Characterization of Zur-dependent genes and direct Zur targets in Yersinia pestis
Characterization of Zur-dependent genes and direct Zur targets in Yersinia pestis
Yingli Li 1
Yefeng Qiu 0
He Gao 1
Zhaobiao Guo 1
Yanping Han 1
Yajun Song 1
Zongmin Du 1
Xiaoyi Wang 1
Dongsheng Zhou 1
Ruifu Yang 1
0 Laboratory Animal Center, Academy of Military Medical Sciences , Beijing 100071 , PR China
1 State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology , Beijing 100071 , PR China
Background: The zinc uptake regulator Zur is a Zn2+-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in Y. pestis. Results: We constructed a zur null mutant of Y. pestis biovar microtus strain 201. Microarray expression analysis disclosed a set of 154 Zur-dependent genes of Y. pestis upon exposure to zinc rich condition. Real-time reverse transcription (RT)-PCR was subsequently used to validate the microarray data. Based on the 154 Zur-dependent genes, predicted regulatory Zur motifs were used to screen for potential direct Zur targets including three putative operons znuA, znuCB and ykgM-RpmJ2. The LacZ reporter fusion analysis verified that Zur greatly repressed the promoter activity of the above three operons. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified Zur protein was able to bind to the promoter regions of the above three operons. The DNase I footprinting was used to identify the Zur binding sites for the above three operons, verifying the Zur box sequence as predicted previously in -Proteobacteria. The primer extension assay was further used to determine the transcription start sites for the above three operons and to localize the -10 and -35 elements. Zur binding sites overlapped the -10 sequence of its target promoters, which was consistent with the previous observation that Zur binding would block the entry of the RNA polymerase to repress the transcription of its target genes. Conclusion: Zur as a repressor directly controls the transcription of znuA, znuCB and ykgM-RpmJ2 in Y. pestis by employing a conserved mechanism of Zur-promoter DNA association as observed in -Proteobacteria. Zur contributes to zinc homeostasis in Y. pestis likely through transcriptional repression of the high-affinity zinc uptake system ZnuACB and two alternative ribosomal proteins YkgM and RpmJ2.
Zinc is an essential trace element for a large number of
enzymes and proteins in bacteria, but it can be toxic at
high levels. It is therefore crucial that intracellular zinc
level over a small concentration range must be tightly
regulated [1-3]. Bacterial zinc homeostasis is achieved
mainly by the coordinated expression of zinc uptake and
export systems that are separately regulated by their own
Bacteria have evolved at least three types of Zn2+ export
systems [2,3] to protect cells from high toxic Zn2+
concentrations, namely cation diffusion facilitators (e.g. CzcD in
Alcaligenes eutrophus), RND type exporters (e.g. CzcABC in
A. eutrophus), and P-type ATPases (e.g. ZntA in Escherichia
coli). CzcD, CzcABC and ZntA are regulated by an
ArsRlike repressor CzrA , a two-component system CzcR/S
, and a MerR-family regulator ZntR , respectively.
Zinc ions are transported into the cytoplasm via high- and
low-affinity zinc uptake systems, which are represented by
ZnuABC of E. coli  and YciABC of Bacillus subtilis [8,9],
respectively. A broad set of zinc uptake systems including
ZnuABC and YciABC are regulated by the zinc uptake
regulator Zur that is a homologous to the well-known Fur
family of metal-dependent regulators .
Yersinia pestis is the causative agent of plague that is a
zoonotic disease primarily affecting rodents .
Maintenance of plague in nature is primarily dependent upon
cyclic transmission between fleas and rodents . Y.
pestis possesses its potential to attack humans, and the
human infection usually occurs with the transmission of
the pathogen from animals by the biting of an infected
flea, but this deadly disease can be transmitted from
person to person by respiratory route. Y. pestis can remain
viable and fully virulent after 40 weeks in soil . Thus, soil
appears a potential telluric reservoir for Y. pestis, which
could represent an alternative mechanism for
maintenance of plague . Zinc homeostasis should be crucial
for survival of Y. pestis in fleas, rodents and soil.
Up to now, regulation of zinc homeostasis by Zur is
poorly understood in Y. pestis. In this study, we
constructed a zur null mutant of Y. pestis biovar Microtus strain
201, and compared its global gene expression profile to
that of the parental strain by using cDNA microarray,
identifying a total of 154 Zur-dependent genes. Three
genes or operons, namely znuA, znuCB and ykgM were
further identified as direct Zur targets. Subsequent
determination of transcription start sites, predicted -10/-35
elements, and Zur binding sites enabled the mapping of
Zur-DNA interactions for these three genes. This study
confirmed that Y. pestis Zur employed a conserved
regulatory mechanism observed in -Proteobacteria.
The wild-type (WT) Y. pestis biovar Microtus strain 201 is
avirulent to humans but highly lethal to mice . It was
grown in Luria-Bertani (LB) broth or chemically defined
TMH medium  at 26 or 37C. E. coli strains BL21
(DE3) was grown in LB broth at 37C. Antibiotics were
added at the following concentrations when required: 100
g/ml for ampicillin, and 50 g/ml for kanamycin.
