Folia Microbiol
DOI 10.1007/s12223-015-0405-z
Transformation and characterization of an arsenic gene operon
from urease-positive thermophilic Campylobacter (UPTC)
in Escherichia coli
M. Matsuda 1 & T. Kuribayashi 1 & S. Yamamoto 1 & B. C. Millar 2 & J. E. Moore 2,3,4
Received: 9 March 2014 / Accepted: 1 June 2015
# Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2015
Abstract An arsenate susceptibility test was performed
with transformed and cultured Escherichia coli DH5α
cells, which carried recombinant DNA of full-length ar-
senic (ars) operon, namely a putative membrane perme-
ase, ArsP; a transcriptional repressor, ArsR; an arsenate
reductase, ArsC; and an arsenical-resistance membrane
transporter, Acr3, from the Japanese urease-positive ther-
mophilic Campylobacter lari (UPTC) CF89-12. The
E. coli DH5α transformant showed reduced susceptibili-
ty to arsenate (~1536 μg/mL), compared to the control.
Thus, these ars four-genes from the UPTC CF89-12
strain cells could confer a reduced susceptibility to arse-
nate in the transformed and E. coli DH5α cells. E. coli
transformants with truncated ars operons, acr3 (acr3)
and arsC-acr3 (ΔarsC-acr3), of the ars operon, showed
an MIC value of 384 μg/mL (~384 μg/mL), similar to
the E. coli cells which carried the pGEM-T vector (con-
trol). Reverse transcription PCR confirmed in vivo tran-
scription of recombinant full-length ars operon and dele-
tion variants (Δacr3 and ΔarsC-acr3) in the transformed
E. coli cells.
* M. Matsuda
matsuda@azabu-u.ac.jp
1
Laboratory of Molecular Biology, Graduate School of Environmental
Health Sciences, Azabu University, Sagamihara 252-5201, Japan
2
Department of Bacteriology, Northern Ireland Public Health
Laboratory, Belfast City Hospital, Belfast BT9 7AD, Northern
Ireland, UK
3
School of Biomedical Sciences, Ulster University, Co. Londonderry,
Coleraine BT52 1SA, Northern Ireland, UK
4
School of Medicine, Dentistry and Biomedical Sciences, Queen’s
University, Belfast BT7 1NN, Northern Ireland, UK
Introduction
To survive arsenic associated toxicity, bacterial organisms
have evolved multiple mechanisms for arsenic detoxification,
reduction, and efflux (Rosen 2002; Qin et al. 2006).
Resistance to arsenic in bacterial organisms results from
energy-dependent efflux of either arsenate or arsenite from
the cell, which is mediated via the ars operon (Mobley and
Rosen 1982; Ji and Silver 1992a, b; Cervantes et al. 1994). In
both Gram-positive and Gram-negative bacteria, the ars genes
are organized frequently in operons (Diorio et al. 1995; Cai
et al. 1998; Sato and Kobayashi 1998; Suzuki et al. 1998;
Butcher et al. 2000; Butcher and Rawlings 2002).
Thermophilic Campylobacter organisms are the major rec-
ognized cause of acute bacterial diarrhea worldwide.
Although the genus Campylobacter currently consists of 17–
18 described species, human gastrointestinal illness is associ-
ated primarily with Campylobacter jejuni and Campylobacter
coli (Lastovica and Skirrow 2000; Debruyne et al. 2009).
Campylobacter lari has also been shown to be the cause of
clinical infections infrequently (Nachamkin et al. 1984;
Martinot et al. 2001; Werno et al. 2002).
Regarding arsenic resistance in C. jejuni, isolates from
poultry are highly resistant to arsenic compounds and the mo-
lecular mechanisms responsible for the resistance have been
determined (Sapkota et al. 2006; Wang et al. 2009; Shen et al.
2014). In addition, Wang et al. and Noormohamed et al. de-
scribed a four-gene operon that contributes to arsenic resis-
tance in C. jejuni (Wang et al. 2009; Noormohamed and Fakhr
2013). This operon encodes a putative membrane permease
(ArsP), a transcriptional repressor (ArsR), an arsenate reduc-
tase (ArsC), and arsenical-resistance membrane transporter
protein (an arsenate efflux pump; Acr3) (Wang et al. 2009).
The presence of arsC and acr3, in addition to arsP and arsR,
was correlated with increased resistance to arsenic compounds

