Molecular Characterization of Transcriptional Regulation of rovA by PhoP and RovA in Yersinia pestis
et al. (2011) Molecular Characterization of Transcriptional Regulation of rovA by PhoP and RovA in Yersinia
pestis. PLoS ONE 6(9): e25484. doi:10.1371/journal.pone.0025484
Molecular Characterization of Transcriptional Regulation of rovA by PhoP and RovA in Yersinia pestis
Yiquan Zhang 0
He Gao 0
Li Wang 0
Xiao Xiao 0
Yafang Tan 0
Zhaobiao Guo 0
Dongsheng Zhou 0
Ruifu Yang 0
Deepak Kaushal, Tulane University, United States of America
0 1 State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology , Beijing , People's Republic of China, 2 State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Centre for Disease Control and Prevention , Beijing , People's Republic of China
Background: Yersinia pestis is the causative agent of plague. The two transcriptional regulators, PhoP and RovA, are required for the virulence of Y. pestis through the regulation of various virulence-associated loci. They are the global regulators controlling two distinct large complexes of cellular pathways. Methodology/Principal Findings: Based on the LacZ fusion, primer extension, gel mobility shift, and DNase I footprinting assays, RovA is shown to recognize both of the two promoters of its gene in Y. pestis. The autoregulation of RovA appears to be a conserved mechanism shared by Y. pestis and its closely related progenitor, Y. pseudotuberculosis. In Y. pestis, the PhoP regulator responds to low magnesium signals and then negatively controls only one of the two promoters of rovA through PhoP-promoter DNA association. Conclusions/Significance: RovA is a direct transcriptional activator for its own gene in Y. pestis, while PhoP recognizes the promoter region of rovA to repress its transcription. The direct regulatory association between PhoP and RovA bridges the PhoP and RovA regulons in Y. pestis.
. These authors contributed equally to this work.
Yersinia pestis is one of the most dangerous bacterial pathogens.
Humans infected with Y. pestis manifest three main forms:
pneumonic, septicemic, and bubonic plagues, and it has a very
high mortality rate without timely and effective antibiotic
treatment . There have been at least three plague pandemics
in human history, including the Black Death, which accounted for
the death of at least one-third of the European population between
1347 and 1353. Plague remains a great threat to public health
because rodent plague epidemics are frequent in various natural
plague foci, especially in Asia, America, and Africa, human plague
infections are reported every year, and Y. pestis can possibly be
used as a biowarfare or bioterrorism agent.
PhoP and PhoQ constitute a classic regulatory two-component
system . The sensor protein PhoQ responds to low
environmental Mg2+, acidic pH, and host-secreted antimicrobial peptides,
and then phosphorylates the response regulator PhoP. As a
transcription factor, phosphorylated PhoP either activates or
represses its target genes through binding to their
promoterproximal DNA regions. Intracellular growth of Y. pestis in
macrophages occurs at early stages of systemic infection . A
phoP null mutant of Y. pestis showed reduced ability to survive in
macrophages and human neutrophils, as well as under in vitro
conditions of low pH, oxidative stress, high osmolarity, and
antimicrobial peptides [4,5,6]; this mutant is slightly attenuated in
mice . As a global regulator, PhoP controls a very complex
regulatory cascade in Y. pestis [7,8,9]. The PhoP regulons in Y.
pestis and Salmonella enterica have considerable differences in terms
of the functional changes in PhoP itself, as well as in the
architecture of PhoP-dependent promoters. This allows the PhoP
regulators to incorporate newly acquired genes into the ancestral
regulatory circuits yet retain control of the core regulon members
in these two bacteria [8,9]. The proven direct PhoP targets in Y.
pestis include several genes that function in detoxification,
protection against DNA damage, resistance to antimicrobial
peptides, and adaptation to magnesium limitation , especially
the mgtCB and udg loci that encodes an Mg2+ transport system and
a UDP-glucuronate decarboxylase for LPS modification,
respectively, required for the replication of Y. pestis in macrophages .
These PhoP-dependent mechanisms used by Y. pestis contribute to
intracellular growth of this pathogen.
As a member of the MarR/SlyA family of transcriptional
regulators that control the virulence of multiple bacterial
pathogens , RovA is required for the virulence of all three
pathogenic yersiniae (Y. pestis, Y. pseudotuberculosis, and Y.
enterocolitica) through regulation of various virulence loci
[12,13,14,15,16]. In Y. pseudotuberculosis and Y. enterocolitica, RovA
stimulates the transcription of inv, which encodes an invasin that
mediates translocation across the intestinal epithelium
[14,15,16,17]. However, the inv gene is naturally inactivated in
Y. pestis due to the insertion of an IS200-like element within its
coding region . The rovA null mutant of Y. pestis is much more
attenuated after subcutaneous inoculation than after intranasal or
intraperitoneal route, indicating a more important role for RovA
in subcutaneous infection than in the pneumonic or systemic one
. In Y. pestis, RovA stimulates the transcription of the psaEF,
psaABC, and CUS-2 prophage loci . The pH6 antigen encoded
by psaABC acts as an antiphagocytic factor  and plays a more
important role in bubonic plague than in the pneumonic and
septicemic forms, closely mimicking the role for RovA . The
CUS-2 prophage is acquired by the Y. pestis ancestor and its
genome forms an unstable episome in Antiqua and Medievalis, and a
stably integrated one in Orientalis [20,21]. The acquisition of this
prophage does not correlate to flea transmission, but contributes to
virulence in mice . The RovA regulator still plays critical roles
in the construction and functioning of the bacterial membrane,
indicating the regulatory functions of RovA in antibiotic resistance
and environmental adaptation .
