Direct and negative regulation of the sycO-ypkA-ypoJ operon by cyclic AMP receptor protein (CRP) in Yersinia pestis
Institute of Laboratory Animal Sciences, Chinese Academy of Medical Peking Union Medical College
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology
Background: Pathogenic yersiniae, including Y. pestis, share a type III secretion system (T3SS) that is composed of a secretion machinery, a set of translocation proteins, a control system, and six Yop effector proteins including YpkA and YopJ. The cyclic AMP receptor protein (CRP), a global regulator, was recently found to regulate the laterally acquired genes (pla and pst) in Y. pestis. The regulation of T3SS components by CRP is unknown. Results: The sycO, ypkA and yopJ genes constitute a single operon in Y. pestis. CRP specifically binds to the promoter-proximate region of sycO, and represses the expression of the sycO-ypkA-yopJ operon. A single CRP-dependent promoter is employed for the sycO-ypkA-yopJ operon, but two CRP binding sites (site 1 and site 2) are detected within the promoter region. A CRP box homologue is found in site 1 other than site 2. The determination of CRP-binding sites, transcription start site and core promoter element (-10 and -35 regions) promotes us to depict the structural organization of CRP-dependent promoter, giving a map of CRP-promoter DNA interaction for sycO-ypkA-yopJ. Conclusion: The sycO-ypkA-yopJ operon is under the direct and negative regulation of CRP in Y. pestis. The sycO-ypkA-yopJ promoter-proximate regions are extremely conserved in Y. pestis, Y. pseudotuberculosis and Y. enterocolitica. Therefore, data presented here can be generally applied to the above three pathogenic yersiniae.
Plague, caused by Yesinia pestis, is a zoonotic disease that
threatened public health seriously. The three pathogenic
Yersinia species, Y. pestis, Y. pseudotuberculosis, and Y.
enterocolitica, share a type III secretion system (T3SS) that is
composed of a secretion machinery, a set of translocation
proteins, a control system, and six Yop effector proteins
[1,2]. Through the T3SS, pathogenic yersiniae inject
effectors into the cytosol of eukaryotic cells when docking at
the surface of host cell. The injected Yops perturb the
signaling cascades that activate the processes of
phagocytosis, cytokine release and respiratory burst. As a result,
phagocytosis is inhibited, recruitment of PMNs and
monocyte-derived macrophages is reduced, and
lymphocyte proliferation is prevented.
The cyclic AMP receptor protein (CRP) is a global
regulator that controls the transcription initiation for more than
100 bacterial genes/operons . CRP is activated by cyclic
AMP (cAMP), forming the cAMP-CRP complex. This
complex binds a symmetrical consensus DNA sequence
TGTGA-N6-TCACA (known as the CRP box sequence)
located within the upstream promoter regions. The
CRPpromoter DNA interaction is crucial for the regulation of
CRP and its homologues are required for virulence and/or
expression of virulence genes in several pathogens,
including Y. pestis , Y. enterocolitica , Vibrio vulnificus ,
Vibrio cholerae  and Mycobacterium tuberculosis . The
crp disruption in Y. pestis attenuates both in vitro and in
vivo growth of the mutant, and leads to a >15,000-fold
loss of virulence after subcutaneous infection, but a less
than 40-fold increase in LD50 by intravenous inoculation
. CRP plays a role in the globally transcriptional
regulation of genes including a wide set of virulence genes in
Y. pestis . Especially, it directly stimulates the expression
of plasminogen activator (Pla) [4,9], a virulence factor
essential for bubonic and primary pneumonic plague
Yersinia protein kinase A (YpkA) and Yersinia outer
protein J (YopJ) are encoded by plasmid pCD1-borne ypkA
and yopJ genes in Y. pestis, respectively. YpkA/YopO is a
serine/threonine protein kinase involved in host actin
cytoskeletal rearrangements and in inhibition of
phagocytosis , while YopJ/YopP acts as an acetyltransferase
inhibiting mitogen-activated protein kinase (MAPK) and
the nuclear factor kappaB (NFB) signaling pathways
used in innate immune response . Both of them are
the effector proteins of T3SS and essentially contribute to
the virulence of Y. pestis [2,14]. SycO is a T3SS chaperone
that increases solubility and secretion efficiency of the
effector YpkA/YopO .