in different C. jejuni strains (Wang et al. 2009). Most recently,
Shen et al. (2013) described the contribution of arsB gene to
arsenic resistance in C. jejuni NCTC11168 by using the inser-
tional mutation procedure of arsB inactivation (Shen et al.
2013).
Regarding the ars operon, containing arsP, arsR, arsC, and
acr3, C. jejuni NCTC11168, 81-176 and RM1221 carry the
full-length ars operon. However, some C. jejuni contain a
truncated ars operon (Wang et al. 2009). Interestingly, the
truncated ars operon is also carried in some C. lari and this
ars operon shows different arsenate resistance.
Although arsP and arsR have recently been identified in
C. lari RM2100 (DDBJ/EMBL/GenBank accession number
NC_012039), according to genome sequencing analysis
(Miller et al. 2008), there has been no report of these two
genes in C. lari.
The human clinical C. lari RM2100 strain lacks genes
encoding ArsC and Acr3 in the operon. During the process
of genome sequence analysis of an environmental Japanese
urease-positive thermophilic Campylobacter (UPTC) isolate,
for the first time, the four-gene operon was found in UPTC
CF89-12 (approximately 4.3 kbp in length) and some other
C. lari isolates (Nakajima et al. 2013). In addition, in vivo
transcription of the operon was confirmed in the UPTC cells.
UPTC CF89-12 and some other UPTC isolates obtained from
the natural environment were identified to be resistant to arse-
nate (Nakajima et al. 2013). Although the functions have not
been found, C. lari isolates also may have various types of ars
operon.
However, the operon that can express ars four-gene in
Escherichia coli has not been constructed in vitro by amplify-
ing the operon from the UPTC CF89-12 strain.
Therefore, the aim of the present study was to construct
recombinant DNA of the UPTC CF89-12 full-length ars op-
eron in vitro and to express the recombinant DNA in E. coli
cells.
Materials and methods
Experimental strategy
Two deletion variants, Δacr3 and ΔarsC-acr3, of the ars op-
eron were constructed in vitro and transcribed at the RNA
level and characterized these in E. coli cells, in order to clarify
the role of these genes in UPTC organisms which are resistant
to arsenate (disodium hydrogenarsenate heptahydrate;
Na2HAsO4·7H2O=312.01).
Bacterial isolates and culture conditions
The Japanese strain, UPTC CF89-12, which was originally
isolated from the water of the Asahigawa River, Okayama
Folia Microbiol
prefecture, Japan (Matsuda et al. 1996), was employed in the
present study.
Following culturing on charcoal-cefazolin-sodium
deoxycholate agar medium (Oxoid, Hampshire, UK), cells
were cultured on blood agar base No. 2 (Oxoid) that contained
defibrinated horse blood (7 % (v/v); Nippon Bio-test, Tokyo,
Japan), supplemented with Butzler Campylobacter selective
medium (Virion, Zurich, Switzerland), under microaerophilic
conditions using BBL Campypak Microaerophilic System
Envelopes (Becton Dickinson, NJ, USA) at 37 °C for 48 h.
Cells were further cultured on Mueller-Hinton agar under the
same microaerophilic conditions.
Genomic DNA preparation and PCR amplification
Genomic DNA was prepared from UPTC CF89-12 cells using
sodium dodecyl sulfate and proteinase K treatment, phenol-
chloroform extraction, and ethanol precipitation (Harrington
et al. 1997).
For the amplification of approximately the 2.7-kbp full-
length ars operon from the UPTC CF89-12 strain (nucleotide
position (np) 553 bp through np 3306 bp; AB611008), a PCR
primer pair of ArsP-F (np 553–573 bp) and Acr3-R (np 3288–
3306 bp) was designed (Fig. 1). PCR amplification and its
product purification were carried out, as described by
Sambrook and Russell (2001) and Nakanishi et al. (2013).
TA cloning of the amplified full-length ars operon using the
pGEM-T Vector Systems I (Promega Corp., Tokyo, Japan)
and E. coli DH5α competent cells prepared by RbCl was also
carried out, as described previously by Sambrook and Russell
(2001) and Nakanishi et al. (2013).
In the present study, two deletion variants of one and two
genes of full-length ars four-gene were constructed, from
UPTC CF89-12 (100 % acr3 deletion and 100 % arsC-acr3
deletion), using their specific PCR primer pair, ArsC-R/T-
Vector-F, and ArsR-R/T-Vector-F, respectively, with inverse
PCR procedures (Fig. 1b). The primer pairs shown in
Fig. 1b for the I-PCR were designed based on the nucleotide
sequence data of approximately a 4.3-kbp full-length ars gene
cluster and its adjacent genetic loci from the UPTC CF89-12
strain.
The PCR mixture contained 1× KOD buffer, 200 μmol/L
each dNTP, 1.2 mmol/L MgSO4, 0.6 mmol/L each primer, a
total of 1 unit KOD-Plus- DNA polymerase (TOYOBO,
Shiga, Japan), and 50 ng genomic template DNA. The PCR
reaction was performed in 25 μL reaction volumes at 94 °C
for 2.0 min, with 30 cycles at 94 °C for 15 s, 55 °C for 30 s,
and 68 °C for 4.0 min, followed by a final extension at 68 °C
for 5.0 min.
Amplified PCR products were separated by 1.0 % (w/v)
agarose gel electrophoresis in 0.5× Tris/borate/EDTA (TBE)
at 100 V and detected by staining with ethidium bromide.
They were purified using a QIAquick PCR Purification Kit
Folia Microbiol

