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ff, r55N 1584-11E? Volume 49 Number 2 Aprit 2016 tt 'rJl rttr I lndexcd in IJIEOLlNE/ lndFx ,tte'. dicur, Aidsline, cancorlit, chemicJl Abstracts, HeatthsTAR, SllC Data Bases The olficial journol ol Taiwan Society o, ,,licrobiotoSy Chinese Society of lmmunotogy The lnfectious Diseases Society of Taiwan Taiwan Society of Parasitotogy Joumal of
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ff,r55N 1584-11E?
Volume 49
Number 2
Aprit 2016
tt 'rJlrttrI
lndexcd in IJIEOLlNE/ lndFx ,tte'. dicur,
Aidsline, cancorlit, chemicJl Abstracts, HeatthsTAR, SllC Data Bases
The olficial journol olTaiwan Society o, ,,licrobiotoSy
Chinese Society of lmmunotogy
The lnfectious Diseases Society of Taiwan
Taiwan Society of Parasitotogy
Joumal of
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ORIGINAL ARTICLE
Regulatory elements of stx2 gene and the
expression level of Shiga-like toxin 2 in
Escherichia coli O157:H7
I Wayan Suardana a,*, Komang Januartha Putra Pinatih b,Dyah Ayu Widiasih c, Wayan Tunas Artama d, Widya Asmara e,Budi Setiadi Daryono f
a Department of Veterinary Public Health, Faculty of Veterinary Medicine, Udayana University,
Denpasar, Indonesiab Department of Microbiology, Faculty of Medicine, Udayana University, Denpasar, Indonesiac Department of Veterinary Public Health, Faculty of Veterinary Medicine, Gadjah Mada University,
Karang Malang, Yogyakarta, Indonesiad Department of Biochemistry, Faculty of Veterinary Medicine, Gadjah Mada University, Karang
Malang, Yogyakarta, Indonesiae Department of Microbiology, Faculty of Veterinary Medicine, Gadjah Mada University, Karang
Malang, Yogyakarta, Indonesiaf Department of Genetic, Faculty of Biology, Gadjah Mada University, Sekip Utara, Yogyakarta,
Indonesia
Received 2 June 2015; received in revised form 19 September 2015; accepted 25 April 2016
Available online - - -
KEYWORDS
Escherichia coli
O157:H7;
promoter;
q gene;
ribosome binding site;
Shiga-like toxin titer
Abstract Background/Purpose: Shiga-like toxin (Stx) is an important factor in the pathogen-
esis of Escherichia coli O157:H7 infection and is responsible for some severe complications.
Stx2 is usually associated with hemolytic uremic syndrome in humans. Its expression is regu-
lated by elements located upstream of the stx2 gene, including stx2-promoter sequence, ribo-
some binding site, and the antiterminator q gene. The present study aimed to find the
correlation between regulatory elements and the expression level of Stx2 in two local isolates
of E. coli O157:H7.
Methods: Two local E. coli O157:H7 strains SM-25(1) and KL-48(2), originating from human and
cattle feces, respectively, and an E. coli reference strain, ATCC 43894, were investigated. The
complete stx2 gene covering the sequences of promoter, ribosome binding site, and open
reading frame and q gene of each strain was analyzed. The magnitude of Stx2 production
was detected with a reverse passive latex agglutination method and Stx mediated cellular
damage was determined with the Vero cell assay.
* Corresponding author. Department of Veterinary Public Health, Faculty of Veterinary Medicine, Udayana University, Denpasar. Jl. PB.
Sudirman Denpasar-Bali, 80232, Indonesia.
E-mail addresses: [email protected], [email protected] (I.W. Suardana).
+ MODEL
Please cite this article in press as: Suardana IW, et al., Regulatory elements of stx2 gene and the expression level of Shiga-like toxin 2 in
Escherichia coli O157:H7, Journal of Microbiology, Immunology and Infection (2016), http://dx.doi.org/10.1016/j.jmii.2016.04.006
http://dx.doi.org/10.1016/j.jmii.2016.04.006
1684-1182/Copyright ª 2016, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Available online at www.sciencedirect.com
ScienceDirect
journal homepage: www.e- jmii .com
Journal of Microbiology, Immunology and Infection (2016) xx, 1e9
Results: A comparison of the complete stx2 gene contained stx2-promoter, ribosome binding
site, and q genes of two local strains KL-48(2) and SM25(1), and the E. coli ATCC 43894 showed
that the amino acid sequences were identical. Both local isolates were Stx negative in the
reverse passive latex agglutination test and nontoxic in the Vero cell assay.
