JOURNAL OF BACTERIOLOGY, Mar. 2009, p. 1595–1603
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 5
Regulation and Activity of a Zinc Uptake Regulator, Zur, in
Kelsy F. Smith,1Lori A. Bibb,2Michael P. Schmitt,2and Diana M. Oram1*
Department of Microbial Pathogenesis, University of Maryland Baltimore, Baltimore, Maryland 21201,1and FDA/CBER,
Bethesda, Maryland 208922
Received 3 October 2008/Accepted 8 December 2008
Regulation of metal ion homeostasis is essential to bacterial cell survival, and in most species it is controlled by
metal-dependent transcriptional regulators. In this study, we describe a Corynebacterium diphtheriae ferric uptake
regulator-family protein, Zur, that controls expression of genes involved in zinc uptake. By measuring promoter
activities and mRNA levels, we demonstrate that Zur represses transcription of three genes (zrg, cmrA, and troA) in
zinc-replete conditions. All three of these genes have similarity to genes involved in zinc uptake. Transcription of zrg
and cmrA was also shown to be regulated in response to iron and manganese, respectively, by mechanisms that are
independent of Zur. We demonstrate that the activity of the zur promoter is slightly decreased under low zinc
conditions in a process that is dependent on Zur itself. This regulation of zur transcription is distinctive and has not
yet been described for any other zur. An adjacent gene, predicted to encode a metal-dependent transcriptional
regulator in the ArsR/SmtB family, is transcribed from a separate promoter whose activity is unaffected by Zur. A
C. diphtheriae zur mutant was more sensitive to peroxide stress, which suggests that zur has a role in protecting the
bacterium from oxidative damage. Our studies provide the first evidence of a zinc specific transcriptional regulator
in C. diphtheriae and give new insights into the intricate regulatory network responsible for regulating metal ion
concentrations in this toxigenic human pathogen.
Metal homeostasis in bacteria is mediated by five families of
metal-dependent transcriptional regulators: DtxR, Fur, ArsR/
SmtB, MerR, and NikR (35). Proteins within each structurally
related family respond to the intracellular levels of essential
metals such as manganese, iron, and zinc. The DtxR, Fur, and
the less-characterized NikR family proteins predominantly
regulate genes required for metal uptake, whereas ArsR/SmtB
and MerR family proteins regulate metal efflux (35). Control
of metal concentrations inside the bacterial cell is essential for
survival and plays a key role in bacterial pathogenesis. For
instance, a large number of bacterial toxins are expressed un-
der metal-depleted conditions, and this expression is con-
trolled by metal-dependent transcriptional regulators. In
Corynebacterium diphtheriae, the etiological agent of the toxin-
mediated upper respiratory tract infection diphtheria, DtxR,
regulates transcription of the diphtheria toxin gene and mul-
tiple other genes involved in iron uptake (18, 38, 40). In addi-
tion to DtxR, the C. diphtheriae genome encodes a second
DtxR-like protein, MntR, which responds to Mn (37, 38), and
it is predicted to encode one Fur, three ArsR/SmtB, four
MerR, and zero NikR homologs.
Iron-dependent global gene regulation is controlled by
DtxR-like proteins in many gram-positive and acid-fast bacte-
rial species, including C. diphtheriae, and by Fur-like proteins
in many gram-negative bacteria. Although DtxR-like and Fur-
like proteins share little sequence homology, they both act as
transcriptional repressors when complexed with a cognate
metal ion. In metal-depleted conditions, apo-Fur and apo-
DtxR (metal-free forms) are unable to bind their target pro-
moters, allowing transcription of the downstream genes (22).
The Fur homolog in C. diphtheriae is uncharacterized but has
similarity to a zinc-dependent regulator found in Mycobacte-
rium tuberculosis, Zur (for zinc uptake regulator) (23).
Zinc is essential for cell survival, serving as a cofactor for
more than 300 enzymes such as superoxide dismutase and
alcohol dehydrogenase. It also functions as a structural scaffold
for RNA polymerase, tRNA synthetases, and approximately 40
additional proteins (10, 11, 31, 41). In fact, Escherichia coli
requires as much zinc as it does iron and calcium for cellular
processes (31). In addition, zinc protects sulfhydryl groups
from free radicals and inhibits free radical formation by com-
peting with redox-active metals such as iron. Alternatively,
high concentrations of zinc can be toxic by blocking thiols and
binding of other metals to their cognate active sites within
enzymes such as cytochrome c oxidase (3, 5, 26).
Unlike iron homeostasis, which has been extensively char-
acterized (1), little research has focused on the uptake and/or
storage of other metals, including zinc, by pathogenic bacteria.
High-affinity zinc uptake is mediated by several ABC trans-
porters, such as the Ycdhi-yceA and zinc uptake ABC systems
(ZnuABC) (16). ZnuA encodes a periplasmic binding protein,
ZnuB encodes an integral protein, and ZnuC encodes the
ATPase of the transporter. In addition, YciC (CobW) may
serve as a low-affinity zinc uptake protein or a metallochaper-
one (16). The total zinc concentration within bacteria is typi-
cally in the millimolar range; however, free unbound intracel-
lular zinc concentrations at femtomolar levels are sufficient to
trigger zinc uptake or efflux (31). Zinc uptake is controlled in
a zinc-dependent manner by Zur in bacteria such as E. coli,
Bacillus subtilis, and M. tuberculosis (14, 23, 34). The impor-
tance of zinc homeostasis in bacterial pathogenesis is only
* Corresponding author. Mailing address: Department of Microbial
Pathogenesis, University of Maryland Dental School, 650 W. Balti-
more St. 7 South, Baltimore, MD 21201. Phone: (410) 706-8705. Fax:
(410) 706-0865. E-mail: email@example.com.