Fig. 1 A schematic
representation of full-length ars
operon and its adjacent genetic
loci in UPTC CF89-12 (a) and the
full-length ars operon and I-PCR
primers and primer locations to
construct two deletion variants of
UPTC CF89-12 ars genes (Δacr3
and ΔarsC-acr3) (b). Location of
the primers and the nucleotide
sequences in this study are also
shown (c)

(TOYOBO). The products were then subjected to cycle se-
quencing with BigDye Terminator (Applied Biosystems,
Tokyo, Japan), using the PCR primers or the I-PCR primers.
The reaction products were separated and detected on an ABI
PRISM® 3100 Genetic Analyzer (Applied Biosystems).
Nucleotide sequence analysis of the full-length ars operon
was carried out using the GENETYX-Windows computer
software version 9 (GENETYX Co., Tokyo, Japan).
Total cellular RNA purification and reverse transcription
PCR
Total cellular RNA was extracted and purified from the
UPTC CF89-12 cells, using RNAprotect Bacteria Reagent
and RNeasy Mini Kit (QIAGEN, Tokyo, Japan). The
primers were designed in silico for reverse transcription
(RT)-PCR amplifications of the transcripts segments from
the UPTC CF89-12 cells, based on the nucleotide
sequence information of full-length ars operon. RT-PCR
analysis was carried out employing two primer pairs
(ArsC-F/-R and Acr3-F/-R; Fig. 1) for the four putative
gene transcripts. Nucleotide sequence alignment analysis
was carried out using CLUSTAL W in order to design the
primers software (1.7 program) (Thompson et al. 1994),
incorporated in the DDBJ. These two primer pairs would
be expected to generate RT-PCR amplicons of approxi-
mately 390 bp (using ArsC-F/-R) and 1030 bp (using
Acr3-F/-R), respectively. The negative control RT-PCR
without reverse transcriptase was not amplified.
RT-PCR was carried out using a QIAGEN OneStep RT-
PCR Kit (QIAGEN) in 10 μL reaction volumes. RT-PCR
mixtures contained 1× QIAGEN OneStep RT-PCR Buffer,
80 μmol/L each dNTP, 60 μmol/L each primer, 20 ng
mRNA, or 20 ng template DNA. RT-PCR was first started
with Hot Start Taq polymerase at 95 °C for 5 min and
followed by the reverse transcription at 55 °C for 30 min,

Fig. 2 PCR amplification A-1) B-1) C-1)

confirmation of full-length ars arsC arsC
arsP acr3 arsP arsP
operon recombinant of the UPTC arsR arsR arsR
CF89-12 (a) and two deletion
variants of UPTC CF89-12 ars
genes, Δacr3 (b) and ΔarsC-acr3
(c), by the colony PCR procedure. A-2) B-2) C-2)
M 1 2 M 1 M 1
Lane 1 full-length ars operon
amplicon in CF89-12, lane 2 bp bp bp
recombinant DNA of full-length
ars operon (a-2), lane 1 acr3
deletion recombinant (b-2), lane 1
4,000
arsC-acr3 deletion recombinant 3,000 3,000
(c-2), and lane M 1-kbp DNA 2,000 2,000
ladder (a-2, b-2, and c-2) 2,000
1,500 1,500
1,000
 Folia Microbiol

