Escherichia coli mechanisms of copper homeostasis in a changing environmen

Escherichia coli mechanisms of copper homeostasis in
a changing environment
Christopher Rensing Ã
, Gregor Grass 1
Department of Soil, Water, and Environmental Science, University of Arizona, Shantz Bld. #38, Rm. 429, Tucson, AZ 85721, USA
Received 1 November 2002; received in revised form 7 January 2003; accepted 7 January 2003
First published online 29 April 2003
Abstract
Escherichia coli is equipped with multiple systems to ensure safe copper handling under varying environmental conditions. The Cu(I)-
translocating P-type ATPase CopA, the central component in copper homeostasis, is responsible for removing excess Cu(I) from the
cytoplasm. The multi-copper oxidase CueO and the multi-component copper transport system CusCFBA appear to safeguard the
periplasmic space from copper-induced toxicity. Some strains of E. coli can survive in copper-rich environments that would normally
overwhelm the chromosomally encoded copper homeostatic systems. Such strains possess additional plasmid-encoded genes that confer
copper resistance. The pco determinant encodes genes that detoxify copper in the periplasm, although the mechanism is still unknown.
Genes involved in copper homeostasis are regulated by MerR-like activators responsive to cytoplasmic Cu(I) or two-component systems
sensing periplasmic Cu(I). Pathways of copper uptake and intracellular copper handling are still not identified in E. coli.
ß 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords: P-type ATPase; Soft metal; Heavy-metal resistance; Multi-copper oxidase; Resistance nodulation cell division family; Membrane fusion
protein; Outer membrane factor; Cu; Periplasm; Copper toxicity; Escherichia coli
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
2. CopA is responsible for cytoplasmic copper homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . 200
3. The proton-driven Cus system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
4. CueO is a multi-copper oxidase protecting the periplasm from copper-induced damage . . . 203
5. The plasmid-encoded pco determinant confers additional copper resistance . . . . . . . . . . . . 204
6. Regulation of genes involved in copper homeostasis re£ects di¡erent demands in a changing
environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
7. Conclusions and perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
1. Introduction
Copper ions undergo unique chemistry due to their abil-
ity to adopt distinct redox states, either oxidized as Cu(II)
or in the reduced state, Cu(I). The stable Cu(II)^N bonds
are often inert while the bonds with oxygen donor ligands
are more labile. Cu(I) is considered a soft metal and pref-
erentially bonds with ligands such as sulfhydryl groups.
Biological systems did not utilize copper before the advent
0168-6445 / 03 / $22.00 ß 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016/S0168-6445(03)00049-4
* Corresponding author. Tel.: +1 (520) 626 8482;
Fax: +1 (520) 621 1647.
E-mail address: rensingc@ag.arizona.edu (C. Rensing).
1
Present address: Institut fu«r Mikrobiologie, Martin-Luther-
Universita«t Halle-Wittenberg, Kurt-Mothes-Str. 3, 06099 Halle, Germany.
FEMSRE 782 2-6-03 Cyaan Magenta Geel Zwart
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of atmospheric oxygen. In the prevailing reducing condi-
tions before this event, copper was in the water-insoluble
Cu(I) state, in the form of highly insoluble sul¢des, and
was not available for biological processes. Cyanobacteria
are thought to be responsible for the beginning of dioxy-
gen production about 109
years ago. The appearance of a
signi¢cant O2 concentration in the atmosphere required
another 200^300 million years because the oxygen pro-
duced was initially consumed by the oxidation of ferrous
iron in the oceans. This event irreversibly changed life on
earth. While enzymes involved in anaerobic metabolism
needed to work in the lower portion of the redox spec-
trum, the presence of dioxygen created the need for a
redox-active metal with E0Mnþ1
/Mn
from 0 to 0.8 V.
The redox potential Cu(II)/Cu(I) of copper is usually high-
er than that of iron Fe(III)/Fe(II). Most copper enzymes
work between 0.25 and 0.75 V. This high potential can be
utilized for direct oxidation of easily oxidized substrates
such as superoxide, ascorbate, catechol or phenolates [1].
Copper proteins are widely distributed in aerobic organ-
isms and mainly have two functions, electron transfer and
dioxygen transport and activation. Copper proteins are
involved in vital processes such as respiration, iron trans-
port, oxidative stress protection, blood clotting and pig-
mentation [2]. Almost all copper proteins, such as multi-
copper oxidases, amine oxidases or lysine oxidase, are
periplasmic or extracellular. In bacteria, cytochrome c ox-
idase is localized in the cytoplasmic membrane whilst in
the mitochondria of eukaryotes it is in the inner mitochon-
drial membrane. In bacteria, including Escherichia coli,
Cu,Zn-superoxide dismutase (Cu,Zn-SOD) is a periplas-
mic enzyme. In contrast, Cu,Zn-SOD is a cytosolic en-
zyme in eukaryotes. These di¡erences are probably the
result of endosymbiosis. Mitochondria are thought to be
descendants of an endosymbiotic K-proteobacterium [3].
Accordingly, the bacterial (mitochondrial) periplasmic
Cu,Zn-SOD would be located outside, i.e. in the eukary-
otic cytoplasm.
Copper requiring proteins are involved in a variety of
biological processes and de¢ciency in these enzymes, or
alteration in their activities, often cause disease states or
pathophysiological conditions [4]. Copper homeostasis is a
requirement for the evolution of aerobic metabolism since
copper is highly toxic even at low concentrations in part
because copper is a redox-active transition metal. Copper
appears to be responsible for the intracellular generation
of superoxide or other reactive oxygen species [5]. In ad-
dition, under anaerobic conditions copper appears to shift
from the Cu(II) to the Cu(I) oxidation state and becomes
much more toxic, possibly because Cu(I) can di¡use
through the cytoplasmic membrane (Fig. 1) [6,7]. There-
fore, intracellular copper concentrations need to be regu-
lated within very narrow limits. Disturbed copper homeo-
stasis has recently been implicated in diseases such as
Alzheimer disease, cystic ¢brosis and Parkinson disease
[8,9].
The mechanisms involved in copper transport and ho-
meostasis in bacteria are only partially understood (Fig.
1). In this review we will attempt to summarize what is
presently known with an emphasis on E. coli since copper
homeostasis and tra⁄cking in bacteria is best understood
in E. coli and in Enterococcus hirae [169]. Relevant ele-
Fig. 1. Copper homeostasis mechanisms in E. coli. Shown are the most relevant homeostatic systems. The individual components are discussed in Sec-
tion 1. CopA is a Cu(I)-translocating P-type ATPase, Ndh-2 a cupric reductase, CueO a multi-copper oxidase and CusCFBA a four-component copper
e¥ux pump. Copper probably traverses the outer membrane through porins, possibly OmpC and OmpF.
C. Rensing, G. Grass / FEMS Microbiology Reviews 27 (2003) 197^213198

ments of copper homeostasis and their function in E. coli
are listed in Table 1. Known copper-dependent proteins in
E. coli, apart from copper homeostasis factors such as
CueO, include the periplasmic zinc, copper SOD (SodC),
NADH dehydrogenase-2 (NDH-2), cytochrome oxidase
(CytBO(3)), aromatic amine oxidase (MaoA) and 3-de-
oxy-Darabino-heptulosonate-7-phosphate synthase (AroF)
[10^13].