?Published ahead of print on 12 December 2008.
beginning to be characterized, but Salmonella enterica serovar
Typhimurium mutants lacking Zur or ZnuC have decreased
virulence in mice, indicating an essential role for zinc in this
model of infection (7).
diphtheriae and demonstrate that it is a zinc-responsive regulator
and is therefore more accurately described as zur (for zinc uptake
regulator). In the presence of zinc, C. diphtheriae Zur represses
transcription of three genes predicted to be involved in zinc up-
take: cmrA, whose product is predicted to be anchored to the
surface of the cell; troA (for transport related operon A), whose
product has homology to metal uptake membrane proteins, such
as Streptococcus pneumoniae psaA (16) and Treponema pallidum
troA (17); and zrg (for zinc-regulated gene), which is similar to
yciC (14). Interestingly, the transcription of zur itself is repressed
in zinc-depleted conditions by a process that involves Zur. This
observation indicates that Zur may have a role in controlling its
own expression by a mechanism that is independent of its char-
acterized activity as a zinc-dependent DNA-binding protein. The
present study is the first molecular characterization of a zinc-
dependent transcriptional regulator in C. diphtheriae, a paradigm
for pathogenesis and gene expression in gram-positive and acid-
MATERIALS AND METHODS
Culture media, strains, and growth conditions. C. diphtheriae strains
NCTC13129 (9), the sequenced isolate of the 1990s outbreak in the former
Soviet Union, and C7(?) (2), a toxigenic strain used extensively for experimental
work since the 1950s, were cultured in PGT, a casein hydrolysate medium (2).
Metal ions were removed from the medium by treating with 10g/liter Chelex-100
(Bio-Rad, Hercules, CA) for 2 h, followed by filter sterilization (42). Where
indicated, supplementation with specific metals was done at the following con-
centrations: 10 ?M FeCl3, 10 ?M MnCl2, 5 ?M CuSO4, and 25 ?M ZnSO4. To
further chelate zinc remaining in the medium following treatment with Chelex-
100, a zinc-specific chelator N,N,N?,N?-tetrakis (2-pyridylmethyl)-ethylene di-
amine (TPEN) was added to a concentration of 20 ?M. E. coli TE1 (20) was used
for cloning, and E. coli S17-1 (39), an RP4 mobilizing strain, was used as a donor
for the conjugative transfer of plasmids into C. diphtheriae. E. coli was cultured
in Luria-Bertani broth (LB) (25). Kanamycin, spectinomycin, and nalidixic acid
were added at concentrations of 20, 100, and 20 ?g/ml, respectively.
Deletion of zur in C. diphtheriae NCTC13129. To construct a deletion within
zur, the plasmid pK19mobsacB, which contains an origin of replication that
functions in E. coli, but not C. diphtheriae, was utilized (36). The extreme 5? and
3? ends of zur were amplified by using PCR (the primers are listed in Table 1),
and the resulting DNA fragments were digested with SalI/ApaI and ApaI/SphI.
The digested PCR fragments were ligated to SalI/SphI-digested pK19mobsacB
to construct pK19mobsacB?zur. This plasmid, which includes a deleted zur gene
(lacking 369 bp of the 426-bp gene), was transformed into E. coli S17-1 and then
mated into NCTC13129 (44). Kanamycin-resistant transconjugants were coun-
terselected for resistance to 10% sucrose, indicating the loss of integrated plas-
mid vector. A deletion of the zur gene on the chromosome of NCTC13129 was
confirmed by PCR and sequencing, and this strain was named NCTC13129?zur.
Complementation of NCTC13129?zur. Full-length wild-type zur, along with its
native promoter and ribosomal binding site, was amplified from NCTC13129 by
using PCR (see primers in Table 1). The resulting fragment was digested with
SacI and ligated into pKPIM (30), which had been identically digested to create
pKPIMzur. Purified pKPIMzur was then transformed into S17-1 and transferred
to NCTC13129 strains via conjugation. The plasmid pKPIM integrates site spe-
cifically at attB2 in NCTC13129. Incorporation of the plasmid into the chromo-
some via an attP site was confirmed by detecting the recombinant attL2 site
created by integration of the plasmid using PCR (30).
Resistance to killing by hydrogen peroxide. Resistance to killing by hydrogen
peroxide, H2O2, was assayed as described previously (28). Briefly, in the zone of
inhibition assay, we measured the diameters of the zones of inhibition of bacte-
rial growth when 20 ?l of 1 M H2O2was applied to 0.6-cm-diameter paper disks
in the centers of plates containing heart infusion agar and lawns of various strains
of C. diphtheriae.
In the percent killing assay, cultures were grown in PGT until the absorbance
of the culture measured at 600 nm was between 1 and 2, at which time H2O2was
added to the growth medium at a final concentration of 10 mM, and the cultures
were then incubated for an additional 10 min. Viable counts from each culture
were then determined by plating dilutions of the culture onto heart infusion agar.