Construction of the zur mutant
The Y. pestis zur mutant strain (zur) was generated by
using the one-step inactivation method based on the
lambda phage recombination system, as previously
described by Datsenko and Wanner . Briefly, the
helper plasmid pKD46 was first transformed into Y. pestis
201. The zur::kana mutagenic cassette was PCR amplified
from plasmid pRS551  with the primers zur-k-F and
zur-k-R and transformed into strain 201/pKD46 (all the
primers used in this study were listed in Additional file 1).
Mutants were selected by plating electroporated cells on
agar plates containing kanamycin. Colonies of resistant
transformants were subsequently selected. Chromosomal
integration of the mutagenic cassette was confirmed by
PCR and sequencing using oligonucleotides external to
the integrated cassette (data not shown). The mutants
were incubated overnight at 37C and then tested for the
loss of the temperature-sensitive plasmid pKD46 by
looking for ampicillin sensitivity. The elimination of the
helper plasmid was verified by PCR (data not shown).
Bacterial growth and RNA isolation
A chemically defined TMH medium  was used to
cultivate strain 201. Both WT and zur were pre-cultivated at
26C to the middle exponential growth phase (OD620
about 1.0) in TMH medium. The cell cultures were then
diluted 1:20 in fresh TMH medium and grown at 26C
until an OD620 of about 1.0. Finally, 5 mM ZnCl2 was
added into each cell culture to ensure zinc rich conditions.
Growth was continued for 30 min at 26C before
harvested for total RNA isolation. This kind of treatment with
Zn had no toxic effect on both WT and zur, according to
the colony counting assay (Additional file 1).
Immediately before being harvested, bacterial cultures
were mixed with two fold of RNAprotect Bacteria Reagent
(Qiagen) to minimize RNA degradation. Total cellular
RNA was isolated using the MasterPure RNA Purification
kits (Epicenter). RNA quality was monitored by agarose
gel electrophoresis and RNA quantity was measured by
DNA microarray analysis
Gene expression profiles were compared between WT and
zur by using a Y. pestis whole-genome cDNA microarray
as described previously . Briefly, RNA samples were
isolated from four individual bacterial cultures, as
biological replicates, for each strain. Total cellular RNA was
isolated and then used to synthesize cDNA in the presence of
aminoallyl-dUTP, genome directed primers (GDPs) and
random hexamer primers . The aminoallyl modified
cDNA was then labelled with Cy5 or Cy3 dye. Microarray
slides spotted in duplicate with 4005 PCR amplicons,
representing about 95% of the non-redundant annotated
genes of Y. pestis CO92  and 91001 , were used for
probe hybridization. The dual-fluorescently (Cy3 or Cy5
dye) labeled cDNA probes, for which the incorporated
dye was reversed, were synthesized from the RNA samples
of the four biological replicates, and then hybridized to
four separated microarray slides, respectively. The
scanning images were processed and the data was further
analyzed by using GenePix Pro 4.1 software (Axon
Instruments) combined with Microsoft Excel software.
The normalized log2 ratio of the zur/WT signal for each
spot was recorded. The averaged log2 ratio for each gene
was finally calculated. Significant changes of gene
expression were identified through the Significance Analysis of
Microarrays (SAM) software (a Delta value of 1.397 and
an estimated False Discovery Rate of 0%) .
Computational analysis of Zur binding sites
The 500 bp promoter regions upstream the start codon of
each Zur-dependent genes as revealed by cDNA
microarray was retrieved with the 'retrieve-seq' program . A
position count matrix was built from the predicted Zur
binding sites in -Proteobacteria by using the
matrices-consensus tool , and displayed by the WebLogo program to
generate a sequence logo . Following this, the
matrices-paster tool  was used to match the Zur position
count matrix within the above promoter regions.
Gene-specific primers were designed to produce a 150 to
200 bp amplicon for each gene (see Additional file 2 for
primer sequences). The contaminated DNA in RNA
samples was further removed by using the Amibion's
DNAfree Kit. cDNAs were generated by using 5 g of RNA and
3 g of random hexamer primers. Using three
independent cultures and RNA preparations, real-time RT-PCR was
performed in triplicate as described previously through
the LightCycler system (Roche) together with the SYBR
Green master mix [22,23]. On the basis of the standard
curves of 16S rRNA expression, the relative mRNA level
was determined by calculating the threshold cycle (Ct)
of each gene by the classic Ct method. Negative controls
were performed by using 'cDNA' generated without
reverse transcriptase as templates. Reactions containing
primer pairs without template were also included as blank
controls. The 16S rRNA gene was used as an internal
control to normalize all the other genes. The transcriptional
variation between WT and zur was calculated for each
Overproduction and purification of Y. pestis Zur protein
The 537 bp entire coding region of zur gene was amplified
by primer Zur-P-F and Zur-P-R from Y. pestis 201 (see
Additional file 2 for primer sequences) and cloned
directionally into the BamHI and HindIII sites of plasmid
pET24a (Novagen), which was verified by DNA
sequencing. The stop codon was introduced in the reverse primer
to make sure that the expressed Zur did not contain
Histag. The resulted recombinant plasmid was transformed
into E. coli BL21 (DE3). For overproduction of Zur, an
overnight culture from a single colony was used to
inoculate 200 milliliter of LB medium. Cells were grown with
vigorous shaking at 37C to an optical density at 620 nm
(OD620) of 0.8 and were induced with 1 mM IPTG
(isopropyl--D-thiogalactoside) for 6 h at 37C. For
purification, harvested cells were treated with BugBuster Protein
Extraction Reagent (Novagen). Inclusion bodies were
recovered by centrifugation and washed twice with the
same reagent. The Zur protein was renaturated and then
concentrated to a final concentration of about 0.6 mg/ml
with the Amicon Ultra-15 (Millipore). The protein purity
was verified by SDS-PAGE with silver staining. All steps
after cell harvest were performed at 4C, and the purified
Zur protein was stored at -80C.