The rovA gene transcribes with two distinct promoters, and the
autoregulation of rovA has been established in Y. pseudotuberculosis, but
whether both of the two promoters are dependent on RovA is unclear
[17,23]. This study indicates that the autoregulatory mechanism is
also conserved in Y. pestis, and further discloses that RovA stimulates
both of the two promoters. In addition, PhoP responses to low
magnesium signals, and recognizes the promoter region of rovA to
repress its transcription in Y. pestis. The direct regulatory association
between PhoP and RovA bridges the two distinct complexes of the
cellular pathways governed by the two regulators.
Materials and Methods
The wild-type (WT) Y. pestis biovar Microtus strain 201, avirulent
to humans but highly virulent to mice, was isolated from Microtus
brandti in Inner Mongolia, China, . The base pairs 41 to 362 of
rovA (432 bp in total length) or 41 to 631 of phoP (672 in total) were
replaced with the kanamycin resistance cassette using the one-step
inactivation method based on the lambda Red phage
recombination system with the helper plasmid pKD46 . This generated
the rovA and phoP mutants of Y. pestis, designated as DrovA and
DphoP, respectively. Chromosomal integration of the mutagenic
cassette was confirmed by PCR and sequencing using
oligonucleotides external to the integrated cassette. The elimination of
pKD46 in the mutants was verified by PCR. All primers used in
this study are listed in Table 1.
A PCR-generated DNA fragment containing the rovA or phoP
coding region with its promoter-proximal region (,500 bp upstream
the coding sequence) and transcriptional terminator (,300 bp
downstream) were cloned into the pACYC184 vector that harbors a
chloramphenicol resistance gene (GenBank accession number
X06403), as verified by DNA sequencing. The recombinant plasmid
was subsequently introduced into DrovA and DphoP, yielding the
complemented mutant strains C-rovA and C-phoP, respectively.
The original chemically defined TMH medium  [called
high magnesium, neutral pH (I)] and its different modifications
Primers (forward/reverse, 53)
GGATCCCTGCTGTGAATAAAGTCTTTGAAC TTGTTGCGTCACCATCTG/GGCTTAACCCGTCTTCAC TTACCACCAGAGCAATCACAG/ATCACGCCATCAACCTGTTC Complementation of mutants
were used for Y. pestis cultivation. The 20 mM MgCl2 in the
original TMH was changed to 10 mM to simulate the low
magnesium (II) condition. To simulate mild acidic pH (III), the
pH value of 7.2 in the original TMH was changed to 5.8.
Overnight cell culture with an optical density (OD620) of about 1.0
in each medium was diluted 1:20 into 18 ml of the corresponding
fresh medium for further cultivation.
Cells were harvested at the middle-exponential or stationary
phase for the followed primer extension or LacZ fusion assay.
For cell harvest at the middle-exponential phase, bacteria were
grown at 26uC with shaking at 230 rpm to enter the exponential
phase; and then, half of the cell cultures were incubated at 37uC
for 3 h and the remaining half were allowed to grow
continuously at 26uC for 3 h. For harvest at the stationary
phase, bacteria were grown at 26uC to enter the stationary
phase; the cell cultures were then divided to grow at 37 and
26uC, respectively, for 3 h as above. The detailed time points for
cell harvest were defined according to the bacterial growth
curves (Fig. 1). The above cultures grown at 37 and 26uC were
designated as shift from 26 to 37uC (#) and 26uC
continuously (&), respectively, so as to determine the effect of
temperature on gene transcription.
Primer extension assay
Total bacterial RNAs were extracted using the TRIzol Reagent
(Invitrogen) [7,25]. Immediately before harvesting, bacterial
cultures were mixed with RNAprotect Bacteria Reagent (Qiagen)
to minimize RNA degradation. RNA quality was monitored by
agarose gel electrophoresis and RNA quantity was determined by
spectrophotometry. For the primer extension assay [7,25], an
oligonucleotide primer complementary to a portion of the RNA
transcript of rovA gene was employed to synthesize cDNAs from
the RNA templates. About 10 mg of the total RNA from each
strain was annealed with 1 pmol of [c232P] end-labeled reverse
primer using a Primer Extension System (Promega) according to
the manufacturers instructions. The same labeled primer was also
used for sequencing with the fmolH DNA Cycle Sequencing
System (Promega). The primer extension products and sequencing
materials were concentrated and analyzed in a 6%
polyacrylamide/8 M urea gel. The result was detected by autoradiography
LacZ fusion and b-galactosidase assay
The 889 bp promoter-proximal DNA region of rovA was
obtained by PCR with the ExTaqTM DNA polymerase (Takara)
using Y. pestis 201 genome DNA as the template. PCR fragments
were then directionally cloned into the EcoRI and BamHI sites of
low-copy-number plasmid pRW50 that harbor a tetracycline
resistance gene and a promoterless lacZ reporter gene .
Correct cloning was verified by DNA sequencing. An empty
pRW50 plasmid was also introduced into each strain tested as the
negative control. The Y. pestis strains transformed with the
recombinant plasmids and the empty pRW50 plasmid were
grown as previously described to measure the b-galactosidase
activity in the cellular extracts using the b-Galactosidase Enzyme
Assay System (Promega) . Assays were performed with at least
three biological replicates.
