Microbiology (2012), 158, 1622–1633
DOI 10.1099/mic.0.055905-0
The ATPases CopA and CopB both contribute to
copper resistance of the thermoacidophilic
archaeon Sulfolobus solfataricus
Christian Völlmecke,1 Steffen L. Drees,1 Julia Reimann,2
Sonja-Verena Albers2 and Mathias Lübben1
Correspondence
Mathias Lübben
[email protected]
Received 26 October 2011
Revised
20 February 2012
Accepted 22 February 2012
1
Lehrstuhl für Biophysik, Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum,
Germany
2
Molecular Biology of Archaea, MPI für Terrestrische Mikrobiologie, Marburg,
Karl-von-Frisch-Straße 10, D-35043 Marburg, Germany
Certain heavy metal ions such as copper and zinc serve as essential cofactors of many enzymes,
but are toxic at high concentrations. Thus, intracellular levels have to be subtly balanced. P-type
ATPases of the PIB-subclass play a major role in metal homeostasis. The thermoacidophile
Sulfolobus solfataricus possesses two PIB-ATPases named CopA and CopB. Both enzymes are
present in cells grown in copper-depleted medium and are accumulated upon an increase in the
external copper concentration. We studied the physiological roles of both ATPases by disrupting
genes copA and copB. Neither of them affected the sensitivity of S. solfataricus to reactive oxygen
species, nor were they a strict prerequisite to the biosynthesis of the copper protein cytochrome
oxidase. Deletion mutant analysis demonstrated that CopA is an effective copper pump at low and
high copper concentrations. CopB appeared to be a low-affinity copper export ATPase, which
was only relevant if the media copper concentration was exceedingly high. CopA and CopB thus
act as resistance factors to copper ions at overlapping concentrations. Moreover, growth tests on
solid media indicated that both ATPases are involved in resistance to silver.
INTRODUCTION
Transition elements such as copper, zinc, nickel and cobalt are
essential trace constituents occurring in many important
cellular processes and structures. At high intracellular
concentrations however, these metals become deleterious
when undergoing Fenton-type redox reactions (Haber &
Weiss, 1932; Goldstein et al., 1993; Liochev & Fridovich,
2002) or blocking the cellular antioxidant defence system. The
highly reactive oxygen species suppress metabolic pathways
by affecting iron–sulfur proteins or damaging other proteins
and DNA (Imlay, 2006). Thus, metal concentrations have to
be equilibrated carefully and cells need powerful mechanisms
to avoid metal accumulation up to toxic levels.
The mechanisms of transition metal uptake into bacterial
cells are largely unknown. Metals may enter the cytoplasm
by using either non-selective pores or membrane-embedded
permeases, or by means of chalkophores (Balasubramanian
et al., 2011). In contrast, export of transition metals is
carried out by P-type ATPases acting as specific transporters
Abbreviations: HMA, heavy-metal-associated; TMPD, N,N,N9,N9-tetramethyl-p-phenylenediamine.
Five supplementary figures and a supplementary table are available with
the online version of this paper.
1622
by coupling the energy of ATP hydrolysis to the ion transfer across cytoplasmic membranes (for reviews, see Møller
et al., 1996; Silver & Phung, 1996; Axelsen & Palmgren, 1998;
Kaplan, 2002; Nies, 2003; Rensing & Grass, 2003; Kühlbrandt,
2004; Argüello et al., 2011). According to an extensive
sequence comparison, they belong to subgroup IB of the Ptype ATPase superfamily whose members are widely distributed in archaea, bacteria and eukaryotes (Axelsen &
Palmgren, 1998). Transition metal transporters of this
subgroup are also referred to as CPx-ATPases because of
their transmembrane ion binding sequence motif Cys-ProXxx (Solioz & Vulpe, 1996).
S. solfataricus is a thermoacidophilic archaeon thriving at
low pH and high temperatures (Brock et al., 1972; de Rosa
et al., 1975) in hot springs which are potentially rich in diverse metals. The organism has developed effective mechanisms to withstand toxic concentrations of metals (Grogan,
1989; Villafane et al., 2009). In this study, we investigated the
copper resistance of S. solfataricus whose genome exhibits
only two P-type ATPases (She et al., 2001), both of which
belong to the heavy metal translocating class IB. We particularly addressed questions regarding substrate specificity
and relative contributions of both P-type ATPases to the
metal resistance.
Downloaded from www.microbiologyresearch.org by
055905 G 2012 SGM
IP: 88.99.165.207
On: Thu, 15 Jun 2017 01:07:05
Printed in Great Britain
Copper resistance of Sulfolobus solfataricus
METHODS
Growth of bacteria. Escherichia coli strain XL1-Blue was grown in
LB medium and used for cloning of plasmids for disruption of S.
solfataricus genes. Unless indicated otherwise, S. solfataricus PBL2025
(Schelert et al., 2004) and its mutants were grown aerobically at 80 uC
and 110 r.p.m. in Brock’s medium (Brock et al., 1972). Strains are
listed in Table 1.
For metal resistance tests, solid media were prepared using Brock’s
medium and Gelrite gellan gum (Shungu et al., 1983) as gelling agent.
Plates poured with 0.54 % (silver) or 0.66 % (copper) Gelrite
provided even surfaces at pH 3 and temperatures of up to 80 uC
and hence were preferred over agar plates for our experiments.
Measurement of growth rates. For measurement of growth rates,
S. solfataricus and its mutant strains were grown in a copperdepleted medium, also referred to as standard medium. This
consisted of a modified basal salt medium (Brock et al., 1972)
containing 1 mM (NH4)2SO4, 2 mM KH2PO4, 25 mM CaCl2, 50 mM
MgSO4, 5 mM FeSO4, 9.1 mM MnCl2, 765 nM ZnSO4, 119 nM
VOSO4, 42 nM CoCl2, 22 nM Na2B4O7, 248 nM Na2MoO4, 1 mM
KI, 0.2 % (w/v) sucrose and 0.2 % (w/v) sterilized ion-depleted (see
below) casein hydrolysate (Oxoid) in quartz-distilled water,
adjusted to pH 3 with 50 % (v/v) H2SO4. A 10-fold concentrated
ion-depleted stock solution was prepared by dissolving 20 g casein
hydrolysate per litre and separating by chromatography on a
Serdolit CHE (Serva) column. For standard growth experiments,
50 ml ion-depleted media supplemented with the desired concentrations of CuSO4 in 100 ml flasks were inoculated to OD600 0.04,
using centrifuged and washed cells from a preculture grown in
Brock’s medium.
Determination of metal resistance of wild-type S. solfataricus.
The sublethal (5maximum tolerated) concentration of a specific
metal was defined as the concentration still allowing 10 % survival of
cells. It was measured by growth in standard medium with the
respective metal ion added at defined concentrations. Cell survival
was quantified by reading OD600. Metal ion resistances were 10 mM
CoCl2, 12 mM NiSO4, 1.4 mM CuSO4, 2 mM CdSO4, 1 mM
Pb(NO3)2, 2 mM ZnSO4, 10 mM AgNO3 and 4 mM mercury acetate.