Gel mobility shift assay (EMSA)
Primers were designed to amplify the DNA region
upstream of the start codon of each gene tested (see
Additional file 2 for primer sequences). EMSA was performed
by using the Gel Shift Assay Systems (Promega) [22,23].
The 5' ends of DNA were labeled using [-32P] ATP and T4
polynucleotide kinase. DNA binding was performed in a
10 l reaction volume containing binding buffer [20 mM
Tris-HCl (pH 8.0), 50 mM KCl, 1 mM DTT, 5% glycerol,
0.05 mg/ml poly-(dI-dC) and 100 M ZnCl2], labeled
DNA and various concentrations of the Zur protein. We
still included three controls in each EMSA experiment: i)
specific DNA competitor (unlabeled promoter region of
the same gene); ii) nonspecific DNA competitor
[unlabeled promoter region of the specific gene without the
predicted binding site. one of the negative controls]; and
iii) nonspecific protein competitor (rabbit anti-F1-protein
polyclonal antibody). After incubation at room
temperature for 30 min, the products were loaded onto a native
4% (w/v) polyacrylamide gel and electrophoresed in
0.5TBE buffer for about 30 min at 220 V. Radioactive
species were detected by autoradiography after exposure
to Kodak film at -70C.
DNase I footprinting
The promoter DNA region was prepared by PCR
amplification performed with the promoter-specific primer pairs
(see Additional file 2 for primer sequences), including a
5'-32P-labeled primer (either forward or reverse) and its
nonlabelled counterpart. The PCR products were purified
by using MinElute reaction cleanup columns (Qiagen).
Increasing amount of Zur was incubated with the labeled
DNA fragment (2 to 5 pmol) for 30 min at room
temperature in a final volume of 10 l containing binding buffer
same as EMSA [22,23]. Before DNA digestion, 10 l of
Ca2+/Mg2+ solution (5 mM CaCl2 and 10 mM MgCl2) was
added, followed by incubation for 1 min at room
temperature. Then, the optimized RQ1 RNase-Free DNase I
(Promega) was added to the reaction mixture, and the
mixture was incubated at room temperature for 50 to 90
s. The cleavage reaction was stopped by adding 9 l of the
stop solution (200 mM NaCl, 30 mM EDTA and 1% SDS)
followed by DNA extraction and precipitation. The
partially digested DNA samples were then analyzed in a 6%
polyacrylamide/8M urea gel. Protected regions were
identified by comparison with the sequence ladders. For
sequencing, the fmol DNA Cycle Sequencing System
(Promega) was used. The result was detected by
autoradiography (Kodak film).
Primer extension assay
For the primer extension assay [22,23], about 10 g of
total RNA from each strain was annealed with 1 pmol of
[-32P] end-labeled reverse primer (see Additional file 2
for primer sequences). The extended reverse transcripts
were generated as described in the protocol for Primer
Extension System-AMV Reverse Transcriptase (Promega).
The yield of each primer extension product would
indicate the mRNA expression level of the corresponding gene
in each strain, and further could be employed to map the
5' terminus of RNA transcript for each gene. The same
labeled primer was also used for sequencing with the
fmol DNA Cycle Sequencing System (Promega). The
primer extension products and sequencing materials were
concentrated and analyzed by 8 M urea-6%
polyacrylamide gel electrophoresis. The result was detected by
autoradiography (Kodak film).
LacZ reporter fusion and -Galactosidase assay
The 500 to 600 bp promoter regions upstream the znuA,
znuCB, and ykgM genes were obtained by PCR with the
Takara ExTaq DNA Polymerase using Y. pestis 201
genome DNA as the template (see Additional file 2 for
primer sequences). PCR fragments were then cloned
directionally into the SmaI (or EcoRI)and BamHI sites of
plasmid pRS551 , which contains a promotorless lacZ
reporter gene. Correct cloning was verified by DNA
sequencing. Both WT and zur were transformed with the
recombinant plasmids and grown as described in
microarray analysis. The empty plasmid pRS551 was also
introduced into both strains as negative control.
Galactosidase activity was measured on cellular extracts
by using the -Galactosidase Enzyme Assay System
(Promega) . Assays were performed in triplicate.
Identification of Zur-regulated genes by cDNA microarray
By the standard dual-fluorescent microarray hybridization
experiments, mRNA level of each gene was compared
between WT and zur upon exposure to zinc rich
conditions. Totally, the transcription of 154 genes was found to
be affected by the zur disruption. Among them, 90 genes
were down-regulated in zur, while 64 genes
up-regulated. According to the genome annotation of Y. pestis
CO92, these Zur-dependent genes were distributed in 15
functional categories (Additional file 3). Their products
included regulators, membrane-related proteins,
transport/binding proteins, biosynthesis and metabolism
related proteins and lots of unknown proteins. Additional
file 4 showed the complete list of differentially regulated
genes, giving an overall picture of the alteration of the
global gene transcription pattern of Y. pestis affected by Zur
with sufficient zinc. The microarray data (GSE15183) had
been deposited in Gene Expression Omnibus (GEO).