Copper uptake systems should be cation speci¢c, so as
to ensure an adequate supply of copper in the presence of
varying levels of related cations such as nickel or zinc.
Once inside the cells, additional proteins are needed for
sequestration and tra⁄cking of copper since intracellular
free cations are toxic. In the case of copper, intracellular
unbound Cu2þ
can result in oxygen-radical damage [14].
However, in yeast intracellular levels of free copper were
not detectable, indicating that all copper under physiolog-
ical, non-toxic conditions is bound to proteins, chelators
and metallochaperones [15]. In addition, cellular e¥ux
systems have been shown to remove excess levels of cati-
ons and to protect the intracellular environment [16].
E. coli is a facultative aerobic, enteric bacterium living
in the digestive tract of warm-blooded animals. The con-
centration of copper can be high in the digestive tract,
particularly in the stomach and duodenum, but even there
it probably does not exceed 10 WM. However, it has been
shown that under acidic anaerobic conditions, which pre-
vail in that part of the digestive tract, copper becomes
much more toxic. Thus, intestinal bacteria may have
evolved elaborate mechanisms for copper homeostasis as
an adaptation to their speci¢c ecological niche, the animal
gut. Recently, two copper-responsive regulatory systems
were identi¢ed. One is a two-component signal transduc-
tion system designated the Cu-sensing or cus locus. The
cusRS genes form a sensor/regulator pair that activates the
adjacent but divergently transcribed genes cusCFBA
[17,18]. The cusCBA gene products are homologous to a
family of proton/cation antiporter complexes involved in
export of metal ions, xenobiotics, and drugs. CusF is a
periplasmic copper-binding protein (S. Franke et al., un-
published). The second system, termed cue (for Cu e¥ux),
is regulated by CueR, a MerR-like transcriptional activa-
tor induced by copper [19^21]. CueR has been shown to
regulate two genes, copA and cueO (formerly yacK) [19^
21]. CopA is a Cu(I)-translocating P-type ATPase while
CueO is a multi-copper oxidase [19,22,23].
Early attempts to elucidate copper homeostasis in E. coli
have been inconclusive. On the basis of a preliminary
characterization of copper-sensitive mutants, it was ini-
tially proposed that six genes (cutA, -B, -C, -D, -E, -F)
are involved in the uptake, intracellular storage, delivery,
and e¥ux of copper in E. coli [24]. However, few of these
genes have been directly linked to copper metabolism,
transport, or regulation. Of these six structural genes,
the cutA locus [25] and the cutC [26], cutE [27] and cutF
[26] genes have been cloned and a putative function has
been assigned to them. These proteins could have an an-
cillary role such as the reduction of methionines in CueO,
altering porins or ensuring proper protein folding in the
periplasm. Whatever their role, they only have a slight
in£uence on copper-resistance levels with the exception
of CutE. The cutE (lnt) gene encodes apolipoprotein N-
acyltransferase. CutE might function in acylation of CusC
and other apolipoproteins required for copper tolerance
[17,28]. However, most of the cut genes are probably
only indirectly involved in copper homeostasis [19,28].
Copper appears to cross the outer membrane through
porins since a copper-resistant, porin-de¢cient mutant
E. coli could be isolated [29]. Similar results could be ob-
tained in laboratory-selected silver-resistant mutants of
E. coli [30]. However, various isogenic OmpF- and/or
OmpC-de¢cient mutants of E. coli did not di¡er signi¢-
cantly in copper or silver resistance [30]. This might indi-
cate that in addition to lowering the permeability of the
outer membrane, cells need to increase their rate of copper
or silver e¥ux. How copper crosses the cytoplasmic mem-
brane has not been conclusively described in bacteria be-
sides the possibility that Cu(I) may equilibrate across this
barrier [7]. Since speci¢c transporters are needed for Cu(I)
uptake in eukaryotes it appears unlikely that this is a
major route of copper entry.
The cupric reductase NDH-2 has been described in
E. coli [13,31] suggesting that Cu(II) is reduced during
uptake as was described in eukaryotes (Fig. 1). Copper
Table 1
Essential elements of copper homeostasis in E. coli
Homeostatic mech-
anism
Regulated by Function
CopA CueR (sensing cytoplasmic Cu[I]) and CpxR (sensing cell envelope
stress)
Detoxi¢cation of cytoplasmic Cu[I]
CusCFBA CusRS (two-component regulation system, sensing periplasmic Cu[I]) Detoxi¢cation of periplasmic (and possibly cytoplasmic)
Cu[I]
CueO CueR (sensing cytoplasmic Cu[I]) Protection of periplasmic proteins from copper-induced
toxicity
PcoABCD PcoRS, (two-component regulation system, sensing periplasmic Cu[I])
(and CusRS)
Protection from extreme periplasmic copper stress
PcoE CusRS (PcoRS) Periplasmic copper chaperone, copper binding
C. Rensing, G. Grass / FEMS Microbiology Reviews 27 (2003) 197^213 199

uptake in Saccharomyces cerevisiae is mediated by sepa-
rate high- and low-a⁄nity systems. High-a⁄nity copper
uptake requires plasma membrane reductases to reduce
Cu(II) to Cu(I). This reduction is mediated by the same
plasma membrane reductases, Fre1 and Fre2, that are in-
volved in iron uptake [32]. Once reduced to Cu(I), the ions
are taken up by separate high-a⁄nity transporter proteins
encoded by the CTR1 and CTR3 genes. High-a⁄nity cop-
per uptake is energy dependent and speci¢c for Cu(I) over
other metals [2]. A third, lower-a⁄nity system, Fet4, for
copper uptake has also been detected in yeast [33^35].
This review will focus on the better-understood copper
homeostatic mechanisms from E. coli. These encompass
the P-type ATPase CopA, the four-part Cus copper e¥ux
complex, the multi-copper oxidase CueO and the plasmid-
borne Pco system. Finally, we will try to combine recent
¢ndings in enzymatic functions of copper detoxi¢cation
with regulatory networks, to form a complex picture of
copper homeostasis in the model organism E. coli within
its changing environment.
2. CopA is responsible for cytoplasmic copper
homeostasis
CopA is the central component of copper homeostasis
in E. coli and is required for intrinsic copper resistance
under both aerobic and anaerobic conditions. The 834-
amino acid (aa) CopA is a Cu(I)-translocating P-type
ATPase that is regulated by CueR. CueR is activated by
intracellular Cu(I) and Ag(I) levels [19^21]. In addition,
the expression of copA may be in£uenced by CpxR, in
response to cell envelope stress [19]. The activity of the
copA promoter is not very di¡erent under both aerobic
and anaerobic conditions. However, the basal level of
transcription is higher under anaerobic conditions indicat-
ing that cytoplasmic copper concentrations might be high-
er than under aerobic conditions [7].
