1. Exploiting the arsenic resistance of the thermophilic
bacterium Thermus thermophilus
Immacolata Antonucci1, Francesca Anna Fusco1, Danila Limauro1, Immacolata Del Giudice1, Josè Berenguer2,
Simonetta Bartolucci1, Gabriella Fiorentino1
1Department of Biology, University of Naples Federico II, Via Cinthia , 80126 Napoli, Italy
2Centro de Biología Molecular Severo Ochoa, UAM-CSIC, 28049 Madrid, Spain
A
B C
TtSmtB acts as transcriptional repressor of the entire operon
in which it is encoded and it is able to sense arsenic.
CONCLUSIONS
References:
1) Sánchez-Rieg AM et al. 2014 PLoS ONE 9(5): e96826. doi:10.1371/journal.pone.0096826
2) Del Giudice I et al. 2013 Biochim Biophys Acta, 1834:2071-9
Microorganisms living in arsenic-rich geothermal environments
act on arsenic using different biochemical strategies. So far, only
limited insights have been obtained on the molecular
mechanisms responsible for the arsenic resistance [1].
Investigation on the thermophilic bacterium Thermus
thermophilus HB27 highlighted its tolerance to millimolar
concentrations of As(V) and As(III). Its genome encodes for an
arsenate reductase, which is an important contributor in the
arsenic resistance system [2].
In this study we focused on the role of the putative
transcriptional regulator TtSmtB (TTC0353), belonging to
ArsR/SmtB family, by in vivo and in vitro analysis.
INTRODUCTION
TtSmtB gene is part of an operon of five genes not
metabolically related with three arsenic responsive
promoters upstream of TTC0351, TtSmtB and
TTC0354.
TTC0351 TTC0354TTC0353 TTC0355TTC0352 TTC0356TTC0350
Cell wall
endopeptidase
1167 bp
Cation
transporting
ATPase
2052 bp
TtSmtB
372bp
PdxT
576 bp
PdxS
882 bp
AsnC
1317 bp
Hyp protein
474 bp
A B C D E F G H
336042 336515 337073 338239 338373 339254 339606 339638 341689 341822 342397 342469 343785
600 bp
400 bp
200 bp
M 1 2 3 4 5 6 7 8 9 10
AB (427 bp) CD (221 bp) EF (295 bp) GH (635 bp)
RT-PCR
analysis
The recombinant protein TtSmtB is a dimer interacting
with the target promoters.
10 Kbp
15 Kbp
SmtB/
DNA
Free
DNA
0351
prom
0353
prom
0354
prom
C -
Target
promoters
- - - -+ + + + SmtB
A mutant strain T. thermophilus ΔSmtB::kat was generated (A)
as confirmed by PCR (B) and RT-PCR (C).
kat
PslpA
smtBTTC0352 TTC0354TTC0351
259 bp
A B
1125 bp
E F
mutant
TTC0352 TTC0354TTC0351 smtB
259 bp
A B
221 bp
C D
372 bp
E F
wt
G H
107 bp
kat
250 bp
500 bp
750 bp
1000 bp
1500 bp
M
T. thermophilus HB27
(wt)
T. thermophilus
ΔsmtB::kat (mutant)
100 bp
200 bp
300 bp
M
RQ
N
o
s
tre
s
s
1
2
m
M
A
s
(V
)
8
m
M
A
s
(II)
0
1
2
3
4
RQ
N
o
s
tre
s
s
1
2
m
M
A
s
(V
)
8
m
M
A
s
(II)
0
1
2
3
TTC0351 TtSmtB
RQ
N
o
s
tre
s
s
1
2
m
M
A
s
(V
)
8
m
M
A
s
(II)
0
2
4
6
8
TTC0354
In T. thermophilus HB27 the presence of arsenic
regulates the expression of the TtSmtB operon.
qRT-PCR analysis
0
0,5
1
1,5
2
0 5 10 15 20 25
NS wt
NS ΔSmtB
12mM As(V) wt
12mM As(V) ΔSmtB
8mM As(III) wt
8mM As(III) ΔSmtB
TtSmtB absence could play a role in the arsenic
resistance mechanism.
The trascriptional role of TtSmtB was confirmed in the
T. thermophilus ΔSmtB::kat strain.
RQ
w
t
S
m
tB
0
2
4
6
TTC0351
RQ
w
t
S
m
tB
0
1
2
3
4
TtArsC
RQ
w
t
S
m
tB
0
1
2
3
4
TTC0354
qRT-PCR analysis
A
B C