Validation of microarray data by Real-time RT-PCR
Microarray results are influenced by various factors, and
thereby should be validated by at least one traditional
method. Accordingly, the real-time quantitative RT-PCR,
using RNA preparations as described in the microarray
analysis, was performed to validate the microarray data.
Based on gene classification, genomic location and
transcriptional changes, 17 genes were chosen for RT-PCR
(Additional file 5). The log-transformed change in relative
quantity of mRNA level between WT and zur was
calculated for each gene. The resulting real-time RT-PCR data
were then plotted against the average log ratio values
obtained by microarray analysis. There was a strong
positive correlation (R2 = 0.796) between the two techniques
(Additional file 5). It should be noted that these 17 genes
gave a 100% consistency for differential regulation
between microarray and RT-PCR data, confirming the
reliability of our microarray data.
Characterization of DNA-binding ability of Zur by EMSA
We prepared a recombinant Y. pestis Zur protein by
overproducing it in E. coli and examined its DNA-binding
activity by EMSA (Fig. 1). Increasing amounts (from 0 to
160 pmol) of the purified Zur protein were incubated
with 10 fmol of32P-labeled znuA promoter region (it
contained a strongly predicted Zur binding site; see Fig. 1a) in
the presence of 100 M ZnCl2 (Fig. 1b). From 1.25 pmol
of Zur, the Zur-DNA complex (i.e. gel retardation)
emerged; with the Zur amount increased, gel retardation
appeared more and more heavily and reached to the peak
at 80 pmol of Zur.
FDiNguArebin1ding ability of Zur
DNA binding ability of Zur. The upstream region of znuA (panel a) or rovA (f), with or without a predicted Zur binding site,
respectively, was amplified by PCR and used as target DNA probe in EMSA. For EMSA, the [-32P]-labeled target DNA probes
(1000 to 2000 c.p.m/l) were incubated with the Zur protein in the presence or absence of 100 M ZnCl2. Increasing amounts
of Zur (b and g), ZnCl2(c), or EDTA (d and e) were employed. The mixtures were directly subjected to 4% polyacrylamide gel
electrophoresis. The rovA gene was used as negative control.
It should be noted that the target DNA was progressively
and continuously retarded (i.e. a gradual increase was
observed in the gel mobility shift) with the increase in Zur
concentration, and that 80 pmol Zur appeared to be a
maximum for DNA binding (Fig. 1b). These results could
be due to a second lower affinity binding site recognized
by Zur at higher concentrations. Alternatively, like
another regulator Fur , larger amounts of Zur proteins
in the buffered environments would promote the
formation of much more dimmers or even polymers, and thus
there might be multiple Zur molecules bound to a single
In assaying EMSA reactions containing either no zinc or
increasing concentrations of zinc (from 0.61 to 2500 M),
5 pmol of Zur was incubated with 10 fmol labeled znuA
promoter region (Fig. 1c). With zinc concentrations
increased, gel retardation occurred more and more heavily
and reach the peak at 78 M; since then, the efficacy of gel
retardation decreased gradually, and a complete
inhibition of Zur-DNA binding was observed when zinc
concentration arising to 1250 M. Accordingly, an optimized
concentration of zinc at 100 M was proposed for EMSA.
Zur bound to target DNA even without added zinc, which
might be due to the contamination of trace amount of Zn
or other bivalent metal ions in the EMSA reactions, or due
to the fact that the purified Zur protein might already
contain some bound zinc with it.
To further validate the effect of zinc, with 5 pmol of Zur
and 10 fmol of target DNA, EDTA at increasing
concentrations (from 0.61 to 2500 M) was added into different
EMSA reactions respectively, so as to chelate zinc or other
contaminated bivalent metal ions in the reaction mixture
(Fig. 1d and 1e). The complete inhibition of Zur-DNA
binding occurred from 78 M EDTA without addition of
zinc (Fig. 1d), while that occurred from 312.5 M EDTA
when 100 M zinc was added (Fig. 1e).
The above results indicated that either zinc or Zur within
a certain range of amounts was crucial for the Zur-DNA
recognition. Generally, contaminated zinc or other
bivalent metal ions was enough to ensure the Zur-DNA
recognition in EMSA, but it would be promoted by addition of
appropriate amounts of zinc into the reaction mixture.
To confirm the specificity of Zur-DNA association in
EMSA, the EMSA experiments still included a rovA
upstream DNA fragment for which no predicted Zur
binding site was found (Table 1 and Fig. 1f). The negative
EMSA results were observed, even though the Zur protein
was increased to 160 pmol in a single reaction mixture
In all of the following EMSA experiments, 10 fmol of
target DNA and 100 M zinc without addition of EDTA were
used in the reaction mixture.
