Characterization of the PIB-Type ATPases Present in Thermus
Lici A. Schurig-Briccio and Robert B. Gennis
Department of Biochemistry, University of Illinois, Urbana, Illinois, USA
60°C to 80°C and which yields sufficient biomass to be a practical
source for thermally stable proteins to be characterized by bio-
can be genetically manipulated and can be used as a host for ex-
pressing recombinant proteins (25, 43). As a source for thermally
stable, recombinant membrane proteins, T. thermophilus can be
particularly attractive. In this report, we explore this potential by
the cloning, purification, and functional characterization, both in
vitro and in vivo, of the three PIB-type ATPases present in T. ther-
mophilus HB27. The PIB-type ATPases are ATP-driven heavy
metal pumps (4, 5, 28, 32, 47), and the three encoded in the ge-
nome of T. thermophilus HB27 (32) are annotated as TTC1358
(CopA, a putative Cu?pump), TTC1371 (CopB, a putative Cu2?
pump), and TTC0354 (a putative Zn2?/Cd2?-ATPase). All three
were isolated as His-tagged proteins and demonstrated to have
metal-stimulated ATPase activity.
The P-type ATPase superfamily includes many cation pumps,
which establish and maintain steep electrochemical gradients of
key cations across membranes at the expense of ATP hydrolysis
(15, 47). They are grouped into five subfamilies, PIthrough PV,
covering a wide range of cationic as well as lipid substrates (e.g.,
flippases) (6, 47, 52). Among the best-studied P-type ATPases are
the Na?/K?pump (44, 54), the plasma membrane H?pump
(48), and the Ca2?-ATPase (the PIIAgroup) (63, 64), commonly
found in eukaryotic cells but also present in some prokaryotes
(21). Structures of several P-type ATPases have been determined
by X-ray crystallography (28, 44, 48, 54, 62).
PIB-type ATPases (3–5, 28, 32, 47) transport heavy metals
(Cu2?, Cu?, Ag?, Zn2?, Cd2?, Co2?) across biomembranes,
playing key roles in metal homeostasis and, by pumping toxic
metals out of cells, increasing their biotolerance of these metals
(42, 50, 58, 66). The PIB-ATPases include the two human Cu?-
son diseases (17). Structures have been reported for the Cu?
hermus thermophilus is an aerobic facultative Gram-negative
bacterium that grows rapidly in the temperature range from
pumps from Legionella pneumophila by X ray (28) and from
Archaeoglobus fulgidus by cryoelectron microscopy and computer
PIB- and PII-type ATPases share common structural and func-
tional features (8, 33, 47). Alignment of sequences and homology
PIB-type ATPases are structurally equivalent to H4, H5, and H6,
respectively, of the PII-type ATPases (3). These transmembrane
unique feature of these PIB-type proteins is the presence of a pu-
brane helix 6 (69). However, the relationship between ion speci-
ficity and CPX sequences remains to be established. Another
(Fig. 1). It has been shown that CXXC N-MBDs are not required
for ion transport, and a regulatory role has been suggested for
these domains (4, 40, 65, 67).
MATERIALS AND METHODS
Construction of expression plasmids. The DNA of the three genes en-
(32) was amplified by PCR from genomic DNA (Table 1). The 8His tag
was introduced at the C terminus of each protein using the QuikChange
site-directed mutagenesis kit (Stratagene). DNA oligonucleotides were
synthesized at either the University of Illinois at Urbana-Champaign
(UIUC) Biotechnology Center or by Integrated DNA Technologies. The
primers to amplify the three PIB-type ATPase genes were designed to
Received 8 April 2012 Accepted 21 May 2012
Published ahead of print 25 May 2012
Address correspondence to Robert B. Gennis, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
August 2012 Volume 194 Number 15Journal of Bacteriologyp. 4107–4113jb.asm.org
introduce a 5= NdeI site and a 3= HindIII site at the end of each gene to
clone the resulting DNA fragments directly in the shuttle vector pMKPb-
cbgaA (Table 1) (provided by J. Berenguer, Universidad Autónoma de
Madrid, Spain). This plasmid contains the promoter from the cyto-
chrome bc1complex from T. thermophilus (41) and a kanamycin resis-
tance gene (Table 1). The different gene sequences were confirmed by
DNA sequencing (UIUC Biotechnology Center). All the genetic manipu-
HB27 cells were transformed with these constructs and selected by the
addition of 30 ?g/ml kanamycin.