In the present work, we disclosed that CRP directly and
negatively regulated the sycO-ypkA-yopJ operon in Y. pestis
under the calcium-rich condition, by using real-time
RTPCR, LacZ reporter fusion, electrophoretic mobility shift
assay (EMSA), and DNase I footprinting assay. Data
presented here further validated the important role of CRP in
virulence of Y. pestis.
The wild-type (WT) Y. pestis strain 201 belongs to a newly
established Y. pestis biovar, Microtus , which was
thought to be avirulent to humans, but highly virulent to
mice. An in-frame deletion of the crp gene was constructed
by using one step inactivation method , generating a
mutant strain referred to as crp . Bacteria were grown in
Luria-Bertani (LB) broth or chemically defined TMH
medium  at 26 or 37C. E. coli was grown in LB broth
at 37C. When needed, antibiotics were added at the
following concentrations: 100 g/ml for ampicillin, 50 g/
ml for kanamycin, and 34 g/ml chloramphenicol.
Bacterial growth and RNA isolation
The WT and crp were grown at 26C in the TMH medium
with the addition of 1 mM cAMP (referred to as
'TMH1mM cAMP') to an OD620 of about 1.0, and then diluted
by 20-fold into the fresh 'TMH-1mM cAMP' medium for
cultivating at 26C until an OD620 of about 1.0, and
finally transferred to 37C for 3 h. Bacterial cells were
harvested for the isolation of total RNA. Immediately before
harvesting, bacterial cultures were mixed with RNAprotect
Bacteria Reagent (Qiagen) to minimize RNA degradation.
Total RNA was isolated using the MasterPure RNA
Purification kit (Epicenter). Contaminated DNA in RNA
samples was removed by using the Amibion's DNA-free Kit.
RNA quality was monitored by agarose gel electrophoresis
and RNA quantity was measured by spectrophotometer.
Gene-specific primers (Table 1) were designed to produce
a 150 to 200 bp amplicon for each gene. 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 PCR was performed in triplicate as
described previously , through the LightCycler system
(Roche) together with the SYBR Green master mix. Based
on the standard curve of 16S rRNA expression for each
RNA preparation, the relative mRNA level was determined
by the classic Ct method. 16S rRNA gene was used to
normalize that of all the other genes. The transcriptional
variation between the WT and crp strains was then
calculated for each gene. A mean ratio of two was taken as the
cutoff of statistical significance.
Primer sequence (5'3')
LacZ reporter fusion and -Galactosidase assay
A 408 bp promoter-proximate of cycO (Table 1) was
cloned directionally into the EcoRI and BamHI sites of
plasmid pRS551 expressing LacZ, which was verified by
DNA sequencing. The recombinant plasmids were
introduced into the WT and crp, respectively. The plasmid
pRS551 was also transformed as negative control. The
resulting strains were grown as described in RNA
isolation. -Galactosidase activity was determined for each
strain by using the Promega -Galactosidase Enzyme
Assay System . Assays were performed in triplicate.
Preparation of purified recombinant His-CRP protein,
electrophoretic mobility shift assay (EMSA) and DNase I
footprinting assay were conducted as described previously
. For EMSA, a 468 bp promoter-proximate region of
cycO (containing a predicted CRP binding site) or the
corresponding cold probe (i.e. unlabeled target DNA) (Table
1) was radioactively labeled, incubated with increasing
amounts of purified His-CRP protein, and then subjected
to 4% (w/v) polyacrylamide gel electrophoresis. In the
DNase I footprinting experiments, coding or noncoding
strand (261 bp in length) containing the predicted CRP
binding site was labeled with [-32P] at the 5' end, then,
incubated with increasing amounts of His-CRP; after
partial digestion with DNase I, the resulting fragments were
analyzed by denaturing gel electrophoresis. Radioactive
species were detected by autoradiography.