Cloning of promoter regions and ?-galactosidase assays. PCR was used to
amplify ?200 bp of the region upstream of arsR (dip1709), zur (dip1710), and zrg
(dip1486) (see primers in Table 1). The resulting fragments were digested with
the restriction enzymes BamHI/SalI (arsR and zur) or SalI/BglII (zrg) and then
ligated into a similarly digested pSPZ (29), a reporter vector with a promoterless
lacZ gene. The upstream regions of cmrA (dip2325) and troA (dip0438) were
TABLE 1. Primers used in this study
Cloning promoter for arsR-like gene
Cloning promoter for arsR-like gene
Cloning promoter for zur
Cloning promoter for zur
Cloning and deletion of zur
Cloning and deletion of zur
Cloning and deletion of zur
Cloning and deletion of zur
Cloning promoter for zrg
Cloning promoter for zrg
Detection of arsR-zur cotranscript
Detection of arsR-zur cotranscript
qRT-PCR for troA
qRT-PCR for troA
qRT-PCR for arsR-like gene
qRT-PCR for arsR-like gene
qRT-PCR for cmrA
qRT-PCR for cmrA
qRT-PCR for zrg
qRT-PCR for zrg
qRT-PCR for zur
qRT-PCR for zur
qRT-PCR for gyrB
qRT-PCR for gyrB
1596SMITH ET AL.J. BACTERIOL.
digested and purified from pcmrA-PO (4) and pdip0438 (M. P. Schmitt, unpub-
lished data), respectively, with the restriction enzymes SphI/XmaI and ligated
into identically digested pSPZ. The constructs were transformed via electropo-
ration into C. diphtheriae NCTC13129 and isogenic ?zur strains. For the ?-ga-
lactosidase assays, strains were inoculated in PGT containing different metal
concentrations and incubated with shaking at 37°C overnight. Twenty micromo-
lar TPEN was added to the medium in zinc depleted conditions. ?-Galactosidase
assays were performed on overnight cultures as previously described (25, 38).
pSPZtox (29) and pSPZcmrA were used as positive controls for iron- and zinc-
depleted conditions, respectively. The plasmid pSPZmntA was constructed by
cloning the mntA promoter containing fragment from pCMmntA (pPO3), and it
was used as a control for manganese conditions (37).
Quantitative reverse transcriptase PCR (qRT-PCR). RNA was isolated, using
the RNApro Blue kit (QBioGene), from NCTC13129, NCTC13129?zur, and
C7(?) grown in zinc-replete and -depleted conditions. DNA was eliminated from
RNA samples by using New England Biolab RNase-free DNase as described by
the manufacturer. cDNA was synthesized from RNA templates by using the
SuperScript III RT (Invitrogen). Negative controls contained RNase-free water
substituted for RT. The cDNA generated was quantitated by quantitative PCR
using a Bio-Rad iQcycler with the Absolute QPCR SYBR green fluorescein mix
(Thermo Scientific). For generation of cDNA and quantitative PCR analysis,
gene-specific primers were used (Table 1). Standard curves were constructed
from serial dilutions of NCTC13129 genomic DNA. The transcript level of gyrB
(a gene that is constitutively expressed in the tested conditions [data not shown])
was used as a control for RNA concentration. To determine the relative tran-
script quantity, the amount of gene-specific transcript was divided by the amount
of gyrB transcript.
Characterization of the zur loci. The genome sequence of C.
diphtheriae NCTC13129 contains a predicted coding sequence
(dip1710) with similarity to genes encoding Fur-like regulators
(12). A BLAST search confirmed that dip1710 is the only
sequence in the genome with similarity to fur-like genes and
that the Zur protein of M. tuberculosis is its closest character-
ized homolog. Based on its homology to the M. tuberculosis
gene and the functional assays described below, we renamed
dip1710 as zur. The genomic loci that include zur in M. tuber-
culosis and C. diphtheriae NCTC13129 are similar in structure
(Fig. 1A). Upstream of zur in both organisms is a gene that
encodes a putative metal-dependent transcriptional regulator
similar to arsR. ArsR family proteins usually repress transcrip-
tion of genes involved in metal efflux (6). In the M. tuberculosis
genome, the zur gene slightly overlaps the 3? end of the arsR-
like gene, and these genes are cotranscribed from a single
zinc-dependent promoter located upstream of the arsR-like
gene (24). In contrast, there is a 179-bp intergenic region
between the arsR-like gene and zur in C. diphtheriae (Fig. 1A).
An aminoacyl-tRNA synthetase glyS and a gene with no iden-
tified homologs are directly upstream and downstream of arsR-
zur, respectively. In the C7(?) strain of C. diphtheriae, which
has been studied for many years as a model for gene regulation
(2, 27, 28, 38), we identified an insertion sequence (IS element)
upstream of the arsR-like gene. DNA sequencing of the region
of the C7(?) genome containing the IS element (GenBank no.
FJ470294) revealed that the 1,445-bp element includes a trans-
posase whose coding sequence encompasses 1,350 bp. Inverted
repeat sequences of 28 bp were found at the ends of the
element (Fig. 1B), and its insertion resulted in the direct target
site duplication of 8 bp (TTTCGATC). The IS element is
located 78 bp upstream of the ATG codon for the arsR-like
gene and 240 bp upstream of the GTG start codon of glyS. The
effect, if any, of this IS element on expression of the genes at
the C7(?) zur locus is unknown. Other than the insertion of the
IS element, the gene organization in the zur loci are identical
in NCTC13129 and C7(?).