Purification of PhoP and RovA proteins
Preparation of the purified PhoP and RovA proteins were
performed as previously described [7,25]. The entire coding
region of the phoP and rovA genes of strain 201 was directionally
cloned into the BamHI and HindIII sites of plasmid pET28a
(Novagen). The recombinant plasmid encoding the 66 His-tagged
PhoP and RovA proteins (His-PhoP and His-RovA, respectively)
Figure 1. Bacterial growth curves at 266C. Overnight cell culture with an OD620 value of about 1.0 in each medium was diluted 1:20 into 18 ml
of the corresponding fresh medium. Bacteria were then grown at 26uC with shaking at 230 rpm, and the OD620 values were monitored for each
culture with a 3 or 4 h interval until the cultures reached the stationary growth phase (a, b, and c). Experiments were done with three biological
replicates. Shown also is the design (d) for cell harvest for subsequent biochemical assays.
were transformed into Escherichia coli BL21lDE3 cells. Expression
of His-PhoP or His-RovA was induced by the addition of 1 mM
IPTG (isopropyl-b-D-thiogalactoside). The overproduced proteins
were purified under native conditions using an Ni-NTA Agarose
Column (Qiagen). The purified protein was concentrated with the
Amicon Ultra-15 centrifugal filter device (Millipore) and the
protein purity was verified by SDS-PAGE.
Gel mobility shift assay (EMSA)
The rovA promoter-proximal regions were amplified by PCR.
For EMSA [7,25], the 5 ends of DNA were labeled using [c232P]
ATP and T4 polynucleotide kinase. DNA binding was performed
in a 10 ml reaction volume containing binding buffer [1 mM
MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM
Tris-HCl (pH 7.5) and 0.05 mg/ml poly-(dI-dC)], labeled DNA
(1000 to 2000 c.p.m/ml), and increasing amounts of the His-PhoP
or His-RovA protein. Three controls were included in each EMSA
experiment: 1) cold probe as specific DNA competitor (the same
promoter-proximal DNA region unlabeled), 2) negative probe as
nonspecific DNA competitor (the unlabeled coding region of the
16S rRNA gene), and 3) nonspecific protein competitor [rabbit
anti-F1-protein polyclonal antibodies]. After incubation at room
temperature for 30 min, the products were loaded onto a native
4% (w/v) polyacrylamide gel and electrophoresed in 0.56 TBE
buffer for about 50 min at 220 V. Radioactive species were
detected by autoradiography after exposure to Kodak film at
DNase I footprinting
For DNase I footprinting [7,25], the rovA promoter-proximal
DNA regions with a single 32P-labeled end were PCR amplified
with either the sense or antisense primer being end-labeled. The
PCR products were purified using MinElute reaction cleanup
columns (Qiagen). Increasing amounts of His-PhoP or His-RovA
were incubated with the purified, labeled DNA fragment (2 to
5 pmol) for 30 min at room temperature, in a final 10 ml reaction
volume containing the binding buffer used in EMSA. Before DNA
digestion, 10 ml of Ca2+/Mg2+ solution (5 mM CaCl2 and 10 mM
MgCl2) was added, followed by incubation for 1 min at room
temperature. The optimized RQ1 RNase-Free DNase I (Promega)
was then added to the reaction mixture, and the mixture was
incubated at room temperature for 40 to 90 s. The reaction was
quenched by adding 9 ml of stop solution (200 mM NaCl, 30 mM
EDTA, and 1% SDS), followed by incubation for 1 min at room
temperature. The partially digested DNA samples were extracted
with phenol/chloroform, precipitated with ethanol, and analyzed
in 6% polyacrylamide/8 M urea gel. Protected regions were
identified by comparison with the sequence ladders. For
sequencing, we used the fmolH DNA Cycle Sequencing System
(Promega). The templates for sequencing were the same as the
DNA fragments of DNase I footprinting assays. Radioactive
species were detected as previously described.
Mutation and complementation
Real-time RT-PCR experiments were performed to assess the
relative mRNA levels of phoP and rovA in the corresponding WT,
mutant, and complemented mutant strains. The phoP transcript
was lacking in DphoP, but was restored in C-phoP relative to WT,
and moreover similar results were observed for the rovA transcript
in WT, DrovA, and C-rovA (data not shown). These data indicates
the successful mutation and complementation of phoP and rovA.
To test whether the phoP or rovA mutation had the polar effect,
the primer extension assays were conducted to detect the yield of
the rovA primer extension product that represents the rovA
transcriptional levels in the corresponding WT, mutant, and
complemented mutant strains (Figure S1). As determined by
several distinct methods (see below), the rovA gene was positively
regulated by RovA under condition I, whereas it was under the
negative control of PhoP under condition II. As determined by the
primer extension assays herein, the rovA transcription under
condition I was significantly repressed in DrovA relative to WT,
and restored in C-rovA; its transcription under condition II was
significantly elevated in DphoP relative to both C-phoP and WT
(Figure S1). The rovA gene yielded almost the same transcriptional
levels between the paired WT/DphoP or WT/DrovA strains. This
complementation analysis confirmed that the observed PhoP or
RovA-dependent transcription of rovA was due to the phoP or rovA
mutation, respectively, rather than a polar mutation.
Growth of WT, DrovA, and DphoP
The growth curves of the WT, DrovA, and DphoP strains grown
at 26uC under three different conditions I, II, and III were
determined (Fig. 1). Under condition I, both DrovA and DphoP
exhibited growth rates lower than WT (Fig. 1a). Under condition
II, growth restriction was observed for DphoP rather than DrovA
relative to WT (Fig. 1b). The three strains showed
indistinguishable growth rates under condition III (Fig. 1c). For each strain,
bacterial growth was impeded under suboptimal conditions II and
III relative to the original condition I. In particular, bacterial cells
exhibited very poor growth when each of strain was grown under
Bacterial cells were harvested at the middle-exponential or
stationary phase for the following cell culture-related biochemical
assays, and the time points for cell harvest were defined strictly
according to the growth curves (Fig. 1d). It should be noted that
bacterial cells grown under different conditions or those of
different isogenic stains were harvested at the identical growth
phase, rather than the identical optical density, which would
devoid the secondary effects of growth rate or phase. In addition,
temperature upshift from 26 to 37uC was designed prior to cell
harvest, generating 26 (26uC continuously) and 37uC (shift
from 26 to 37uC) grown cells.