The superfamily of P-type ATPases is a ubiquitous
group of proteins involved in transport of charged sub-
strates across biological membranes [36]. One branch con-
tains the phylogenetically related subgroup of P-type
ATPases that catalyze transport of transition- or heavy-
metal ions. They have previously been described as CPx-
type ATPases [37] or soft-metal-transporting P-type ATP-
ases [16]. These ATPases have eight transmembrane do-
mains [38], in contrast to the hard-metal P-type ATPases,
which have 10 transmembrane segments. Soft-metal-trans-
porting P-type ATPases can be further divided into sub-
groups that contain Cu(I)/Ag(I)-translocating ATPases
and Zn(II)/Cd(II)/Pb(II)-translocating ATPases [16,39,
40]. Well-studied prokaryotic copper transport systems
are the copper ATPases of E. hirae, CopA and CopB
[41], and CopA from Helicobacter pylori [42] and E. coli
[22]. Eukaryotes possess similar copper ATPases, such as
Ccc2 in S. cerevisiae, RESPONSIVE-TO-ANTAGO-
NIST1 in Arabidopsis thaliana [43] and ATP7A [44] and
ATP7B [45] in humans. The results of in vitro copper or
silver transport experiments and copper-stimulated ATP-
ase activity demonstrate that the transported species are
Cu(I) and Ag(I). ATP7A (Menkes disease-related protein),
E. hirae CopB and E. coli CopA were shown to transport
copper only in the presence of dithiothreitol (DTT), con-
sistent with transport of Cu(I). Recent results demon-
strated that CopA formed a acylphosphate intermediate
with [Q-32
P]ATP only in the presence of Cu(I) or Ag(I)
but not with Cu(II), Zn(II), or Co(II) [46]. ATPase activity
of puri¢ed CopA also was only stimulated by Cu(I) and
Ag(I). CopA from the thermophile Archaeoglobus fulgidus
required both DTT and cysteine for copper-dependent
stimulation of ATPase activity. DTT was needed for re-
duction of copper to Cu(I) [47].
CopA possesses two putative CXXC motifs in the N-
terminal domain that are potential metal-binding sites.
However, aa substitutions by site-directed mutagenesis
showed that these motifs are not required for function
and do not confer metal speci¢city [48]. Similar results
were also obtained with ATP7A, ATP7B, CadA from Lis-
teria monocytogenes and ZntA from E. coli [49^52]. While
these metal-binding motifs might not be required for func-
tion they might have a regulatory e¡ect. The apparent Km
of Cu(I) in acylphosphate intermediate formation in wild-
type CopA is 1.5 WM but in a quadruple mutant with all
cysteines substituted by serines the Km is lowered to
0.45 WM in an ATPase assay with puri¢ed CopA. How-
ever, there could be an alternative explanation of these
counterintuitive results. It is possible that the CXXC mo-
tifs bind copper in this in vitro assay and the apparent Km
is higher than in the absence of these motifs [46].
3. The proton-driven Cus system
In contrast to Gram-positive bacteria such as Bacillus
subtilis, a Gram-negative bacterium not only needs to safe-
guard the cytoplasm but also has to translocate metals
across the outer membrane to protect the vital periplasmic
compartment from metal-induced damage. A family of
related transport systems exclusively found in Gram-neg-
ative bacteria is that of the CBA-transport systems. These
are involved in export of metal ions, xenobiotics, and
drugs [53^55], and usually contain three di¡erent structur-
al proteins encoded by a single operon. The central pump
protein belongs to the resistance nodulation cell division
family (RND). RND proteins are secondary transporters
probably energized by proton^substrate antiport [54,56^
58]. The two other components are a membrane fusion
protein (MFP) [59] and an outer membrane factor
(OMF). MFPs were also grouped into the family of peri-
plasmic e¥ux proteins [60] or periplasmic adaptor proteins
[61]. OMFs are associated with the outer membrane and
span the periplasmic space [53,62]. Recently, crystalliza-

tion of the OMF TolC has revealed that TolC, as a trimer,
assembles into a transperiplasmic channel-tunnel em-
bedded in the outer membrane [63]. Therefore, according
to a recent model RND, MFP and OMF interact to form
an active channel spanning the periplasm and thus connect
the cytoplasm to the outer membrane (Fig. 2). Prominent
metal-transporting CBA-type systems have been charac-
terized in Pseudomonas, Ralstonia, Synechococcus, Salmo-
nella and E. coli. Thus, they seem to be widespread in
Gram-negative bacteria and probably confer additional
resistance to certain heavy metals. Currently, the czc de-
terminant is the best-characterized metal CBA-like trans-
porter. In the highly metal-resistant bacterium Ralstonia
metallidurans strain CH34, resistance to zinc, cadmium,
and cobalt is catalyzed by the gene products of the co-
balt^zinc^cadmium (czc)-resistance operon [56,58]. The
Czc system is an e¥ux pump that functions as a chemios-
motic divalent cation/proton antiporter [56,58,64]. The czc
determinant encodes three structural proteins, CzcA, CzcB
and CzcC. These proteins have become the prototype
CBA-type family of three-component chemiosmotic ex-
porters, including members that e¥ux toxic cations or
organic compounds [65]. An in-depth look into CBA
transporters is provided by a review by Nies [170].
The RND proteins confer substrate speci¢city [66,67]
whereas the OMF proteins are often interchangeable since
members of the OMF family could functionally substitute
for each other. A R. metallidurans strain deleted in cnrC
encoding the OMF protein of the cobalt^nickel-resistance
determinant was functionally substituted in trans by the
related czcC or nccC gene [68]. A similar result was ob-
tained with antibiotic-resistance systems from Pseudomo-
nas aeruginosa [69^74].
In E. coli the copper (and silver)-translocating Cus sys-
tem is encoded on the chromosome as a determinant com-
prising two operons transcribed in opposite directions.
One operon encodes the regulators CusRS that form a
two-component regulatory system. CusS is a histidine ki-
nase located at the cytoplasmic membrane probably sens-
ing copper ions in the periplasm. CusR acts as a response
regulator that activates transcription of cusCFBA. The cus
determinant is induced by copper (and to a lesser extent by
silver) and both genetic and phenotypical analysis strongly
suggests that the products of cusCFBA form a copper-ex-
truding complex ([7,17,75]; S. Franke et al., unpublished)
(Fig. 2).
In our opinion the Cus system transports copper di-
rectly from the periplasm across the outer membrane. Ini-
tial evidence suggesting that cations are transported from
the periplasm and not the cytoplasm came from genetic
analysis in E. coli. Although transcription of the cus de-
terminant was induced by copper, deletion of cusCFBA
failed to show a copper-sensitive phenotype under aerobic
conditions. Cells disrupted in copA (and no longer encod-
ing the Cu(I)-translocating P-type ATPase) are more cop-
per sensitive than wild-type cells. A double mutant, where
both copA and cusCFBA are disrupted, is no more copper
sensitive than a strain only disrupted in copA [75]. This
indicates that cusCFBA is not an alternative pump for the
removal of copper ions from the cytoplasm. However, a
disruption of both cueO, encoding the multi-copper oxi-
dase in E. coli (see Section 4), and cusCFBA showed a
Fig. 2. Functional model of the Cus e¥ux complex. The four-part Cus complex consists of the inner membrane pump CusA, the periplasmic protein
CusB and the outer membrane protein CusC forming a channel bridging the periplasmic space. Entry of copper may occur from the periplasm (A),
from the cytoplasm (B) or via the copper-binding chaperone CusF from the periplasm (C).