Screening for potential direct Zur targets by
computational promoter analysis
We further performed computational pattern matching
analysis to predict direct Zur targets from the
Zur-dependent genes disclosed by microarray. The regulatory
consensus elements of Zur were analyzed (Fig. 2), and a position
count matrix (Fig. 2c) was generated to statistically
represent the conserved signals recognized by Zur, and
subsequently used to screen for the potential Zur binding sites
within the promoter sequences of the Zur-dependent
genes uncovered by cDNA microarray. This analysis
generated a score value for each promoter sequence, and the
larger numbers of these scores would corresponded to the
more highly consensus-like sequences in the promoters,
i.e., the higher probability of Zur direct binding .
Four genes (ykgM, znuC, znuA and astA) giving the largest
score values (Table 1) were picked out for further
investigation. The former three genes represent the first genes of
three distinct putative operons, namely ykgM-rpmJ2,
znuCB and znuA, respectively. ykgM and rpmJ2 encoded
ribosomal proteins, while znuA, znuC and znuB encoded
the Zn2+ uptake system ZnuABC. The znuCB and znuA
operons were transcribed with opposite direction and
Computational marching of the Zur consensus
Position of DNA fragment used
, The numbers indicated the nucleotide position upstream the start code of each gene tested.
TFhigeuZreur2regulatory consensus in -Proteobacteria
The Zur regulatory consensus in -Proteobacteria. (a) Original putative Zur binding sites were derived from Panina et
al's study . They were predicted from 13 genes in -Proteobacteria including E. coli, Klebsiella pneumoniae, Salmonella typhi, Y.
pestis, and Vibrio cholerae, by the comparative genomics analysis . Both coding and non-coding sequences of the above Zur
sites were trimmed into 19 bp inverted repeat sequences and then aligned to generate a sequence-logo with a Zur box
sequence (AATGTTATAWTATAACATT). (b) A position count matrix was generated as well from the alignment, where each
row represented a position and each column a nucleotide. This matrix was subsequently used for the computational pattern
arated by a short intergenic region (73 bp in length in Y.
pestis) . astA is the second gene of the astCADBE
operon responsible for the arginine succinyltransferase
pathway of arginine catabolism.
Zur binds to DNA regions upstream znuA, znuCB and
The real-time RT-PCR validated that Zur repressed the first
gene of each of the three operons, znuA, znuCB and
ykgMrpmJ2 (Additional file 5). Herein, the DNA regions
upstream these first genes (generated as indicated in Fig.
1a) were subjective to EMSA. It was demonstrated that the
purified Zur protein bound to each of these potential
target promoter regions in a Zur dose-dependent manner in
vitro (Fig. 3). Thus, a direct association of Zur with the
promoter regions of znuA, znuCB and ykgM-rpmJ2 was
The EMSA experiments still included three additional
genes, astC, astA and rovA (Fig. 3). As expected, the
negative control rovA gave negative EMSA result. astC and astA
were the first and second genes of the astCADBE operon,
respectively. The whole operon was induced by Zur as
determined by cDNA microarray, and real-time RT-PCR
confirmed the up-regulation of astC by Zur (Additional
file 5). astA gave a high score value (8.2) in the
computational promoter analysis, while astC presented a very low
value of 4.4 (Table 1). Both of astC and astA gave the
negative EMSA results (Fig. 3). Herein, neither astCADB nor
astADB was thought to be under the direct control of Zur
by directly binding to a cis-acting element within
corresponding upstream promoter region.
Zur represses promoter activity of znuA, znuCB and
To further validate the effect of Zur on the promoter
activity of znuCB, znuA and ykgM-rpmJ2, we constructed the
znuC::lacZ, znuA::lacZ and ykgM::lacZ fusion promoters
each consisting of an upstream DNA of the corresponding
gene, and then each of them was transformed into WT and
zur, respectively. The -galactosidase production of these
lacZ fusions was measured in both WT and zur, which
represented the promoter activity of the corresponding
gene in each strain.
It should be noted that the zur mutation had an effect on
the copy number of recombinant or empty pRS551
plasmid, and accordingly a normalized Miller unit was used to
calculate the fold change in the activity of each fusion
promoter in zur in relative to WT (Table 2). For each of the
three genes, there was a significant increase of
-galactosidase activity in zur compared to WT when they grew in
TMH with the addition of zinc. Thus, Zur repressed the
promoter activities of znuC, znuA and ykgM. Taken all the
gBFeingndeuisrneg o3f Zur to the promoter regions of its potential target
Binding of Zur to the promoter regions of its
potential target genes. The [-32P]-labeled upstream region of
each genes (10 fmol of target DNA probes) were incubated
with the purified Zur protein in the presence of 100 M
ZnCl2. 0, 1.25, 2.5, 5, 5, 5 and 0 pmol of Zur were used in
lanes 1 to 4 and C1 to C3, respectively. The mixtures were
directly subjected to 4% polyacrylamide gel electrophoresis.
For lanes 1 to 4, the retarded DNA band with decreased
mobility turned up, which presumably represented the
ZurDNA complex. To confirm the specificity of the binding
complexes, either a 200-fold molar excess of nonspecific
competitor (2 pmol of unlabeled znuA DNA without its predicted
binding region in lane C1) or a 200-fold molar excess of
specific competitor (2 pmol of unlabeled target DNA probe in
lane C2) was added to the binding mixture. 2 pmol of an
unrelated protein, i.e., purified rabbit anti-F1 antibody, were
included in lane C3. Both znuA and znuC gave positive EMSA
results. Since these two genes had overlapped upstream
regions and shared a single predicted Zur site, the EMSA data
of only znuA rather than znuC was presented herein.
above results together, it could be rational to say that
znuA, znuCB and ykgM-rpmJ2 were under the direct and
negative control of Zur.