Transcription of rovA under different temperatures
The primer extension experiments (Fig. 2) were then conducted
to determine the yields of primer extension product of rovA (i.e., the
relative rovA transcription levels, or the relative rovA promoter
activities) in WT upon the above temperature upshift. The primer
extension assay detected two transcriptional start sites located at
343 and 78 bp upstream of rovA (Fig. 2); therefore, two promoters
(named P2 and P1, respectively) were transcribed for rovA. At the
middle-exponential growth phase (Fig. 2a), the P2 promoter
activity showed no obvious change upon the temperature upshift
under conditions I, II, and III; yet, the P1 promoter activity
decreased upon temperature upshift under all the three growth
conditions. At the stationary growth phase (Fig. 2b), the promoter
activities of both P2 and P1 decreased upon temperature upshift
under all the three growth conditions. In conclusion, the
temperature shift from 26 to 37uC triggered the down-regulation
of both P2 and P1 promoters of rovA at the stationary growth
phase. However, the down-regulatory effect occurred only for P2
at the middle-exponential growth phase.
For subsequent experiments, only bacterial cultures of shift
from 26 to 37uC were analyzed, as 37uC is the temperature
during human infections.
Autoregulation of RovA
A rovA-lacZ fusion vector, containing the 889 bp
promoterproximal region of rovA and the promoterless lacZ, was
transformed into both WT and DrovA to compare the rovA
promoter activities in these two strains grown under conditions I,
II, and III, respectively (Fig. 3a). Under all the three conditions,
the expression of rovA significantly decreased in DrovA relative to
WT. In addition, the primer extension experiments (Fig. 3b) were
conducted to compare the yields of primer extension product of
rovA in WT and DrovA; the activities of both P1 and P2 promoters
were under the positive control of RovA under all the three growth
conditions, which was consistent with the above lacZ fusion data.
Interestingly, the rovA transcription in WT were up-regulated
under condition III relative to the other two conditions (Fig. 2a
and 2b), and thus mild acid appeared to stimulate the rovA
The 889 bp promoter-proximal region of rovA tested in the
LacZ fusion assay was amplified, radioactively labeled, and
subjected to EMSA with a purified His-RovA protein (Fig. 3c).
The results show that His-RovA is able to bind to this DNA
fragment in a dose-dependent manner in vitro (Fig. 3c). As further
determined by DNase I footprinting (Fig. 3d), the purified
HisRovA protected two distinct regions upstream of rovA against
DNase I digestion in a dose-dependent manner. These two
footprints, located from 592 to 502 bp (RovA site 2) and from 39
to 6 bp (Site 1) upstream of rovA, respectively, were considered
RovA sites. Taken together, RovA is able to recognize
all the promoters of its own gene to stimulate their activity in
To test the affinity of RovA to Sites 1 and 2, EMSA was
performed with two distinct rovA upstream DNA fragments
containing Sites 1 and 2, respectively (Fig. 4). DNA retardation
occurred at 0.74 pmol of His-RovA for the fragment containing
Site 2 (Fig. 4a), whereas it was observed at 2.46 pmol for that
containing Site 1 (Fig. 4b). This indicated that RovA had a much
higher affinity to Site 2 than Site 1. The RovA proteins at all
amounts used could not bind to the 16S rDNA fragment as the
negative control, confirming the specificity of EMSA in this study
Negative regulation of rovA by PhoP
The rovA-lacZ fusion vector (Fig. 3a) was transformed into both
WT and DphoP to compare the rovA promoter activities in the two
strains grown under conditions I, II, and III (Fig. 5a). Under
conditions I and III, there was no significant difference in the rovA
promoter activities in the WT and DphoP strains. Under condition
Figure 4. Affinity of RovA to its Sites 1 and 2. Labeled DNA fragments (a and b), which contained RovA Sites 1 and 2 respectively, were
incubated with increasing amounts of purified His-RovA. The EMSA experiment was conducted with a coding region of the 16S rRNA gene (c) as the
negative control. Also shown is the schematic representation of the EMSA design (d).
II, the expression of rovA was significantly enhanced in the DphoP
relative to the WT. Further primer extension experiments for rovA
(Fig. 5b) again detected the two promoters, P1 and P2, when the
bacterial cells were grown under the three conditions. P1 activity
was under the negative control of PhoP under condition II, and
independent of this regulator under the other two growth
conditions. PhoP had no effect on P2 activity under all the three
conditions. The EMSA assay disclosed that the purified His-PhoP
protein was able to bind to the 889 promoter-proximal region of
rovA in a dose-dependent manner in vitro (Fig. 5c). Subsequent
DNase I footprinting experiments (Fig. 5d) indicated that
HisPhoP protected a single region located from 102 to 47 bp
upstream of rovA. This footprint was considered the PhoP site.
Therefore, the rovA transcription is negatively controlled by the
PhoP regulator under the low magnesium conditions through the
PhoP-promoter DNA association.
Promoter structure of rovA
In this study, DNase I footprinting experiments precisely
determined the PhoP and RovA sites for rovA. The primer
extension assays mapped two promoters (P1 and P2) for rovA.