Conclusion: The expression level of Shiga-like toxin of the two local isolates of E. coli O157:H7
did not only depend on the regulatory elements of the stx2 gene.
Copyright ª 2016, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Introduction
Escherichia coli O157:H7, as one of the enterohemorrhagic
E. coli serotypes, is known as an important food-borne
pathogen that can cause severe gastrointestinal and sys-
temic diseases, which in some patients are followed by
death.1,2 The clinical symptoms of some patients are in part
due to the Shiga-like toxins (Stx) produced by such path-
ogen strains. Stx, which are encoded by stx genes, are
divided into two immunologically distinct serogroups: Stx1
and Stx2. Stx1 is nearly identical to Stx produced by
Shigella dysenteriae, differing in only one amino acid in the
A-chain. However, Stx2 has less amino acid similarity to Stx
(w55%).3 An in-vivo study showed that Stx2b and Stx2c
have potencies similar to that of Stx1, while Stx2a, Stx2d,
and elastase-cleaved Stx2d are 40e400 times more potent
than Stx1.4
Stx2 is most frequently associated with human diseases,
especially hemolytic uremic syndrome. This condition is
more frequent in children and the elderly.5 The molecular
mechanisms that regulate the initial transcription of stx2
gene are critical to numerous Stx2 productions. Mutation at
stx2-promoter sequences transcribing the stx2 gene could
result in little or no Stx2 production.6
Specifically, in the stx2 gene, the stx2A and stx2B sub-
units genes are located downstream of the phage late
promoter (pR) on the genomes of resident prophages.
Removal of the repression, which can occur when DNA
damage activates a cascade of regulatory events beginning
with expression of the N transcription antitermination
protein.7 In the presence of N, transcription transcends the
termination barrier and consequently, more distal genes
are expressed including q gene. N-modified transcription
from pR passes through tR1, tR2, and tR3 barriers before
extending into q gene. The q gene product allows tran-
scription initiating at the pR0 promoter, immediately beyond
q, to extend downstream to include the lytic genes.8 The Q
protein acts at qut (Q utilization sequence) and promotes a
read through of the transcription terminator tR0 that acti-
vates pR.9 Furthermore, Q protein binds to DNA in the re-
gion between the �10 and �35 promoter elements of stx2
gene and interacts with RNA polymerase paused just
downstream at þ16 relative to the start of transcription.8
As a result, induction of the stx-bearing phage is associ-
ated with enhanced Stx production. According to these
phenomena, the purpose of this study was to prove the
regulatory elements of stx2 gene among E. coli O157:H7
local strains originating from human and animal that
correspond to magnitude of Stx2 production in order to
corfirm the zoonotic potency of local strains.
Methods
Bacterial strains
Two local strainsdSM-25(1) originating from feces of Bali
cattle, and KL-48(2) originating from feces of a patient
suffering renal failure at the SanglahGeneral Hospital Centre
(Denpasar-Bali)dwere investigated in this study. Both
strains had been pheno- and genotypically identified as E.
coli O157:H7 by previous study.10e13 An E. coli reference
strain, ATCC 43894, and a wild isolate E. coli, DS-16(2), from
the institute strain collection were used as control strains.