dramatic increase in copper sensitivity even in the presence
of CopA. These results suggest that CusCFBA transports
periplasmic copper. We also assume that this is true for
homologous systems such as CzcCBA. This hypothesis is
supported by the observation that other transporters of
the RND superfamily such as AcrB and MexB can also
transport substrates that do not cross the cytoplasmic
membrane [76]. These ¢ndings indicate that binding of
the substrate can occur on the periplasmic side of the
transporter [77].
A previous model of the well-studied RND pump,
CzcA, suggested that cations are pumped out of the cyto-
plasm while pumping protons in, since CzcA possesses
both a putative proton channel and a putative cation
channel. However, we think this model is only partially
correct because the Km measured in transport experiments
is not physiological at 10 mM for zinc [57]. Rather, cations
could also be transported from the periplasm or the peri-
plasmic side of the cytoplasmic membrane across the outer
membrane. The recent crystal structure of AcrB, a related
RND transporter, suggests the presence of two possible
pathways. Substrates could be transported from the cyto-
plasm or the periplasm [78].
Data obtained with CzcA suggested a protein topology
encompassing 12 transmembrane domains and two large
periplasmic loops that probably also applies to CusA
(Figs. 2 and 3) [57]. The periplasmic loops potentially con-
tain metal-binding sites and may prevent metal cations
from entering the cell by pumping them across the outer
membrane while they are at or near the cytoplasmic mem-
brane. Recent mutational analysis of CusA indicated that
some methionine residues located in the second periplas-
mic loop of CusA are important for function (Fig. 3; S.
Franke et al., unpublished). Those methionines are not
present in Zn(II)- or Ni(II)-transporting RND proteins.
However, they are conserved in other homologous, puta-
tive copper-translocating CBA-like transporters. There-
fore, it is tempting to speculate that some of those methio-
nine residues are involved in copper binding or transport.
Moreover, transmembrane segment four of monovalent
metal cation (Cu[I], Ag[I])-translocating RND transport-
ers distinctively di¡ers from those transporting divalent
metal cations or antibiotics (Table 2).
The CusA protein (1047 aa) is thought to be the central
component, responsible for copper transport. Additional-
ly, the putative channel-forming CusC protein (457 aa)
and the clamping CusB (407 aa) are necessary for full
Cus-mediated copper resistance (S. Franke et al., unpub-
lished). An in-frame deletion of either cusB or cusC re-
Table 2
Di¡erent motifs in transmembrane segment IV of RND proteins prob-
ably determine substrate entry
Transport of Amino acid
sequencea
Examples
Monovalent metals AX5DX6E CusA (E. coli)
SilA (S. enterica)
Divalent metals DX5DX6E NccA (A. xylosoxidans)
CzcA (R. metallidurans)
Antibiotics AX5DDX5E AcrB (E. coli)
MexB (P. aeruginosa)
a
Binding motifs were derived from Goldberg et al. [57], Guan and Na-
kae [79] and Aires et al. [80].
Fig. 3. Two-dimensional topological model of CusA with e¡ects of aa substitutions. Topology of CusA was modeled by analogy to other RND trans-
porters such as CzcA (from R. metallidurans), AcrB (from E. coli), MexB and MexD from P. aeruginosa). Changed aa residues are boxed and the ef-
fects of these mutations are shown on the right. Aa residues A399, D405 and E412 in transmembrane segment IV denote the signature motif of mono-
valent cation-transporting RND proteins. PP, periplasm; CPM, cytoplasmic membrane; CP, cytoplasm.

sulted in nearly complete loss of function of the Cus sys-
tem. OMFs are thought to be associated with the outer
membrane. Some OMFs such as OprM are predicted to be
lipoproteins and possess a cysteine residue at their N-ter-
minal end after signal peptidase processing. This lipopro-
tein box is responsible for thiol^ether linkage to diacylglyc-
eride. Since these proteins do not contain an aspartate at
position two of the mature protein they are localized to
the outer membrane [81^83]. CusC contains both a signal
sequence and a cysteine residue at aa residue position 18
[17] and is closely related to OprM and MtrE, both in-
volved in drug e¥ux. Other OMFs of metal-resistance
determinants such as CzcC or NccC are only distantly
related and do not have a cysteine residue at their N-ter-
minus ([63]; G. Grass and D.H. Nies, unpublished). If
attachment to the outer membrane is accomplished in
both cases, the underlying mechanisms might be di¡erent.
However, the activity and expression of OprM with a
mutation of the highly conserved N-terminal cysteine res-
idue was not a¡ected indicating acylalation is not impor-
tant for function [84].
A unique feature of copper/silver CBA-type transporters
is the presence of a small (110-aa) periplasmic protein,
CusF, in the Cus system. CusF was characterized recently
(S. Franke et al., unpublished). It could be shown that the
mature protein binds one copper per polypeptide and that
the puri¢ed copper-containing protein is pink in color. A
non-polar deletion of cusF led to a decrease in Cus-medi-
ated copper resistance. Mutational analysis indicated that
copper binding in CusF is accomplished in part by two
conserved methionine residues. Other potential metal-
binding residues such as ¢ve histidine residues, a con-
served phenylalanine or a conserved aspartate residue
seem not to be involved in copper binding. Possibly
CusF is a periplasmic metallochaperone transporting cop-
per to the CusCBA e¥ux complex and thus facilitating
copper detoxi¢cation of the periplasm (Fig. 2). Since the
cus determinant is also responsible for a small increase in
Ag(I) resistance [85], it is likely that the transported cop-
per species is Cu(I).
In contrast to the CueO multi-copper oxidase that re-
quires oxygen for function, the CusCBA e¥ux complex
works under both anaerobic and aerobic conditions. Re-
cent results from Outten et al. [17] clearly showed Cus-
mediated copper detoxi¢cation is especially important
under anaerobic conditions. Under aerobic conditions
copper sensitivity is only observed when both cueO and
cusCFBA are deleted.
4. CueO is a multi-copper oxidase protecting the periplasm
from copper-induced damage
The copA gene encoding the copper e¥ux P-type ATP-
ase and cueO are both regulated by CueR. Together they
constitute the Cue system. CueO (516 aa) is a multi-copper
oxidase that possesses laccase-like activity. Recently, sev-
eral other microbial protein sequences showing similarities
to fungal laccases were identi¢ed [86]. These bacterial mul-
ti-copper oxidases have been implicated in antibiotic bio-
synthesis, sporulation, copper tolerance, morphogenesis
and manganese oxidation [23,87^89]. Multi-copper oxi-
dases couple the one-electron oxidation of substrate(s) to
full reduction of molecular oxygen to water by employing
a functional unit formed by three types of copper-binding
sites with di¡erent spectroscopic and functional properties
[90]. Type 1 blue copper (T1) is the primary electron ac-
ceptor for the substrate, while a trinuclear cluster formed
by type 2 copper and binuclear type 3 copper (T2/T3) is
the oxygen-binding and -reduction site. Prominent mem-
bers of this group are mammalian ceruloplasmin, plant
ascorbate oxidases and fungal laccases. By de¢nition, lac-
cases (p-diphenol:O2 oxidoreductase EC 1.10.3.2) catalyze
the oxidation of p-diphenols with the concurrent reduction
of dioxygen to water, although the actual substrate specif-
icities of laccases are often quite broad and vary with the
enzyme source. These multi-copper oxidases are widely
found in fungi and plants and numerous laccases have
been cloned from these sources [91^93]. In contrast to
our understanding of the electron transfer reactions that
occur in laccases, relatively little is known about their
physiological function. In fungi, laccases have been impli-
cated in processes such as pigmentation, fungal sporula-
tion, lignin degradation and biosynthesis, and pathogene-
sis [94]. However, very few of these functions have been
experimentally proven.