Construction of plasmids. For copA disruption, the upstream
flanking region of Sso2651 was amplified using the primers 59UFRA_KpnI/39UFR-A_NcoI; the downstream flanking region of Sso2651
was amplified using the primers 59DFR-A_BamHI/39DFR-A_NotI
(Table S1, available with the online version of this paper). For copB
disruption, the upstream flanking region of Sso2896 was amplified
using the primers 59UFR-B_KpnI/39UFR-B_NcoI; the downstream
flanking region of Sso2896 was amplified using the primers 59DFRB_BamHI/39DFR-B_NotI. The obtained fragments (400–1000 bp)
were cloned into plasmid pET2268 (Albers & Driessen, 2008),
containing the lacS cassette flanked by upstream (NcoI and KpnI) and
downstream (BamHI and NotI) fragment insertion sites. The
plasmids used are listed in Table 1.
Transformation of S. solfataricus and screening for recombinants with defective copA and copB genes. Deletion mutants
were obtained as described by Albers & Driessen (2008). In brief,
linearized deletion plasmids were electroporated in PBL2025. The
transformants were selected in lactose minimal medium for 12–
14 days. After reinoculation in lactose minimal medium, cells were
streaked on 0.1 % tryptone/Gelrite plates and incubated at 76 uC for
6 days. Colonies were sprayed with X-Gal solution as described
previously (Jonuscheit et al., 2003) and blue colonies were inoculated
in tryptone liquid medium. Successful deletion of the target genes was
determined as described below.
PCR analysis of deletion mutants. Analysis of S. solfataricus DcopA
and DcopB deletion mutants was performed by PCR using the primers
59UFR-A/39lacS-A and 59lacS-A/39DFR-A or 59-UFR-B/39lacS-B and
59lacS-B/39DRF-B yielding a product only in case of successful insertion
of lacS gene into the copA or copB loci. Mutants were double-checked
using the primers 59copA/39copA or 59copB/39copB yielding a product
only in the case of non-successful deletion of copA or copB loci.
Isolation of genomic DNA. Genomic DNA from S. solfataricus was
isolated from 200 ml cultures by addition of 200 ml phenol/Tris and
chloroform/isoamyl alcohol (24 : 1) and centrifugation. DNA was
precipitated from the aequeous phase by 200 mM NaCl and 30 %
(v/v) ethanol and dissolved in 10 mM Tris/HCl, 1 mM EDTA, pH 8.0
(TE buffer).
Table 1. Strains and plasmids used in this study
Strain or plasmid
Strains
E. coli
XL1 blue
DC194
S. solfataricus
PBL2025
PBL2025 DcopA
PBL2025 DcopB
Plasmids
pBAD His/MycA
pBAD-Sso_copA
pBAD-Sso_copB
pBAD-Eco_copA
pET2268
pET2268 DcopA
pET2268 DcopB
http://mic.sgmjournals.org
Genotype or feature
Reference or source
endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F9[ : : Tn10 proAB+
2
lacIq D(lacZ)M15] hsdR17(r2
K mK )
LMG194 DcopA
Rensing et al. (2000)
98/2 del(SSO3004–3050)
PBL2025 copA : : lacS
PBL2025 copB : : lacS
Schelert et al. (2004)
This work
This work
bla
S. solfataricus copA
S. solfataricus copB
E. coli copA
bla lacS cassette containing its own promoter and terminator region
copA : : lacS
copB : : lacS
Invitrogen
This work
This work
This work
Szabó et al. (2007)
This work
This work
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 01:07:05
Stratagene
1623
C. Völlmecke and others
Membrane preparation. Cells were disrupted by using a Branson
sonicator for 5 min at level 7 and 50 % pulse length. Homogenates
were centrifuged twice for 15 min at 5000 g. Membranes were
pelleted from supernatants for 1 h at 150 000 g and suspended in
TEN-buffer [TE buffer plus 110 mM NaCl and 10 % (w/v) glycerol].
Protein concentrations were determined as described by Smith et al.
(1985).
Immunoblotting. Antisera to purified CopA of S. solfataricus and E.
coli (unpublished data) were raised commercially (Seqlab). Rabbit
antisera to CopB of S. solfataricus were produced by immunization
with the recombinant fragment CopB-A or CopB-B (Deigweiher
et al., 2004). CopB-A antiserum exhibits cross-reactivity to CopA of S.
solfataricus and hence was used to display comparative expression
profiles of both ATPases.
0.0004 % L-arabinose and 3 mM CuSO4. Growth was measured by
recording OD600 after approximately 12 h.
Sequence alignment and phylogenetic tree. Multiple sequence
alignments were done using the computer program CLUSTAL W
(Larkin et al., 2007). The PHYLIP suite (Felsenstein, 1989), version
3.69, was used to calculate and to graphically display the phylogenetic
tree of PIB-ATPases from distance matrix analysis. Bootstrapping
based on 1000 samples was used to determine the phylogenetic
branching order.
RESULTS AND DISCUSSION
Gene organization of copA and copB
Cytochrome oxidase assay and redox-difference spectroscopy.
The specific oxidase activity was measured spectroscopically at
546 nm at room temperature in a buffer containing 20 mM 2-(Nmorpholino)ethanesulfonic acid adjusted to pH 6.0 with KOH,
10 mM potassium sulfate, 1 mM EDTA (buffer A) and 0.1 mM of
the artifical substrate N,N,N9,N9-tetramethyl-p-phenylenediamine
(TMPD). The reaction was started by addition of 10 ml cytoplasmic
membrane fraction, and inhibited by 0.2 mM potassium cyanide.
Reduced-minus-oxidized absorbance difference spectra in the visible
region were recorded with a Jasco V-650 spectrophotometer at final
protein concentrations of 1.5 mg ml21 in buffer A, supplemented
with 0.1 % (w/v) b-D-dodecylmaltoside. Spectra of samples oxidized
with 1 mM ferricyanide were subtracted from those obtained in the
presence of 10 mM sodium dithionite.
Cloning of copA and copB in E. coli expression vectors. copA
and copB were amplified from S. solfataricus genomic DNA using
primer pairs 59copA_NcoI/39copA_BamHI and 59copB_NcoI/
39copB_BamHI (Table S1). E. coli copA was amplified from genomic
DNA using primers 59EcopA_NcoI/39EcopA_BamHI. The resulting
PCR products were cloned into pBADHis/MycA (Invitrogen) yielding
plasmids pBAD-Sso_copA, pBAD-Sso_copB and pBAD-Eco_copA.
Heterologous complementation of E. coli copA mutants.