Accordingly, the core promoter 210 and 235 elements for RNA
polymerase recognition were predicted. Collection of data on the
translation/transcription start sites, Shine-Dalgarno sequence (a
ribosomal binding site in the mRNA), promoter 210 and 235
elements, as well as PhoP and RovA sites enabled us to depict the
organization of PhoP and RovA-dependent promoters of rovA
characterized herein (Fig. 6). The Y. pestis rovA promoter-proximal
region is essentially identical to the Y. pseudotuberculosis one .
The two RovA sites within the rovA promoter-proximal region in
Y. pestis are very similar to, although slightly different from, those
determined in Y. pseudotuberculosis . Y. pseudotuberculosis and Y.
pestis have the same P2 promoter, but P1 in Y. pestis is 2 bp
upstream of that in Y. pseudotuberculosis. The slight differences
observed in the P1 sites and the RovA site sequences might be due
to the personal inclination during DNA sequence reading.
Bacterial growth under magnesium-limitation conditions
The magnesium cation (Mg2+) is one of the essential elements
for bacterial cell growth due to its function as a cofactor of
enzymes. When grown under the condition II, Y. pestis cells
exhibited poor growth, and moreover a extremely heavy
restriction of growth was observed for DphoP. The Mg2+ transport
systems are positively controlled by the Mg2+-responsive PhoP
regulator in Y. pestis [7,29], and the phoP mutation will impair the
magnesium homeostasis of DphoP under Mg2+-limiting
Regulation of rovA by growth temperature
The three pathogenic yersiniae Y. pestis, Y. pseudotuberculosis, and
Y. enterocolitica are ranked at different linkages in the evolution of
the Yersinia genus . The Y. pseudotuberculosisY. pestis clade
diverged from Y. enterocolitica hundreds of millions of years ago,
whereas Y. pestis from Y. pseudotuberculosis within thousands of years
[31,32]. Consistently, Y. pseudotuberculosis and Y. pestis are very
divergent from Y. enterocolitica, but share a very high level of
genomic homology with each other.
As previously shown [17,33], two (P1 and P2) and three
promoters are transcribed for rovA in Y. pseudotuberculosis and Y.
enterocolitica, respectively, at room temperature (20 to 26uC), but a
down-regulation was observed at 37uC. In addition to the
mechanism of transcriptional regulation, the temperature control
of rovA expression occur also at the post-transcriptional level in Y.
pseudotuberculosis . In this study, P1 and P2 were also detected
for rovA in Y. pestis. The temperature shift from 26 to 37uC
triggered the down-regulation of P1 promoter in Y. pestis at both
stationary and middle-exponential growth phase. However, the
down-regulation of P2 upon temperature upshift occurred only at
the stationary phase rather than the middle-exponential one,
indicating a growth phase-dependent effect for P2.
Regulation of rovA by transcriptional regulators
This study confirms that the autoregulation of rovA in Y. pestis is
identical to that reported in Y. pseudotuberculosis, and further
discloses that RovA stimulates the activity of both of the two
promoters of its own gene in Y. pestis.
The nucleoid-associated protein H-NS silences target genes by
selectively targeting their upstream DNA sequences with GC
contents lower than that of the resident genome . Similarly, Y.
pseudotuberculosis H-NS binds to a long DNA region upstream of P2
(Fig. 6), and represses the rovA transcription [17,23]. In addition, a
LysR-type regulator RovM specifically binds to a short region
closely upstream of the 235 element of P1 and far downstream of
P2 (Fig. 6), and participates in the repression of rovA in Y.
pseudotuberculosis . Interestingly, the cooperation of RovM and
H-NS is required for efficient silencing of rovA transcription . It
seems that the interaction of RovM and H-NS on the rovA
promoter-proximal regions, which is accompanied by H-NS/
RovM-DNA association, promotes the formation of a stable
repressor complex to silence the rovA transcription .
The RovA Site 2 overlaps the H-NS site for rovA (Fig. 6), and
RovA alleviates the H-NS-mediated repression of rovA by
antagonizing the H-NS-promoter DNA association [17,23]. The
RovA Site 2 is upstream of the 235 elements of both P1 and P2,
and thus the transcriptional activation of P1 and P2 by RovA is a
Figure 6. Organization of the rovA promoter-proximal region. The DNA sequence was derived from the genomic data of Y. pestis 91001 and
the start codon was shown at the 3 terminal. The bent arrows indicate the two promoters P1 and P2 (transcription start sites). Predicted promoters
210 and 235 elements, and Shine-Dalgarno box are enclosed in boxes. The RovA, PhoP, H-NS, and RovM sites are underlined with different lines. The
H-NS [17,23] and RovM  sites are derived from those determined in Y. pseudotuberculosis, since the Y. pestis rovA promoter-proximal region is
essentially identical to the Y. pseudotuberculosis one.
Class I stimulation dependent on the RNA polymerase a subunit
C-terminal domain (aCTD) for function [23,37]. With the
development of RovA autostimulation, the cellular RovA reaches
a certain level; in this case, the low-affinity Site 1 is occupied by
RovA. Notably, Site 1 is downstream of both P1 and P2, and
accordingly the RovA Site 1 association blocks the entry of the
RNA polymerase, which destroys the endless RovA-mediated
activation of rovA transcription [23,37,38]. This RovA
concentration-dependent regulation of its own gene allows the bacterium to
finely modulate the cellular RovA levels for the most favorable
production of RovA-dependent virulence factors [23,37,38].