Extraction of DNA and polymerase chain reaction
Bacterial DNA was extracted by using QIAamp DNA Mini Kits
(Qiagen) according to the manufacturer’s instructions. The
complete stx2 gene covering stx2-promoter, ribosome
binding site (RBS), and open reading frame sequences and
the q gene of each strain were amplified using Fast Start
PCR Master Mix (Roche, Mannheim, Germany) on MJ Mini
Personal Thermal cycler PTC-1148 (Bio-Rad). The poly-
merase chain reaction (PCR) program was carried out in
27 mL reaction volumes containing 1.5 mL DNA template
(300 ng/mL), 17 mL Fast Start PCR Master, and 1 mL (20 pmol)
of each primer. The oligonucleotide primers Stx2(F): 50-GCC
ATT AGC TCA TCG GGA TA-30 and Stx2(R): 50-CGA ATG CTC
AGT CTG ACA GG-30 were used for the complete stx2 gene
amplification.14 Primers QWS(F): 50-GTA GTC GCA ACA CGG
TCA GA-30 and QWS(R): 50-GAC TGC GTG GCA ATG TAA CC-30
were designed according to the published sequences of the
antiterminator Q protein to amplify complete q gene. PCR
amplification of stx2 had initial DNA denaturation at 94�C
for 7 minutes, followed by 35 cycles of denaturation at 94�C
for 1 minute, annealing at 60�C for 35 seconds, and elon-
gation at 72�C for 1.5 minutes, which was followed by a
final extension at 72�C for 5 minutes. Amplification of the q
gene had similar procedure but with 1 minute for the
elongation step at 72�C. Following PCR, 4 mL of each
product was analyzed by electrophoresis in 1% agarose gel
(Gibco BRL) at 100 volts for 35 minutes followed by staining
with 1% solution of ethidium bromide. Subsequently, gel
was visualized by using a UV transilluminator to confirm the
presence of PCR products.
2 I.W. Suardana et al.
+ MODEL
Please cite this article in press as: Suardana IW, et al., Regulatory elements of stx2 gene and the expression level of Shiga-like toxin 2 in
Escherichia coli O157:H7, Journal of Microbiology, Immunology and Infection (2016), http://dx.doi.org/10.1016/j.jmii.2016.04.006
Sequencing and phylogenetic analysis
The purification of PCR products was performed using the
QIAquick PCR Purification Kit (Qiagen) according to the
manufacturer’s instructions. The sequencing of stx2 gene
was performed using genetically analyzer (ABI 3130 XL Ge-
netic Analyzer) at the Eijkman Institute for Molecular
Biology, Jakarta, Indonesia. For the sequencing, the same
primer pairs were used as described previously. The se-
quences were analyzed using MEGA version 5.2 software
(Arizona State University). The nucleotide sequences of E.
coli O157:H7 strains such as 98-16 (AB168106), V20
(AB071845), Thai-13 (AB168111), Fu40 (AB168103), 144
(AB168104), Thai-12 (AB168110), Thai-1 (AB168108), phage
933(NC_000924), and wus (EU700491) determined in this
study will appear in the NCBI nucleotide sequence database.
The phylogenetic analysis was constructed using
neighbor-joining algorithm with 1000-replication multiple
bootstrap test.15,16
Detection of Stx 2 production
For the detection of magnitude of Stx 2 production, a
reverse passive latex agglutination assay (VTEC-RPLA;
Denka Seiken) was performed according to the manufac-
turer’s instructions.6 The local strains, positive control E.
coli ATCC 43894, and a negative control DS-16(2) strain
were grown in LuriaeBertani liquid medium with shaking
(3 g) at 37�C overnight. The bacterial culture was centri-
fuged at 2000 g for 10 minutes and the agglutination test
was carried out in 96-well V-bottom for 24 hours at room
temperature. The reciprocal of the highest dilution of the
test sample that gave a positive reaction was defined as
RPLA titer.
Analysis of toxicity through cytopathic effect
production on Vero cells
Each of the E. coli strains was inoculated in 30 mL of Lur-
iaeBertani medium and incubated at 37�C for 24 hours on a
low speed shaker. The bacterial cells were pelleted by
centrifugation (3000 g) at 4�C for 40 minutes. The super-
natant was taken and 15 mL were mixed by adding of 5.97 g
ammonium sulfate gradually in order to obtain 65% satu-
ration. The solution was centrifuged again as described
above. The precipitate obtained was dissolved in 3 mL of
sterile physiological saline and dialyzed at 4�C overnight.