Physiological importance of the Zur protein. To investigate
the function of C. diphtheriae zur, a nonpolar deletion mutation
was constructed in the genome of NCTC13129 using the mo-
bilizable vector pK19mobsacB?zur (see Materials and Meth-
ods and Fig. 1), creating NCTC13129?zur. To complement this
strain, we cloned wild-type zur into integration vector pKPIM
(30), resulting in pKPIMzur. We then compared the growth
curves of NCTC13129 to those of NCTC13129?zur in the
presence or absence of zinc, iron, and manganese to determine
whether the absence of Zur affects the growth of C. diphtheriae.
Similar growth curves were observed for the NCTC13129 and
NCTC13129?zur regardless of the presence or absence of 10
?M iron, 10 ?M manganese, or 25 ?M zinc (data not shown).
Zinc concentrations of ?200 ?M inhibited growth of both the
NCTC13129 and NCTC13129?zur strains of C. diphtheriae to
comparable extents, demonstrating the toxic effect of high
zinc concentrations on this organism (data not shown). Also,
no differences were observed between NCTC13129 and
NCTC13129?zur in protein profiles or cell morphology, via
whole-cell protein lysates and light microscopy, respectively
(data not shown). Thus, the absence of Zur had no detectable
effect on C. diphtheriae in these experiments.
Since zinc has been shown to protect bacteria from oxidative
FIG. 1. Genetic arrangement of the C. diphtheriae and M. tuberculosis zur loci. (A) The dark gray arrow represents glyS, a glycyl-tRNA
synthetase beta subunit. The gray striped arrow represents an arsR-like gene, a putative metal-dependent transcriptional regulator. The black arrow
represents zur, a zinc-dependent transcriptional regulator. dip1711 and Rv2360c are genes with unknown function. The insertion site of the IS
element in C. diphtheriae C7(?) is shown as a triangle, and the region deleted in the ?zur strain is indicated as a dashed line. The percentages of
amino acid sequence similarities between genes are indicated between the loci. (B) The sequences of the 28-bp inverted repeats at the ends of the
IS element are shown. Bases that are interruptions in the inverted repeat are shown in lowercase, and the rest of the IS element is indicated as
a dashed line.
VOL. 191, 2009ZINC UPTAKE REGULATOR IN C. DIPHTHERIAE1597
stress (13), we also tested the ability of NCTC13129 and
NCTC13129?zur to survive challenge with H2O2. H2O2sus-
ceptibility was determined by using two methods: growth inhi-
bition assays and killing assays. Both C7(?) and C7(?)?dtxR
strains have been assessed for levels of H2O2stress resistance
in previous studies and were used as controls (29). The
NCTC13129?zur pKPIM strain showed a slightly larger zone
of inhibition (Table 2) and a higher percentage of killing (Ta-
ble 2) than both NCTC13129 pKPIM and complemented
NCTC13129?zur pKPIMzur, indicating that it was more sen-
sitive to H2O2stress than its wild-type parent. Although the P
value for the difference between the wild-type and ?zur strain
zones of inhibition was ?0.05 (i.e., 0.08), a trend of higher
peroxide sensitivity is observed in the zur mutant. In the killing
assay, the difference between the ?zur strain and the
NCTC13129 pKPIM or NCTC13129?zur pKPIMzur strains
had P values of ?0.05 (0.01 and 0.034, respectively). There-
fore, the absence of Zur in NCTC13129 resulted in a slight
increase in sensitivity to H2O2.
Identification of the zur promoter(s). To identify the pro-
moter(s) of zur, we cloned the region immediately upstream of
zur into a ?-galactosidase promoter reporter vector that repli-
cates in C. diphtheriae, pSPZ (29). Since the promoter of the
M. tuberculosis zur gene is located upstream of rv2358/arsR
(Fig. 1, (24), the upstream region of the NCTC13129 arsR-like
gene was also tested for promoter activity. In contrast to M.
tuberculosis, C. diphtheriae NCTC13129 contains promoters di-
rectly upstream of both the arsR-like gene and zur (Fig. 2). We
also investigated whether there was an arsR/zur cotranscript
using RT-PCR with primers that annealed within the inter-
genic region (Table 1). No arsR-zur cotranscript was observed
but transcripts that contained only the arsR-like gene or zur
were detected (data not shown).
Regulation of zur transcription. In many organisms, tran-
scription of metal-dependent transcriptional regulators is reg-
ulated in response to the presence of their cognate metal (24).
Therefore, we tested the activity of the promoters upstream of
the arsR-like gene and zur in the presence or absence of zinc.
The promoter upstream of the arsR-like gene showed no sig-
nificant difference in response to differential zinc conditions or
to the presence of Zur (Fig. 2). In contrast, the zur promoter
had lower activity in zinc depleted versus replete conditions in
both the NCTC13129 and NCTC13129?zur pKPIMzur with P
values ?0.05 (?0.001). Strikingly, zur promoter activity re-
mained high in the NCTC13129?zur background irrespective
of the presence or absence of zinc, suggesting that Zur has a
role in the regulation in its own expression. This phenomenon
was confirmed by the observation that the zinc dependence of
the zur promoter was restored in NCTC13129?zur pKPIMzur
(Fig. 2). Both the arsR-like gene promoter and zur promoter
activities were unaltered by the presence or absence of either
iron or manganese (data not shown). These data indicate that
the zur promoter is repressed specifically in zinc-depleted con-
ditions in a Zur-dependent manner.