Structural organization of Zur-dependent znuCB, znuA
and ykgM-rpmJ2 promoters
Primer extension assay was performed to determine the
transcription start sites of znuC, znuA and ykgM (Fig. 4). A
strong primer extension product was detected for both
znuC and ykgM, while three primer extension products
were detected for znuA. Since the shorter extension
products might represent the premature stops due to
difficulties of polymerase in passing difficult sequences, only the
longest product was chosen for the transcription start site
of znuA. Accordingly, a transcription start site was
identified for each of the three genes, and thereby a
Zur-dependent promoter was transcribed for each of them. The
nucleotide number of each transcription start site was
taken as '+1', and the -10 and -35 core promoter elements
recognized by sigma factor 70 were predicted upstream
the transcription start sites.
To precisely determine the Zur binding sites of znuCB,
znuA and ykgM-rpmJ2, DNase I footprinting assay was
performed in the presence of zinc (both coding and
noncoding strands) (Fig. 5). DNase I footprinting results
confirmed the binding of Zur to these promoter regions in
vitro. Zur protected a distinct DNA region (i.e. Zur binding
site) against DNase I digestion in a dose-dependent
pattern for ykgM (Fig. 5). As expected, the Zur box was found
in this footprint region. znuCB and znuA are transcribed
with opposite direction. Two separated footprint regions
(sites 1 and 2) were detected within the znuCB-znuA
intergenic region. The Zur box was found in site 1 rather than
The DNase I footprinting assay still included two
additional genes astA and gst. The gst upstream DNA region
gave no predicted Zur site (Table 1), while EMSA
indicated that Zur could not bind the astA promoter region in
vitro (Fig. 3). As expected, no Zur-protected region was
detected within the promoter DNA regions for both astA
and gst (Fig. 5).
The determination of Zur binding sites, transcription start
sites, and core promoter elements (-10 and -35 regions)
promoted us to depict the structural organization of
Zuractivated znuCB, znuA and ykgM-rpmJ2 promoters (Fig. 6),
giving a map of Zur-promoter DNA interaction for these
Global characterization of Zur-dependent genes
Zur senses the intracellular levels of zinc ions, and
mediates a transcriptional response aimed at restoring
homeostasis [1,7]. Under zinc-rich conditions, Zur binds the
divalent zinc ion and inhibits the transcription of target
genes. Under zinc-restricted conditions, Zur does not bind
to the corresponding genes and the zinc homeostasis
functions are expressed.
The microarray expression analysis is able to compare the
expression profiles between a WT strain (Reference
sample) and the isogenic mutant (Test sample) of Zur.
Accordingly, the detecting Zur-dependent genes included
various functional categories of genes, as characterized in
a variety of bacteria including B. subtilis , Mycobacterium
tuberculosis , Streptococcus suis  and Xanthomonas
Normalized Miller Units
Notes: The promoter DNA regions upstream znuC, znuA and ykgM were cloned into the pRS551 plasmid, respectively, to fuse with the
promoterless lacZ gene. -Galactosidase activity (miller units) was detected to represent the promoter activity. Copy number of recombinant
pRS551 was determined by real-time quantitative PCR, with the primers specific for the borne lacA gene. The detecting fold change of plasmid copy
number was set to be 1 to generate a normalization factor that was subsequently used for generating the normalized fold change of promoter
activity (miller units) in WT in relative to zur.
campestris . In the present work, a total of 154 genes
were found to be regulated by Zur in Y. pestis.
When a score value of 8 was taken as the cutoff, the
computational pattern matching analysis revealed that only
four Zur-dependent genes/operons (ykgM-rpmJ2, znuCB,
znuA and astA) contained the predicted Zur binding sites
within their upstream regions, and further EMSA
experiments confirmed that Zur bound to the target promoters
for the former three, rather than astA with a score value of
Primer extension assays. Primer extension assays were
performed for znuC, znuA and ykgM, by using RNA isolated
from the exponential-phase of both WT and zur grown in
TMH medium with 5 mM of Zn2+. An oligonucleotide primer
complementary to the RNA transcript of each gene was
designed from a suitable position. The primer extension
products were analyzed with 6% acrylamide sequencing gel.
Lanes C, T, A and G represented the Sanger sequencing
reactions. On the right side, DNA sequences were shown
from the bottom (5') to the top (3'), and the transcription
start sites were underlined.
8.2 that was the lowest one compared to those of the other
three. Thus, most of these differentially regulated genes
were affected by Zur indirectly due to the following
reasons : i) the zur mutant could accumulate more zinc
than the wild type, which could cause the transcriptional
changes in some genes as a side-effect, and ii) Zur affected
some regulatory genes and thus indirectly regulate
downstream genes through these local regulators.