Subsequently, the toxin was filter sterilized with 0.22-mm
filters (Millipore). Toxin concentrations (mg/mL) were
measured with a solution of Bio-Rad Protein Assay with
spectrophotometric absorbance at a wavelength of 595 nm.
Cytotoxicity assay
The cytotoxicity assay was performed as described by Piaru
et al with a minor modification.17 Confluent monolayers
were removed with trypsin EDTA, resuspended to approxi-
mately 1 � 104 cells/mL in M199 medium (Invitrogen) and
50 mL toxin were pipetted into each well of a 96-well mi-
crotiter plate. After incubation at 37�C in 5% CO2 for
15 minutes, the medium was replaced with 100 mL M199
medium and incubated for 48 hours at 37�C in 5% CO2. Each
sample was tested in triplicate. Vero cell monolayer
morphology was observed under an inverted microscope
and checked for cytotoxic effect for 24 hours and 48 hours
of induction.
MTT assay
After 24 hours and 48 hours incubation at 37�C in a hu-
midified air atmosphere containing 5% CO2, 25 mL of 5 mg/
mL of MTT in saline was added into each well and further
incubated at 37�C for 4 hours. To dissolve formazan pre-
cipitate, 80 mL of a buffer containing 10% sodium dodecyl
sulfate (Sigma Chemical Co.) and 50% N,N-
dimethylformamide (pH 4.7) was added into each well.
Further incubation was performed overnight at 37�C. Op-
tical densities (OD) of dissolved formazan were read at
550 nm using a microplate spectrophotometer (Model 680
XR). Viability ratio (%) of living cells for each treatment was
determined using the formula:
[Mean OD of treated cells/Mean OD of control
cells] � 100%.17,18 (1)
Statistical analysis of cytotoxicity
Potential cytotoxicity was evaluated with linear regression
analysis using SPSS version 14 (SPSS Inc., Chicago, IL, USA)
and the significant difference between control and treated
cells was statistically analyzed by paired Student t test
(p < 0.05).
Results
Analysis of the stx2 gene
Two E. coli O157:H7 strains, KL-48(2) and SM-25(1), origi-
nating from human and animal, respectively, and one of E.
coli reference strain (ATCC 43894) as a positive control
were examined in the study. The stx2 gene and upstream
sequences in the local strains, and E. coli reference strain
were analyzed by PCR product of 1587 bp. The sequences
(1241 bp) of open reading frame of stx2 gen of the local
strains have been deposited in the National Center for
Biotechnology Information with accession numbers KC
659956.1 and JQ 411011.1, respectively. The sequence
analysis of the open reading frame of stx2 gene covering A
and B subunits showed an identical sequence in all three
strains. We also compared the stx2 gene sequences of the
two local strains against those of other well-characterized
strains from the gene database. The sequence alignment
showed that both local strains had a sequence homology of
100% with E. coli strain Enterobacteria phage 933W, and
98.31% compared with the stx2 gene of three E. coli strains:
Fu40, 144, and Thai-12 (21 out of 1241 nucleotides
different). Both local strains also differed in 22 out of 1241
nucleotides (98.23% similarity) compared with E. coli strain
Thai-1, 23 nucleotides (98.15% similarity) against two
further E. coli strains (98-16 and V20), and 25 nucleotides
Regulatory elements and expression level of Stx2 3
+ MODEL
Please cite this article in press as: Suardana IW, et al., Regulatory elements of stx2 gene and the expression level of Shiga-like toxin 2 in
Escherichia coli O157:H7, Journal of Microbiology, Immunology and Infection (2016), http://dx.doi.org/10.1016/j.jmii.2016.04.006