?-Galactosidase assays determine promoter strength with-
out being impacted by mRNA stability, whereas qRT-PCR,
although affected by transcript stability, more precisely quan-
tifies the transcripts present at a single time point. Therefore,
we used qRT-PCR to further analyze the arsR-like gene and
zur transcript levels in NCTC13129 and NCTC13129?zur un-
der zinc-replete and -depleted conditions. The arsR-like gene
transcripts, although barely detectable, remained constant in
NCTC13129 and NCTC13129?zur regardless of the zinc con-
centration, confirming the promoter fusion data described
above (Fig. 3A and C). In contrast, the zur transcript level was
4.5-fold higher in zinc-depleted conditions (Fig. 3B and C),
which correlated with the observed promoter activity (Fig. 2).
The zur transcript was only assayed in NCTC13129 because the
majority of the zur gene was deleted in NCTC13129?zur, pre-
venting its detection in this assay. With a weakly transcribed
constitutive promoter, the accumulation of ?-galactosidase
may indicate a higher promoter activity compared to transcript
levels at a single time point detected in qRT-PCR data (com-
pare arsR in Table 3 and Fig. 2).
To determine whether zinc-dependent regulation of zur oc-
curs in other strains of C. diphtheriae, transcription of zur and
the arsR-like gene was assayed in the C7(?) strain by using
qRT-PCR. The transcript levels of arsR were lower in C7(?)
than in NCTC13129 but remained unchanged in zinc-replete
and -depleted conditions (Table 3). Interestingly, not only was
the transcript of zur at lower levels in C7(?), the differential
regulation observed between zinc-replete and -depleted con-
FIG. 2. arsR and zur promoter activity. NCTC13129 (indicated as
wild type [WT]) and NCTC13129?zur (indicated as ?zur) strains with
either vector control, pKPIM, or complementing wild-type zur,
pKPIMzur (shown as ?zur), were inoculated into zinc-replete (?Zn) or
-depleted (–Zn) conditions and tested for arsR and zur promoter ac-
tivity. ?-Galactosidase activity is expressed in Miller units. The cmrA
promoter is a positive control for zinc-depleted conditions. The aster-
isks denote statistical significance (?0.05) between zinc-replete and
-depleted conditions, as determined by a one-way analysis of variance,
followed by the Holm-Sidak method.
TABLE 2. Peroxide stress susceptibility of C. diphtheriae strains
Avg ? SDa
% of wild-type zone
% Killing in exposure
10038 ? 11
68 ? 11*
24 ? 5
18 ? 15
50 ? 9*
30 ? 10
170 ? 15*
87 ? 7
110 ? 8
91 ? 14
aValues are given as averages for at least three samples. *, Statistically sig-
nificant difference from the wild-type parent strain (P ? 0.05) as determined by
a one-way analysis of variance, followed by the Holm-Sidak method.
1598 SMITH ET AL. J. BACTERIOL.
ditions was not seen in this strain. Thus, the regulation and
accumulation of the zur transcript differed in these C. diphthe-
Transcriptional regulation by Zur. We performed a BLAST
search of the C. diphtheriae genome to identify genes that may
be regulated by Zur. Sequence probes that were used for the
BLAST search included genes that are known to be regulated
by Zur in other bacteria and genes that encode proteins in-
volved in metal transport. This analysis identified a gene we
named zrg (dip1486), with homology to cobW/yciC genes from
a variety of bacterial species and which encodes a putative
low-affinity zinc uptake protein, as well as a gene we named
troA (dip0438), with sequence similarity to T. pallidum troA
and to S. pneumoniae psaA, that is predicted to encode a
component of an ABC-type metal transporter. The cmrA gene,
which was previously shown to be zinc regulated (4), is pre-
dicted to encode a sortase anchored cell wall protein. The
mechanism of the zinc-dependent repression of cmrA has not
previously been investigated. We thus sought to determine
whether Zur had a role in regulating transcription of these
We cloned the upstream regions of zrg, cmrA, and troA, into
the promoter reporter vector pSPZ and assayed the activity of
each in NCTC13129 and NCTC13129?zur. All three promot-
ers were repressed in zinc-replete conditions and derepressed
in zinc-depleted conditions in NCTC13129 (Fig. 4A). In
NCTC13129?zur, the activity of all three promoters was con-
stitutive in response to zinc. Zinc-dependent promoter activity
was restored when zur was provided from pKPIMzur in
NCTC13129?zur. These data indicate that the activities of
these three promoters were controlled by Zur in response to
changes in zinc concentration.
We tested each of the promoters for activity in the presence
or absence of iron and manganese. When the activity of each
promoter was compared under iron-replete or -depleted con-
ditions in the wild-type NCTC13129 strain, no activity was
observed. Identical results were obtained when manganese was
substituted for iron (Fig. 4B and C, first two columns). This
result was not unexpected since the assay medium contained
zinc, which represses the expression of all three promoters.