Heterologous expression in E. coli of the Zn2?/Cd2?-ATPase was
done using the C43 (DE3) strain with the expression vector pET22b (Ta-
ble 1). The Cys16Ala mutant of the Zn2?/Cd2?-ATPase was generated
using QuikChange (Strategene) and also expressed in E. coli.
Cell growth, enzyme expression, and purification. T. thermophilus
HB27 was grown in 1 liter of culture medium in a 2-liter flask using rich
medium (8 g/liter yeast extract, 6 g/liter tryptone, 3 g/liter NaCl, 3 ml
glycerol, pH 7.4) at 60°C for 16 h in an incubator shaker (final optical
density at 600 nm [OD600] ? 1.4). E. coli C43 was grown in LB medium
plus 100 ?g/ml ampicillin at 37°C, and gene expression was induced by
the addition of 1 mM IPTG (isopropyl-D-thiogalactoside) when the cell
medium reached an A600of ?0.7. All the purification procedures were
carried out at 0 to 4°C. Cells were harvested and then resuspended in
buffer A (25 mM Tris, pH 7.5, 100 mM NaCl, 10% glycerol, and 2 mM
?-mercaptoethanol) with the addition of 3 mM MgSO4, DNase I, and a
passing the suspension through a Microfluidizer at a pressure of 80,000
lb/in2. Membranes were collected by centrifugation, resuspended in buf-
fer A (plus the protease inhibitor cocktail), and then solubilized by the
tion of 1%. After being stirred gently for 2 h at 4°C, the suspension was
cleared by centrifugation to remove insoluble material. To this solution
with buffer A plus 0.05% DDM and 35 mM imidazole. The protein was
eluted with buffer A plus 0.05% DDM and 250 mM imidazole. Fractions
were concentrated using an Amicon spin filter with a molecular mass
FIG 1 Conserved amino acid motifs which provide signature sequences that predict the metal selectivities on the indicated P-type ATPase.
TABLE 1 Strains and plasmids used in this work
Strain or plasmidRelevant genotype or description
F?mcrA ?(mrr-hsdRMS-mcrBC) ?80lacZ?M15 ?lacX74 nupG recA1 araD139
?(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 ??
F?ompT gal dcm hsdSB(rB
Derived from pACYC184
Source or reference
E. coli TOP 10Invitrogen
E. coli C43
E. coli C43Zn/Cd
T. thermophilus HB27
Y. Koyama (ATCC BAA-163)
pET22b containing zntA
pET22b containing zntA C16A
bc1complex promoter kanamycin resistance gene; derived from pMHPfbcbgaA
pMKPbc copA cloned in bgaA region
pMKPbc copB cloned in bgaA region
pMKPbc zntA cloned in bgaA region
slpA promoter and bleomycin resistance gene
pWUR112 with 750 bp upstream and downstream fragments of copA gene
pWUR112 with 750 bp upstream and downstream fragments of copB gene
pWUR112 with 750 bp upstream and downstream fragments of pacL gene
pWUR112 with 750 bp upstream and downstream fragments of zntA gene
Schurig-Briccio and Gennis
jb.asm.org Journal of Bacteriology
cutoff of 100 kDa, and imidazole was removed by dialysis with buffer A
plus 0.05% DDM. The protein solution (approximately 4 mg/ml) could
be flash frozen and stored at ?80°C.
Protein concentration was determined using the BCA kit (Thermo
Scientific, Pierce Protein Research Products). SDS-PAGE was done using
precast 4 to 20% gels (Nusep), and proteins bands were visualized with a
and with Coomassie brilliant blue (Thermo Scientific).
pNPP activity assay. Phosphatase activity was measured colorimetri-
cally using para-nitrophenyl phosphate (pNPP) as the substrate (12, 49).
Reactions were performed at 65°C for 20 min. About 0.01 mg/ml of pro-
6.5], 100 mM NaCl, 3 mM MgSO4, 0.05% DDM) and various metal salt
concentrations. The reaction was started by addition of the substrate and
stopped by adding 1 M NaOH. Absorbance at 410 nm was used to deter-
mine the amount of p-nitrophenol product using a ε410value of 17.000
mg?1of protein min?1.
indicated in the figure legends and table footnotes. Dithiothreitol (DTT;
was measured at room temperature, and the pH value at 65°C was calcu-
lated using a pKa/°C conversion factor for Tris of ?0.031 (27). Metal
content of the buffer used for ATP and pNPP activity was determined by
atomic absorption, and neither copper nor zinc was detected.