Plasmids pBADHis/MycA, pBAD-Sso_copA, pBAD-Sso_copB and
pBAD-Eco_copA were transformed into the copper-sensitive E. coli
strain DC194 (Rensing et al., 2000), resulting in strains DC194-pBAD,
DC194-Sso_copA, DC194-Sso_copB and DC194-Eco_copA. Starter
cultures were grown overnight at 37 uC in LB medium containing
ampicillin (100 mg ml21), chloramphenicol (20 mg ml21) and
kanamycin (50 mg ml21). For the complementation experiments,
cultures were diluted 1 : 1000 in the same medium supplemented with
SSO10823
copT
SSO2652
SSO2651
copR
copA
lacS
The structural gene encoding CopA (Sso2651) is in a
typical operon structure together with a gene encoding the
small copper binding protein CopT which bears the metal
coordinating ligands within the so-called TRASH domain
(Ettema et al., 2003) (Fig. 1). It is associated with a
transcriptional unit containing the copper-responsive regulator gene copR (Villafane et al., 2011). Both copT and copA
have been shown to be transcribed in response to increased
copper concentrations (Ettema et al., 2006; Villafane et al.,
2009). The copB gene (Sso2896) is organized in a different
region (Fig. 1). Next to the translation initiation site of the
presumed monocistronic copB, the two genes copY (encoding
a regulator-type protein) and copZ [encoding a small copper
chaperone of the heavy-metal-associated (HMA) group] are
arranged in opposite orientation to copB. Notably, two pairs
of insertion sequence elements are located at positions
adjacent to the copYZ/copB cluster (Fig. 1).
Assignment of CopA and CopB to the PIB-ATPase
group
In microbes, translocation of heavy metals across cytoplasmic membranes is primarily related to membraneassociated transport ATPases of the P-type (Solioz &
Vulpe, 1996; Palmgren & Axelsen, 1998). The number of
genes encoding these transporters in micro-organisms
varies between one, as in Sulfolobus acidocaldarius (Chen
SSO11417 SSO2897
SSO2898 SSO11412
IS elements copZ
copY
copA
SSO2894 SSO2893
IS elements
SSO2896
copB
lacS
copB
500 bp
Fig. 1. Organization of cop genes. copR and copY (red arrows) are putative regulator genes; copT and copZ (light blue arrows)
encode small copper-binding proteins probably acting as copper chaperones; copA and copB (grey arrows) are structural
genes for copper ATPases. Pairs of insertion sequence elements (IS elements; dark blue arrows) are flanking the genes related
to the transcriptional units of copB. In the single mutant strains DcopA and DcopB, the respective genes are replaced by lacS
(green arrows) of S. solfataricus.
1624
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 01:07:05
Microbiology 158
Copper resistance of Sulfolobus solfataricus
et al., 2005), and nine, as in Haloarcula marismortui (Baliga
et al., 2004). This variability could be a response to environmental challenges by diverse heavy metals such as
copper, zinc, cobalt or cadmium. As mentioned above,
the genome of S. solfataricus (She et al., 2001) has two loci
encoding P-type ATPases. Fig. S1 shows a sequence
alignment of both S. solfataricus ATPases, with the PIBATPases from E. coli and Archaeoglobus fulgidus being
well-characterized copper translocators. Sequence features
clearly demonstrate the characteristic P-type domain
arrangement in the order of A-, P- and N-domains. In
addition, the N-terminal HMA-domains displaying a
CXXC metal binding motif allow assignment of CopA
and CopB of S. solfataricus to subclass type IB according to
Palmgren’s nomenclature (Axelsen & Palmgren, 1998).
CopA exhibits a canonic CPC sequence motif within the
putative hydrophobic region 6, and it is recognized as
contributing to the transmembrane metal binding site. On
the other hand, CopB holds the very rare sequence YPC at
this position. According to a BLAST search, this feature
prevails in only 18 out of more than 1000 known PIB-type
ATPases, indicating its unusually rare occurrence in metal
transport.
Phylogenetic relationship of CopA and CopB
The relationships of the S. solfataricus ATPases can be
studied by comparing a larger series of sequences. Pairwise
distances are calculated from the multiple sequence
alignment, and a phylogenetic tree of the PIB-ATPases
was constructed by distance matrix analysis (Fig. 2, Fig. S2
and Table S2). The unrooted tree divides the ATPases in
two major subgroups named CopA-1 and CopA-2/FixI
according to a recent analysis (González-Guerrero et al.,
2010). In another publication (Coombs & Barkay, 2005),
the assignment to CopA-1 and CopA-2 is contrary to that
outlined by González-Guerrero et al. (2010). The CopA-2
subgroup contains ATPases such as FixI that are described
as being assembly factors of metalloproteins such as
cytochrome oxidases (Kahn et al., 1989; Preisig et al.,
1996; Koch et al., 2000; Hassani et al., 2010). Recent
investigations have demonstrated that CopA-2 of
Pseudomonas aeruginosa is an ATP-driven copper export
system operating at low rates (González-Guerrero et al.,
2010). The CopA-2 lineage and other branches are not
shown in detail in Fig. 2, because both ATPases of S.
solfataricus belong to the other subgroup CopA-1, which
comprises ATPases in charge of copper homeostasis.
CopA-1-type ATPases are assigned as resistance factors
exporting copper at high rates (Argüello et al., 2007;
Osman & Cavet, 2008), whereas a few ATPases have been
previously considered to be copper importers due to their
phenotypic characteristics (Odermatt et al., 1993; Tottey et
al., 2001; Lewinson et al., 2009). These apparently
conflicting views have been lately reconciled by the
statement that ‘‘all Cu+-ATPases drive cytoplasmic Cu+efflux, albeit at quite different transport rates’’ (Raimunda
et al., 2011).
http://mic.sgmjournals.org
Although the tree topology is only weakly supported by low
bootstrapping values at the deep branching points, the S.
solfataricus ATPases (Fig. 2, shaded in purple) are grouped
separately from the proven export ATPases such as
Archaeoglobus CopA, Enterococcus hirae CopB or E. coli
CopA. Another cluster, the YPC-group (shaded in pink),
splits off from the same precursor as the Sulfolobus group
in a stable branching order. Members of the latter family
carry the unusual YPC instead of the CPX signature in
transmembrane helix 6. All CopA-ATPases of the Sulfolobales hold the classical CPC motif, whereas CopB of S.
solfataricus and of another strain of S. islandicus have YPC
instead (hatched shading). Three lines of evidence let us
speculate about CopB-ATPases being taken up preferably
from an organism belonging to the parallel YPC-group by
lateral gene transfer: (i) these genes occur exclusively in
these two strains but not in the other Sulfolobus species; (ii)
these genes are uniquely embedded into the divergently
oriented gene cluster flanked by insertion sequence
elements (Fig. 1); and (iii) the copB gene has a low GCcontent (30.7 %) compared with the copA gene (37.7 %)
and the GC-content of the entire S. solfataricus genome
(35.8 %). One reason for the assumed gene transfer could
be an unknown biological function of CopB which might
be related to the unusual YPC sequence pattern. On the
other hand, the copB cassette could be an evolutionary
remnant of a presently needless function that is barely
retained due to a slow evolutionary clock.
Construction of copA and copB deletion mutants
For independent disruption of copA and copB, their coding
DNA sequences have been entirely exchanged for the
endogenous lacS gene encoding b-galactosidase in the
lacS2 strain S. solfataricus PBL2025 via allelic replacement
(Albers & Driessen, 2008). Mutants carrying a disruption in
either copA or copB were identified on Gelrite plates after
incubation at 76 uC by blue/white screening based on the XGal hydrolase activity of the incorporated lacS gene product.