It has been shown that CueO protects periplasmic en-
zymes from copper-induced damage [23]. Sequence analy-
sis of CueO revealed a twin-arginine motif within the lead-
er sequence. In fact, CueO possesses a perfect signal
peptide sequence recognized by the Tat translocation ma-
chinery. The Tat pathway transports fully folded proteins,
with their cofactors already bound before translocation
into the periplasm [95]. Therefore, loading of copper
into CueO in the cytoplasm with subsequent transport
into the periplasm could contribute to cytoplasmic copper
detoxi¢cation. However, the impact of this CueO-depen-
dent copper translocation on copper resistance would only
be marginal. The P-type ATPase CopA can transport cop-
per much more e⁄ciently across the cytoplasmic mem-
brane. CueO function is strictly oxygen dependent even
though expression of the cueO gene also occurred under
anaerobic conditions. However, the cueO promoter re-
vealed a strong dependence on oxygen. Furthermore, ex-
pression of cueO is very sensitive to copper. Compared to
other copper homeostasis mechanisms examined in E. coli,
expression of cueO showed the lowest half maximum in-
duction (3 WM) by copper [17].
The three-dimensional structure of CueO was recently
solved at 1.4 Aî resolution [96]. Four copper ions are
bound in the CueO monomer, forming an active center
typical for multi-copper oxidases such as ascorbate oxi-

dase, fungal laccases, Fet3 or ceruloplasmin [96]. Enzy-
matic activity of CueO is regulated by copper and shows
enhanced oxidase activity only in the presence of addition-
al copper. Recently, the crystal structure of CueO with
exogenous copper bound has been determined at 1.85 Aî
resolution, identifying a ¢fth copper-binding site (Fig. 4).
A copper (II) ion is found to bind to the protein 7.5 Aî
from the T1 copper site in a region rich in methionine
residues. Copper was bound to two methionine sulfur
atoms, Met 441 and Met 359, and two aspartate carbox-
ylic acid oxygens, Asp 439 and Asp 460. The other car-
boxylate oxygen of Asp439 is only 2.9 Aî apart from and
apparently hydrogen bonded to His 443. The ND1 atom
of this histidine ligates the T1 copper. Thus, this bound
copper is in a position to mediate electron transfer from
substrates to the T1 copper (S.A. Roberts et al., unpub-
lished).
At this point the mechanism by which CueO protects
cells from copper-induced damage is still unknown. Char-
acterization of puri¢ed CueO demonstrated that it is also
able to oxidize ferrous iron [23,97]. Iron oxidation prior to
uptake was experimentally determined to be the function
of Fet3, a multi-copper oxidase from yeast S. cerevisiae
[98]. However, it is not clear if this is one of the physio-
logical roles of CueO. CueO is able to oxidize enterobactin
and related catechols in vitro, producing a colored precip-
itate [97]. This suggested that the formation of the colored
precipitate might be related to polymerization reactions
involved in microbial melanization processes. Synthesis
of a copper-binding compound would be an excellent
way to decrease toxic copper concentrations and could
be one of the physiological roles of CueO. Another hy-
pothesis is that CueO oxidizes periplasmic Cu(I) to the less
permeable and thus less toxic Cu(II) [7,75]. This activity
could stop both membrane damage and Cu(II) reduction,
thus protecting the cells from copper-induced damage by
preventing the Fenton-like reaction of redox-active cop-
per.
All multi-copper oxidases with a function in copper re-
sistance possess an extensive methionine-rich region prox-
imal to the copper centers (Fig. 4). The role of this domain
in enzymatic function is yet unknown. It could be a reg-
ulatory domain that binds copper and then activates the
enzyme or it could be involved in protein^protein interac-
tions [99]. Another possibility is that this region has a
protective role for the enzyme preventing fragmentation
of the multi-copper oxidase by copper-generated oxygen
radicals [100].
5. The plasmid-encoded pco determinant confers additional
copper resistance
Copper can be introduced into soils via sewage sludge,
mine e¥uents and industrial waste. For more than 100
years copper has been used as the active ingredient in
bacteriocides and fungicides on fruits and vegetables.
Since the mid-1980s, copper-resistant bacterial pathogens
have been detected repeatedly [101]. Copper resistance was
the most frequently found resistance in bacteria from pre-
antibiotic era isolates [102]. Moreover, copper can inhibit
soil organisms and has recently been implicated in induc-
tion of the viable-but-not-cultivable condition [103].
Many soils are de¢cient in essential metals such as cop-
per or cobalt, resulting in mineral disorders that a¡ect
livestock at pasture. A well-documented example is copper
de¢ciency termed ‘swayback’ in sheep from Australia. The
condition appears to occur as a result of copper interact-
ing with other dietary components, particularly molybde-
num and sulfur, resulting in a decrease in copper absorp-
tion. This has led to supplementing the diet of sheep with
copper. Paradoxically, sheep are very susceptible to copper
toxicity and even moderate dietary supplements to prevent
de¢ciency can result in elevated hepatic copper concentra-
tions, liver failure and death of the animal [104]). All
breeds of sheep have a reduced ability to excrete copper
in the bile and will accumulate copper in the liver [105].
However, sheep display a variant copper susceptible phe-
notype when compared with other mammals. For exam-
ple, copper and antibiotics have been used as growth pro-
moters in pig diets for at least 45 years [106]. This practice
has continued because pigs fed such supplemented diets
exhibited an improved average daily weight gain and
food conversion e⁄ciency. It has been proposed that cop-
Fig. 4. CueO with bound exogenous copper. Ribbon drawing of CueO,
with the T1 copper, the Cu3O cluster and the exogenous copper shown
as space-¢lling spheres. The three related azurin-like domains are col-
ored separately, and the methionine-rich helix is located at the lower
right. The regulatory copper lies near the T1 copper site at one end of
a methionine-rich helix. The concentration of methionine side chains in
that region is demonstrated with the methionine side chains shown in
ball-and-stick representation (S.A. Roberts et al., unpublished).

per elicits an antibacterial e¡ect on the bacteria of the pig
gut [107].
Some strains of E. coli can survive in copper-rich envi-
ronments that would normally overwhelm the chromo-
somally encoded copper homeostatic systems. Such strains
possess additional plasmid-encoded genes that confer cop-
per resistance. Copper-resistance operons have been char-
acterized from E. coli, Pseudomonas syringae pv. tomato
and Xanthomonas campestris [108]. The genes from these
copper-resistant determinants are largely homologous and
probably have similar functions. The conjugative plasmid
pRJ1004 confers copper resistance and was isolated from
E. coli in the gut £ora of pigs fed a diet supplemented with
copper sulfate as a growth promoter [109]. The copper
resistance speci¢ed by this plasmid involves the pco gene
cluster, which contains seven genes, pcoABCDRSE [110].