ATPase activity on activating metal ions or ATP was fit to the Michaelis-
Menten equation, ? ? Vmax([L]/[L] ? K0.5), where [L] is the concentra-
tion of variable ligand. Data analysis was done using Origin 8.0 software.
P-type ATPases were replaced by the bleomycin resistance gene under the
control of the surface layer protein A gene promoter (slAp) (22). Upstream
and downstream regions flanking the P-type ATPases (flank A and flank B)
A the forward primers introduced an EcoRI site and the reverse primers in-
and the reverse primers introduced a HindIII site. The resulting products
of ligation into pWUR112 (Table 1). Each suicide plasmid was transformed
Testing the sensitivity of T. thermophilus growth in the presence of
metals. The cells were grown in liquid media (8 g/liter yeast extract, 6
g/liter tryptone, 3 g/liter NaCl, 3 ml glycerol, pH 7.4) supplemented with
the desired metal concentration (CuCl2, ZnCl2, or CdCl2). Cell cultures
were inoculated from overnight growths to an initial A600of 0.1, and the
A600was obtained again after different times of growth at 60°C.
tagged forms of each of the three PIB-type ATPases were success-
fully expressed homologously in T. thermophilus HB27 using the
pMKPbcbgaA expression plasmid. Between 5 and 10 mg of each
PIB-type ATPase was obtained from 12 liters of culture medium.
shows that this procedure routinely yielded a ?95% pure protein
the expressed protein (Fig. 2, lanes 4 to 7*). The SDS-PAGE pat-
tern of the Zn2?/Cd2?-ATPase indicates heterogeneity which
terminus (Fig. 2, lines 3* and 7*).
Metal-stimulated ATPase activity. Figure 3 shows the influ-
ence of the presence of 1 ?M and 10 ?M metal cations on the
ATPase activities of the three different PIB-type ATPases. CopA is
stimulated to the greatest extent (about 3.5-fold) by Cu?but also
is stimulated more by Cu2?(more than 4-fold) than by Cu?, as
expected (Fig. 3B). Surprisingly, the putative Zn2?/Cd2?-ATPase
not by Zn2?and Cd2?.
For comparison, heterologous expression of the Zn2?/Cd2?-
purified recombinant protein from E. coli does not exhibit any
evidence of proteolytic fragmenting at the N terminus (Fig. 2,
lanes 3* and 7*). The ATPase activity of the Zn2?/Cd2?-ATPase
expressed in E. coli was increased by the addition of Zn2?and
Cd2?but also by Cu?(Fig. 3D). Since the sequence of the
CXXC metal binding motif (starting at Cys16) at the N terminus,
the C16A mutation was made to test if perturbing this motif al-
in the metal selectivity was observed compared to the wild type
(Fig. 3D), and 75% of ATPase activity was maintained.
Cu?, Cu2?, and Zn2?appear to have the largest effects on the
ATPase activities of CopA, CopB, and the Zn2?/Cd2?-ATPase
of ATPase activity by each enzyme with its activating metal was
determined, showing K0.5values in the submicromolar range for
each (Table 2). In all cases, the addition of the activating metal
form of the enzyme, as previous shown for the sodium pump (9).
The Kmfor ATP was also determined for each enzyme in the
presence of the activating metal, and the values were in the sub-
millimolar range (Table 2), similar to those for other P-type
FIG 2 SDS-PAGE of the three PIB-ATPases from T. thermophilus. Each lane
contains ?5 ?g solubilized protein purified by Co2?-nitrilotriacetic acid col-
umn chromatography. The lane on the extreme left shows molecular mass
8 are stained for the presence of the C-terminal His tag. Lanes 1, 2, and 4
expressed in T. thermophilus, as do the corresponding lanes 5, 6, and 8. Lanes
PIB-Type ATPases in T. thermophilus
August 2012 Volume 194 Number 15jb.asm.org 4109
the chromogenic substrate para-nitrophenyl phosphate (pNPP) in-
temperatures the background hydrolysis rate of pNPP was too high.