As shown in this study, the PhoP regulator recognizes a single
site within the rovA promoter-proximal region, and negatively
controls the rovA transcription under magnesium-limiting
conditions. It is further confirmed that PhoP as the responsive regulator
of the PhoP/PhoQ two-component system responds to low
magnesium signals [7,8,9] rather than magnesium-rich or acidic
pH conditions. An 18-bp PhoP box sequence
(TGTTTAWN4TGTTTAW), which is consisted of a direct repeat of the
heptanucleotide consensus (underlined), has been established previously
in Y. pestis . This box consensus represents the conserved signals
for PhoP recognition in Y. pestis. Herein, a PhoP box-like sequence
(TGTGTTTTTAATGTTAAT) is found in the PhoP site for rovA.
Notably, the promoter activity of P1, but not P2, is dependent on
PhoP. The PhoP site overlaps the 210 region of the P1 promoter,
and thus the PhoP-promoter association is thought to block RNA
polymerase-DNA association, thereby repressing the transcription
of rovA. This mode of regulator-promoter DNA interaction for
transcriptional repression is frequently observed in transcriptional
repressors, such as Fur  and Zur  in Y. pestis. PhoP and
RovA control distinct complexes of cellular pathways, especially
including those involved in virulence and host-adaptation
[7,8,9,12,17,41]. The two regulons governed by PhoP and RovA,
respectively, have evolved to merge into a single global regulatory
circuit, due to the direct transcriptional association between PhoP
The rovA upstream DNA regions are identical in Y. pestis and Y.
pseudotuberculosis, and moreover all the four regulators (RovA,
PhoP, H-NS, and RovM) involved in the regulation of rovA are
extremely conserved in these two bacteria. Therefore, the
mechanisms that regulate rovA discussed above are conserved in
these two bacteria.
S. typhimurium has the homolgous gene (named slyA) of rovA. SlyA
and PhoP formed a complex positive feedback circuit in S.
typhimurium . The slyA transcription is activated by the PhoP/
PhoQ system under low Mg2+ conditions [43,44], and PhoP
footprints the slyA upstream region , which indicating that
PhoP stimulates slyA directly through PhoP-promoter DNA
association. H-NS binds to the phoP upstream region to silence
the transcription of phoPQ operon under high Mg2+ conditions
. PhoP binds to the phoP upstream region and activates (i.e.,
autoregulates) the phoP transcription under low Mg2+ conditions
[46,47]. SlyA also footrpints with the phoP upstream region, and
competes with H-NS since they share the same footprint that is
adjacent to the PhoP site within the phoP upstream region .
Thus, the association between the phoP upstream region and SlyA
will facilitate PhoP binding to the PhoP site by reducing the
inhibitory activity of the H-NS protein . Whether Y. pestis
employs a regulatory feedback circuit involving in RovA and
PhoP/PhoQ needs to be elucidated. In addition to Y. pestis and Y.
pseudotuberculosis, the genus Yersinia still contains another pathogenic
species, i.e., Y. enterocolitica, which shows a widely genetic diversity
from the Y. pseudotuberculosis/Y. pestis clade. The DNA upstream of
rovA in Y. enterocolitica differs greatly from the relevant Y.
pseudotuberculosis/Y. pestis DNA. The Y. enterocolitica rovA gene is
transcribed with the P1 and P2 promoters that are identical to
those in Y. pseudotuberculosis/Y. pestis, and moreover a third
promoter P3 downstream of P1 is detected. The differences in
the promoters result in significantly lower levels of rovA
transcription in Y. enterocolitica. H-NS binds to two regions
upstream of rovA to repress the rovA transcription in Y. enterocolitica.
H-NS shows much lower affinities for either of the two sites than
for the reported single Y. pseudotuberculosis/Y. pestis site. RovA
stimulates the rovA transcription in Y. enterocolitica although the lack
of observable RovA binding to the Y. enterocolitica promoter. RovM
binds to a single region upsteam of rovA to repress the rovA
transcription, as reported for Y. pseudotuberculosis. Together, the
cisacting DNA region of rovA has undergone great genetic variation
between Y. enterocolitica and Y. pseudotuberculosis/Y. pestis, which will
lead to the remodeling in the mechanisms for controlling the rovA
transcription, although the relevant trans-acting factors (H-NS,
RovA, and RovM) are highly conserved in the three pathogenic
Figure S1 Primer extension assay for validation of
nonpolar mutation. The rovA or phop null mutant (DrovA or Dphop,
respectively) was generated from the wild-type strain 201 (WT),
and then the corresponding complemented mutant strain (C-rovA
or C-phop, respectively) was constructed. As determined by several
distinct methods (see the text of manuscript), the P1 promoter of
rovA was positively regulated by RovA when the bacteria were
grown in the original TMH medium, but negatively controlled by
PhoP when grown in the TMH containing 10mM MgCl2. Herein,
an oligonucleotide primer, which was complementary to the RNA
transcript of rovA, was employed to detect the primer extension
product that represented the relative P1 promoter activity in the
corresponding strains. The primer extension products were
analyzed with 8 M urea26% acrylamide sequencing gel. Lanes
C, T, A, and G represent the Sanger sequencing reactions. Shown
on the right side of the image is the transcription start site
(nucleotide T, corresponding to the P1 promoter) that was located
at 78 bp upstream of rovA. The P1 promoter was significantly
repressed in DrovA relative to both C-rovA and WT gown in the
original TMH; yet, it was significantly enhanced in DphoP relative
to both C-phoP and WT grown in the TMH containing 10mM
MgCl2 The P1 promoter was transcribed at almost the same level
in every paired WT and complemented mutant. These results
confirmed that the phoP or rovA mutation was nonpolar.
The English writing of the manuscript was polished by EnPapers.
Conceived and designed the experiments: DZ RY. Performed the
experiments: YZ HG LW XX YT ZG DZ. Analyzed the data: YZ DZ.
Contributed reagents/materials/analysis tools: YZ DZ. Wrote the paper:
DZ RY YZ.