The correct genotypes of these strains were verified by PCR
and sequencing of isolated genomic DNA (Fig. S3). In
addition to the single mutants DcopA and DcopB, the DcopA/
DcopB double mutant would be highly desirable for our
studies. However, its construction is difficult to perform at
present with respect to the limited availability of screening
or selection markers working in S. solfataricus.
Phenotypes of copA and copB deletion mutants
by growth analysis
Based on the sequence similarity of CopA and CopB to the
other members of the PIB-ATPase family, we predicted that
both are copper-specific transporters. Initial growth
experiments had been carried out using Gelrite plates with
copper sulfate- or silver nitrate-soaked filters. With copper,
no inhibition (wild-type) or a small, diffuse zone of
inhibition (DcopB mutant) was visible, indicating relatively
high resistance (Fig. 3e). In contrast, no significant growth
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 01:07:05
1625
C. Völlmecke and others
81
34
65
Enterococcus hirae CopA
Lactococcus lactis
Pseudomonas putida CueA
Pseudomonas fluorescens CueA
Pseudomonas aeruginosa CopA
Agrobacterium tumefaciens CopA
Rhizobium leguminosarum bv. viciae ActP
Sinorhizobium meliloti Actp
Acidithiobacillus ferrooxidans CopB
Streptococcus mutans CopA
Bacillus subtilis CopA
Mycobacterium tuberculosis CtpV
Synechococcus elongatus PacS
20
Synechocystis PacS
Enterococcus hirae CopB
Staphylococcus aureus CopA
Archaeoglobus fulgidus CopB
Listeria monocytogenes CtpA
Salmonella typhimurium CopA
Escherichia coli CopA
Shewanella oneidensis CopA
Legionella pneumophila CopA
100
Sulfolobus solfataricus CopB
Sulfolobus islandicus Y.N.15.51 CopB
89
Sulfolobus islandicus Y.N.15.51 CopA
27
Sulfolobus islandicus L.D.8.5 CopA
100
Sulfolobus solfataricus CopA
Sulfolobus tokodaii CopA
Sulfolobus acidocaldarius CopA
Halogeometricum borinquense
Halorubrum lacusprofundi
48
Haloarcula marismortui
99
Halorubrum lacusprofundi
Haloterrigena turkmenica
88
Marinithermus hydrothermalis
Thermaerobacter marianensis
96
64
Sphaerobacter thermophilus
Acidiophilum multivorum
100
Nitrobacter hamburgensis
28
Oligotropha carboxidovorans
Methylobacterium extorquens
94
Afipia sp. 1NLS2
Thermobaculum terrenum
100
Acidobacterium capsulatum
Pseudonocardia dioxinivorans
Archaeoglobus fulgidus CopA
Helicobacter pylori CopA
Helicobacter felis CopA
Campylobacter jejuni CopA
Synechococcus elongatus CtaA
Pirellula sp.
Bdellovibrio bacteriovorus
Gramella forsetii
65
other ATPases
(including CopA-2s)
Fig. 2. Unrooted phylogenetic tree of type PIB-ATPases showing the subgroup of CopA-1-ATPases involved in copper
homeostasis. Sequences of other PIB-ATPases, including the subgroup of CopA-2-ATPases involved in the biosynthesis of
metal proteins are cut away for clarity (for a more comprehensive graph and further details, see Fig. S2 and Table S2). The tree
was calculated and displayed by using CLUSTAL W and the PHYLIP suite; intermediate horizontal branch lengths assign sequence
diversity, and the stability of tree topology is denoted by bootstrapping values (ranging from 0 to 100). Archaeal and eubacterial
ATPases, which have the unusual YPC sequence pattern instead of the most frequently occurring CPC pattern in the putative
transmembrane helix 6, form a cluster (shaded in pink). The PIB-ATPases of different Sulfolobus species form another cluster
(shaded in purple) related to it. Herein, solely the CopB ATPases of S. solfataricus and of S. islandicus carry the YPC pattern
(hatched shading).
was observed for the DcopA mutant, demonstrating pronounced copper sensitivity. In the case of silver (Fig. 3f),
both DcopA and DcopB mutants produced broad cell-free
surroundings suggesting that CopA and CopB contribute
to silver detoxification at comparable extents.
1626
However, due to the insolubility of silver chloride and lack
of a fully synthetic culture medium for S. solfataricus, our
quantification of metal resistance in liquid media was
limited to copper. For this purpose, we used depleted
media containing residual copper at a concentration of
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 01:07:05
Microbiology 158
Copper resistance of Sulfolobus solfataricus
1.2 (a)
0.8
(b)
1.0
Growth (OD600)
Growth (OD600)
0.6
0.8
0.6
0.4
0.2
0.0
0.2
0.0
0
10
20
30
Time (h)
40
50
60
0
1.4 (c)
1.4
1.2
1.2
1.0
1.0
10
20
30
40
Time (h)
50
60
(d)
Final OD600
Final OD600
0.4
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
0
2
4
6
8
Cu(II) concentration (µm)
(e)
WT
WT
(f)
10
copA
copB
copA
copB
0
500
1000 1500 2000
Cu(II) concentration (µm)
2500
Fig. 3. Sensitivity of S. solfataricus wild-type, DcopA and DcopB mutants to copper and silver. (a, b) Time courses of cell
growth measured in copper-depleted medium, supplemented with CuSO4 at the indicated concentrations, by measurement of
OD600 against uninoculated medium. The respective control curves without added Cu are shown in Fig. S5. $, 2 mM CuSO4,
DcopA (a) or 1400 mM CuSO4, DcopB (b); &, wild-type. (c, d) Toxicity of copper, shown in cells exposed to various
concentrations of CuSO4, measured by the final OD600 reached after 48 h of growth. $, DcopA (c) or DcopB (d); &, wild-type.
(e, f) Growth of S. solfataricus on solid media in the presence of metal ions. Gelrite plates with 20 mM CuSO4-soaked (e) or
6 mM AgNO3-soaked (f) filters (dark circles). WT, Wild-type.
substantially lower than 1 mM, as determined by a chemical
assay (Brenner & Harris, 1995) (note that a fivefold
concentrated sample of our depleted medium was still
below the reported detection limit of 1 mM Cu+ of this
method), to which CuSO4 was added at defined concentrations. Fig. 3(a and b) show the growth curves of wildtype, DcopA and DcopB mutants. Wild-type cells reach
stationary phase after about 45 h in 2 mM CuSO4 (Fig. 3a)
or 1400 mM CuSO4 (Fig. 3b). The DcopA mutant showed
http://mic.sgmjournals.org
dramatically reduced growth at 2 mM (Fig. 3a), whereas for
the DcopB deletion strain, 1400 mM CuSO4 is needed to
slow the onset and beginning of stationary phase at lower
levels (Fig. 3b). The high toxicity of copper to the DcopA
mutant is obvious by the I50 (inhibitor concentration at
which growth, measured by final OD, was reduced to 50 %
of that of the uninhibited culture) of less than 2 mM (Fig.