Relative activities of metal stimulation of pNPPase activity (not
34), and the pNPPase activity of each of the three T. thermophilus
FIG 3 Activation of the purified PIB-type ATPases by metals. The relative ATPase activities of CopA expressed in T. thermophilus (A), CopB expressed in T.
expressed in E. coli. Data are expressed as averages ? standard deviations (SD) of three independent experiments.
TABLE 2 pNPP hydrolase activity, vanadate inhibition, and Kmfor ATP and K0.5for the different metalsa
pNPP activityb(nmol mg?1s?1) with:
P-type ATPaseKmATP (mM)c
0.11 ? 0.02
0.15 ? 0.03
0.30 ? 0.05
1.30 ? 0.25 (Cu?)
0.40 ? 0.05 (Cu2?)
0.18 ? 0.02 (Zn2?)
No vanadate100 ?M vanadate
27.5 ? 3.2
31.2 ? 4.1
30.5 ? 2.6
7.4 ? 2.1
3.6 ? 1.5
8.2 ? 3.1
aData are expressed as averages ? SD of three independent experiments.
bActivity assays were done in the presence of different metals: CopA, 1 ?M Cu?; CopB, 1 ?M Cu2?; Zn2?/Cd2?-ATPase expressed in E. coli, 1 ?M Zn2?.
cKmfor ATP and K0.5for the different metals were determined with malachite green as described in Materials and Methods.
Schurig-Briccio and Gennis
jb.asm.orgJournal of Bacteriology
ferent metals on the growth of T. thermophilus were compared in
overproduced or in which the chromosomal gene was knocked
indicated concentration of the metals (Fig. 4) and the OD was
is shown in Fig. 4 because it illustrates the biggest difference in
note that the cellular growth rates in rich medium without metal
addition were not modified in cells either overexpressing or lack-
ing the P-type ATPases separately (not shown).
the putative Zn2?/Cd2?-ATPase, respectively, increases the Cu2?
tolerance for growth compared to that of the wild type. Consistent
with these results, the growth of the corresponding mutant strains
T. thermophilus, was deleted. That deficiency has no effect in copper
tolerance in vivo, as is shown in Fig. 4C. These experiments demon-
strate that the putative Zn2?/Cd2?-ATPase plays a role in copper
detoxification, along with CopA and CopB, and are consistent with
Zn2?/Cd2?-ATPase (Fig. 3). The dosage effect of the Zn2?/Cd2?-
Zn2?/Cd2?-ATPase is overproduced, but gene knockout does not
Each of the three membrane-bound PIB-type ATPases encoded in
the genome of Thermus thermophilus has been expressed and pu-
sion system. Two of the enzymes are homologous to copper
pumps CopA and CopB, while the third is homologous to Zn2?/
5 to 10 mg from 12 liters of culture. All three enzymes exhibited
thermophilus CopA [TtCopA] and TtCopB) are among the small
number of copper ATPases that have been purified in an active
form (31), and only a few CopB enzymes have been purified and
characterized (16, 38, 57). Based on metal stimulation of both the
ATPase and pNPPase activities (Fig. 3), the current work shows
that in vitro TtCopA favors Cu?whereas TtCopB favors Cu2?,
similar to the pattern observed with CopA and CopB from Ar-
chaeoglobus fulgidus (38, 39, 68), with the exception that the
to 20 mM) in the presence of 1 ?M Cu?. By contrast, the CopB
from Enterococcus hirae has been shown to act as an ATP-driven
pump for Cu?and Ag?in inverted membrane vesicles (57). In
role in detoxification of copper and their presence increases the
4). Hence, both of these pumps are likely to pump copper out of
the cell. The copper affinity in vitro is higher for CopB than for
CopA. This is consistent with the higher copper sensitivity of the
growth of the CopB knockout strain than of that of the CopA
functionally relevant at lower copper concentrations in the me-
One would expect that any copper present in the bacterial cy-
toplasm would be in the reduced Cu?form, which is potentially
very deleterious (19, 37). Indeed, there appear to be no copper-
DNA binding proteins which are parts of the systems for actively
ridding the cytoplasm of copper. Although CopA or CopB are
stimulated to different extents by Cu2?in vitro, it is not clear that
in vivo they would ever encounter Cu2?but, rather, would utilize
chaperone-bound Cu?in the bacterial cytoplasm as a substrate.