Table 1 Similarity value (%) and the number of nucleotides different of stx2 gene among Escherichia coli ATCC 43894, KL-48(2), and SM-25(1) strains against several isolates
available in GenBank.a
E. coli
O157:H7
98-16
E. coli
O157:H7
V20
E. coli
O157:H7
Thai-13
E. coli
O157:H7
Fu40
E. coli
O157:H7
144
E. coli
O157:H7
Thai-12
E. coli
O157:H7
Thai-1
Enterobacteria
phage 933
E. coli
ATCC
43894
E. coli
KL-48(2)
Humanb
E. coli
SM-25(1)
Cattleb
E. coli
WUS
E. coli O157:H7 98-16 d 4/1241 6/1241 2/1241 2/1241 2/1241 5/1241 23/1241 23/1241 23/1241 23/1241 21/1241
E. coli O157:H7 V20 99.68 d 6/1241 2/1241 2/1241 2/1241 5/1241 23/1241 23/1241 23/1241 23/1241 21/1241
E. coli O157:H7 Thai-13 99.52 99.52 d 4/1241 4/1241 4/1241 7/1241 25/1241 25/1241 25/1241 25/1241 23/1241
E. coli O157:H7 Fu40 99.84 99.84 99.68 d 0/1241 0/1241 3/1241 21/1241 21/1241 21/1241 21/1241 19/1241
E. coli O157:H7 144 99.84 99.84 99.68 100 d 0/1241 3/1241 21/1241 21/1241 21/1241 21/1241 19/1241
E. coli O157:H7 Thai-12 99.84 99.84 99.68 100 100 d 3/1241 21/1241 21/1241 21/1241 21/1241 19/1241
E. coli O157:H7 Thai-1 99.60 99.60 99.44 99.76 99.76 99.76 d 22/1241 22/1241 22/1241 22/1241 20/1241
Enterobacteria phage 933 98.15 98.15 97.99 98.31 98.31 98.31 98.23 d 0/1241 0/1241 0/1241 9/1241
E. coli ATCC 43894 98.15 98.15 97.99 98.31 98.31 98.31 98.23 100 d 0/1241 0/1241 9/1241
E. coli KL-48(2) Humanb 98.15 98.15 97.99 98.31 98.31 98.31 98.23 100 100 d 0/1241 9/1241
E. coli SM-25(1) Cattleb 98.15 98.15 97.99 98.31 98.31 98.31 98.23 100 100 100 d 9/1241
E. coli WUS 98.31 98.31 98.15 98.47 98.47 98.47 98.39 99.27 99.27 99.27 99.27 d
a The lower left corner shows the percentage (%) of nucleotide similarity, upper right corner shows the difference of nucleotides (bp) per total nucleotides (bp). The nucleotides of E.
coli O157:H7 used for alignment obtainable from GenBank with each accession number in parentheses such as: 98-16 (AB168106), V20 (AB071845), Thai-13 (AB168111), Fu40 (AB168103),
144 (AB168104), Thai-12 (AB168110), Thai-1 (AB168108), phage 933 (NC_000924), and WUS (EU700491).b Local isolates studied.
E. coli Z Escherichia coli.
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(97.99% similarity) against E. coli strain Thai-13. The results
of similarity value (%) and the number of nucleotides
different of stx2 gene among isolates are summarized in
Table 1, and the phylogenetic tree is shown in Fig. 1.
The regulatory elements covering promoter and
ribosome-binding site regions of the stx2 gene in both local
strains, and E. coli reference ATCC 43894 as a positive
control were studied in comparison to those of E. coli
E. coli O157:H7 98-16
E. coli O157:H7 V20
E. coli O157:H- Thai-13
E. coli O157:H7 144
E. coli O157:H7 Thai-12
E. coli O157:H7 Fu40
E. coli O157:H7 Thai-1
Clade 1
E. coli WUS
E.phage 933W
E. coli SM 25(1) cattle*
E. coli ATCC 43894
E. coli KL 48(2)human*
Clade 2
100
100
67
22
20
0.002
Figure 1. Phylogenetic tree was constructed using the neighbor-joining algorithm of open reading frame of stx2 gene (1241 bp).
The number in the branch of the phylogram indicates bootstrap value (%) by 1000-replication multiple, and the scale indicates two
per 1000 substitutions of the nucleotide sequence of stx2 gene. E. coli Z Escherichia coli.
Figure 2. Comparison of the nucleotide sequence of the promoter region and ribosome binding site among stx2 genes of
Enterobacteria phage 933W, Escherichia coli ATCC 43894, KL-48(2) human, SM25(1) cattle, and E. coli Thai-12. Position of start
codon, ribosome binding site, and �10 and �35 promoter regions are indicated by underlines. The hyphen and the asterisk indicate
identical base and absence of base, respectively.