1. Perry RD , Fetherston JD ( 1997 ) Yersinia pestis-etiologic agent of plague . Clin Microbiol Rev 10 : 35 - 66 .
2. Groisman EA ( 2001 ) The pleiotropic two-component regulatory system PhoPPhoQ . J Bacteriol 183 : 1835 - 1842 .
3. Lukaszewski RA , Kenny DJ , Taylor R , Rees DG , Hartley MG , et al. ( 2005 ) Pathogenesis of Yersinia pestis infection in BALB/c mice: effects on host macrophages and neutrophils . Infect Immun 73 : 7142 - 7150 .
4. Oyston PC , Dorrell N , Williams K , Li SR , Green M , et al. ( 2000 ) The response regulator PhoP is important for survival under conditions of macrophageinduced stress and virulence in Yersinia pestis . Infect Immun 68 : 3419 - 3425 .
5. Hitchen PG , Prior JL , Oyston PC , Panico M , Wren BW , et al. ( 2002 ) Structural characterization of lipo-oligosaccharide (LOS) from Yersinia pestis: regulation of LOS structure by the PhoPQ system . Mol Microbiol 44 : 1637 - 1650 .
6. O'Loughlin JL , Spinner JL , Minnich SA , Kobayashi SD ( 2010 ) Yersinia pestis two-component gene regulatory systems promote survival in human neutrophils . Infect Immun 78 : 773 - 782 .
7. Li YL , Gao H , Qin L , Li B , Han YP , et al. ( 2008 ) Identification and characterization of PhoP regulon members in Yersinia pestis biovar Microtus . BMC Genomics 9 : 143 .
8. Perez JC , Shin D , Zwir I , Latifi T , Hadley TJ , et al. ( 2009 ) Evolution of a bacterial regulon controlling virulence and Mg(2+) homeostasis . PLoS Genet 5 : e1000428 .
9. Perez JC , Groisman EA ( 2009 ) Transcription factor function and promoter architecture govern the evolution of bacterial regulons . Proc Natl Acad Sci U S A 106 : 4319 - 4324 .
10. Grabenstein JP , Fukuto HS , Palmer LE , Bliska JB ( 2006 ) Characterization of phagosome trafficking and identification of PhoP-regulated genes important for survival of Yersinia pestis in macrophages . Infection and immunity 74 : 3727 - 3741 .
11. Ellison DW , Miller VL ( 2006 ) Regulation of virulence by members of the MarR/SlyA family . Curr Opin Microbiol 9 : 153 - 159 .
12. Cathelyn JS , Crosby SD , Lathem WW , Goldman WE , Miller VL ( 2006 ) RovA, a global regulator of Yersinia pestis, specifically required for bubonic plague . Proc Natl Acad Sci U S A 103 : 13514 - 13519 .
13. Dube PH , Handley SA , Revell PA , Miller VL ( 2003 ) The rovA mutant of Yersinia enterocolitica displays differential degrees of virulence depending on the route of infection . Infect Immun 71 : 3512 - 3520 .
14. Nagel G , Lahrz A , Dersch P ( 2001 ) Environmental control of invasin expression in Yersinia pseudotuberculosis is mediated by regulation of RovA, a transcriptional activator of the SlyA/Hor family . Molecular microbiology 41 : 1249 - 1269 .
15. Revell PA , Miller VL ( 2000 ) A chromosomally encoded regulator is required for expression of the Yersinia enterocolitica inv gene and for virulence . Molecular microbiology 35 : 677 - 685 .
16. Ellison DW , Lawrenz MB , Miller VL ( 2004 ) Invasin and beyond: regulation of Yersinia virulence by RovA . Trends in microbiology 12: 296 - 300 .
17. Heroven AK , Nagel G , Tran HJ , Parr S , Dersch P ( 2004 ) RovA is autoregulated and antagonizes H-NS-mediated silencing of invasin and rovA expression in Yersinia pseudotuberculosis . Molecular microbiology 53 : 871 - 888 .
18. Simonet M , Riot B , Fortineau N , Berche P ( 1996 ) Invasin production by Yersinia pestis is abolished by insertion of an IS200-like element within the inv gene . Infect Immun 64 : 375 - 379 .
19. Huang XZ , Lindler LE ( 2004 ) The pH 6 antigen is an antiphagocytic factor produced by Yersinia pestis independent of Yersinia outer proteins and capsule antigen . Infect Immun 72 : 7212 - 7219 .
20. Derbise A , Chenal-Francisque V , Pouillot F , Fayolle C , Prevost MC , et al. ( 2007 ) A horizontally acquired filamentous phage contributes to the pathogenicity of the plague bacillus . Mol Microbiol 63 : 1145 - 1157 .
21. Li Y , Dai E , Cui Y , Li M , Zhang Y , et al. ( 2008 ) Different region analysis for genotyping Yersinia pestis isolates from China . PLoS ONE 3 : e2166 .
22. Yang F , Ke Y , Tan Y , Bi Y , Shi Q , et al. ( 2010 ) Cell membrane is impaired, accompanied by enhanced type III secretion system expression in Yersinia pestis deficient in RovA regulator . PLoS One 5.e12840.
23. Tran HJ , Heroven AK , Winkler L , Spreter T , Beatrix B , et al. ( 2005 ) Analysis of RovA, a transcriptional regulator of Yersinia pseudotuberculosis virulence that acts through antirepression and direct transcriptional activation . J Biol Chem 280 : 42423 - 42432 .
24. Zhou D , Tong Z , Song Y , Han Y , Pei D , et al. ( 2004 ) Genetics of metabolic variations between Yersinia pestis biovars and the proposal of a new biovar, microtus . J Bacteriol 186 : 5147 - 5152 .