Primer extension analysis
For the primer extension assay , an oligonucleotide
primer (Table 1) complementary to a portion of the RNA
transcript of each gene was employed to synthesize
cDNAs from the RNA templates. Electrophoresis of
primer extension products was performed with a 6%
polyacrylamide/8M urea gel. 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
The sycO, ypkA and yopJ genes constitute a single operon
The RT-PCR assay indicated that the sycO, ypkA and yopJ
genes (designated as pCD12, pCD13 and pCD14 in Y.
pestis 91001 , respectively) were transcribed as a single
primary RNA (Fig. 1), and thereby these three genes
constituted a single operon in Y. pestis Microtus strain 201.
CRP greatly represses transcription of the
Our previous cDNA microarray analysis showed that the
transcription of sycO, ypkA and yopJ was repressed by CRP
. Herein, the real-time RT-PCR assays confirmed that
these three genes were up-regulated by more than 50 folds
in the crp mutant in relative to the WT strain (Fig. 2).
Taken together, transcription of the sycO-ypkA-yopJ operon
was under the negative control of CRP.
FTirgaunsrceri1ptional organization of the sycO-ypkA-yopJ operon
Transcriptional organization of the sycO-ypkA-yopJ
operon. Arrows represent the length and direction of
transcription of sycO, ypkA and yopJ on pCD1. The horizontal
arrow depicts the putative primary RNA transcript. The
arrowheads indicate the location of primer pair and the
expected amplicons. Genomic DNA and cDNA generated by
RT were used as the templates for PCR and RT-PCR,
respectively. To ensure that there was no contamination of
genomic DNA in the RT reactions, negative controls of
RTPCR were performed using 'cDNA' generated without
reverse transcriptase as templates. Reactions containing
primer pairs without template were also included as blank
controls. As expected, both negative and blank controls of
RT-PCR gave no amplicon (data not shown).
FCiRgPu-rdeep2endent transcription of sycO, ypkA and yopJ
CRP-dependent transcription of sycO, ypkA and yopJ.
Shown was the mean log2 ratio ( crp versus WT) of mRNA
level for each gene.
CRP greatly represses promoter activity of
To test the action of CRP on the sycO-ypkA-yopJ promoter
activity, we constructed the sycO::lacZ fusion promoter
consisting of a 690 bp promoter-proximate region of sycO
and promoterless lacZ, and then transformed into the WT
and crp, respectively. Empty vector pRS551 was also
introduced into them, respectively, as controls. -galactosidase
activity was measured for evaluating the sycO-ypkA-yopJ
promoter activity in each strain. Since the crp mutation
had an effect on the copy number of recombinant or
empty pRS551 plasmid , a normalized fold change in
the activity of each fusion promoter in WT in relative to
crp was calculated to avoid the influence of copy number
of pRS551 (Table 2).
Accordingly, the -galactosidase activity in the crp
increased compared to the WT when they grew in the
'TMH-1mM cAMP' medium, indicating that CRP greatly
repressed the promoter activity of sycO-ypkA-yopJ (Table
CRP binds to promoter-proximate region of
A CRP box-like sequence was found in the
promoter-proximate region of sycO-ypkA-yopJ , indicating the direct
association of CRP with the sycO-ypkA-yopJ promoter
region. Further EMSA experiments showed that the
cAMPCRP complex bound to the sycO-ypkA-yopJ promoter
region in a CRP dose-dependent manner (Fig. 3a). CRP
could not bind to the target DNA in the absence of cAMP.
To validate the specificity of CRP-DNA interaction,
YPO0180 and YPO1099 [gene IDs in CO92 ] were
used as negative controls (Fig. 3b). The PCR-generated
upstream DNA of YPO0180 did not harbor the predicted
CRP binding site, while the YPO1099 upstream region
gave an extremely low score value of 0.96 during the
pattern matching analysis using the CRP consensus (sycO
gave a score value of 8.57) . Both of them gave negative
EMSA result, even the CRP protein was increased to 4 g
in a single reaction mixture (Fig. 3b).