Regulatory elements and expression level of Stx2 5
+ MODEL
Please cite this article in press as: Suardana IW, et al., Regulatory elements of stx2 gene and the expression level of Shiga-like toxin 2 in
Escherichia coli O157:H7, Journal of Microbiology, Immunology and Infection (2016), http://dx.doi.org/10.1016/j.jmii.2016.04.006
strains EDL933 and Thai-12, which are known to have pos-
itive and negative Stx2 production, respectively. Identical
homology was detected between the sequences of both
local strains and E. coli reference ATCC 43894 as well as E.
coli strain EDL933 in these regions. However, both local
strains also have some base differences compared with
Thai-12 regulatory elements sequences that may influence
Stx2 production: two base differences in the stx2-promoter
region, one in the �35 region, and the other at a site
adjacent to the �10 region. In addition, one base insertion
between the ribosome binding site and the start codon was
detected in Thai-12. The upstream sequences of stx2-reg-
ulatory elements among strains are shown in Fig. 2.
Analysis of the antiterminator q gene
Within both local strains, and E. coli reference strain ATCC
43894, the q gene could be amplified with unique PCR prod-
uct size of 583 bp. The q gene sequences of two local strains,
KL-48(2) and SM-25(1), were aligned with those of the
reference strain and an E. coli strain Enterobacteria phage
933 from thenucleotidedatabase. Thealignmentof 474bpof
q geneamong isolates showed identically of both local strains
against E. coli reference ATCC 43894 as well as Enter-
obacteria phage 933 (data not shown). These results sug-
gested the possibility of strongly antitermination activity of
both Q protein of local strains at qut sites as well as the 933W
genome that was studied previously by another researcher.6
Detection of Stx 2 production
The Stx2 production could not be detected in the culture
supernatant of either local strain examined by RPLA assay,
whereas the production levels of Stx2 were largely lower
(<2 ng/mL) than those of other positive E. coli strain ATCC
43894. The data in Table 2 summarize Stx2 titer of both
local strains, E. coli ATCC 43894, and E. coli negative
isolate DS-16(2).
Analysis of cytopathic effect production on Vero
cells
Escherichia coli O157 has a tissue tropism to Vero cells,
causing a cytopathic effect (CPE); therefore, it is a good cell
line for the evaluation of toxicity.19 During the 24 hours and
48 hours of exposure, the viability of the Vero cells had
reduced significantly (p < 0.05) only in the positive control
E. coli reference ATCC 43894, but contrary against the
treatment using both local strains which showed no signifi-
cant effect (p > 0.05) to the formation of CPE as well as
negative control. The illustration of CPE formation on Vero
cells line of isolates before and after induced by Stx are
presented in Fig.3, and the detail percentage of CPE for each
isolate for 24 hours and 48 hours is summarized in Table 3.
Discussion
The nucleotide similarity and the number of nucleotides
different (Table 1) as a result of alignment of 1241 nucle-
otides covering open reading frame of stx2 gene were
identical between local strains KL-48(2) and SM-25(1)
against E. coli reference ATCC 43894, and Enterobacteria
phage 933, which is known as a highly virulent strain that
caused an outbreak in the USA in 1982.20 Identical nucleo-
tide sequence among local strains of E. coli O157:H7 with
Enterobacteria phage 933 as well as E. coli reference ATCC
43894 indicate the probability of local strains to cause
similar clinical symptoms as an effect of Stx2.