25. Zhan L , Han Y , Yang L , Geng J , Li Y , et al. ( 2008 ) The cyclic AMP receptor protein, CRP, is required for both virulence and expression of the minimal CRP regulon in Yersinia pestis biovar microtus . Infect Immun 76 : 5028 - 5037 .
26. Straley SC , Bowmer WS ( 1986 ) Virulence genes regulated at the transcriptional level by Ca2+ in Yersinia pestis include structural genes for outer membrane proteins . Infect Immun 51 : 445 - 454 .
27. El-Robh MS , Busby SJ ( 2002 ) The Escherichia coli cAMP receptor protein bound at a single target can activate transcription initiation at divergent promoters: a systematic study that exploits new promoter probe plasmids . Biochem J 368 : 835 - 843 .
28. Chain PS , Carniel E , Larimer FW , Lamerdin J , Stoutland PO , et al. ( 2004 ) Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis . Proc Natl Acad Sci U S A 101 : 13826 - 13831 .
29. Zhou D , Han Y , Qin L , Chen Z , Qiu J , et al. ( 2005 ) Transcriptome analysis of the Mg2+-responsive PhoP regulator in Yersinia pestis . FEMS Microbiol Lett 250 : 85 - 95 .
30. Chen PE , Cook C , Stewart AC , Nagarajan N , Sommer DD , et al. ( 2010 ) Genomic characterization of the Yersinia genus . Genome Biol 11 : R1 .
31. Achtman M , Zurth K , Morelli G , Torrea G , Guiyoule A , et al. ( 1999 ) Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis . Proc Natl Acad Sci U S A 96 : 14043 - 14048 .
32. Skurnik M , Peippo A , Ervela E ( 2000 ) Characterization of the O-antigen gene clusters of Yersinia pseudotuberculosis and the cryptic O-antigen gene cluster of Yersinia pestis shows that the plague bacillus is most closely related to and has evolved from Y. pseudotuberculosis serotype O:1b . Mol Microbiol 37 : 316 - 330 .
33. Lawrenz MB , Miller VL ( 2007 ) Comparative analysis of the regulation of rovA from the pathogenic yersiniae . J Bacteriol 189 : 5963 - 5975 .
34. Herbst K , Bujara M , Heroven AK , Opitz W , Weichert M , et al. ( 2009 ) Intrinsic thermal sensing controls proteolysis of Yersinia virulence regulator RovA . PLoS Pathog 5 : e1000435 .
35. Stoebel DM , Free A , Dorman CJ ( 2008 ) Anti-silencing: overcoming H-NSmediated repression of transcription in Gram-negative enteric bacteria . Microbiology 154 : 2533 - 2545 .
36. Heroven AK , Dersch P ( 2006 ) RovM, a novel LysR-type regulator of the virulence activator gene rovA, controls cell invasion, virulence and motility of Yersinia pseudotuberculosis . Mol Microbiol 62 : 1469 - 1483 .
37. Ishihama A ( 2000 ) Functional modulation of Escherichia coli RNA polymerase . Annu Rev Microbiol 54 : 499 - 518 .
38. Heroven AK , Bohme K , Rohde M , Dersch P ( 2008 ) A Csr-type regulatory system, including small non-coding RNAs, regulates the global virulence regulator RovA of Yersinia pseudotuberculosis through RovM . Mol Microbiol 68 : 1179 - 1195 .
39. Gao H , Zhou D , Li Y , Guo Z , Han Y , et al. ( 2008 ) The iron-responsive Fur regulon in Yersinia pestis . J Bacteriol 190 : 3063 - 3075 .
40. Li Y , Qiu Y , Gao H , Guo Z , Han Y , et al. ( 2009 ) Characterization of Zurdependent genes and direct Zur targets in Yersinia pestis . BMC Microbiol 9 : 128 .
41. Cathelyn JS , Ellison DW , Hinchliffe SJ , Wren BW , Miller VL ( 2007 ) The RovA regulons of Yersinia enterocolitica and Yersinia pestis are distinct: evidence that many RovA-regulated genes were acquired more recently than the core genome . Mol Microbiol 66 : 189 - 205 .
42. Song H , Kong W , Weatherspoon N , Qin G , Tyler W , et al. ( 2008 ) Modulation of the regulatory activity of bacterial two-component systems by SlyA . J Biol Chem 283 : 28158 - 28168 .
43. Norte VA , Stapleton MR , Green J ( 2003 ) PhoP-responsive expression of the Salmonella enterica serovar typhimurium slyA gene . Journal of bacteriology 185 : 3508 - 3514 .
44. Shi Y , Latifi T , Cromie MJ , Groisman EA ( 2004 ) Transcriptional control of the antimicrobial peptide resistance ugtL gene by the Salmonella PhoP and SlyA regulatory proteins . The Journal of biological chemistry 279 : 38618 - 38625 .
45. Kong W , Weatherspoon N , Shi Y ( 2008 ) Molecular mechanism for establishment of signal-dependent regulation in the PhoP/PhoQ system . J Biol Chem 283 : 16612 - 16621 .
46. Soncini FC , Vescovi EG , Groisman EA ( 1995 ) Transcriptional autoregulation of the Salmonella typhimurium phoPQ operon . J Bacteriol 177 : 4364 - 4371 .
47. Lejona S , Aguirre A , Cabeza ML , Garcia Vescovi E , Soncini FC ( 2003 ) Molecular characterization of the Mg2+-responsive PhoP-PhoQ regulon in Salmonella enterica . J Bacteriol 185 : 6287 - 6294 .