Copper resistance in P. syringae pv. tomato is speci¢ed by
the cop determinant, which contains six genes, cop-
ABCDRS, arranged in two operons, copABCD and
copRS, respectively. This arrangement is also found in
the pco determinant but with an additional gene, pcoE,
further downstream [110^112]. In all cases copper resis-
tance has been shown to be inducible [113,114]. Radio-
active 64
Cu uptake experiments suggested that an energy-
dependent copper e¥ux mechanism is associated with the
pco copper-resistance genes from plasmid pRJ1004 [110].
However, critical evaluation of these experiments shows
that reduced uptake does not necessarily imply an addi-
tional e¥ux mechanism.
Our current model of the resistance mechanism of the
pco determinant is illustrated in Fig. 5. PcoA (605 aa) is
the central protein of the pco determinant. The pcoA gene
encodes a 605-aa protein of the multi-copper oxidase fam-
ily. Since periplasmic extract containing PcoA showed
copper-inducible oxidase activity [115], an indication on
function of the products of the pco determinant might
be gained from the recent results with CueO. In vitro,
catechols, especially enterobactin, were the substrate with
the lowest Km of all substances tested ([97]; G. Grass et
al., unpublished). CueO-mediated oxidation of enterobac-
tin (and its precursors) led to the formation of colored
precipitates. Tetaz and Luke [109] already noticed that
E. coli strains harboring the Pco-plasmid pRJ1004 pro-
duced brown colonies with dark brown centers when
streaked on medium containing CuSO4. In addition,
PcoA could functionally substitute for CueO in E. coli
indicating they have a similar function. Similar to CueO,
PcoA also has a twin-arginine motif in its leader sequence
and is probably translocated by the TAT pathway with
copper bound to its active sites.
PcoB is a 296-aa protein predicted to be an outer mem-
brane protein. In a copper-sensitive E. coli strain pcoA and
pcoB could confer copper resistance at a much lower ex-
pression level compared to pcoA alone, indicating that
they might interact. These ¢ndings are similar to results
obtained in P. syringae, where expression of copA with
copB alone could also confer copper resistance. The pres-
ence of CopC and CopD was only needed for maximal
Fig. 5. Proposed mechanism of Pco-mediated copper detoxi¢cation. Copper enters the periplasm by an unknown mechanism, possibly through OmpC
and OmpF. The function of the outer membrane protein PcoB has not been elucidated. Consequently, copper might be detoxi¢ed by sequestration on
oxidized catechol siderophores or Cu(I) could be oxidized to the less toxic Cu(II) by the multi-copper oxidase PcoA. In order to load copper into cata-
lytic sites within PcoA, PcoC and PcoD might transport copper across the cytoplasmic membrane with PcoC delivering copper to PcoD. PcoE binds
copper in the periplasm and possibly shuttles copper to PcoA.

resistance. These observations are re£ected when looking
at genomic sequences. Often, only homologs of PcoA and
PcoB are encoded on a genome but not PcoC and PcoD.
However, in both the Cop and the Pco system PcoC and
PcoD are required for full resistance. In P. syringae studies
indicated that CopC and CopD may function together in
copper uptake [116]. The homologous PcoC and PcoD
might have a similar function. PcoC containing 126 aa
was shown to bind one copper atom per molecule and
might act as a dimer [115,117]. PcoC could regulate copper
uptake by PcoD, a 309-aa integral membrane protein with
eight putative membrane-spanning domains as predicted
by topology modeling according to Klein et al. [118]. In-
terestingly, in B. subtilis homologs of pcoC and pcoD are
not encoded as separate genes but as a fusion gene termed
ycnJ. The YcnJ protein consists of 541 aa with the N-
terminus being homologous to PcoC and the C-terminus
to PcoD. This suggests that in E. coli PcoC and PcoD
might also interact to form a functional unit (Fig. 5). In
our current model PcoC would deliver periplasmic copper
to PcoD for uptake into the cytoplasm, probably for load-
ing into PcoA.
The pco determinant contains another gene, pcoE, re-
quired to confer full resistance. The pcoE gene is not part
of the pcoABCD operon but is located further downstream
on plasmid pRJ1004. Expression of pcoE is regulated by
its own copper-regulated promoter and is under the con-
trol of the two-component systems CusRS [17,119]. A re-
lated protein, SilE, is encoded by the sil determinant
[120,171]. Expression of pcoE alone led to copper accumu-
lation in the periplasm indicating a role in binding of
copper in the periplasm [121].
Copper resistance in P. syringae pv. tomato is speci¢ed
by the cop determinant, which contains six genes, cop-
ABCDRS, homologous to the equivalent pco genes
[111]. The pco and the cop determinants are regulated by
a plasmid-encoded and a chromosomally encoded two-
component system. In P. syringae alginate synthesis is in-
duced by copper [122]. Alginate was shown to protect cells
from reactive oxygen stress. However, a similar system
does not exist in E. coli. This di¡erence might be one of
the reasons why copper-resistant pseudomonads turn
bright blue whereas copper-resistant E. coli turn brown
[110,111]. Another possibility is that copper bound to oxi-
dized siderophores from Pseudomonas exhibits a blue col-
or, and in E. coli, with di¡erent siderophores, this might
look brown.
Interestingly, homologs of these plasmid-encoded gene
products are also present on the E. coli chromosome.
CueO is related to PcoA and probably has a similar func-
tion. A PcoC homolog, YobA (124 aa), does not contain
any methionines and its function in copper homeostasis is
unknown. In addition, a homolog of PcoD, YebZ (290
aa), with unknown function is present on the E. coli chro-
mosome (Table 3). Like pcoCD, the adjacent yabA and
yebZ genes are probably translated as a dicistronic tran-
script.
6. Regulation of genes involved in copper homeostasis
re£ects di¡erent demands in a changing environment
Copper homeostasis in E. coli is a multi-layered process
not only regulating uptake and e¥ux but also periplasmic
copper sequestration. Genes participating in these process-
es are regulated di¡erently re£ecting di¡erent environmen-
tal demands. Cells are continuously challenged to keep
copper in a delicate balance. While a hypersensitive regu-
lation of expression of genes involved in resistance of a
solely toxic metal, such as mercury, would be bene¢cial
[172], this mechanism would be only inappropriate for
homeostasis of an important trace metal like copper [20].
Three copper-dependent transcriptional regulatory sys-
tems have been identi¢ed in E. coli, the DNA-binding
CueR regulator, and the chromosomal (CusRS) and plas-
mid-encoded (PcoRS) two-component regulators [121].
CueR is a member of the MerR family of metal-responsive
regulators and regulates copA and cueO [19]. Metal-re-
sponsive MerR-like regulators are broadly distributed in
bacteria. Previously characterized members include the ar-
chetype mercury-responsive MerR [123,124], ZntR respon-
sible for zinc-dependent expression of ZntA, the Zn(II)
e¥ux ATPase from E. coli [125], and PbrR, regulating
the R. metallidurans CH34 lead-resistance determinant
[126]. MerR-like regulators are discussed more extensively
in a review by Brown et al. [172]. Even though members of
Table 3
Function of plasmid-encoded copper resistancea
and chromosomal homologs in E. coli
Homologs Localization Function
PcoA CueO Periplasm, monomeric Multi-copper oxidase, copper binding, Cu[I] oxidation?, oxidation of catechols
PcoB ^ Outer membrane Copper binding?