Interestingly, we did observe higher levels of activity from the
zrg promoter under iron-depleted conditions in the NCTC
13129?zur strain (23 Miller units in iron-replete medium ver-
sus 33 Miller units in iron-depleted medium). Similarly, we
FIG. 3. Detection of arsR and zur transcripts. RNA isolated from
NCTC13129 (WT) and NCTC13129?zur (?zur) in zinc-replete and
-depleted conditions was analyzed for relative transcript levels (nor-
malized to gyrB mRNA levels) of arsR (A) and zur (B). (C) Fold
change of these genes from NCTC13129 in zinc-replete conditions.
TABLE 3. Detection of arsR and zur transcripts
Relative transcript quantity
(avg ? SD)a
0.01 ? 0.00
0.03 ? 0.02
0.01 ? 0.00
0.05 ? 0.03
0.58 ? 0.13
4.8 ? 2.7
0.56 ? 0.06
1.1 ? 0.67
aNormalized with gyrB. Values are averages for at least three samples.
bThe fold change is calculated as ?Zn/–Zn.
FIG. 4. Activity of Zur-regulated promoters. NCTC13129 (indi-
cated as WT) and NCTC13129?zur (indicated as ?zur) strains
with either vector control, pKPIM, or complementing wild-type zur,
pKPIMzur (shown as ?zur), inoculated into zinc-replete (?Zn) or -de-
pleted (–Zn) conditions were used to test the activity of promoters (zrg,
cmrA, and troA) in the reporter plasmid pSPZ. (A) The ?-galactosi-
dase activity was determined under differential conditions. The activity
of the zrg promoter under iron and zinc stress conditions (B) and
activity of the cmrA promoter under manganese and zinc stress con-
ditions (C) are also presented. The promoters of tox, mntA, and cmrA
genes (cloned into the reporter vector) were used as positive controls
for iron-, manganese-, and zinc-depleted conditions, respectively.
VOL. 191, 2009ZINC UPTAKE REGULATOR IN C. DIPHTHERIAE 1599
observed higher activity from the cmrA promoter in manga-
nese-depleted medium in the NCT13129?zur strain back-
ground (28 Miller units in manganese-replete versus 92 Miller
units in manganese-depleted medium). The activity of the pro-
moter for troA was unchanged when the medium was depleted
of either iron or manganese in the NCTC13129?zur back-
ground. These observations led us to hypothesize that the
effects of iron and manganese on the zrg and cmrA promoters,
respectively, might be epistatic to the effects of zinc and Zur.
We next examined the activities of the zrg and cmrA pro-
moters in medium depleted of both iron and zinc or both
manganese and zinc. As shown in Fig. 4B (columns 3 and 4),
depletion of both iron and zinc resulted in a 1.5 increase in the
activity of the zrg promoter compared to the activity in medium
depleted only for zinc. As a control we used the well-charac-
terized tox promoter, whose activity is responsive only to iron
and not to zinc (38). The activity of the cmrA promoter in-
creased sixfold when the growth medium was depleted of both
zinc and manganese compared to activity in medium depleted
only of zinc (Fig. 4C, columns 3 and 4). For this assay, we used
the mntA promoter as a control for manganese-dependent
regulation since it is controlled by MntR in response to man-
ganese (37). Interestingly, the mntA promoter showed an in-
crease in activity when the growth medium was depleted of
both manganese and zinc (compared to medium lacking only
manganese), indicating that MntR-dependent manganese reg-
ulation is epistatic to the effect of zinc.
Our observations imply that the zrg promoter is controlled
first by zinc in a process that requires Zur and secondarily by
the presence of iron in a process that is independent of Zur.
The cmrA promoter is controlled first by zinc and Zur and
secondarily by manganese in a Zur-independent mechanism.
Clearly, the regulation of the zrg, cmrA, and mntA promoters is
more complex than the one-metal, one-regulator models pro-
posed for many metal-dependent promoters.
To confirm that the transcript levels correlated with the
promoter activity, we used qRT-PCR to assay cmrA, zrg, and
troA transcription in NCTC13129 and NCTC13129?zur under
zinc-replete and -depleted conditions. The transcript levels of
all three genes were significantly lower in NCTC13129 under
zinc-replete conditions than the levels in NCTC13129 in zinc-
depleted conditions and in NCTC13129?zur in all conditions
(Fig. 5A). The fold change was calculated by dividing all tran-
script levels by the corresponding NCTC13129 wild-type strain
transcript level under zinc-replete conditions (Fig. 5B). The
largest transcript level fold change was observed for zrg with an
average of 250. The cmrA transcript had an average of a 170-
fold change, and the troA transcript level had an average fold
change of 36. Large differences between the fold changes of
zrg, cmrA, and troA transcript levels correlate with the differ-
ences in transcripts present under induced conditions (i.e., zinc
depleted or lacking Zur), rather than a difference in the re-
pression or uninduced state (i.e., zinc replete and wild-type
Zur levels). These data confirm that Zur represses the tran-
scription of zrg, cmrA, and troA in zinc-replete conditions.