Table 1 Details of the two

deletion variants (Δacr3 and Deletion variant Deletion
ΔarsC-acr3) of UPTC CF89-12
ars operon Nucleotide position (AB611008) Amino acid residue

acr3 deletion variant (Δacr3) 2024–3037 337

arsC and acr3 deletion variant (ΔarsC-acr3) 1615–3037 473

and PCR was carried out with 30 cycles at 94 °C for
0.5 min, at 50 °C for 0.5 min, and at 72 °C for 1.0 min
followed by a final extension at 72 °C for 2 min.
Amplified RT-PCR products were separated by 1 % (w/
v) agarose gel electrophoresis in 0.5× TBE at 100 V and
detected by staining with ethidium bromide.
Arsenate susceptibility tests
Arsenate (Na2HAsO4) susceptibilities (256 to 1536 μg/mL)
were performed against the transformed E. coli DH5α cells
carrying the recombinant DNA of the full-length ars operon,
and two deletion variants of ars operon were determined on
the agar medium, namely the blood agar base NO. 2, that
contained defibrinated horse blood. E. coli DH5α strain cells
that solely carry the T-vector were employed as a control for
the arsenate susceptibility tests.
Results and discussion
In vitro construction of full-length ars operon and two
deletion variants
Recombinant DNA of full-length ars operon was con-
structed in vitro, from the UPTC CF89-12 isolate, repre-
sented schematically in Figs. 1b and 2(a-1). Then, the
construction of the recombinant DNA of full-length ars
operon in the transformed E. coli DH5α cells was con-
firmed by the colony hybridization procedure (Fig. 2(a-
2)). In addition, the construction of two deletion variants
of UPTC CF89-12 ars four-genes was confirmed, as
schematically shown in Fig. 2(b-1, c-1). Figure 2(b-2, c-
2) clearly confirms the constructions of two deletion var-
iants of UPTC CF89-12 ars four-genes, Δacr3 and
ΔarsC-acr3. The details of two deletion variants are
shown in Table 1.
ars operon were highly resistant to arsenate (~1536 μg/
mL) (Table 2), similar to UPTC CF89-12 strain cells, as
described already (Nakajima et al. 2013). Although, in the
previous paper, the UPTC CF89-12 strain was shown to
be resistant to arsenate (~1024 μg/mL) (Nakajima et al.
2013); the strain was not resistant to arsenate of 1536 μg/
mL (data not shown). Therefore, our study shows that
arsenic resistance (~1536 μg/mL) was additive in the
transformed E. coli DH5α cells containing the recombi-
nant full-length molecule of UPTC CF89-12 strain ars
operon. Consequently, within the two deletion variants
of UPTC CF89-12 ars operon, both Δacr3 and ΔarsC-
acr3 recombinant variants showed an MIC value of
384 μg/mL (Table 2).
Since the transformed E. coli DH5α cells which carry
the recombinant full-length ars genes from UPTC CF89-
12 ars operon were shown to be highly resistant to arse-
nate, the ars operon from the UPTC CF89-12 strain could
confer high arsenate resistance in E. coli DH5α cells
(~1536 μg/mL). In addition, recombinant three ars genes,
arsP-arsR-arsC (Δacr3), and two ars genes, arsP-arsR
(ΔarsC-acr3), could not confer an additive resistance to
arsenate in the transformed E. coli DH5α cells (~384 μg/
mL), as shown in Table 2.
Three UPTC isolates, UPTC CF89-12, UPTC 237, and
UPTC NCTC12894, were highly resistant to arsenate
(~1024 μg/mL) (Nakajima et al. 2013). Some other
UPTC isolates examined in that study (UPTC158 and
497) were also resistant to arsenate (~256 or ~512 μg/
mL) (Nakajima et al. 2013). In addition, other C. lari
isolates (i.e., UPTC A1, A3, 89049, 92251, UN C. lari
JCM2530T, 264, 299, 448, 84C-1) were less resistant to
arsenate (Nakajima et al. 2013).
Table 2 Arsenate susceptibility of the transformed E. coli DH5α cells
containing the recombinant variants of UPTC CF89-12 ars operon

