]
Sofia
H.J.
Burland
V.
Daniels
D.L.
Plunkett
G.
III
Blattner
F.R.
(
1994
)
Analysis of the Escherichia coli genome. V. DNA sequence of the region from 76.0 to 81.5 minutes
.
Nucleic Acids Res.
 
22
,
2576
2586
.
[28]
Yamaguchi
A.
Udagawa
T.
Sawai
T.
(
1990
)
Transport of divalent cations with tetracycline as mediated by the transposon Tn10-encoded tetracycline resistance protein
.
J. Biol. Chem.
 
265
,
4809
4813
.
[29]
Rosenberg
H.
Gerdes
R.G.
Chegwidden
K.
(
1977
)
Two systems for the uptake of phosphate in Escherichia coli
.
J. Bacteriol.
 
131
,
505
511
.
[30]
Van Veen
H.W.
Abee
T.
Kortstee
G.J.J.
Konings
W.N.
Zehnder
A.J.B.
(
1994
)
Translocation of metal phosphate via the phosphate inorganic transport system of Escherichia coli
.
Biochemistry
 
33
,
1766
1770
.
[31]
Van Veen
H.W.
Abee
T.
Kortstee
G.J.J.
Konings
W.N.
Zehnder
A.J.B.
(
1993
)
Mechanism and energetics of the secondary phosphate-transport system of Acinetobacter-johnsonii-210A
.
J. Biol. Chem.
 
268
,
19377
19383
.
[32]
Van Veen
H.W.
(
1997
)
Phosphate transport in prokaryotes: molecules, mediators and mechanisms
.
Antonie van Leeuwenhoek
 
72
,
299
315
.
[33]
Keasling
J.D.
(
1997
)
Regulation of intracellular toxic metals and other cations by hydrolysis of polyphosphate
.
Ann. N.Y. Acad. Sci.
 
829
,
242
249
.

Author notes

1School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK.