Microbiology (2013), 159, 1340–1351
DOI 10.1099/mic.0.066803-0
Comparative proteomic analysis reveals new
components of the PhoP regulon and highlights a
role for PhoP in the regulation of genes encoding
the F1F0 ATP synthase in Edwardsiella tarda
Yuanzhi Lv, Kaiyu Yin, Shuai Shao, Qiyao Wang and Yuanxing Zhang
Correspondence
Qiyao Wang
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology,
Shanghai 200237, China
[email protected]
Received 4 February 2013
Accepted 5 May 2013
Edwardsiella tarda is an important cause of haemorrhagic septicaemia in fish and also of gastroand extra-intestinal infections in humans. We have recently demonstrated that the PhoP-PhoQ
two-component regulatory system plays important roles in both virulence and stress tolerance in
E. tarda. In this study, the proteomes of the WT and phoP mutant strains were compared to define
components of the PhoP regulon in E. tarda EIB202. Overall, 18 proteins whose expression levels
exhibited a twofold or greater change were identified; 13 of these proteins were found to require
the presence of PhoP for full expression, while five were expressed at a higher level in the phoP
mutant background. Identified proteins represented diverse functional categories, including
energy production, amino acid metabolism and oxidative stress defence. Quantitative real-time
PCR analysis of the mRNA levels for the identified proteins confirmed the proteomics data.
Interestingly, the b subunit of the F1F0 ATP synthase, playing an important role in growth and
virulence of E. tarda, was listed as one of the proteins whose expression was greatly dependent
on PhoP. The F1F0 ATP synthase was encoded in a gene cluster (atpIBEFHAGDC) and the nine
genes were transcribed as an operon. PhoP positively regulated the transcription of the nine ATP
synthase genes and exerted this effect through direct binding to the promoter of atpI. Overall, the
results provide new insights into the PhoP regulon and unravel a novel role for PhoP in the
regulation of the F1F0 ATP synthase.
INTRODUCTION
Edwardsiella tarda, a Gram-negative bacterium of the
Enterobacteriaceae family, inhabits a broad range of hosts,
including fish, birds, reptiles, amphibians and humans
(Abbott & Janda, 2006). It has been associated with
gastroenteritis, septicaemia, liver abscess, meningitis, skin
and soft tissue infection, and wound infection (Mohanty &
Sahoo, 2007). In recent years, increasing outbreaks of E.
tarda-associated edwardsiellosis in various piscine species
have occurred throughout the world and caused enormous
losses to marine and freshwater aquaculture industries
(Park et al., 2012). Thus, an understanding of the virulence
mechanisms of this pathogen will facilitate the development of an effective vaccine to control edwardsiellosis.
PhoP-PhoQ is a two-component regulatory system that
controls the expression of a large number of genes and
Abbreviations: AspC, aspartate aminotransferase; CAMP, cationic
antimicrobial peptide; 2-DE, 2D gel electrophoresis; DeoB,
phosphopentomutase; GshB, glutathione synthetase; PfkA, 6phosphofructokinase; SspA, stringent starvation protein A; TrxB,
thioredoxin reductase; TSA, tryptic soy agar; TSB, tryptic soy broth.
1340
governs virulence and survival in several bacterial species,
including Salmonella enterica, Escherichia coli, Shigella
flexneri and Yersinia pestis (see Groisman, 2001, for a
review). Upon receipt of specific environmental signals, the
sensor kinase PhoQ activates its cognate response regulator
PhoP, which then modulates the expression of a set of
genes directly or indirectly (Kato et al., 2008). Owing to the
attenuated phenotype and immunogenic potential, the
phoP/phoQ-deleted S. enterica serovar Typhi has been
shown to be safe and immunogenic in humans (Hohmann
et al., 1996).
Recently, systematic analysis of the two-component
systems of E. tarda revealed that PhoP and EsrB are the
major virulence regulators (Lv et al., 2012a). In E. tarda,
the PhoP-PhoQ system is able to sense host body
temperature (23–35 uC) and low Mg2+ concentration,
allowing PhoP to regulate expression of the type III and
type VI secretion systems through direct activation of esrB
(Chakraborty et al., 2010; Lv et al., 2012a). A phoP null
mutant is sensitive to clindamycin, thermal stress and
cationic antimicrobial peptides (CAMPs) and shows
attenuated virulence (Lv et al., 2012a). LD50 of the phoP
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Proteomic analysis of the PhoP regulon in E. tarda
mutant in zebrafish is increased 316-fold in comparison
with that of the WT strain (Lv et al., 2012a). Moreover,
PhoP is activated by polymyxin B and then induces the
expression of UDP-glucose dehydrogenase, which is
essential for LPS integrity and CAMP resistance (Lv
et al., 2012b).
In view of the roles played by PhoP in stress tolerance and
virulence, a more comprehensive understanding of the PhoP
regulon in E. tarda is needed. In particular, elucidating
the PhoP regulon is likely to shed light on how E. tarda
withstands harsh environmental conditions and may also
help us understand the virulence of this pathogen. In the
present study we employed a proteomics approach to study
the PhoP regulon in E. tarda. This comparative proteomic
analysis led us to identify 18 differentially expressed proteins
involved in different cellular processes. The possible roles of
these proteins in the biology of E. tarda are discussed.
Additionally, for the first time to our knowledge, we report
that PhoP regulates the expression of the b subunit of the
F1F0 ATP synthase (AtpD) through directly binding to the
promoter of the atp (atpIBEFHAGDC) operon, which
contributes to growth and virulence of E. tarda.
METHODS
Bacterial strains and culture conditions. Bacterial strains used in
this study are listed in Table 1. To prepare bacteria for proteomic
analysis, we grew the WT E. tarda EIB202 and a phoP-deletion mutant
DphoP (Lv et al., 2012a) in 100 ml of Dulbecco’s modified Eagle’s
medium (DMEM,) at 28 uC for 24 h, as previously described (Lv
et al., 2012a). For routine culture, strains were grown in tryptic soy
broth (TSB, Becton Dickinson) or on tryptic soy agar (TSA, Becton
Dickinson) at 28 uC (for E. tarda) or 37 uC (for E. coli). When
required, appropriated antibiotics (Sigma-Aldrich) were added at the
following final concentrations (mg ml21): ampicillin 100, polymyxin B
10, kanamycin 50 or streptomycin 100.
Whole-cell protein extraction. Cells were harvested by centrifugation at 5000 g for 10 min at 4 uC and washed three times with
30 ml of ice-cold low-salt washing buffer (2.5 mM KH2PO4 and
5 mM NaH2PO4). Cell pellets from triplicate cultures of each strain
were individually suspended in 500 ml of lysis buffer (7 M urea, 2 M
thiourea, 4 % CHAPS, 40 mM DTT) containing complete protease
inhibitors (Roche Diagnostics). The cells were sonicated on ice for
3610 s and centrifuged at 20 000 g under 4 uC for 1 h to pellet the
insoluble debris. The supernatants were collected and protein
concentrations were assayed using the 2-D Quant kit (GE
Healthcare). The prepared samples were stored at 270 uC in
800 mg aliquots.
Two-dimensional gel electrophoresis. IEF was performed on the
Ettan IPGphor III system (GE Healthcare) according to the guidelines
of the manufacturer. Samples containing a total of 800 mg of protein
were loaded into 24 cm IPG strips (GE Healthcare), providing a
nonlinear pH 3–10 gradient. Strips were rehydrated for 12 h at 30 V
and then focused sequentially at 200 V for 1 h, 1000 V for 1 h, 1000–
8000 V gradient for 3 h and 8000 V for 6 h. After IEF, strips were
equilibrated for 15 min in 10 ml of equilibration buffer (6 M urea,
30 % glycerol, 50 mM Tris/HCl pH 8.8, 2 % SDS, 0.001 % bromophenol blue) containing 1 % DTT, followed by a second equilibration
with 2.5 % iodoacetamide in the same buffer. Then, separation on the
second dimension was performed on 12.5 % polyacrylamide gels,
using the Ettan DALTsix electrophoresis system (GE Healthcare). The
gels were run with 3 W per gel for 30 min and then with 20 W per gel
for 4 h at 20 uC. After SDS-PAGE, the gels were stained with
Coomassie brilliant blue R-250 (Amresco) and then scanned with an
ImageScanner (GE Healthcare). The resulting images were analysed
with PDQuest Advanced 8.0.1 2-D analysis software (Bio-Rad
Laboratories). The relative volume of each spot was determined
from the spot intensity in pixel units and normalized to the sum of
the intensities of all the spots in the gel. Proteins with at least a
twofold change in relative volume and P,0.05 were considered to be
significantly differentially expressed.
Table 1. Strains and plasmids used in this study
PB, polymyxin B; Sm, streptomycin; Km, kanamycin.
Strain or plasmid
E. tarda
EIB202
DphoP
Daur
DatpD
E. coli
TOP10F9
BL21(DE3)
CC118 lpir
SM10 lpir
Plasmids
pDMK
pDMK-aur
pDMK-atpD
pET28a
pET-phoP
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Relevant characteristics
Source or reference
WT, PBr, Smr, Kms, CCTCC no. M 208068
EIB202, in-frame deletion of phoP from bp 135 to 491
EIB202, in-frame deletion of aur from bp 115 to 1341
EIB202, in-frame deletion of atpD from bp 103 to 1353
Xiao et al. (2008)
Lv et al. (2012a)
This study
This study
General cloning strain
Host strain for PhoP expression
Host for p requiring plasmids
Host for p requiring plasmids, conjugal donor
Invitrogen
Novagen
Herrero et al. (1990)
Herrero et al. (1990)
Suicide vector, pir dependent, R6K, sacBR, Kmr
pDMK with aur fragment deleted 115 to 1341 nt, Kmr
pDMK with atpD fragment deleted 103 to 1353 nt, Kmr
Expressing vector, Kmr
pET28a expression PhoP, Kmr
Xiao et al. (2009)
This study
This study
Novagen
This study
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Y. Lv and others
In-gel digestion and protein identification by MALDI TOF/TOF
MS/MS. The protein spots of interest were cut out of the gels and
destained with 100 mM ammonium bicarbonate in 30 % acetonitrile
(ACN). The destained gel pieces were completely dried in a Speedvac
vacuum concentrator (Savant Instruments). The samples were
digested at 37 uC using 1 mg sequencing grade trypsin (Promega)
per 100 mg protein overnight. The supernatant was transferred into a
new microtube. Peptides were extracted with 100 ml of 0.1 %
trifluoroacetic acid in 60 % ACN for 20 min. The two supernatants
were pooled, dried and resolubilized with 2 ml of 20 % ACN. One
microlitre of the sample solution was spotted onto a MALDI target
plate and air-dried at room temperature. Then, 0.5 ml of a saturated
solution of a-cyano-4-hydroxycinnamic acid in 50 % ACN and 0.1 %
trifluoroacetic acid was added to the dried peptide digest spots and
allowed to air-dry at room temperature. All MS and MS/MS analyses
were performed in a MALDI TOF/TOF 4800 Proteomics Analyser
(Applied Biosystems) in the m/z range 800–4000. The instrument was
operated in reflector positive ion mode, with an accelerating voltage
of 20 000 V. For each sample, one MS spectrum was acquired and the
eight most intense precursors were subsequently selected for MS/MS
analysis. Spectra were processed and analysed by the Global Protein
Server Workstation (Applied Biosystems), which uses internal
MASCOT software (Matrix Science) for searching the peptide mass
fingerprints and MS/MS data. The data obtained were screened
against the NCBI nonredundant protein database. The search
parameters were as follows: fixed modifications of carbamidomethyl,
peptide mass tolerance of ±100 p.p.m., fragment mass tolerance of
±0.4 Da, peptide charge state of 1+, one missed cleavage per
peptide. Identifications were accepted if they included a peptide ion
score above the MASCOT identity threshold (95 % confidence).
Gene expression analysis by quantitative real-time PCR. Total
RNA was extracted from E. tarda strains grown in DMEM using an
RNA isolating kit (Tiangen). RNA samples were then treated with
RNase-free DNase I (Promega) to remove any contaminating
genomic DNA. RNA quantity and quality were assessed using ND2000 Spectrophotometer (Nanodrop Technologies). One microgram
of the total RNA was used as a template for the first strand cDNA
synthesis with PrimeScript reverse transcriptase (Takara). Quantitative real-time PCR (qRT-PCR) analysis was performed in a total
volume of 20 ml, using 2 ml of the diluted cDNA, 1 ml of each primer
(10 mM stock) (Table 2) and 10 ml of FastStart Universal SYBR Green
Master (ROX) (Roche). qRT-PCR was performed on the 7500 RealTime PCR System (Applied Biosystems) under the following conditions: 95 uC for 10 min, 40 cycles of 95 uC for 15 s, and 60 uC for
1 min. Melting curve analysis of amplification products was performed
at the end of each PCR to confirm that only one PCR product was
amplified. Relative quantification was performed using the comparative CT (22DDCT) method (Livak & Schmittgen, 2001), with the
housekeeping 16S rRNA gene as an internal standard in each strain.
Reverse transcription-PCR. The PCR amplification reaction was
performed in a total volume of 20 ml containing 10 ml of Ex-Taq
(Takara), 2 ml of cDNA and 1 ml of each primer indicated in Fig. 3(a)
and Table 2. The genomic DNA was used as a positive control while
the RNA was used as a negative control. The PCR program was 95 uC
for 5 min followed by 30–35 cycles at 95 uC for 30 s, 60 uC for 30 s,
and 72 uC for 90 s; and then one cycle of 72 uC for 5 min to complete
the reaction. The PCR products were analysed on 1.2 % agarose gel
and visualized by ethidium bromide staining.
Construction of deletion mutants of aur and atpD. To create the
deletion allele of aur, the primer pairs Aur-A/Aur-B and Aur-C/
Aur-D (Table 2) were used to amplify the upstream and downstream
fragments of aur from E. tarda EIB202 genomic DNA, respectively.
These two fragments containing a 20 bp overlap were used as
templates in a second PCR using primers Aur-A and Aur-D, creating
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an in-frame deletion fragment from bp 115 to 1341 in aur. A deletion
fragment from bp 103 to 1353 in atpD was created in the same way
using primer pairs AtpD-A/AtpD-B and AtpD-C/AtpD-D (Table 2).
The fused fragments were sequenced and cloned into suicide vector
pDMK (Xiao et al., 2009) that was linearized with BglII and SphI. The
resulting plasmids, pDMK-aur and pDMK-atpD, were mated from E.
coli SM10 lpir into E. tarda EIB202 by conjugation. The transconjugants with the plasmids integrated into the chromosome by
homologous recombination were selected on TSA plates containing
kanamycin and streptomycin. To complete the allelic exchange for inframe deletions, double-crossover events were counter-selected on
TSA plates containing 10 % sucrose. The aur and atpD deletion
mutants were confirmed by PCR amplification of the respective DNA
loci, and subsequent DNA sequencing of each PCR product.
Growth curve analysis. Overnight cultures of WT, DphoP, Daur and
DatpD were adjusted to an OD600 of 1.0. One millilitre of this
suspension was added to 100 ml of TSB medium and incubated at
28 uC with shaking at 200 r.p.m. for 40 h. A 500 ml aliquot of each
culture was taken every 4 h for turbidity measurements at 600 nm.
CAMP sensitivity assays. Bacterial strains were cultured to the mid-
exponential growth phase and diluted to 16106 c.f.u. ml21 in TSB
medium. Equal volumes of bacteria culture and peptides diluted in PBS
were mixed together, and 100 ml aliquots were placed in a 96-well plate
and incubated at 28 uC for 1 h. Control cultures were treated with PBS
alone. Each sample was then serially diluted and plated on TSA plates to
assess bacterial viability. Data were presented as per cent survival
relative to the control cultures. The peptides used included cecropin P1
(5 mg ml21, GL Biochem), LL-37 (200 mg ml21, GL Biochem),
magainin 2 (100 mg ml21, GL Biochem), mastoparan (80 mg ml21,
GL Biochem) and polymyxin B (25 mg ml21, Sigma-Aldrich).
Virulence assay. The LD50 values of the strains were determined in a
zebrafish (Danio rerio) infection model (Pressley et al., 2005). The
healthy zebrafish, each weighing approximately 0.25 g, were raised in
reverse osmosis-purified water in a flow-through system at 25 uC.
Fish were anaesthetized with tricaine methanesulfonate (MS-222,
Sigma-Aldrich) at a concentration of 80 mg l21 before infection.
Groups of ten fish were injected intramuscularly with bacterial doses
ranging from 101 to 107 c.f.u. fish21. Fish mortality was monitored
over a period of 14 days post-infection, and then all fish were
euthanized with an overdose of MS-222. Fish injected with PBS served
as negative controls. The LD50 values were calculated by the method
described by Reed & Muench (1938). The animal work presented here
was approved by the Animal Care Committee, East China University
of Science and Technology.
Preparation of N-terminal His6-tagged PhoP protein. E. coli
BL21(DE3) cells harbouring plasmid pET28a-phoP (pET-phoP) were
grown in LB medium until OD600 reached 0.5. Expression of His6PhoP was induced with 0.5 mM isopropyl b-D-thiogalactoside, and
the cells were grown at 30 uC overnight. The recombinant protein was
purified by Ni-NTA affinity chromatography (Qiagen). Protein purity
was verified by SDS-PAGE.
Electrophoretic mobility shift assay. Electrophoretic mobility shift
assay (EMSA) was performed as previously described (Zhao et al.,
2008), with some modifications. Briefly, 50 ng of atpI promoter DNA
fragment was mixed with PhoP protein at concentrations of 0, 1, 2
and 3 mM in 20 ml of an EMSA buffer containing 10 mM Tris/HCl,
pH 7.4, 150 mM KCl, 0.1 mM dithiothreitol and 0.1 mM EDTA.
The promoter region of phoP was included as a positive control
(Chakraborty et al., 2010), while a DNA fragment within the atpD
ORF was used as a negative control. Mixtures were incubated at 25 uC
for 30 min, and subsequently separated on 8 % nondenaturing
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Microbiology 159
Proteomic analysis of the PhoP regulon in E. tarda
polyacrylamide gel in 0.56TBE buffer at 100 V. Signals were detected
by ethidium bromide staining.
Statistics. Statistical analyses were performed using GraphPad Prism
version 5.01 for Windows (GraphPad Software). A two-tailed Fisher’s
exact test was used to compare the numbers of surviving animals in
different groups. Two-tailed Student’s unpaired t test was used to
compare gene expression between two groups.
RESULTS
Overview of the comparative proteome
In order to analyse the effect of phoP mutation on the E.
tarda proteome, we cultured WT EIB202 and its derivative
phoP-deletion strain DphoP in DMEM in triplicate
experiments. The whole-cell protein expressions of WT
and DphoP were visualized on two-dimensional gel
electrophoresis (2-DE) gels (Fig. 1). Only proteins with a
difference of at least twofold in spot intensity volume and
P,0.05 were considered as differentially expressed proteins. Based on these criteria, a total of 19 spots showed
significantly different expression between WT and DphoP
strains (Fig. 1). Generally, more of the observed proteins
showed downregulated expression (n 5 13) than upregulated expression (n 5 6) in the DphoP strain in relation to
the WT strain. These spots were cut out from the 2-DE gels
and subjected to in-gel digestion, followed by MS/MS
analysis. All spots were successfully identified, yielding a
total of 18 different proteins (spots B2 and B5 were
identified as the same protein), which were classified
according to their putative biological functions (Table 3).
protein spots (B2 and B5) with increased expression were
identified as aldehyde/alcohol dehydrogenase. The experimental molecular masses of the two spots were significantly
lower than the theoretical values (Fig. 1), which might be a
result of post-translational proteolytic processing.
Prediction of the subcellular organization of proteins using
PSORTb demonstrated that 13 of these differentially
expressed proteins were cytoplasmic proteins. TonB-dependent haemin receptor was anchored to the outer membrane.
Aldehyde/alcohol dehydrogenase, TrxB and the b subunit of
the F1F0 ATP synthase might be located in multiple sites.
ETAE_2757, which belongs to the a/b-hydrolase fold family,
could not yet be characterized for its cell location.
Transcriptional analysis of differentially
expressed genes
To confirm the proteomics data, qRT-PCR analyses were
carried out to examine the expression levels of mRNAs for
differentially expressed proteins. Ten of the 18 proteins
were selected for verification, and the housekeeping 16S
rRNA gene was taken as the internal control. As depicted in
Table 4, the qRT-PCR analyses generally confirmed both
the up- and downregulation in the transcriptional level of
the differentially expressed proteins shown in the 2-DE
gels. The change in gene expression for dnaK failed to
correlate with its protein expression (Fig. 1 and Table 3),
indicating either that this protein is not increased in
expression in DphoP or that a post-transcriptional
regulatory mechanism is responsible for the increase in
protein expression.
Three of the 13 proteins with decreased expression in
DphoP were involved in energy production and conversion.
These proteins included the b subunit of the F1F0 ATP
synthase (spot A1), phosphopentomutase (DeoB) (A3) and
glycerol dehydrogenase (A5). Three proteins involved in
amino acid transport and metabolism [aspartate aminotransferase (AspC) (A4), cysteine synthase A (A8) and zinc
metalloprotease aureolysin (A2)] were also downregulated
in DphoP. Also, two proteins associated with carbohydrate
transport and metabolism showed decreased expression in
DphoP. These were 6-phosphofructokinase (PfkA) (A6)
and mannose-specific phosphotransferase system enzyme
IIAB component (A7). Additionally, two proteins involved
in protein turnover, thioredoxin reductase (TrxB) (A10)
and stringent starvation protein A (SspA) (A12), were
downregulated in DphoP. Other proteins with decreased
expression in DphoP were glutathione synthetase (GshB)
(A9), endoribonuclease L-PSP (A13) and a/b-hydrolase
fold enzyme (A11).
Of the five upregulated proteins in DphoP, two had
homology to phosphoglycerate kinase (B3) and glyceraldehyde-3-phosphate dehydrogenase (B6), which are key
enzymes of glycolysis and gluconeogenesis. In addition, the
molecular chaperone DnaK (B4) and TonB-dependent
haemin receptor (B1) were upregulated in DphoP. Two
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Construction and characterization of the aur and
atpD deletion mutants
Among the differentially expressed proteins, Aur and AtpD
displayed the most significant changes in both protein
expression and gene expression (Fig. 2a and Table 4). These
two proteins, encoded by ETAE_2022 and ETAE_3533,
showed maximum identities of 39 % and 96 % to
aureolysin and the b subunit of the F1F0 ATP synthase of
S. enterica, respectively. Aureolysin is structurally similar to
the zinc metalloprotease thermolysin, and both belong to
the M4 family of metallopeptidases, which typically
undergo autocatalytic activation (Banbula et al., 1998).
The F1F0 ATP synthases of bacteria synthesize the majority
of cellular energy in the form of ATP, of which the b
subunit is responsible for nucleotide binding and catalysis
(von Ballmoos et al., 2009). In order to characterize the
role of Aur and AtpD in E. tarda virulence, the deletion
mutants lacking aur or atpD were constructed. Daur grew
at a rate similar to that of WT in TSB medium at 28 uC
(Fig. 2b). However, DatpD displayed a longer doubling
time in the exponential phase and had a lower cell density
at the stationary phase compared to WT (Fig. 2b),
demonstrating that AtpD is essential for growth of E.
tarda. It also indicates that the downregulation of atpD
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Table 2. Primers used in this study
Name
Mutant construction
Aur-A
Aur-B
Aur-C
Aur-D
AtpD-A
AtpD-B
AtpD-C
AtpD-D
qRT-PCR
2757-qFor
2757-qRev
atpD-qFor
atpD-qRev
gldA-qFor
gldA-qRev
aur-qFor
aur-qRev
pfkA-qFor
pfkA-qRev
manX-qFor
manX-qRev
adhE-qFor
adhE-qRev
gapA-qFor
gapA-qRev
dnaK-qFor
dnaK-qRev
hemR-qFor
hemR-qRev
16S-qFor
16S-qRev
RT-PCR
gi1
gi2
ib1
ib2
bf1
bf2
fa1
fa2
aa1
aa2
ag1
ag2
gd1
gd2
dc1
dc2
cg1
cg2
Protein expression
phoP-expFor
phoP-expRev
EMSA
atpI-emsaFor
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Sequence (5§–3§)*
GGAAGATCTAGTTTCTGAGCGATGATGAGGCGGT
ATTCAAAGGGACCGCCGATGGTTTTTGCTTTG
CATCGGCGGTCCCTTTGAATAAGCGTCACA
ACATGCATGCGTCAGCTAAGCGATCTGTTGCCTCA
GGAAGATCTTGTGGAATCGCAGGTTTATCAGGGC
CCACGGCTTCGCCTTTTACCTCCAGTGCGTT
GGTAAAAGGCGAAGCCGTGGAAAAAGCCAAG
ACATGCATGCAGCTCTGCAGATGCCTGAGCATAGT
GAGTGACGAGGAGTCCAAGGC
TAGCGAACGGTTTCCAGCA
AGATCGGCGAAGAAGAGCG
CCCGAACAGACCCACCTTAC
CCATCCACAACGGGCTAACG
GCACGGTTTCAATCTGCTCACT
CCATCGGCGGTGCTTTATC
TGCGCCAGGGTTGTGAATT
GCGTTACTGTGGCGACCTG
GCGTGTTTCTTGCCTTTAGTG
CCGCATCATCGTCGTCAGT
TGGCGTATTTCGGGTTATTG
AGATGCTTTCGACGATCAGTG
AACGGTCTTGCTTTCTTCCTG
CGTTATGGGCGTAAACCAC
GGTAGCGTGAACGGTGGTC
GTGAAAGACGTACTGCTGCTGG
GCTTGGTCGGAATGGTGGT
TCTACAACGATGCCCGCCACT
CGTCGAAGCGTAACCCAAAGC
ACTGAGACACGGTCCAGACTCCTAC
TTAACGTTCACACCTTCCTCCCTAC
CCTCTTTGCAGGATATGGTGTCTTGG
TAAACCACCCCAGCGACACGATAAG
TATTGCTTGGGCATTCGCTCTGG
CATGGAACGTTGAGCAACCACTGTG
TTAAGGAGTTGACCCTGCAGCCGTT
AGCGACCAGTTTATCGACGATGTCG
TGAAGAGCTGCGTAAACAGGTCG
GCCTACCGGAACTTCCAGAATAC
AACCGCTACGCTATCGCACTGAAC
TCGGCGCATACTGTTTCTGCTTGAG
AGTTTGCCTCTGATCTGGACGATGC
CGAGCCTTGTTGTAGACCAACTGCA
CGGAGCTGAAACGGAAATCATGGGA
CAGAGGTGATAGAGCCAGTTTTCG
ATCTACCGTTATACCCTGGCCGGTA
TACGCGAAGTTTGGCAATCGCCTTG
TGCGATGGAGTCGAAGCGTAAAGCA
CGCCGTACAGCATCAGGATATCTTC
CGCGGATCCATGCGTATCTTAGTCGTCGAA
CCCAAGCTTTCATGCCGGCACGTCGAA
AAATGTCTATATTGGCAATATTGGC
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Proteomic analysis of the PhoP regulon in E. tarda
Table 2. cont.
Name
Sequence (5§–3§)*
atpI-emsaRev
phoP-emsaFor
phoP-emsaRev
con-emsaFor
con-emsaRev
CAATAGTGCGGACTCTCCCCAGCCT
ATACCACAAGAGACAGTCTACGACA
CATGTATAGGTTCTTTCACTCCCGA
TCGACATGAAGGGCGAGATCGGCGA
TCCGTCATCTCGTGGTAGAAGTCGT
*Restriction sites introduced for cloning purposes are underlined.
may be responsible for the growth defect of Dphop reported
in a previous study (Lv et al., 2012a).
It was reported recently that E. tarda is highly resistant to
the action of CAMPs, and PhoP is a major regulator of this
resistance (Lv et al., 2012a, b). To examine whether aur and
atpD are involved in CAMP resistance, we performed
liquid incubation assays to compare the resistance of Daur
and DatpD to structurally different CAMPs (cecropin P1,
LL-37, magainin 2, mastoparan and polymyxin B) to that
of WT. However, no significant difference was observed
between WT, Daur or DatpD mutants (data not shown).
To determine the effects of aur and atpD on the virulence of
E. tarda, we evaluated the LD50 values of the mutants on a
zebrafish infection model. Daur displayed a LD50 value of
(a)
kDa
pH 3
pH 10
3.026102 c.f.u. fish21, similar to that of WT (3.166102 c.f.u.
fish21). DatpD, in contrast, had a LD50 value of
3.686103 c.f.u. fish21. At a dose of 104 c.f.u. fish21, WT
and Daur killed all fish within 4 days (Fig. 2c). Deletion of
atpD delayed the lethality towards zebrafish, and four DatpDinfected fish were still alive by day 7 post-infection (Fig. 2c).
Dphop did not kill any fish during 7 days post-infection at the
same dose (Lv et al., 2012a).
PhoP directly regulates the transcription of
F1F0-type ATP synthase genes
The complete genome sequence of E. tarda EIB202 (Wang
et al., 2009a) reveals a gene cluster of 6820 bp that would
encode subunits of the F1F0 ATP synthase. This cluster
(b)
pH 3
pH 10
97
66
A1 A2
A5
B1
B2
A3
44
A6
A7
A4
B3
A8
B4
A9 A10
A11
B6
B5
29
A12
20
14
A13
Fig. 1. 2-DE patterns of the whole-cell protein lysate of E. tarda strain EIB202 WT (a) and DphoP (b) cultured in DMEM
medium. The identified spots are labelled and were identified by MALDI TOF/TOF MS/MS. Downregulated proteins in DphoP
are numbered from A1 to A13, while upregulated proteins in DphoP are numbered from B1 to B6. Protein spot numbers are
related to the information provided in the text and Table 3. The experiments were performed in triplicate in at least three
independent periods, and representative gels are shown.
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Y. Lv and others
1346
Table 3. List of differentially expressed proteins (.twofold) between E. tarda WT and DphoP
Spot
no.
NCBI accession no.