3c). These data are clearly compatible with the notion that
CopA is an export ATPase with high affinity to the copper
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 01:07:05
1627
C. Völlmecke and others
ion and functions as a copper and silver resistance factor.
CopB, although being strongly involved in silver resistance,
has a less evident role in regard to copper. The latter has
to be added at very high concentrations to elicit the DcopB
phenotype, showing a 700-fold higher copper resistance than DcopA (Fig. 3d). Moreover, the DcopB mutant
(I50~1400 mM) was slightly, but reproducibly, more sensitive to copper when compared with the wild-type
(I50~1600 mM). This suggests that CopB can be assigned
as an export pump, which becomes relevant at high
copper concentrations.
Expression of copA and copB
The expression level of integral membrane proteins CopA
and CopB in S. solfataricus can be semiquantitatively
demonstrated by immunoblots of cytoplasmic membranes
developed with polyclonal antisera. We used heterologously expressed CopA and CopB as molecular size
markers (Fig. 4a, lanes 8 and 9). As expected, CopB is
lacking in the DcopB mutant (Fig. 4a, lane 6), but is present
in wild-type and DcopA mutant membranes of cells grown
in copper-deficient medium (Fig. 4a, lanes 2 and 4). The
CopB levels were significantly increased in the presence of
copper (Fig. 4a, lanes 3 and 5). It is worth noting that
0.5 mM CuSO4 already suffices to induce CopB accumulation (Fig. 4a, lane 5). CopA was undetectable in copperdepleted medium (Fig. 4a, lanes 2 and 6) because of its
weak immunological cross-reactivity at low concentrations.
However, its accumulation was sufficient to show up in
blots after stimulation of wild-type and DcopB mutant cells
with copper (Fig. 4a, lanes 3 and 7). Copper induction of
CopA biosynthesis is well supported by transcriptional data
obtained for the wild-type (Ettema et al., 2006). Increased
expression in the presence of silver could only be demonstrated for CopB (Fig. 4b, lanes 3 and 5). Combined, these
data underline the classification of CopA as a copper and
silver export ATPase and support the notion that tolerance of S. solfataricus to these metals is controlled by
both PIB-ATPases.
To further clarify the role of CopB, membranes of wildtype cells exposed to various transition metals at sublethal
concentrations have been analysed by immunoblotting.
Compared with cells grown in standard (i.e. copperdepleted) growth medium, the band intensities in Fig. 4(c)
showed a clear increase of CopB levels in the presence of
copper, but the other tested metals had no effect. It is thus
probable that external copper specifically induces the
build-up of CopB-ATPase.
Lack of heterologous functional complementation
of an E. coli copA mutant
In order to prove the biological functions of the S.
solfataricus ATPases, the cloned copA and copB genes were
expressed in the E. coli strain DC194 (Rensing et al., 2000)
which is deficient in the PIB-type copper export ATPase
1628
copA. Growth levels of the transformed E. coli strains in the
presence of sublethal copper concentrations are shown in
Fig. 5. Strain DC194 transformed by a complementation
plasmid containing E. coli copA served as reference and
restored growth at 3 mM CuSO4. Cells transformed with
vectors carrying the S. solfataricus genes copA and copB
yielded reproducible, but only slightly better growth than
the (mock-transformed) control, which at best indicated a
partial complementation. The increase in temperature
resulted in a somewhat higher growth of the copA- and
copB-transformed strains (Fig. 5). The absent complementation at 37 uC is not due to deficient CopA and CopB
expression, as proven by immunostaining (Fig. 5, insert).
In contrast with many PIB-ATPases (Rensing et al., 2000;
Lewinson et al., 2009), both S. solfataricus ATPases were
incapable of compensating for the copper transport defect
of the E. coli copA mutant. The small increase in growth
rate at elevated temperatures may indicate weakly developing complemention. The lack of full restoration may be
explained by reduced catalytic activities of the thermophilic
proteins at the relatively low-temperature living conditions
of E. coli. However, this does not hold for all thermophiles,
because the PIB-ATPase of Pyrococcus furiosus has been
reported to achieve functional complementation at 37 uC
(Lewinson et al., 2009). Although Pyrococcus can grow at
100 uC (Fiala & Stetter, 1986), the different natural di- and
tetra-ether compositions of S. solfataricus and Pyrococcus
membranes may prescribe specific lipid requests for CopA
and CopB to attain functional complementation in E. coli
membranes.
Sensitivity of cop mutants towards reactive
oxygen species (ROS)
It has been reported that copA-2 mutants of P. aeruginosa
are much more sensitive to ROS than wild-type or copA-1
mutants (González-Guerrero et al., 2010). In order to
assign a possible role for the CopA and CopB ATPases, we
investigated the ROS sensitivity of S. solfataricus. We used
Paraquat to generate ROS in our experiments. It is evident
by the growth characteristics in Fig. S4 that S. solfataricus is
highly sensitive to low concentrations of this compound,
giving rise to 50 % survival at about 750 nM. This notable
sensitivity is caused by its chemical reactivity leading to
increased ROS formation at high growth temperatures.
However, the ROS sensititivity of both DcopA and DcopB
mutants is virtually imperceptible from the S. solfataricus
wild-type strain. In contrast, wild-type P. aeruginosa and
DcopA-1 strains exhibit significantly higher resistance to
ROS than the copA-2 mutants (González-Guerrero et al.,
2010). It may be speculated that the high sensitivity to ROS
is related to impaired biosynthesis of detoxifying Cu–Zn
superoxide dismutase, which is located in the periplasm of
Gram-negative bacteria (Kroll et al., 1995). However,
enzymes of the Cu–Zn class did not appear to be involved
in ROS metabolism of S. solfataricus, whereas an Fe
superoxide dismutase has been isolated from culture fluids
(Cannio et al., 2000).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 01:07:05
Microbiology 158
Copper resistance of Sulfolobus solfataricus
(a)
copA copA copB copB
Sample:
WT
WT
Cu added: (no) (800 M) (no) (0.5 M) (no) (800 M)
Control
130
100
70
CopA
CopB
55
40
35
25
15
kDa
1
2
3
4
5
6
7
9
8
(b)
WT
Sample:
Ag added: (no)
WT copA copA copB copB
(10 M) (no) (0.25 M) (no) (10 M)
Control
130
100
70
CopA
CopB
55
40
35
25
15
kDa
1
2
3
4
5
Cu
Ni
Co
6
7
9
8
(c)
No
Pb
Cd
Zn
Hg
Control
130
100
CopB
70
55
40
35
25
15
kDa
1
2
3
http://mic.sgmjournals.org
4
5
6
7
8
9
10
Fig. 4. (a, b) Western blots of cytoplasmic
membranes (100 mg each) from S. solfataricus
cells, developed with polyclonal antiserum
raised to the actuator domain of CopB (named
CopB-A), which is cross-reactive with CopA.