Remarkably, the most strongly Zur-repressed genes
(Additional file 2) included znuA, ykgM-rpmJ2, rovA (a
virulence-required regulator to induce psaEF),psaEF (a
regulator to induce psaABC), psaA (the virulence
determinant pH6 antigen), ail (YPO2190, a putative attachment
invasion locus protein), YPO13431348
(transport/binding proteins) and YPO40184021 (phosphoribosyl
transferase proteins). In addition to major zinc homeostasis
functions (the zinc transport system ZnuABC, and two
ribosomal proteins YkgM and RpmJ2; see below), several
virulence-related genes (rovA, psaEF, psaA and ail) were
greatly repressed by Zur under zinc-rich conditions. It was
thought that Y. pestis responded to zinc limitations, and
thereby modulated the expression of not only zinc
homeostasis-related functions but also some virulence
functions required for infection. The in vivo regulatory cascade
between Zur and these virulence-related genes needs to be
elucidated in Y. pestis.
Cis-acting DNA consensus of the repressor Zur
Native Zur is a dimer, even in the absence of zinc or other
metal ions [1,7]. Zur contains two zinc binding motifs,
and binds at least two Zn2+ per dimer specifically [1,7].
Mainly acting as a negative regulator, Zur with Zn2+ as a
cofactor binds to an consensus sequence (called 'Zur box')
overlapping either the -35 region or the entire -10/-35
region of its target promoters, to block the entry of the
RNA polymerase and thereby to repress the transcription
of its target genes [24-28].
FDiNguasree I5footprinting assays
DNase I footprinting assays. Both the coding and noncoding strands of the promoter DNA fragments were generated by
PCR. Labeled DNA probe was incubated with various amounts of purified Zur (lanes 1, 2, 3 and 4 contained 0, 2.5, 5 and 10
pmol, respectively). After partial digestion with DNase I, the resulting fragments were analyzed with 6% acrylamide sequencing
gel. Lanes C, T, A and G represented the Sanger sequencing reactions. On the right side, the Zur protected regions were
labeled with bold lines, and the footprint sequences were shown below. Positive and minus numbers flanking the bold lines
indicate the nucleotide positions downstream and upstream the transcriptional site (taken as +1), respectively.
FOirgguarneiza6tion of Zur-dependent promoters for znuC, znuA and ykgM
Organization of Zur-dependent promoters for znuC, znuA and ykgM. The DNA sequences derived from the genomic
data of Y. pestis CO92 and the start codon (ATG) of each gene was shown at the 3' terminus. The bent arrows indicated the
transcription start sites and the corresponding nucleotide numbers were shown by taking the transcription start site as "+1".
The predicted promoter -10 and/or -35 elements were boxed. Zur binding sites were underlined. The invert repeats in the
Zur box was showed with two invert arrows.
Computational comparative genomics analysis 
identified the Zur box sequences of
GAAATGTTATANTATAACATTTC for -proteobacteria, GTAATGTAATAACATTAC
for the Agrobacterium group of -proteobacteria,
GATATGTTATAACATATC for the Rhodobacter group of
-proteobacteria, and TAAATCGTAATNATTACGATTTA for the
Bacillus group of Gram-positive bacteria. The above Zur
binding motifs differs from each other in nucleotide
sequence, but all of them are about 20 bp AT-rich
sequences and consist of two imperfect inverted repeat.
In the present study, a 19 bp palindrome sequence
(AATGTTATANTATAACATT) was identified to be the Zur box
in Y. pestis, which confirmed those predicted in
-Proteobacteria (see above). In our previous study [12,22], the
iron-responsive Fur regulon was characterized in Y. pestis.
Fur and Zur represent the two members of the Fur-family
regulators in Y. pestis. The Y. pestis Fur box sequence is a
91-9 inverted repeat (5'-AATGATAATNATTATCATT-3')
[12,22]. The conserved signals recognized by Fur and Zur
show a high level of similarity in nucleotide sequence
Direct Zur targets
As collectively identified in E. coli , B. subtilis [27,28],
M. tuberculosis , S. coelicolor [31,32] and X. campestri
, direct targets of the repressor Zur include primarily
zinc transport systems (e.g. ZnuABC) and other
membrane-associated transporters, protein secretion
apparatus, metallochaperones, and a set of ribosomal proteins.
The repressor Zur generally binds to a Zur box-like
cis-acting DNA element within its target promoter regions (see
above). Zur still acts as a direct activator of a Zn2+ efflux
pump in X. campestris; in this case, Zur binds to a 59 bp
GC-rich sequence with a 20 bp imperfect inverted repeat
overlapping the -35 to -10 sequence of its target
In the present work, Zur as a repressor directly regulated
znuA, zunBC and ykgM-rpmJ2 in Y. pestis. Zur binds to the
Zur box-like sequences overlapping the -10 region within
the target promoters (Fig. 6), and thus Y. pestis Zur
employed a conserved mechanism of Zur-promoter DNA
association as observed in -Proteobacteria (see above).
Regulation of zinc homeostasis by Zur
The high-affinity zinc uptake system ZnuABC belongs to
the ABC transporter family and is composed of the
periplasmic binding protein ZnuA, the ATPase ZnuC, and the
integral membrane protein ZnuB . Only in the presence
of zinc or other divalent metal cations, Zur binds to a
single cis-acting DNA element within the bidirectional
promoter region of znuA and znuCB [24-26]. In this work,
two separated DNase I footprint regions (sites 1 and 2)
were detected within the znuCB-znuA intergenic region.