Locus
Energy production and conversion
ETAE_3533
A1
YP_003297575
ETAE_0488
A3
YP_003294546
ETAE_0899
A5
YP_003294955
B2
YP_003295560
ETAE_1508
ETAE_1508
B5
YP_003295560
Amino acid transport and metabolism
ETAE_2022
A2
YP_003296070
ETAE_1238
A4
YP_003295292
ETAE_1134
A8
YP_003295190
Carbohydrate transport and metabolism
ETAE_3450
A6
YP_003297492
ETAE_1559
A7
YP_003295611
B3
B6
YP_003297001
YP_003295535
ETAE_2957
ETAE_1483
Protein
Protein description
Theor. MW
(kDa)/pI
Score
Sequence
coverage (%)
Peptides
matched
Location*
Fold
changeD
AtpD
DeoB
GldA
AdhE
AdhE
b Subunit of F1F0 ATP synthase
Phosphopentomutase
Glycerol dehydrogenase
Aldehyde/alcohol dehydrogenase
Aldehyde/alcohol dehydrogenase
50.1/4.94
44.7/5.38
39.0/5.12
96.4/6.33
96.4/6.33
401
454
397
899
1070
36
45
29
29
16
13
14
7
21
8
U*
C
C
U*
U*
211.1
22.1
22.9
2.5
2.5
Aur
AspC
Zinc metalloprotease aureolysin
Aspartate aminotransferase, PLPdependent
Cysteine synthase A
49.0/5.21
43.4/5.50
124
542
12
57
6
16
C
C
227.5
23.9
34.0/6.03
358
78
19
C
W
6-Phosphofructokinase
Mannose-specific phosphotransferase
system enzyme IIAB component
Phosphoglycerate kinase
Glyceraldehyde-3-phosphate
dehydrogenase
35.2/5.41
35.0/5.40
487
378
60
40
18
12
C
C
210.6
29.2
41.1/5.33
35.7/6.60
163
846
30
65
8
18
C
C
2.4
2.1
35.4/5.40
304
44
9
C
25.1
13.4/5.19
255
38
5
C
28.1
34.9/5.47
24.5/5.23
68.4/4.78
1100
404
89
71
59
28
17
13
16
U*
C
C
22.8
24.2
3.1
72.8/6.29
727
53
29
OM
3.6
33.7/5.37
654
54
12
U
CysK
PfkA
ManX
Pgk
GapA
Coenzyme transport and metabolism
ETAE_0278
GshB
Glutathione synthetase
A9
YP_003294336
Translation, ribosomal structure and biogenesis
ETAE_2752
TdcF
Endoribonuclease L-PSP
A13
YP_003296796
Post-translational modification, protein turnover, chaperones
ETAE_2202
TrxB
Thioredoxin reductase (NADPH)
A10
YP_003296248
ETAE_0517
SspA
Stringent starvation protein A
A12
YP_003294575
ETAE_0576
DnaK
Molecular chaperone
B4
YP_003294634
Inorganic ion transport and metabolism
ETAE_1797
HemR TonB-dependent haemin receptor
B1
YP_003295845
General function prediction only
ETAE_2757
a/b-Hydrolase fold enzyme
A11
YP_003296801
211.4
Microbiology 159
*Abbreviation of cellular location. Protein cellular location is annotated by PSORTb V.3.0 (http://www.psort.org). C, Cytoplasmic; OM, outer membrane; U, unknown. U* indicates that the protein
may have multiple localization sites.
DThe changes indicate the protein expression ratio for DphoP and WT (DphoP/WT), expressed as a negative reciprocal for proteins that were present at reduced levels in DphoP. Unique protein
spots in WT are marked with ‘W’.
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Proteomic analysis of the PhoP regulon in E. tarda
Table 4. qRT-PCR analysis of gene expression for the
identified differentially expressed proteins
(a)
The changes indicate the ratio of mRNA expression in DphoP to WT
(DphoP/WT), expressed as a negative reciprocal for genes that were
present at reduced levels in DphoP.
gi|269140100
gi|269140874
gi|269138255
gi|269139369
gi|269140791
gi|269138910
gi|269138859
gi|269138834
gi|269137934
gi|269139144
Gene
Fold change
ETAE_2757
atpD
gldA
aur
pfkA
manX
adhE
gapA
dnaK
hemR
227.34±1.43*
230.21±2.30*
22.30±0.24*
21942±169*
22.06±0.17*
22.31±0.25*
1.98±0.27*
1.71±0.10*
1.07±0.05
1.52±0.03*
AtpD Aur
(b)
4.00
3.00
OD600
NCBI GI
DphoP
WT
2.00
1.00
0.00
*P,0.05.
0
4
8
12
16 20 24
Time (h)
28
2
4
3
Time (days)
5
32
36
40
(c)
Based on the organization of the ATP synthase gene cluster,
atpI through atpC may be polycistronic, and under the
control of the same promoter. Reverse transcription-PCR
(RT-PCR) was used to determine whether the nine ATP
synthase genes are co-transcribed. RT-PCR products of
expected sizes were obtained with primers spanning the
regions from atpI to atpC (Fig. 3a, b), suggesting that these
nine genes are co-transcribed. In contrast, no RT-PCR
product was observed with primer pairs gi1/2 or cg1/2.
And the corresponding PCR product could be produced
using genomic DNA as the template (Fig. 3a, b). Taken
together, the results indicate that the atp operon uses the
promoter of atpI for transcription.
To further characterize the role of PhoP in the regulation
of atp operon expression, we analysed the interaction of
PhoP protein and atpI promoter DNA by EMSA. The
results show that the N-terminal His6-tagged PhoP can
bind to a 317 bp DNA fragment (from nt 2314 to +3)
derived from the promoter region of atpI, at an affinity
lower than that between PhoP and its own promoter region
(from nt 2293 to +3), which served as a positive control
(Fig. 4b). There was no interaction observed between PhoP
and a DNA fragment within the atpD ORF, which was
included as a negative control (Fig. 4b).
http://mic.sgmjournals.org
100
80
Survival (%)
consists of four F0 genes and five F1 genes encoding the
subunits I (atpI), a (atpB), c (atpE), b (atpF), d (atpH), a
(atpA), c (atpG), b (atpD) and e (atpC) in that order (Fig.
3a). As described above, the transcription of atpD in DphoP
was reduced 30-fold compared with that in WT (Table 4).
To investigate whether the other ATP synthase genes are
regulated by PhoP, we performed qRT-PCR analyses to
evaluate the mRNA levels of each gene in WT and DphoP.
The results show that the transcription of the ATP synthase
genes was significantly reduced 1.6- to 30-fold in DphoP
(Fig. 4a).
60
40
20
0
0
1
6
7
Fig. 2. Effects of AtpD and Aur on the growth and virulence of E.
tarda. (a) Enlarged partial 2-DE gels showing downexpression of
AtpD and Aur in DphoP in relation to WT. (b) Growth curves of WT
(#), DphoP (m), DatpD (X) and Daur (&) in TSB at 28 6C with
growth monitored by OD600. (c) Survival curves for zebrafish
injected intramuscularly with WT (#), DphoP (m), DatpD (X) and
Daur (&) at a dose of 104 c.f.u. per fish. For (b) and (c), the
experiments were performed three independent times, and a
representative result is displayed.
DISCUSSION
In this study, we analysed protein expression profiles to
define the potential target proteins affected by PhoP in E.
tarda. A total of 18 proteins were identified as being
expressed in a PhoP-dependent manner; 13 proteins were
found to be positively regulated by PhoP, while five
proteins were found to be negatively influenced by PhoP.