These results are also supported by both strains having
distinct nucleotide sequence compared with some isolates
of E. coli O157:H7/e, which are known to originate from
nonclinical sources such as: strain 98-16 isolated from
human feces; strains 144 and Fu40 from cattle feces6; Thai-1
and Thai-12 from bovine feces; and Thai-13 from beef.21 The
data in Table 1 also could be used to cluster of each isolate
as showed in the phylogenetic tree (Fig. 1). The data in Fig. 1
show both strains sharing a similar clade with E. coli refer-
ence ATCC 43894 and Enterobacteria phage 933W with its
bootstrap value 100%, and placed it in a different clade,
especially to Thai-12 strain. The results showed the local
strains of E. coli O157:H7 were identical to E. coli reference
ATCC 43894 as well as Enterobacteria phaga 933W based on
the stx2 sequences. The identically of Stx2 nucleotide
sequences among isolates concluded of those were poten-
tially had similar potency to produce a toxin (Stx2) as well as
their potency to colonize and to cause a CPE on Vero cells.10
The assumption was base on theoretically, the complete
protein (Stx2) is thus the product of stx2 gene, and mutation
in any of which may lead to the abnormally of functional
product.22 The mutation in the promoter sequences tran-
scribing the stx2 gene could result in little or no Stx2 pro-
duction.6 Furthermore, the analysis of regulatory elements
of stx2 gene (Fig. 2) showed that the nucleotide sequence of
�10 and�35 promoter regions among Enterobacteria phage
933W, E. coli ATCC 43894, and both local strains were
identical, as well as the RBS region and gap between RBS and
start transcription site. The mutation and substitution of
regulatory elements among strains was just different against
E. coli Thai-12, which was assumed to be responsible for the
little or no detected Stx2 production.6
Analysis of the q gene, which is known to have an
important role in the initial transcription of stx2 gene, was
also conducted referring to previous research.6 The nucle-
otide alignment showed that the sequence of the q gene of
both local strains was conserved against Enterobacteria
phage 933 and E. coli reference ATCC 43894. The q gene
Table 2 Detection of Stx2 production among Escherichia
coli ATCC 43894, KL-48(2), SM-25(1), and DS-16(2) by using
RPLA assay with reference data as a comparison.
Strain Serotype Source stx2
gene
Stx2
titerb
ATCC 43894a O157:H7 Human, USA þ 64
KL-48(2) O157:H7 Human, Indonesia þ <2
SM-25(1) O157:H7 Cattle feces,
Indonesia
þ <2
DS-16(2)a O157:H7 Beef, Indonesia e <2
a Control strain for Stx2 production.b Reciprocal of the dilution that gave positive reaction in the
a reverse passive latex agglutination assay.
6 I.W. Suardana et al.
+ MODEL
Please cite this article in press as: Suardana IW, et al., Regulatory elements of stx2 gene and the expression level of Shiga-like toxin 2 in
Escherichia coli O157:H7, Journal of Microbiology, Immunology and Infection (2016), http://dx.doi.org/10.1016/j.jmii.2016.04.006
sequence also showed difference with E. coli Thai-12,
which has known lack in Stx2 production. These results
could be assumed that both local strains also theoretically
potential producing high level of Stx2 toxin as equal as E.
coli strains ATCC 43894 and Enterobacteria phage 933.
On the order hand, by contrast the production of Stx2 of
both local strains using the RPLA assay showed a lower
level compared with E. coli ATCC 43894 although they had
similar nucleotide sequences including complete gene of
stx2, promoter sequence, RBS, as well as q gene. The Stx2
titer of both local strains was lower than the 2 mg/mL
detection limit of the RPLA assay (Table 2). According to
the study of Koitabashi et al,6 the strain with similar
nucleotide sequence of the regulatory elements of stx2
gene as well as EDL 933 strain would produce high Stx2
titer. In this study the phenomenon was just exist to E. coli
ATCC 43894, but contrary with both local strains. Strain
KL-48(2) and SM-25(1) were known to have intact stx2 gene
sequences including the regulatory elements, but no Stx2
production, was detected contrary with ATCC 43894 as a
control. Furthermore, the deeply confirmation to evaluate
the correlation between the production levels of Stx
against their capability to induce CPE also showed parallel
results. Local strains showed slight or no effect compared
with E. coli ATCC 43894 and only resulted as equally as
A A1
B C
D E
Figure 3. The viability and proliferation of vero cell after MTT cell proliferation assay 48 hours postinduction. Images taken with
a phase contrast microscope (Olympus, Shinjukuku, Tokyo, Japan, type IMT-2/605029) with a magnification of 10 � 40. The arrow
shows the stained living cells. (A, A1) nonetoxin-induced cells; (B) cells induced by Escherichia coli ATCC 43894 toxin as a positive
control; (C) cells induced by KL-48(2) toxin; (D) cells induced by SM-25(1) toxin; (E) cells induced by DS-16(2) toxin as a negative
control.