The Zur box was found in only site 1 other than site 2. It
was postulated that a Zur molecule might recognize the
conserved Zur box (site 1) and further cooperatively
associate with another Zur molecule to help the later one to
bind to a less conserved (or completely different) binding
site (site 2). Further reporter fusion experiments and/or in
vitro transcription assays, using znuCB-znuA intergenic
promoter regions with different mutations/deletions
within sites 1 and 2, should be done to elucidate the roles
of site 1 and site 2 in Zur-mediated regulation of znuCB
More than 50 ribosomal proteins together with three
rRNAs (16S, 23S, and 5S rRNA) constitute the prokaryotic
ribosome that is a molecular machine for protein
biosynthesis. Most prokaryotic ribosomal proteins conserved
highly, and their genes are assigned as single-copy genes
in the genomes of many bacteria and are indispensable for
cell viability. Some ribosomal protein genes (e.g. L36,
L33, L31 and S14) have their paralogous pairs in many
bacterial genomes, and it remains unclear why many
bacteria possess these duplications in their genomes .
posed regulatory cascade would contribute to bacterial
zinc homeostasis under zinc-deficient conditions.
Zinc controls transcription of L36, L33, L31 and S14 .
Each paralogous pairs can be classified into two types; one
type contains a CxxC zinc binding motif (generally a pair
of conserved cysteines; designated C+), whereas the other
does not (C-) . The C- forms have lost the Zn ribbons
in contrast to their original ribosomal proteins . It was
predicted that an ancient duplication of the C+ forms took
place before the divergence of major bacterial lineages.
Subsequently, loss of the C+ form or loss of the CxxC
motif after the duplication generated the C-form) [33,34].
The C+ form is stable in cell when it contains a zinc ion
bound to its CxxC motif [34,35].
The paralogous pairs of L31 protein are RpmE (C+) and
YtiA (C-) in B. subtilis [34,35]. Expression of ytiA is
repressed by Zur using zinc as its cofactor . Liberation
of RpmE from ribosome is triggered by the expression of
ytiA, which is induced by the de-repression of Zur under
zinc-deficient conditions . The paralogous pairs of
L31 protein are RpmE (YPO0111) and YkgM (YPO3134)
in Y. pestis, while those of L36 protein are RpmJ
(YPO0230) and RpmJ2 (YPO3135) . YkgM and
RpmJ2 are the C- forms of corresponding ribosomal
proteins. ykgM and rpmJ2 constitutes a putative ykgM-rpmJ2
operon in Y. pestis . It was shown herein that the
ykgMrpmJ2 operon was repressed by Zur. As expected, Zur
bound to a Zur box-like element within the ykgM
Almost all the L36, L33, L31, and S14 protein genes are
regulated by zinc in S. coelicolor, and their C- paralogs was
negatively regulated by Zur [31,32]. Similar findings have
been reported in M. tuberculosis .
Taken the above together, a regulatory cascade was
proposed herein on the basis of the previous notions [31-35].
Zinc was a key factor in controlling changes in the
composition of L36, L33, L31 and S14 proteins in ribosome.
Under zinc rich conditions, original L36, L33, L31 and
S14 proteins (C+) bound with zinc ions were stable and
functional in ribosome, and expression of their C-
counterparts was repressed by Zur using zinc as its cofactor.
Under zinc starvation conditions, these C+ proteins
would not contain a zinc ion and would thus no longer be
stable in the cell, while the zinc starvation would cause a
de-repression of expression of their C- counterparts and
would be associated with the ribosome instead of
corresponding C+ proteins. The above alternation between C+
and C- ribosomal proteins might be helpful to increase
the concentration of zinc ions available for other
zincrequiring proteins in the cell. Therefore, the above
A zur null mutant of Y. pestis microtus strain 201 was
constructed in the present work. Microarray expression analysis
disclosed a set of 154 Zur-dependent genes of Y. pestis upon
exposure to zinc rich condition, and the microarray data was
validated by real-time RT-PCR. Further biochemical assays,
including LacZ reporter fusion, EMSA, DNase I footprinting,
and primer extension, revealed that Zur as a repressor
directly controlled the transcription of znuA, znuCB and
ykgM-RpmJ2 in Y. pestis by employing a conserved
mechanism of Zur-promoter DNA association as observed in
-Proteobacteria. It was thought that Zur contributed to zinc
homeostasis in Y. pestis through transcriptional repression of
the high-affinity zinc uptake system ZnuACB and two
alternative ribosomal proteins YkgM and RpmJ2.
DZ and RY conceived the study and designed the
experiments. YL performed all the experiments as well as data
mining. YQ and HG contributed to LacZ reporter analysis,
primer extension assay, and DNA binding assays. HG and
ZG were involved in protein expression and purification.
DZ and YH participated in microarray analysis. DZ, YS,
ZD and XW assisted in computational analysis and figure
construction. The manuscript was written by YL and DZ,
and revised by RY. All the authors read and approved the
Additional file 4
Additional file 5
Comparison of transcription measurements by microarray and
realtime PCR assays. The relative transcriptional levels for 17 genes selected
from Supplementary Table S1 were determined by real-time RT-PCR. The
log2 values were plotted against the microarray data log2 values. The
correlation coefficient (R2) for comparison of the two datasets is 0.796.
Click here for file
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