the Cu-dependent transcriptional regulator CopY (CsoR), fully
consistent with a role of CopA in Cu?detoxification of the bacte-
rial cytoplasm (4, 46, 56). The copB gene in T. thermophilus is
adjacent to the gene encoding the periplasmic multicopper oxi-
dase (53), suggesting a functional relationship between these two
proteins. One of the reactions catalyzed by the multicopper oxi-
FIG 4 Role of the P-type ATPases in metal tolerance. The effect of increasing
metal concentrations on the growth of different T. thermophilus strains is
function of metal concentration: HB27 strain (wild type; squares), a strain
overexpressing the indicated P-type ATPases (solid triangles), and mutant
pressing the copA gene or lacking it. (B) Cells overexpressing the copB gene or
lacking it. (C) Cells lacking the pacL gene. (D, E, and F) Cells overexpressing
the zntA gene or lacking it. Data are expressed as averages ? SD of four inde-
PIB-Type ATPases in T. thermophilus
August 2012 Volume 194 Number 15jb.asm.org 4111
tified in the genome of T. thermophilus. PCuAC appears to deliver
Cu?into subunit II of the T. thermophilus cytochrome ba3respi-
ratory oxygen reductase (1), and a role for the equivalent protein
in Rhodobacter sphaeroides in the assembly of copper-containing
respiratory oxidases has also been reported (60). Similarly, the
Sco1/PrrC/SenC family of proteins has been implicated in copper
acquisition by bacteria and in the assembly of copper-containing
proteins, including respiratory oxygen reductases (11, 23, 24, 59,
60). Since PIB-type copper pumps have been implicated in the
assembly of the copper-containing cbb3-type respiratory oxygen
reductases (26, 35), it is reasonable that the periplasmic copper
CopA and/or CopB in the assembly of the ba3-type and caa3-type
copper-containing respiratory oxygen reductases present in T.
thermophilus (55, 61).
The putative Zn2?/Cd2?-ATPase. The stimulation by metal
binant protein is obtained from the T. thermophilus or E. coli ex-
pression system. The expected activation by Zn2?and Cd2?is
observed only for the enzyme expressed heterologously in E. coli.
This difference in metal selectivity is likely due to proteolytic
cleavage for the enzyme isolated from T. thermophilus but does
not appear to be due to an alteration of the single CXXC metal-
binding sequence motif near the N terminus. Mutagenesis of the
first Cys of the motif to Ala has no influence on metal selectivity
and only a minor influence on the ATPase activity (80% of the
wild-type activity). Similar results were obtained upon mutagen-
esis of the Zn-ATPases from E. coli (45) and from Arabidopsis
The recombinant TtZn2?/Cd2?-ATPase protein expressed ei-
of T. thermophilus growth to copper when the gene encoding this
enzyme is knocked out and the increased resistance to copper
toxicity upon overexpressing the putative Zn2?/Cd2?-ATPase
(Fig. 4). The data on whether the putative Zn2?/Cd2?-ATPase
not support a clear conclusion. Interestingly, a PIB-ATPase regu-
lator (CsoR) in T. thermophilus has been shown to respond to
either Cu?or Zn2?(51), suggesting an interaction in the detoxi-
fication responses of the organism to these two cations. Further
work will be needed to clarify these interactions.
One caveat about the test used to evaluate the physiological
role of the metal pumps is that the influence of either the overex-
pression or deletion of the genes expressing any individual pump
systems that influence metal homeostasis. This is emphasized by
the response of the CsoR regulator protein to either Cu?or Zn2?
(51). In short, the measured sensitivity to growth could be due to
Along the same lines, it is important to mention that there are
several different systems encoded in the genome of T. thermophi-
ing both permeases and ABC-type transporters. The activity of
the P1B-type transporters. Nevertheless, taken at face value, the
data strongly suggest that the Zn2?/Cd2?-ATPase does assist in
pumping copper out of the cytoplasm of T. thermophilus.
We thank J. Berenguer and the late J. Fee for providing plasmid pMKPb-
cbgaA and the HB27 strain, respectively. We are also indebted to James
Hemp, Young Ahn, Ranjani Murali, and G. Gokce Yildiz for their help
and for useful discussions.
This research was supported by grant GM095600 from the National
Institutes of Health.
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