PcoC YobAb
Periplasm, dimeric? Copper binding
PcoD YebZb
Inner membrane Copper uptake?
PcoE ^ Periplasm Copper chaperone?, copper binding
PcoR CusR Cytoplasm Transcriptional activator DNA binding,
PcoS CusS Inner membrane Histidine kinase, periplasmic copper (Cu[I]) sensing
a
Plasmid pRJ1004 carries the pco determinant [110].
b
YobA (P76279) and YebZ (P76278) are putative ORFs located on the E. coli chromosome.

the MerR family share crucial features such as DNA and
metal binding they recognize distinct metal substrates re-
sulting in activation of cognate promoters.
Initially an unusually long spacing in PcopA suggested a
possible copper-responsive signal transduction system of a
MerR-like regulator [19]. Consequently, an E. coli strain
deleted in cueR lost copper-dependent PcopA induction. In
contrast to the regular 17-bp spacer in RpoD-dependent
promoters, a palindromic sequence in an extended 19-bp
spacer between the 335 and 310 sites of PcueO and Pcop-
A could be identi¢ed [20]. This sequence is reminiscent of
those found within promoters regulated by other members
of the MerR family of transcriptional regulators. The
dyad sequence has 7 bp of perfect symmetry and is shared
with the binding motif of MerR. However, there is no
obvious similarity between the CueR- and MerR-binding
7-bp motifs [20].
Upstream of the 335 site a weak consensus UP element
can be identi¢ed next to a CpxR-binding site [19]. CpxR is
the cell envelope stress response regulator [127]. A deletion
of this site led to reduced induction of PcopA under high
copper concentrations, indicating PcopA is not only regu-
lated by CueR but also by CpxR [19]. In Salmonella typhi
and Salmonella typhimurium cueR and copA are tran-
scribed divergently but in E. coli they are separated by
two open reading frames (ORFs) [20]. It is not known if
those ORFs are involved in copper homeostasis or, more
likely, represent recent events of genomic rearrangement.
CueR senses cytoplasmic copper and silver, and tran-
scription of PcopA is activated in response to elevated
concentrations [20]. CueR-dependent induction of PcopA
exhibits a hypersensitive response to silver ions, but a lin-
ear response to copper ions. Such a regulation mechanism
makes biological sense considering that silver occurs as
Ag(I) only but copper is in a steady state between Cu(I)
and Cu(II) and that both Ag(I) and Cu(I) are toxic for
cells. CueR shares a helix-turn-helix motif for DNA bind-
ing at the N-terminal end with other MerR-like regulators
such as ZntR [128] or MerR [129] and a C-terminal metal-
binding site. It could be demonstrated that puri¢ed CueR
bound to PcopA between 335 and 310 in a DNase I
footprint assay [19^21]. Interestingly, a conserved cysteine
residue in ZntR and MerR is absent in CueR. Addition-
ally, spacing between domains within the proteins is di¡er-
ent which might be responsible for speci¢city towards cop-
per [19].
Several promoters of copper (and silver)-inducible genes
are preceded by a palindromic sequence, termed ‘cop-box’.
Cop-boxes are the sites of DNA binding of a cognate
response regulator. On the E. coli chromosome cop-boxes
can be found upstream of cusC and on plasmid pAP173
upstream of pcoA and pcoE [17,119]. In other organisms
such as Salmonella enterica serovar Typhimurium or
P. syringae similar cop-boxes can be identi¢ed preceding
silC and silE or copA and copH, respectively [130,131].
Whereas the copA, cueO and cusCFBA regulation re£ects
copper handling under ‘daily life’ circumstances for E.
coli, expression of the plasmid-borne pco determinant is
only required when these housekeeping systems are over-
whelmed. Accordingly, expression of the pco structural
genes is not hypersensitive to external copper concentra-
tions [119]. The highly e¡ective pco determinant is only
expressed under elevated copper concentrations.
The regulators PcoRS are required for copper-inducible
expression of copper resistance mediated by the pco deter-
minant [110]. PcoS possesses two transmembrane segments
with peptide loops extending into the periplasm. With in-
creasing copper levels, this kinase phosphorylates the cog-
nate response regulator PcoR. It in turn can control tran-
scription. Mutation disrupting pcoRS abolishes high-level
copper resistance [110]. However, copper-dependent ex-
pression from PpcoA and PpcoE was not completely lost
[119]. Yet, the regulators PcoRS are required for maxi-
mum copper-inducible expression from PpcoA. In con-
trast, the pcoRS genes are not required for copper-induc-
ible expression of PpcoE located downstream of
pcoABCD. PcoE reduces the time required for E. coli
strains to recover from copper-mediated stress [17]. Rouch
and Brown [119] recognized that the two promoters of pco
remain regulated by a chromosomal determinant, now
identi¢ed as CusRS, even when the pcoRS genes were
deleted.
The similarity between CusR and PcoR is 61% and
between CusS and PcoS 38%. In the absence of CusS,
CusR might be activated by (an)other histidine kinase(s).
In a vcusRS strain pcoRS were able to provide copper-
inducible expression of a L-galactosidase reporter under
the control of PpcoA, but not from PpcoE or PcusC.
Thus, the regulators CusRS provide copper-inducible ex-
pression of PpcoE and PcusC and an increased basal level
of PpcoA. On the other hand, the regulators PcoRS regu-
late PpcoA but not PpcoE and PcusC. Taken together the
related two-component regulator pairs CusRS and PcoRS
are able to function independently [17].
CutC is a cytosolic protein of 248 aa and a vcutC dele-
tion strain showed copper sensitivity at high copper con-
centrations. Previous studies have implicated CutC in cop-
per e¥ux, suggesting a role of CutC in intracellular
tra⁄cking of Cu(I), since an increase in cytosolic Cu(I)
would undoubtedly lead to intracellular damage [5,132].
The cutC gene is probably regulated by the extracytoplas-
mic function sigma factor RpoE [133,134]. The RpoE-de-
pendent extracytoplasmic stress response in E. coli is in-
duced by excessive amounts of unfolded proteins in the
envelope of the cell, particularly unfolded outer membrane
porins [135^138].
7. Conclusions and perspective
Considerable progress in understanding the basic mech-
anisms of copper handling in E. coli has been made in

recent years. However, large pieces of the puzzle are still
missing. We are only beginning to understand the metal-
lochaperones involved in cytoplasmic and periplasmic cop-
per tra⁄cking, and not much is known about copper up-
take systems in bacteria including E. coli. While copper
uptake mechanisms in E. coli still await discovery, in the
Gram-positive bacterium E. hirae copper homeostasis is
accomplished by the cop operon, which consists of the
four genes copY, copZ, copA and copB [41,169]. CopA
and CopB are both copper-translocating P-type ATPases
and earlier data suggested that CopA serves in copper
uptake under conditions of copper limitation, whereas
CopB serves in copper extrusion when intracellular copper
reaches toxic levels [139]. However, this has not been
shown for CopA through direct transport measurements.
In contrast, in E. coli the chromosomally encoded P-type
ATPase CopA exclusively functions as an e¥ux transport-
er.