To regulate metal homeostasis, bacteria use multiple metal-
dependent transcriptional regulators, each controlling differ-
ent regulons in response to different inducers. We describe
here the characterization of a zinc-dependent Fur family ho-
molog in C. diphtheriae. To our knowledge, Zur, is the first
zinc-dependent transcriptional regulator described in Coryne-
bacteria. We identified three genes, cmrA, zrg, and troA whose
transcription is repressed by Zur. In addition, we demonstrated
that the zur gene is transcribed from a promoter that is re-
pressed by Zur when zinc is scarce. Our data also indicate that
C. diphtheriae is capable of integrating signals from different
metals to exert exquisite control over the transcription of spe-
Although C. diphtheriae lacking Zur do not have a growth
defect compared to the wild-type, all C. diphtheriae strains
exhibited slowed growth rates in medium containing concen-
trations of zinc greater than 200 ?M, thus indicating that zinc
toxicity occurs in C. diphtheriae. Due to uncontrolled uptake of
zinc, one might expect the C. diphtheriae zur mutant strain to
be more sensitive to high concentrations of zinc than its wild-
type parent, as is observed for Xanthomonas campestris zur
mutants (43). This was not the case. In X. campestris Zur
controls the expression of both zinc uptake and efflux; thus,
when Zur is absent, X. campestris not only undergoes unregu-
lated uptake of zinc, but it also fails to activate zinc efflux
systems (19). Our data suggest that, unlike X. campestris Zur,
C. diphtheriae Zur is not required for the activation of zinc
efflux. In addition, given the roles of zinc as an antioxidant and
as a cofactor for enzymes such as superoxide dismutase, we
tested the ability of a ?zur mutant strain to survive challenge
with H2O2. The C. diphtheriae NCTC13129?zur strain demon-
strated an increased susceptibility to peroxide stress, suggest-
ing that Zur is required for expression of oxidative stress de-
Interestingly, in C. diphtheriae NCTC13129, zur transcript
levels are affected by zinc concentrations, but the levels of the
adjacent arsR-like gene transcript are not. This distinguishes C.
diphtheriae zur from its homolog in M. tuberculosis, where the
ArsR-like protein represses the arsR-zur cotranscript in re-
sponse to zinc-depleted conditions (8, 24). In addition, the
FIG. 5. RNA levels of Zur-regulated genes. RNA isolated from
NCTC13129 and NCTC13129?zur under zinc-replete and -depleted
conditions was analyzed by using qRT-PCR. (A) Relative level of
transcripts (normalized to gyrB mRNA levels) of cmrA, zrg, and troA.
(B) Fold change of these genes from NCTC13129 under zinc-replete
1600 SMITH ET AL. J. BACTERIOL.
repression of zur transcription in C. diphtheriae NCTC13129 in
zinc-depleted conditions is dependent on the presence of Zur.
These variations highlight important differences and suggest
that Zur may play different roles in the physiology of these two
species. The regulation of C. diphtheriae zur transcription by
zinc and Zur is the first indication of Zur activity in zinc-
depleted conditions. However, in other species, Fur proteins
have been shown to exhibit uncharacteristic activity, such as
functioning as a direct DNA-binding transcriptional activator
in Neisseria meningitidis and as a repressor in the absence of
metal in Helicobacter pylori (22). DNA binding experiments are
currently in progress to determine whether this effect on the
zur transcript is a result of direct Zur binding to its promoter.
We observed differences in transcription of zur in C. diph-
theriae strains C7(?) and NCTC13129, demonstrated by both
the absence of zinc-dependent zur regulation and the lower
overall zur transcript levels in C7(?). Using qRT-PCR, the
Zur-regulated genes zrg, cmrA, and troA were all shown to be
regulated by zinc and Zur in C7(?) and, unlike the finding for
the zur transcript levels, the transcript levels of zrg, cmrA, and
troA in C7(?) were all extremely similar in NCTC13129 under
identical growth conditions (data not shown). It is unlikely that
the zinc stress conditions used were significantly different or
that transcription of all genes is generally lower in C7(?) com-
pared to NCTC13129. Although the IS element upstream of
arsR in C7(?) could result in lower arsR transcription, it is
unlikely that this IS element is affecting zur transcription di-
rectly, since zur has its own promoter. In addition, we deter-
mined the DNA sequence of the region between arsR and zur
in C7(?), and it was identical to that in NCTC13129. There
remains the possibility that there are other differences between
these strains that account for the loss of zinc-dependent reg-
ulation of zur transcript in C7(?), including unknown trans-
acting factors. C7(?) is a lab strain originally isolated in the
1950s, while NCTC13129 is a recent clinical isolate represen-
tative of an outbreak that occurred in the late 1990s (2, 9). The
differences in zur regulation and in the sequence of the loci in
these two strains highlight the value of assaying recent clinical
isolates and the importance of genomic sequencing of multiple
strains of a single bacterial species.
In both strains of C. diphtheriae the zrg transcript is strongly
repressed in high zinc conditions in a Zur-dependent manner.
This is also the case for its homologs in B. subtilis and M.