Therefore, CRP specifically bound to the sycO-ypkA-yopJ
promoter region and directly repressed the transcription
Structural organization of CRP-dependent
In order to locate the precise CRP binding site within the
sycO-ypkA-yopJ promoter region, DNase I footprinting
assay was performed with both coding and non-coding
strands. As shown in Fig. 4, CRP protected two distinct
Fold change ( crp/WT)
Normalized fold change of promoter activity in crp in relative to WT
-Galactosidase activity (miller units) was detected as the promoter activity. An extremely low promoter activity was detected for the crp or WT
transformed with empty pRS551 (data not shown). Copy number of recombinant pRS551 (PsycO-lacZ) was determined by real-time quantitative
PCR, 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 the crp in relative to the WT. Each experiment was done in triplicate.
EFliegcutroep3horetic mobility shift assay
Electrophoretic mobility shift assay. The band of DNA fragment containing the promoter region of sycO disappeared with
increasing amounts of CRP protein, and a retarded DNA band with decreased mobility turned up (Fig. 3a), which presumably
represented the CRP-DNA complex. But for YPO0180 and YPO1099, the CRP-DNA complex did not appear even His-CRP
was increased to 4 g for each reaction mixture (Fig. 3b).
FDiNguasree I4footprinting assay
DNase I footprinting assay. The labeled DNA probe was incubated with various amounts of purified His-CRP (lanes 1, 2, 3,
4, and 5 contained 0, 500, 1000, 2000 and 3000 ng, respectively), and subjected to DNase I footprinting assay. Lanes G, A, T
and C represented the Sanger sequencing reactions. On the right-hand side was indicated the protected regions (bold line).
The DNA sequences of footprints were shown from the top (3') to the bottom (5').
DNA regions (sites 1 and 2) against DNase I digestion in
a dose-dependent pattern. Only site 1 contained the CRP
The transcription start site of sycO was determined by
primer extension assay. A single primer extension product
was detected and thus a single CRP-dependent promoter
was transcribed for sycO-ypkA-yopJ (Fig. 5). Compared to
the WT, a much stronger primer extension product was
detected in the crp. Since the yield of primer extension
product would indicate the mRNA expression level of
sycO in each strain, data presented here confirmed the
repression of sycO-ypkA-yopJ by CRP.
The primer extension results could be also employed to
map the 5' terminus of RNA transcript for sycO (i.e. the
transcription start site of sycO-ypkA-yopJ) (Fig. 6). The -10
The determination of CRP-binding sites, transcription
start site, and core promoter element (-10 and -35
regions) promoted us to depict the structural organization
of CRP-dependent promoter, giving a map of
CRP-promoter DNA interaction for sycO-ypkA-yopJ (Fig. 6).
CRP and the sycO-ypkA-yopJ operon
CRP specifically bound to the sycO promoter-proximate
region and directly repressed the expression of
sycO-ypkAyopJ in Y. pestis biovar Microtus strain 201. The
sycO-ypkAyopJ promoter-proximate regions were extremely
conserved in Y. pestis (including all the four biovars Antiqua
, Mediaevalis , Orientalis  and Microtus ),
Primer extension analysis. Electrophoresis of the primer
extension products was performed with a 6%
polyacrylamide/8M urea gel. Lanes C, T, A and G represented the
Sanger sequencing reactions. The transcriptional start sites
Y. pseudotuberculosis  and Y. enterocolitica .
Therefore, data presented in Y. pestis biovar Microtus can be
generally applied to the above three pathogenic yersiniae.
A single CRP-dependent promoter transcribed for the
sycO-ypkA-yopJ operon, but two CRP-binding sites (site 1
and site 2) were detected within its promoter region. A
CRP box-like sequence (TAGATATCACC) was found in
site 1 rather than in site 2. It was speculated that site 2 was
a non-specific or non-functional CRP-binding site.
Further reporter fusion experiments and/or in vitro
transcription assays, using the sycO promoter-proximate regions
with different mutations/deletions within sites 1 and 2,
should be done to elucidate the roles of site 1 and site 2 in
CRP-mediated regulation of sycO-ypkA-yopJ.