Transport of copper across the outer membrane into the
periplasm has been shown for NosA in Pseudomonas stut-
zeri and OprC in P. aeruginosa [140,141]. At this point, no
protein in bacteria responsible for copper uptake across
the cytoplasmic membrane has conclusively been identi¢ed
and characterized.
No intracellular copper chaperone has yet been identi-
¢ed in E. coli but we believe that in the future, metallo-
chaperones with speci¢city for copper will be identi¢ed in
E. coli and other related bacteria. The E. coli genome
contains many ORFs of unknown function containing a
CXXC motif that could potentially be involved in copper
binding. However, other proteins that do not possess a
CXXC motif such as CutC might also be copper chaper-
ones. Eukaryotes possess speci¢c copper chaperones re-
sponsible for intracellular copper tra⁄cking [142,143].
These proteins possess no obvious sequence similarities
except for a conserved CXXC metal-binding site
[15,144]. In yeast, the copper metallochaperone Atx1 de-
livers copper to the Cu(I)-translocating P-type ATPase
Ccc2 which delivers copper into the Golgi apparatus for
insertion into the multi-copper oxidase Fet3 [145]. In some
prokaryotes, Atx1-related proteins have also been identi-
¢ed. Examples include Atx1 from the cyanobacterium
Synechocystis PCC 6803 [146] and CopZ from E. hirae
and B. subtilis [147,148]. CopZ from E. hirae and B. sub-
tilis might interact with the N-terminus of the Cu(I)-trans-
locating P-type ATPases in these organisms [149,150]. So
far no copper-speci¢c cytoplasmic chaperone has been
identi¢ed in E. coli. It was speculated that the cytosolic
N-terminal part of the copper-ATPase CopA is cleaved o¡
and acts as a copper chaperone [151]. However, the ab-
sence of a phenotype in a CopA mutant with all four
cysteines substituted by serines suggests that this region
is not required for function and is probably not a chaper-
one [48]. In several Streptomyces species the copper chap-
erone MelC1 delivers copper to a binuclear copper center
(CuA and CuB) in tyrosinase that is required for melanin
biosynthesis. The chaperone MelC1 maintains the state of
apotyrosinase, which facilitates copper incorporation and
secretion [152,153].
These results indicate copper chaperones in bacteria are
widespread and this might also be true for zinc. Recently,
it could be shown that several bacterial species harbor
zinc-speci¢c metallothioneins [154]. The bacterial metallo-
thioneins are cysteine-rich small proteins that bind multi-
ple metal ions. A deletion of the metallothionein gene
smtA in Synecchococcus PCC 7942 resulted in a ¢ve-fold
decrease in zinc resistance [155]. GatA, a related protein,
has recently been identi¢ed in E. coli. GatA binds one
Zn(II) through its cysteine residues whereas SmtA from
Synecchococcus is able to sequester four zinc cations
[154]). A role for GatA in the sequestration of excess
zinc appears unlikely since GatA contains an SmtA-like
zinc ¢nger fold but a metallothionein cluster is not
present. These results also illustrate that caution needs
to be exercised in the prediction of speci¢c roles for pro-
teins by sequence comparison. A protein might bind cop-
per or zinc but this ability to bind metals is without phys-
iological consequence. We might also discover metal
chaperones with previously unknown metal-binding mo-
tifs.
Copper is required in electron transport systems and as
a cofactor in enzymes such as oxidases and hydrolases.
However, it is toxic in its cuprous form for its ability to
generate hydroxyl radicals in the presence of superoxide.
This reaction is analogous to the Fenton reaction of fer-
rous iron [155,156]. The soxRS regulon of E. coli is ex-
pressed under oxidative stress. The SoxR protein is the
superoxide-sensitive activator of the soxS gene. The con-
stitutively expressed SoxR protein has homology to the
MerR-type family of regulators and is a homodimer
with two [2Fe^2S] centers per dimer. The oxidation of
the reduced [2Fe^2S]1þ
form of SoxR to a [2Fe^2S]2þ
form leads to SoxR activation [157]. This active form of
SoxR leads to enhanced soxS transcription and increased
levels of SoxS activate expression of the soxRS regulon
[158]. It was shown that in the presence of molecular oxy-
gen, CuSO4 acted as an inducer of SoxR-dependent soxS
expression. This indicates that copper toxicity in E. coli is
caused by the generation of reactive oxygen species. There-
fore, an E. coli strain lacking either oxidative DNA repair
enzymes or SODs was hypersensitive to killing by copper
[5]. Additionally, copper toxicity might be enhanced by
enzymes such as NADH reductase-2, which was shown
to be capable of reducing cupric to cuprous ions in E.
coli [31]. Copper (CuSO4) might a¡ect NADH dehydroge-
nase or other respiratory enzymes by causing a redox im-
balance [5]. However, the same properties that make cop-
per toxic help copper-containing enzymes achieve rapid
rates of catalysis.
Pathogens often have to ward o¡ toxicity due to the
production of reactive oxygen species as part of the host
defense. There has been clear evidence of copper-contain-

ing enzymes such as Cu,Zn-SOD or multi-copper oxidases
as virulence factors. Expression of Cu,Zn-SOD enhances
intracellular survival of E. coli and S. enterica Typhimu-
rium [159,160]. The E. coli multi-copper oxidase CueO
might be an excellent model system to understand how
related enzymes protect pathogenic organisms. In the fun-
gus Crypotococcus neoformans the multi-copper oxidase
laccase and its product melanin were identi¢ed as impor-
tant virulence factors. Host cellular immunity might be
compromised by melanin and its ability to act as an anti-
oxidant or to protect the cell wall surface [161]. Melanized
cells of C. neoformans were more resistant to antibody-
mediated phagocytosis and the antifungal e¡ects of murine
macrophages than non-melanized cells [162]. Because mel-
anins are e⁄cient radical scavengers, melanized cells were
less susceptible to killing by oxygen- or nitrogen-derived
radicals [163,164]. Electron spin resonance spectra indi-
cated electron transfer between melanin and chemically
generated free radicals. Furthermore, melanin appears to
contribute to virulence by protecting fungal cells against
host defense attack [165].
Some of the copper-regulated genes can accomplish sur-
prising additional tasks. One example is IbeB (CusC) from
E. coli K1. E. coli K1 is the most common Gram-negative
organism causing neonatal meningitis. Invasion of brain
microvascular endothelial cells is a prerequisite for pene-
tration into the human central nervous system. It was
demonstrated that the IbeB protein of E. coli K1 is an
important contributing determinant [166,167]. However,
at this moment it is not apparent how IbeB functions in
bacterial penetration.
Elucidation of copper homeostasis in the bacterial mod-
el organism E. coli will result in understanding of the
delicate balance of this important yet toxic redox-active
metal. We anticipate great progress in the near future,
not only through genomics and proteomics, but also
from an ambitious, global project to model E. coli called
the International E. coli Alliance [168].
Acknowledgements
We would like to thank Sylvia Franke and Dietrich H.
Nies for their kind permission to share recent data on the
Cus system prior to publication. Research in the labora-
tory of C.R. was supported by Hatch project 136713 and
by grant 5P42 4940-13 from the NIEHS Basic Research
Superfund.
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