(a) Accumulation of CopA or CopB in wildtype and Dcop mutants in the presence of
copper. (b) CopB accumulation in the presence of AgNO3. The strain and amount of
CuSO4 or AgNO3 added to each sample are
indicated above each lane. Lanes: 1, molecular
size marker; 8, CopA size marker; 9, CopB size
marker. (c) CopB accumulation in wild-type S.
solfataricus triggered by the presence of
various metal ions during growth, shown by
Western blotting with antiserum against the
CopB-B domain, which does not cross-react
with CopA. Membranes were isolated from
cells grown in standard medium (lane 2) or in
the presence of metal concentrations that have
been previously determined to be sublethal to
S. solfataricus (see Methods), namely 1 mM
Cu2+ (lane 3), 10 mM Ni2+ (4), 8 mM Co2+
(5), 0.8 mM Pb2+ (6), 1.5 mM Cd2+ (7),
1.5 mM Zn2+ (8), 3 mM Hg2+ (9). Molecular
size markers (lane 1) and purified CopB (lane
10) were added as references.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 01:07:05
1629
C. Völlmecke and others
Fig. 5. Complementation test of copA and copB
genes of S. solfataricus in E. coli strain DC194,
which is deficient in copA, at two different
temperatures. Growth of DC194 transformed
with expression plasmids encoding copA of E.
coli (positive control, black bars), copA (dark
grey) or copB (light grey) of S. solfataricus, or the
plasmid pBAD without insert (negative control,
white) in the presence of 3 mM CuSO4 was
recorded by measuring OD600 after approximately 12 h. The OD600 values of reference
cultures (Eco_copA) were set to 100 %,
amounting to 1.7 at 37 6C, and to 1.0 at
41 6C. Insert: immunostained membranes of E.
coli Eco_copA, Sso_copA and Sso_copB.
Probing the role of CopA and CopB in the
biosynthesis of cytochrome oxidase
As mentioned above, ATPases of the CopA-2 subclass have
been demonstrated to be involved in the biosynthesis of
metal proteins by copper export (González-Guerrero et al.,
2010). In order to probe the S. solfataricus CopA- and
CopB-ATPases with respect to haem-copper cytochrome
oxidase production, membranes of all strains have been
subjected to cytochrome analysis by reduced-minusoxidized absorbance difference spectroscopy (Fig. 6). In
copper-depleted medium, wild-type cells produced the
typical cytochrome profile of a-bands observed in the
closely related S. acidocaldarius strain, at low but sufficient
levels to sustain normal aerobic growth rates. It consists of
the complex III analogon apocytochrome b bound to haem
AS which absorbs at 585 nm (SoxC and SoxG), and of the
two haem-copper terminal oxidases of the SoxM-type
cytochrome bb3 and the SoxB-type aa3, which absorb at
562 and 604 nm (Lübben et al., 1992, 1994; Komorowski
et al., 2002). Interestingly, both DcopA and DcopB mutant
membrane spectra displayed higher oxidase peaks than the
wild-type, particularly pronounced by the increase of
cytochrome aa3 at 604 nm (Fig. 6). Membranes of the
wild-type and DcopB mutant from cells supplemented with
400 mM CuSO4 were analysed as a control, demonstrating
the potential of cytochrome synthesis under conditions of
copper repletion. Whereas the terminal cytochrome oxidase
levels at 562 and 604 nm were only slightly enhanced
compared with the copper-limiting case, the apocytochrome/haem AS peak at 585 nm was strongly raised.
The bb3- and aa3-type cytochrome oxidase activities of
membranes, measured by the fully cyanide-sensitive electron
transfer from the pseudo-substrate TMPD (Lübben et al.,
1994; Gleissner et al., 1997) further supported the abovementioned spectroscopic observations. As shown in Table 2,
1630
0.0025 OD
585
604
562
copB (+)
WT (+)
copB (–)
copA (–)
WT (–)
10 nm
Fig. 6. Redox absorbance difference spectra of cytoplasmic
membranes, solubilized with 0.1 % (w/v) b-D-dodecylmaltoside. As
indicated, the spectra of dithionite-reduced minus ferricyanideoxidized membranes (1.5 mg ml”1) were recorded from wild-type,
DcopA mutant and DcopB mutant, grown in ion-depleted medium
as well as from wild-type and DcopB mutant, grown in the
presence of 400 mM CuSO4 (+).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 01:07:05
Microbiology 158
Copper resistance of Sulfolobus solfataricus
Table 2. TMPD-oxidase activity of membranes from S. solfataricus strains
2Cu, No copper in growth medium; +Cu, growth medium was supplemented with 400 mM CuSO4.
Strain
Wild-type 2Cu
DcopA 2CuD
DcopB 2Cu
Wild-type +Cu
DcopB +Cu
Specific activity* (mmol mg”1 min”1)
Relative activity
0.082±0.003
0.108±0.004
0.159±0.015
0.408±0,044
0.408±0.013
1.0
1.3
1.9
5.0
5.0
*Activities were fully inhibited by 200 mM KCN, n53.
DNo data could be obtained for membranes of the DcopA mutant supplemented with copper as 400 mM CuSO4 was lethal to this strain.
the cytochrome oxidase activities of the DcopA and DcopB
mutant membranes are higher than the wild-type, but
significantly smaller than after copper repletion. In relation
to the bb3- and aa3-type cytochrome levels, our catalytic data
suggest that several copper-involving steps are required to
attain full oxidase activity which are rather speculative.
Our observations demonstrated that it is quite unlikely that
any of the PIB-ATPases play an essential role in cytochrome
oxidase biosynthesis, because loss-of-function phenotypes
lacking cytochrome oxidase would be expected from disruption of either copA or copB genes. The copA and copB
double mutant would of course fully prove this statement.
Nevertheless, both single cop disruptions led to higher
cytochrome oxidase levels at limiting copper concentration.
Although the mechanism of CuA or CuB incorporation in S.
solfataricus is unknown, our data underline the importance
of internal copper to this process. Deletion of either copperATPase would reduce the export activity and in turn raise
the cytoplasmic level of copper. Higher internal concentrations could increase the copper loading of assembly
factors that may act from the outside, as postulated for the
membrane-anchored mitochondrial proteins Sco1/Sco2 and
Cox11 (Horng et al., 2004; Carr et al., 2005; Banci et al.,
2011). Consistent with this interpretation is the increase in
cytochrome levels observed in S. solfataricus after addition of
CuSO4, because external copper is similarly assumed to
result in higher internal levels. Alternatively, the putative
assembly factors may also be copper-loaded directly from
the outside.
Taken together, the increase in cytochrome oxidase
absorbance and activity in the absence of either coppertranslocating ATPase strongly discredits any crucial role in
oxidase assembly by delivering copper to the active site in
S. solfataricus.
tolerance. CopA is a very efficient copper export pump,
because a disruption in its encoding gene led to severely
increased copper sensitivity. The biological role of CopB is
less clear. Its gene may have been acquired by lateral gene
transfer, as with only one known exception its gene does not
exist in other relatives of S. solfataricus. With respect to
copper, the phenotype of DcopB differed slightly from the
wild-type. Apparently, like a booster module, CopB supports
the cellular resistance machinery by expanding the copper
tolerance range by about 0.2 mM. In the search for a possible
alternative role of CopB, for example as a copper-exporting
assembly factor, we could rule out its direct involvement in
the biosynthesis of Cu-proteins. The copper/silver ATPase
CopB may either have a physiological assignment yet to be
discovered or be a leftover from an earlier function lost
during evolution.