Table 3 Percentage of cytopathic effect (CPE) on Vero
cells after induction of Stx for 24 hours and 48 hours.a
Isolates % CPE (24 h) % CPE (48 h) Averages
1 2 1 2
E. coli ATCC 43894 44.52 65.24 60.57 51.275b
E. coli KL-48(2) 17.06 32.13 19.36 17.91 21.615a
E. coli SM-25(1) 12.01 33.90 17.00 29.81 23.180a
E. coli DS-16(2) 10.75 38.79 7.92 37.91 23.843a
a Superscripts toward column with the same letter indicate
nonsignificant difference (p > 0.05), and with different letter
indicated significantly difference (p < 0.05).
E. coli Z Escherichia coli.
Regulatory elements and expression level of Stx2 7
+ MODEL
Please cite this article in press as: Suardana IW, et al., Regulatory elements of stx2 gene and the expression level of Shiga-like toxin 2 in
Escherichia coli O157:H7, Journal of Microbiology, Immunology and Infection (2016), http://dx.doi.org/10.1016/j.jmii.2016.04.006
negative control E. coli DS-16(2), which was identified
negative stx2 gene.
According to these results, it can be concluded that the
expression level of Stx2 of both local strains was not only
regulated by the element of q gene, promoter, and RBS but
this also was predicted depending on the other elements.
This assumption is supported by findings of other researchers
who found that PhoB, as a protein of transcription regula-
tory, is known to have an important role for the virulence of
E. coli O157:H7. PhoB activates the transcription, produc-
tion, and release of Stx2.23 Its mechanism such as in extra-
cellular phosphate concentration (Pi-limited conditions)
falls below 4 mM, PhoR undergoes conformational change,
resulting in its release from repressor complex and auto-
phosphorylation. The PhoR is a histidine kinase/phosphatase
that donates a phosphanyl group to PhoB so that the PhoB
activates. Active PhoB will bind to DNA sequences upstream
of Pho regulon genes, which are known as Pho boxes,
located within Pho-dependent promoter region.24,25 PhoB
indirectly inhibits the transcription of the cI gene repressor,
which in return releases the consecutive transcription of cro
that should induce a lytic phage cycle. Prophages ultimately
induce the Q-modified transcription, which were initiated at
PR0 that transcends the tR0 terminator, leading to expression
of downstream genes including stx2 thereby increasing stx2
transcription. The other scenario could be direct PhoB
binding onto sequences upstream of the stx2 promoter,
activating stx2 transcription.23 By contrast, if the inorganic
phosphate concentration is high (>4 mM), PhoR might
interact with the PstSCAB-phoU complex, which represses its
autophosphorylation and acts as a PhoB phosphatase so that
the PhoB is deactivated.24 Further research will be needed
to clarify the facts that have not been revealed, especially
to study the role of PhoB in the Stx2 production of both local
strains as well as measurement of Stx2-related to mRNA
expression according to the study by Kawano et al26 so that
this phenomenon will be revealed completely.
Conflicts of interest
The authors declare that there is no conflict of interests
related to the work in this article.
Acknowledgments
The authors gratefully acknowledge the Directorate Gen-
eral of Higher Education, Ministry of National Education for
providing financial support in the form of National Strategic
Research Grant under Grant no. 239.14/UN 14.2/
PNL.01.03.00/2014, dated May 14, 2014. Special thanks to
Dr. med. vet. Omer Akineden, Professur fur Milchwissen-
schaften, Institut fur Tierarztliche Nahrungsmittelkunde,
Justus-Liebig Universitat Gießen, Germany for his motiva-
tions and revisions.
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Regulatory elements and expression level of Stx2 9
+ MODEL
Please cite this article in press as: Suardana IW, et al., Regulatory elements of stx2 gene and the expression level of Shiga-like toxin 2 in
Escherichia coli O157:H7, Journal of Microbiology, Immunology and Infection (2016), http://dx.doi.org/10.1016/j.jmii.2016.04.006
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