Differential expression was verified by qRT-PCR for nine
out of ten selected proteins. For the 18 proteins that were
under the control of PhoP, five proteins, TrxB (Charles
et al., 2009), DeoB, PfkA and SspA (Yu & Guo, 2011), and
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Y. Lv and others
gidB
(a)
atp I B
E F H
A
G
1
2
3 1
2
1
2
fa1/2
3
cg1/2
gd1/2
ATP synthase F1
bf1/2
3
dc1/2
fa1/2
ATP synthase F0
ib1/2
1
glmU
ag1/2
bf1/2
gi1/2
gi1/2
C
aa1/2
ib1/2
(b)
D
2
aa1/2
3
1
2
Flanking ORF
ag1/2
3
1
2
gd1/2
3
1
dc1/2
2
3
1
2
cg1/2
3
1
2
3
2000 bp
1200 bp
800 bp
500 bp
Fig. 3. Genetic organization of the F1F0 ATP synthase gene cluster as an operon. (a) Schematic representation of the ATP
synthase gene cluster in E. tarda EIB202. (b) RT-PCR analysis of the co-transcription of the ATP synthase genes. Primer pairs
indicated in (a) were used to generate RT-PCR products with cDNA (1), RNA (2) or genomic DNA (3) as the template.
Most of the 18 differentially expressed proteins can be
classified into two broad functional categories, metabolism
and stress-related functions. Five proteins whose expression
is under the control of PhoP are likely involved in stress
response and survival in limiting conditions. TrxB and GshB
were found to be expressed at a lower level in the DphoP
mutant. The thioredoxin and glutathione/glutaredoxin
systems are ubiquitous mechanisms that shuttle redox
potential from NADPH to cytosolic substrates, thereby
providing a repair system for oxidized thiol groups in
cytosolic proteins and maintaining the cytosol redox status
(Carmel-Harel & Storz, 2000; Koháryová & Kolárová, 2008).
It has been shown that S. Typhi trxB mRNA is upregulated
within human macrophages and an S. enterica serovar
Typhimurium trxB mutant shows decreased intra-murinemacrophage survival and attenuated virulence in nematodes
(Bjur et al., 2006; Faucher et al., 2006; Sem & Rhen, 2012).
Expression of E. coli gshB is induced by H2O2 and chlorine
and a gshB mutant is sensitive to oxidative stress (CarmelHarel & Storz, 2000; Wang et al., 2009b). Both thioredoxin
and glutathione contain a redox-active cysteine residue.
Interestingly, cysteine synthase A (CysK) was absent in
1348
(a)
1.2
Relative fold expression
DnaK (Zhou et al., 2005), were previously reported to be
associated with the PhoP-PhoQ system in other organisms.
PhoP deploys largely different targets of regulation across
the family Enterobacteriaceae, the result of both regulation
of species-specific targets and rewiring of shared genes,
which may contribute to phenotypic differences between
organisms (Perez et al., 2009). Furthermore, for the first
time, we revealed that PhoP contributes to the expression
of an entire cluster of genes encoding F1F0 ATP synthase,
which is present in various living organisms.
DphoP
WT
1.0
0.8
*
*
0.6
*
0.4
*
*
0.2
0.0
*
*
*
*
atpl atpB atpE atpF atpH atpA atpG atpD atpC
(b)
PhoP
(mM)
0
1
2
4
0
1
2
3
–
0
+
3
Bound DNA
DNA only
atpl promoter
phoP promoter
Control
Fig. 4. PhoP protein promotes the transcription of the atp operon
through direct binding with the atpI promoter region. (a) qRT-PCR
detection of transcription of the ATP synthase genes in WT and
DphoP cultured in DMEM medium. Results were presented as
mean±SD (n53). *, P,0.05. (b) EMSA analysis of the atpI promoter
region (317 bp, from nt ”314 to +3) and phoP promoter region
(296 bp, from nt ”293 to +3). The amounts of PhoP protein used
were 0, 1, 2 and 3 mM. Binding of PhoP (3 mM) to a DNA fragment
within the atpD ORF was included as a negative control.
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Microbiology 159
Proteomic analysis of the PhoP regulon in E. tarda
DphoP. Cysteine is the most common amino acid residue
acting as a thiolate ligand in the Fe–S clusters and plays a
critical role in the thiol-redox system (Wang et al., 2009b).
The inability to synthesize cysteine limits the ability to
respond to oxidative stress in S. Typhimurium (Turnbull &
Surette, 2010). Alkhuder et al. (2009) also showed that the
ability of Francisella tularensis to utilize the available
glutathione as a source of cysteine in the host cytosol is
essential to multiply and survive in macrophages and to
virulence in mice. TrxB has previously been shown to be
positive regulated by PhoP in S. Typhi and S. Typhimurium
(Charles et al., 2009). Moreover, a microarray analysis
revealed seven genes (sodC, sodB, sodA, katA, katY, ahpC and
gst) involved in protection against oxidative stress are under
positive regulation by PhoP in Y. pestis (Zhou et al., 2005).
Our findings, together with those of previous reports,
indicate that PhoP has an important role in regulating the
expression of proteins involved in oxidative stress defence.
In E. tarda, the ability to withstand reactive oxygen is
required for resistance to phagocyte-mediated killing
(Srinivasa Rao et al., 2003). Thus, downregulation of
TrxB, GshB and CysK supports the observation of impaired
intracellular survival (data not shown) and attenuated
virulence of the DphoP mutant (Lv et al., 2012a). Under
oxidative stress, proteins are subject to conformational
changes leading to protein mis-folding and aggregation, and
this oxidation would titrate out the molecular chaperones
DnaK, DnaJ and GrpE, which aid refolding of denatured or
mis-folded proteins (Calloni et al., 2012). It is possible that
the increased expression of DnaK may represent an attempt
to minimize the oxidative damage caused by the down
expression of TrxB, GshB and CysK in the DphoP mutant. Di
Pasqua et al. (2010) previously found that the exposure of S.
enterica serovar Thompson to thymol carries the effects
related to an oxidative stress, where increased expression of
chaperone proteins DnaK and GroEL in the treated cell was
contemporaneous with decreased expression of TrxA, CysK
and SodB. Another down-expressed protein involved in
stress response was matched with SspA. SspA is an RNA
polymerase-associated protein that is required for the stress
response of E. coli during the stationary phase and affects
expression of virulence genes and pathogenesis of several
Gram-negative pathogens (Hansen et al., 2005).