ACKNOWLEDGEMENTS
The authors are grateful to Dr Bernd Masepohl (Ruhr-Universität
Bochum) for helpful discussions. This work was supported by grants
to M. L. from the Deutsche Forschungsgemeinschaft (grant LU405/3-1)
and the Protein Research Department (RUB). S. V. A. was supported by
a VIDI grant from the Dutch Science Organization (NWO). S. V. A.
and J. R. received intramural funding from the Max Planck Society and
the BMBF (SulfoSYS).
REFERENCES
Albers, S. V. & Driessen, A. J. (2008). Conditions for gene disruption
by homologous recombination of exogenous DNA into the Sulfolobus
solfataricus genome. Archaea 2, 145–149.
Argüello, J. M., Eren, E. & González-Guerrero, M. (2007). The
structure and function of heavy metal transport PIB-ATPases.
Biometals 20, 233–248.
Argüello, J. M., González-Guerrero, M. & Raimunda, D. (2011).
Conclusion
Bacterial transition metal P(IB)-ATPases: transport mechanism and
roles in virulence. Biochemistry 50, 9940–9949.
The ATPases CopA and CopB of S. solfataricus both belong
to the CopA-1 group of PIB-ATPases. They are synthesized
at low levels under standard growth conditions, i.e. under
copper limitation, and the expression levels increase after
addition of copper. Both enzymes impart copper and silver
Axelsen, K. B. & Palmgren, M. G. (1998). Evolution of substrate
http://mic.sgmjournals.org
specificities in the P-type ATPase superfamily. J Mol Evol 46, 84–101.
Balasubramanian, R., Kenney, G. E. & Rosenzweig, A. C. (2011).
Dual pathways for copper uptake by methanotrophic bacteria. J Biol
Chem 286, 37313–37319.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 01:07:05
1631
C. Völlmecke and others
Baliga, N. S., Bonneau, R., Facciotti, M. T., Pan, M., Glusman, G.,
Deutsch, E. W., Shannon, P., Chiu, Y., Weng, R. S. & other authors
(2004). Genome sequence of Haloarcula marismortui: a halophilic
archaeon from the Dead Sea. Genome Res 14, 2221–2234.
Banci, L., Bertini, I., Cavallaro, G. & Ciofi-Baffoni, S. (2011). Seeking
the determinants of the elusive functions of Sco proteins. FEBS J 278,
2244–2262.
Hassani, B. K., Astier, C., Nitschke, W. & Ouchane, S. (2010).
CtpA, a copper-translocating P-type ATPase involved in the biogenesis of multiple copper-requiring enzymes. J Biol Chem 285,
19330–19337.
Horng, Y. C., Cobine, P. A., Maxfield, A. B., Carr, H. S. & Winge, D. R.
(2004). Specific copper transfer from the Cox17 metallochaperone to
Brenner, A. J. & Harris, E. D. (1995). A quantitative test for copper
both Sco1 and Cox11 in the assembly of yeast cytochrome C oxidase.
J Biol Chem 279, 35334–35340.
using bicinchoninic acid. Anal Biochem 226, 80–84.
Imlay, J. A. (2006). Iron-sulphur clusters and the problem with
Brock, T. D., Brock, K. M., Belly, R. T. & Weiss, R. L. (1972). Sulfolobus:
oxygen. Mol Microbiol 59, 1073–1082.
a new genus of sulfur-oxidizing bacteria living at low pH and high
temperature. Arch Mikrobiol 84, 54–68.
Jonuscheit, M., Martusewitsch, E., Stedman, K. M. & Schleper, C.
(2003). A reporter gene system for the hyperthermophilic archaeon
Cannio, R., D’angelo, A., Rossi, M. & Bartolucci, S. (2000). A
Sulfolobus solfataricus based on a selectable and integrative shuttle
vector. Mol Microbiol 48, 1241–1252.
superoxide dismutase from the archaeon Sulfolobus solfataricus is an
extracellular enzyme and prevents the deactivation by superoxide of
cell-bound proteins. Eur J Biochem 267, 235–243.
Carr, H. S., Maxfield, A. B., Horng, Y. C. & Winge, D. R. (2005).
Functional analysis of the domains in Cox11. J Biol Chem 280, 22664–
22669.
Chen, L., Brügger, K., Skovgaard, M., Redder, P., She, Q.,
Torarinsson, E., Greve, B., Awayez, M., Zibat, A. & other authors
(2005). The genome of Sulfolobus acidocaldarius, a model organism of
Kahn, D., David, M., Domergue, O., Daveran, M. L., Ghai, J., Hirsch, P.
R. & Batut, J. (1989). Rhizobium meliloti fixGHI sequence predicts
involvement of a specific cation pump in symbiotic nitrogen fixation.
J Bacteriol 171, 929–939.
Kaplan, J. H. (2002). Biochemistry of Na,K-ATPase. Annu Rev
Biochem 71, 511–535.
Koch, H. G., Winterstein, C., Saribas, A. S., Alben, J. O. & Daldal, F.
(2000). Roles of the ccoGHIS gene products in the biogenesis of the
the Crenarchaeota. J Bacteriol 187, 4992–4999.
cbb(3)-type cytochrome c oxidase. J Mol Biol 297, 49–65.
Coombs, J. M. & Barkay, T. (2005). New findings on evolution of
Komorowski, L., Verheyen, W. & Schäfer, G. (2002). The archaeal
metal homeostasis genes: evidence from comparative genome analysis
of bacteria and archaea. Appl Environ Microbiol 71, 7083–7091.
de Rosa, M., Gambacorta, A. & Bu’lock, J. D. (1975). Extremely
thermophilic acidophilic bacteria convergent with Sulfolobus acidocaldarius. J Gen Microbiol 86, 156–164.
Deigweiher, K., Drell, T. L., IV, Prutsch, A., Scheidig, A. J. & Lübben,
M. (2004). Expression, isolation, and crystallization of the catalytic
domain of CopB, a putative copper transporting ATPase from the
thermoacidophilic archaeon Sulfolobus solfataricus. J Bioenerg Biomembr
36, 151–159.
Ettema, T. J., Huynen, M. A., de Vos, W. M. & van der Oost, J. (2003).
TRASH: a novel metal-binding domain predicted to be involved in
heavy-metal sensing, trafficking and resistance. Trends Biochem Sci 28,
170–173.
Ettema, T. J., Brinkman, A. B., Lamers, P. P., Kornet, N. G., de Vos,
W. M. & van der Oost, J. (2006). Molecular characterization of a
respiratory supercomplex SoxM from S. acidocaldarius combines
features of quinole and cytochrome c oxidases. Biol Chem 383, 1791–
1799.
Kroll, J. S., Langford, P. R., Wilks, K. E. & Keil, A. D. (1995). Bacterial
[Cu,Zn]-superoxide dismutase: phylogenetically distinct from the
eukaryotic enzyme, and not so rare after all! Microbiology 141, 2271–
2279.