CRP and T3SS
The crp mutation caused a reduced secretion of YOP
proteins in both Y. enterocolitica  and Y. pestis  grown
under calcium-depleted conditions. This indicated that
CRP is a positive regulator for the YOP secretion by Y.
pestis. It is well known that the YOP secretion phenotype is
only observable under calcium depleted conditions.
Herein, the direct and negative regulation of
sycO-ypkAyopJ by CRP was observed at transcriptional level under
calcium-rich conditions. How CRP controls T3SS is
essentially unclear yet. It needs to investigate the
mRNA/protein pools of T3SS that are regulated by CRP under
calcium depleted or rich conditions and upon cell contact,
and to answer whether CRP has a regulatory action on
T3SS in general or on SycO, YpkA and YopJ specifically.
CRP and virulence
The crp deletion attenuated Y. pestis much more greatly by
subcutaneous route of infection in relative to an
intravenous inoculation, and a reduced in vivo growth phenotype
of the crp mutant was observed . CRP seemed more
important for the infection at the subcutaneous site and in
the lymph other than the later systemic infection, while
the reduced in vivo growth of the crp mutant should
contribute to its attenuation by intravenous infection. The crp
disruption led to a great defect of pla expression . Since
Pla specifically promoted Y. pestis dissemination from
peripheral infection routes, the defect of pla expression in
the crp mutant will contribute to the huge loss of virulence
of this mutant strain after subcutaneous infection.
Expression of Pla, Pst, F1 antigen and T3SS are dependent
on CRP, and this regulator appears to control a wide set of
virulence-related factors in Y. pestis . All the above
CRPregulated genes are harbored in plasmids that are required
through horizontal gene transfer. Either the CRP protein
itself or the mechanism of CRP-promoter DNA
association is extremely conserved between E. coli and Y. pestis.
Therefore, the above laterally acquired genes have evolved
to integrate themselves into the 'ancestral' CRP regulatory
cascade. It has been shown recently that the histone-like
protein H-NS mediates the silencing of laterally acquired
genes with low G+C contents scattered on the bacterial
genome (these H-NS-dependent genes often contribute to
virulence or host adaptation in corresponding pathogens)
[25,26]. Herein, regulation (either activation or
repression) of foreign genes in plasmids was mediated by the
ancient regulator CRP in the host, Y. pestis.
Three T3SS genes, sycO, ypkA and yopJ, constitute a single
operon in Y. pestis. The CRP regulator binds to the
upstream DNA region of sycO, and represses the
expression of the sycO-ypkA-yopJ operon. The sycO
promoterproximate regions are extremely conserved in Y. pestis, Y.
pseudotuberculosis and Y. enterocolitica, indicating that the
CRP-dependent expression of sycO-ypkA-yopJ can be
generally applied to the above three pathogenic yersiniae.
SFtirguucrteur6al organization of the sycO-ypkA-yopJ promoter region
Structural organization of the sycO-ypkA-yopJ promoter region. The sycO-ypkA-yopJ promoter-proximate sequences
(100 bp upstream to 50 bp downstream the start codon of sycO) from Y. pestis Antiqua (biovar Antiqua), KIM5 (Mediaevalis),
CO92 (Orientalis) and 91001(Microtus), as well as those from Y. pseudotuberculosis IP32953 and Y. enterocolitica 8081, were
aligned and conserved nucleotide sites were labeled with asterisks. The CRP binding sites were underlined, and the invert
repeats in the CPR box was showed with two invert arrows. Showed also were transcriptional/transcriptional start sites and
promoter -10 and/or -35 elements.
DZ and RY conceived the study and designed the
experiments. LJZ and LY performed all the experiments. LZ, YL
and HG contributed to RT-PCR, primer extension assay
and DNA binding assays. ZG participated in protein
expression and purification. DZ, LFZ, CQ and DZ assisted
in computational analysis and figure construction. The
manuscript was written by LJZ and DZ, and revised by RY.
All the authors read and approved the final manuscript.
Financial support for this work came from the National Natural Science
Foundation of China for Distinguished Young Scholars (30525025), the
National Natural Science Foundation of China (30771179), and the
National Key Program for Infectious Disease of China (2009ZX10004-103