Arsenate susceptibility determination of C. lari isolates Recombinant Arsenate concentration (μg/mL)

variant
0 128 256 384 512 768 1024 1536 2048
Then, in order to clarify contribution differences of the ars
locus from the UPTC CF89-12 strain against arsenate, arse- T-vector + + + + − − − − −
nate susceptibility of the transformed E. coli cells was deter- full-length + + + + + + + + −
mined and shown in Table 2. Δacr3 + + + + − − − − −
The transformed E. coli DH5α cells containing the re- ΔarsC-acr3 + + + + − − − − −
combinant full-length molecule of UPTC CF89-12 strain
Folia Microbiol
A) M 1 2 3 4 5
bp
1,000
500
C) M 1 2 3 4 5
bp
1,000
500
Fig. 3 RT-PCR analyses of the transcript segments from the in vitro
constructed recombinant variants of full-length ars operon (a) and two
deletion variants of ars genes (acr3 deletion, Δacr3 (b) and ΔarsC-acr3
deletion, ΔarsC-acr3 (c)) from UPTC CF89-12. Lane M 100 bp DNA
ladder (New England Biolabs Inc., Tokyo, Japan), lanes 1 and 4 with
RT-PCR analyses for the full-length ars operon and two
deletion variants of ars genes
RT-PCR analyses were carried out on RNA components
extracted and purified from the transformed E. coli DH5α
cells containing the recombinant full-length ars operon
and two deletion variants (Δacr3 and ΔarsC-acr3) from
UPTC CF89-12, in order to confirm the in vivo transcrip-
tion. The in vivo transcription from three recombinant
variants was examined, using the primer pair of ArsC-F/
-R for the arsC transcript and another primer pair of
Acr3-F/-R for the acr3 transcript. As shown in Fig. 3a,
in the transformed E. coli cells containing the recombi-
nant full-length ars operon, in vivo transcription of the
arsC and acr3 genes was confirmed (lanes 2 and 5 in
Fig. 3a). In addition, the in vivo positive transcription of
arsC and the negative transcription of acr3 were con-
firmed in the deletion variants of the acr3 gene (the
Δacr3; lanes 2 and 5 in Fig. 3) and both in vivo negative
transcriptions of the arsC and acr3 genes in the deletion
variants of the arsC-acr3 (ΔarsC-acr3 genes; lanes 2 and
5 in Fig. 3c). Thus, the in vivo transcriptions of the re-
maining ars genes were confirmed in the two deletion
variants of UPTC CF89-12 ars operon in the transformed
E. coli cells.
In this study, firstly, full-length ars operon was iden-
tified in the representative C. lari UPTC CF89-12 strain.
Reduced arsenate susceptibility (~1536 μg/mL) was
conferred to E. coli DH5α, by transforming the ars op-
eron of Japanese environmental C. lari UPTC CF89-12
strain.
6 B) M 1 2 3 4 5 6
bp
1,000
500
6
recombinant genomic DNA and with Hot Start Taq DNA polymerase,
lanes 2 and 5 with total cellular RNA and with reverse transcriptase, lanes
3 and 6 with total cellular RNA and without reverse transcriptase, lanes 1
to 3 with the primer pair of ArsC-F/-R, and lanes 4 to 6 with the primer
pair of Acr3-F/-R
RT-PCR confirmed in vivo transcription of those corre-
sponding genes in the three recombinant variants.
Conclusions
The recombinant DNA of full-length ars operon, arsP, arsR,
arsC, and acr3, from the UPTC CF89-12 strain could confer
high arsenate resistance (1536 μg/mL) to the transformed
E. coli DH5α cells. In addition, two deletion variants, acr3
deletion and arsC-acr3 deletion, had similar resistance as the
E. coli cells carrying the pGEM-T vector, as a control.
Acknowledgments This research was partially supported by a Grant-
in-Aid for Scientific Research (C) (no. 20580346) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan (to MM).
MM and JEM were funded through a Great Britain Sasakawa Foundation
(Butterfield) Award to jointly examine the clinical significance of
Campylobacter infection in the UK and Japan.
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