Kühlbrandt, W. (2004). Biology, structure and mechanism of P-type
ATPases. Nat Rev Mol Cell Biol 5, 282–295.
Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan,
P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A. & other
authors (2007). CLUSTAL W and CLUSTAL_X version 2.0. Bioinformatics
23, 2947–2948.
Lewinson, O., Lee, A. T. & Rees, D. C. (2009). A P-type ATPase
importer that discriminates between essential and toxic transition
metals. Proc Natl Acad Sci U S A 106, 4677–4682.
conserved archaeal copper resistance (cop) gene cluster and its
copper-responsive regulator in Sulfolobus solfataricus P2. Microbiology
152, 1969–1979.
Liochev, S. I. & Fridovich, I. (2002). The Haber–Weiss cycle – 70 years
Felsenstein, J. (1989). PHYLIP – phylogeny inference package (version
Lübben, M., Kolmerer, B. & Saraste, M. (1992). An archaebacterial
3.2). Cladistics 5, 164–166.
terminal oxidase combines core structures of two mitochondrial
respiratory complexes. EMBO J 11, 805–812.
Fiala, G. & Stetter, K. O. (1986). Pyrococcus furiosus sp. nov. represents
a novel genus of marine heterotrophic archaebacteria growing
optimally at 100uC. Arch Microbiol 145, 56–61.
Gleissner, M., Kaiser, U., Antonopoulos, E. & Schäfer, G. (1997). The
archaeal SoxABCD complex is a proton pump in Sulfolobus
acidocaldarius. J Biol Chem 272, 8417–8426.
Goldstein, S., Meyerstein, D. & Czapski, G. (1993). The Fenton
reagents. Free Radic Biol Med 15, 435–445.
González-Guerrero, M., Raimunda, D., Cheng, X. & Argüello, J. M.
(2010). Distinct functional roles of homologous Cu+ efflux ATPases
in Pseudomonas aeruginosa. Mol Microbiol 78, 1246–1258.
later: an alternative view. Redox Rep 7, 55–57, author reply 59–60.
Lübben, M., Arnaud, S., Castresana, J., Warne, A., Albracht, S. P. &
Saraste, M. (1994). A second terminal oxidase in Sulfolobus
acidocaldarius. Eur J Biochem 224, 151–159.
Møller, J. V., Juul, B. & le Maire, M. (1996). Structural organization,
ion transport, and energy transduction of P-type ATPases. Biochim
Biophys Acta 1286, 1–51.
Nies, D. H. (2003). Efflux-mediated heavy metal resistance in
prokaryotes. FEMS Microbiol Rev 27, 313–339.
Odermatt, A., Suter, H., Krapf, R. & Solioz, M. (1993). Primary
structure of two P-type ATPases involved in copper homeostasis in
Enterococcus hirae. J Biol Chem 268, 12775–12779.
Grogan, D. W. (1989). Phenotypic characterization of the archaebacterial genus Sulfolobus: comparison of five wild-type strains.
J Bacteriol 171, 6710–6719.
Osman, D. & Cavet, J. S. (2008). Copper homeostasis in bacteria. Adv
Haber, F. & Weiss, J. (1932). Über die Katalyse des Hydroperoxydes.
Palmgren, M. G. & Axelsen, K. B. (1998). Evolution of P-type
Naturwissenschaften 20, 948–950.
ATPases. Biochim Biophys Acta 1365, 37–45.
1632
Appl Microbiol 65, 217–247.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 01:07:05
Microbiology 158
Copper resistance of Sulfolobus solfataricus
Preisig, O., Zufferey, R. & Hennecke, H. (1996). The Bradyrhizobium
Silver, S. & Phung, L. T. (1996). Bacterial heavy metal resistance: new
japonicum fixGHIS genes are required for the formation of the highaffinity cbb3-type cytochrome oxidase. Arch Microbiol 165, 297–305.
surprises. Annu Rev Microbiol 50, 753–789.
Raimunda, D., González-Guerrero, M., Leeber, B. W., III & Argüello,
J. M. (2011). The transport mechanism of bacterial Cu+-ATPases:
Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner,
F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. &
Klenk, D. C. (1985). Measurement of protein using bicinchoninic
distinct efflux rates adapted to different function. Biometals 24, 467–475.
acid. Anal Biochem 150, 76–85.
Rensing, C. & Grass, G. (2003). Escherichia coli mechanisms of
Solioz, M. & Vulpe, C. (1996). CPx-type ATPases: a class of P-type
copper homeostasis in a changing environment. FEMS Microbiol Rev
27, 197–213.
ATPases that pump heavy metals. Trends Biochem Sci 21, 237–241.
Rensing, C., Fan, B., Sharma, R., Mitra, B. & Rosen, B. P. (2000).
Szabó, Z., Sani, M., Groeneveld, M., Zolghadr, B., Schelert, J., Albers,
S. V., Blum, P., Boekema, E. J. & Driessen, A. J. (2007). Flagellar
CopA: An Escherichia coli Cu(I)-translocating P-type ATPase. Proc
Natl Acad Sci U S A 97, 652–656.
motility and structure in the hyperthermoacidophilic archaeon
Sulfolobus solfataricus. J Bacteriol 189, 4305–4309.
Schelert, J., Dixit, V., Hoang, V., Simbahan, J., Drozda, M. & Blum, P.
(2004). Occurrence and characterization of mercury resistance in the
Tottey, S., Rich, P. R., Rondet, S. A. & Robinson, N. J. (2001). Two
hyperthermophilic archaeon Sulfolobus solfataricus by use of gene
disruption. J Bacteriol 186, 427–437.
She, Q., Singh, R. K., Confalonieri, F., Zivanovic, Y., Allard, G.,
Awayez, M. J., Chan-Weiher, C. C., Clausen, I. G., Curtis, B. A. & other
authors (2001). The complete genome of the crenarchaeon Sulfolobus
Menkes-type atpases supply copper for photosynthesis
Synechocystis PCC 6803. J Biol Chem 276, 19999–20004.
in
Villafane, A. A., Voskoboynik, Y., Cuebas, M., Ruhl, I. & Bini, E.
(2009). Response to excess copper in the hyperthermophile Sulfolobus
solfataricus strain 98/2. Biochem Biophys Res Commun 385, 67–71.
solfataricus P2. Proc Natl Acad Sci U S A 98, 7835–7840.
Villafane, A., Voskoboynik, Y., Ruhl, I., Sannino, D., Maezato, Y.,
Blum, P. & Bini, E. (2011). CopR of Sulfolobus solfataricus represents a
Shungu, D., Valiant, M., Tutlane, V., Weinberg, E., Weissberger, B.,
Koupal, L., Gadebusch, H. & Stapley, E. (1983). GELRITE as an Agar
novel class of archaeal-specific copper-responsive activators of
transcription. Microbiology 157, 2808–2817.
Substitute in Bacteriological Media. Appl Environ Microbiol 46, 840–
845.
Edited by: D. H. Nies
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 01:07:05
1633