tuberculosis (14, 23, 32). Sequence homology suggests that Zrg
is a low-affinity zinc transporter protein, or a metallochaper-
one, that “passes” zinc ions between transport proteins and
enzymes. Our data support the notion that Zrg is a low-affinity
zinc transporter protein given that repression of a zinc chap-
erone under high zinc concentrations would not likely be of
value to the cell. The evidence that Zrg is a zinc transporter is
supported by observations that the B. subtilis Zrg homolog
YciC is important for zinc utilization (14, 15). Similar to zrg,
the transcript of cmrA is highly repressed by Zur under zinc-
replete conditions in C. diphtheriae. Based on this observation
and the prediction that cmrA encodes a sortase anchored cell
wall protein, we hypothesize that CmrA is a surface protein
associated with an ABC transporter involved in the uptake of
The final C. diphtheriae gene that we characterized was troA,
which is repressed by Zur in zinc-replete conditions. In con-
trast to the observations with cmrA and zrg, troA promoter
activity was observed in zinc-replete conditions, albeit at low
levels (Fig. 5A). We also observed that induction of troA tran-
scription occurs in the presence of higher concentrations of
zinc than the transcription of either zrg or cmrA (data not
shown). These observations suggest that the troA promoter is
released from Zur repression when the concentrations of zinc
are sufficient to allow binding of Zur at the zrg and cmrA
promoters. The troA gene encodes a membrane protein with
homology to S. pneumoniae PsaA and is the first gene in a
seven gene operon. The genes of this operon are predicted to
encode the components of an ABC transporter, a putative
surface-anchored sortase protein, and three putative mem-
brane proteins of unknown function. Some of these genes have
homology to genes in the ZnuABC metal uptake machinery
family. The similar gene cluster in S. pneumoniae is thought to
be a manganese transporter, but recent evidence suggests that
it may transport zinc (16). In addition to its role in metal ion
transport, S. pneumoniae PsaA also contributes to virulence
and oxidative stress resistance (21).
One of the most intriguing findings from our investigation of
C. diphtheriae Zur-dependent gene regulation is the identifi-
cation of two genes whose transcription is affected by more
than one metal ion. Zur is required for the zinc-dependent
primary regulation of both zrg and cmrA. We demonstrated
that the zrg promoter is secondarily regulated by the availabil-
ity of iron and that the cmrA promoter is secondarily regulated
by the presence of manganese. In both cases, the secondary
regulation is independent of Zur, suggesting that a second
metal-dependent regulator may be involved. There are exam-
ples of bacterial promoters regulated by more than one metal-
dependent regulator, including the promoter for rv0282, a
gene of unknown function in M. tuberculosis, which undergoes
dual zinc- and iron-dependent regulation by Zur and IdeR (a
DtxR homolog), respectively (23). Similarly, E. coli mntH, a
homolog of the eukaryotic natural resistance associate macro-
phage protein (NRAMP), is regulated by iron and manganese
by mechanisms that require Fur and MntR (33). Since zrg and
cmrA are secondarily regulated by iron and manganese, re-
spectively, we hypothesize that DtxR, the only iron-dependent
transcriptional regulator, and MntR, the only manganese-de-
pendent transcriptional regulator characterized in C. diphthe-
riae, are involved. The binding sites for DtxR and MntR have
been characterized (37, 38), and we have searched the regions
upstream of zrg and cmrA for sequences with similarity to these
sites. The DNA region with highest similarity (10/19) to the
consensus DtxR binding site (TTAGGTTAGGCTAACC
TAA) upstream of zrg is 74 bp upstream of the start codon
(Fig. 6). The binding site for MntR is less well characterized
(37), but there are two possible binding motifs for MntR up-
stream of cmrA. A five-out-of-seven match to the direct repeat
(TGAACAA) found in the MntR binding site is located 19 bp
upstream, and a 19-bp sequence with 63% identity to the
MntR inverted repeat is located 3 bp upstream of the cmrA
start codon (Fig. 6). The roles of MntR and DtxR in control-
ling transcription of cmrA and zrg are under active investiga-
The binding sites for C. diphtheriae Zur have not been de-
fined, but given its similarity to M. tuberculosis Zur, we
searched regions upstream of cmrA, zrg, and troA for sequences
VOL. 191, 2009ZINC UPTAKE REGULATOR IN C. DIPHTHERIAE 1601
similar to the 21 bp at the center of the M. tuberculosis Zur
binding site (TATTGAAAATNATTTTCAATA) (23). There
are two putative Zur binding sites upstream of cmrA (Fig. 6).
The first site overlaps the start codon (running from ?19 to
?2) and matches the M. tuberculosis consensus site at 16 out of
20 conserved positions. The second site is located 26 bp up-
stream of the cmrA start codon and is a 65% match to the
consensus. We identified a single putative Zur binding site
located 5 bp upstream of the zrg start codon that matches the
consensus sequence at 13 of 20 positions (Fig. 6). Finally,
upstream (70 bases from the start codon) of troA there is a
single putative Zur binding site (14 of 20 bases identical). The
locations of the putative Zur binding sites upstream of cmrA,
zrg, and troA are consistent with the notion that they overlap
the promoter sequences in these regions.
In summary, we have characterized zinc-dependent Zur reg-
ulation of three genes and the interplay between zinc and other
metals in controlling transcription in C. diphtheriae. The posi-
tions of the putative binding sites for Zur, DtxR, and MntR in
the promoter regions of zrg, cmrA, and troA is suggestive that
the regulators act at these promoters. DNA-binding assays are
in progress to confirm the roles of each regulator. In addition,
we characterized the transcription of zur and observed that,
unlike most other fur family genes, zur is repressed under
low-zinc conditions in a process requiring Zur (in strain
NCTC13129). Finally, we observed increased sensitivity of the
C. diphtheriae zur mutant strain to H2O2stress and, although
the exact mechanism of this sensitivity is unknown, this indi-
cates a role for Zur both in metal homeostasis and in protec-
tion against host defenses that utilize oxygen radicals to kill
We thank Mark Strauch, Mark Oram, and Kelley Hovis for their
careful reading and helpful comments of the manuscript and Dean
Dessem for assistance with statistical analysis.
This study was supported by research grant NIH/NIAID K22
AI60882 to D.M.O.
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