Alterations in the expression of metabolism-associated
proteins (ten proteins) are more numerous than for any
other COG category. PfkA is a key enzyme regulating the
glycolysis pathway, which catalyses the phosphorylation of
fructose-6-phosphate. The enzyme cannot catalyse the
reverse reaction in vivo. When cells are exposed to
oxidative stress, they need excessive amounts of the
antioxidant cofactor NADPH (Muñoz-Elı́as & McKinney,
2006). Thus, the downregulation of PfkA in DphoP may reroute the metabolic flux from glycolysis to the pentose
phosphate pathway, allowing the cell to generate more
NADPH. It has been reported that amino acids and purines
appear to be limited in the host cells, and mutant strains of
several Gram-negative pathogens with the corresponding
http://mic.sgmjournals.org
auxotrophies are impaired for intracellular survival and
virulence (Eisenreich et al., 2010). AspC is a multifunctional enzyme that catalyses the synthesis of aspartate,
phenylalanine, tyrosine and other compounds through a
transamination reaction (Powell & Morrison, 1978). DeoB
interconverts ribose 1-phosphate and ribose 5-phosphate,
which bridges glucose metabolism and RNA biosynthesis
(Tozzi et al., 2006). The importance of this enzyme has
been underscored by the observation that targeted deletion
of deoB in F. tularensis results in markedly decreased
virulence (Horzempa et al., 2008). Downregulation of
AspC and DeoB in DphoP implies that PhoP affects E. tarda
metabolism and may promote adaptation to in vivo
environments. Meanwhile, the large amount of enzymes
among the identified proteins indicates that the ability to
adapt to and switch metabolic processes is of importance
during infection.
The E. tarda DphoP mutant is highly susceptible to a variety
of CAMPs, including the human-derived LL-37, the frogderived magainin 2, the wasp-derived mastoparan, the
Ascaris suum-derived cecropin P1 and the peptide antibiotic
polymyxin B, produced by the soil bacterium Paenibacillus
polymyxa (Lv et al., 2012a). In Salmonella, different PhoPregulated determinants often mediate resistance to different
antimicrobial peptides. Some of these determinants modify
the LPS, whereas others encode proteases with the capacity
to cleave CAMPs (Groisman, 2001). We previously reported
that UDP-glucose dehydrogenase, a PhoP-activated protein,
contributes to LPS synthesis and CAMP resistance in E.
tarda (Lv et al., 2012b). Proteomic and transcriptional
analysis indicated that the expression of aur is highly
dependent on PhoP (Fig. 2a, Table 4). In Staphylococcus
aureus, Aur has the ability to cleave and inactivate LL-37,
one of the few human CAMPs (Sieprawska-Lupa et al.,
2004). S. aureus strains that produce significant amounts of
Aur are less susceptible to the antimicrobial fragment LL-1737 than strains that do not express Aur (Sieprawska-Lupa et
al., 2004). Moreover, Aur is essential for cleaving the central
complement protein C3, serves as a potent complement
inhibitor and effectively inhibits phagocytosis and killing of
bacteria by neutrophils (Laarman et al., 2011). However, in
this study, we found that E. tarda Aur is not essential for the
resistance to the tested CAMPs and virulence in vivo. It
remains to be explored whether E. tarda Aur is responsible
for degrading other CAMPs or serves as a complement
inhibitor.
The F1F0 ATP synthase plays a central role in energy
transduction, and therefore generating ATP from ADP and
inorganic phosphate via oxidative phosphorylation is vital
to numerous cellular processes (von Ballmoos et al., 2009).
In Mycobacterium smegmatis, atpD is essential for growth
on nonfermentable and fermentable carbon sources (Tran
& Cook, 2005). In Listeria monocytogenes, the increased
activity of the ATP synthase upon acid addition depletes
the cell’s supply of ATP, resulting in cell death (McEntire et
al., 2004). In S. Typhimurium, the atp genes are involved in
the acid-tolerance response and an atp mutant is avirulent
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Y. Lv and others
in the mouse typhoid model when assayed by oral and
intraperitoneal routes (Garcia-del Portillo et al., 1993). An
S. Typhimurium mutant lacking the entire atp operon has
impaired survival in the liver and spleen of intravenously
infected mice and confers protection against subsequent
oral rechallenge (Northen et al., 2010). However, the
regulation of the ATP synthase expression remains largely
unknown. In E. tarda, the genes encoding the F1F0 ATP
synthase are located in an operon, atpIBEFHAGDC, similar
to that in E. coli (Jones et al., 1983). Our proteomic analysis
showed that PhoP positively regulates the expression of the
F1F0 ATP synthase subunit b (AtpD), which is essential for
virulence of E. tarda. This attenuated virulence could be
due to the defective growth of the mutant strain that was
observed in vitro. Moreover, we revealed that the
transcription of the nine ATP synthase genes is dependent
on PhoP and PhoP directly regulates the expression of the
atp operon through interaction with the atpI promoter.
This regulation mechanism may also exist in other
members of the family Enterobacteriaceae due to the
universal distribution of the PhoP-PhoQ system and F1F0
ATP synthase.
A number of limitations exist in our proteomic analysis.
Firstly, gel-based proteomics is limited by the pI and MW
boundaries of the gels. As a consequence, only a subset of
the total proteome of E. tarda was visualized and
investigated. Secondly, we used a total cell lysate for
separation on the 2-DE gels. Membrane proteins are
known to be underrepresented in such protein samples
(Cordwell, 2006). This could explain why there are fewer
integral membrane proteins or membrane-associated
proteins in the identified proteins in this study. Thirdly,
regulatory proteins whose expression is dependent on
PhoP, such as EsrB (Chakraborty et al., 2010; Lv et al.,
2012a), are missing in the identified proteins. This is
probably due to the intrinsic low-abundance nature of
these regulatory proteins.
In conclusion, the comparative proteomic analysis presented here identified 18 proteins whose expression was
influenced by PhoP in E. tarda. Although this study is not a
full description of the PhoP regulon, the findings provide
new evidences that PhoP functions as a global regulator in
E. tarda, indicating PhoP plays very significant roles in
modulating the expression of proteins involved in
oxidative stress response and metabolic processes.
Furthermore, this study highlights a regulatory function
of PhoP on atp (F1F0 ATP synthase) operon expression and
extends our understanding of the PhoP regulon.
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Technology Research and Development Program of China
(2013AA093101), Ministry of Agriculture of China (CARS-50), the
National Special Fund for State Key Laboratory of Bioreactor
Engineering (2060204), as well as the Shanghai Leading Academic
Discipline Project (B505).
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