(Received for publication, October 18, 1994; and in revised form, December 1, 1994 )
From the
Enterococcus hirae possesses two P-type ATPases, CopA and CopB, that are involved in copper homeostasis. These enzymes are induced by extracellular copper concentrations that are either too low or too high for optimal growth. To identify the regulatory proteins involved in induction, the DNA upstream of copA was cloned and sequenced. Following a putative promoter region, it contains two genes, copY and copZ, that encode proteins of 145 and 69 amino acids, respectively. Both proteins contain metal binding motifs and exhibit significant sequence similarity to known regulatory proteins. Gene disruption of copY by reverse genetics caused constitutive overexpression of CopA and CopB, generating a copper-dependent phenotype. In contrast, disruption of copZ suppressed the expression of the two copper-ATPases, rendering the cells copper-sensitive. Both null mutations could be complemented in trans with plasmids bearing copY or copZ. Thus, copY and copZ encode trans-acting metalloregulatory proteins that are required for induction of the cop operon by copper. In this mechanism, CopY apparently acts as a metal-fist type repressor and CopZ as an activator.
As a cofactor of many enzymes, copper is an essential heavy metal ion. Yet, copper can be very toxic to both eukaryotic and prokaryotic cells. A series of homeostatic mechanisms has evolved to balance copper detoxification mechanisms with the need to acquire essential copper at low levels. One such mechanism that has only recently been discovered is ATP-driven copper transport by P-type ATPases, first described for the Gram-positive bacteria Enterococcus hirae in 1992(1) . In this organism, two similar P-type ATPases, CopA and CopB, are transcribed from the same operon, and the data suggest that CopA serves in the uptake and CopB in the extrusion of copper by the cell(2) .
In an exploding
fashion, a number of other suspected copper-ATPases have since been
identified. In humans both the Menkes and the Wilson genes were found
to encode putative
copper-ATPases(3, 4, 5, 6, 7, 8) .
The gene pacS of the cyanobacteria Synechococcus species PCC7942 was also found to encode a copper-ATPase that is
located in the thylakoid membrane(9) . Similar ATPase genes
reported are fixI from Rhizobium
meliloti(10) , CCC2 and ORF YBR295w from Saccharomyces cerevisiae, copA from Helicobacter
pylori, ORF o732 from Escherichia coli, and another gene, synA, from Synechococcus (GenBank/EMBL
Data Bank accession numbers X15079, L36317, Z36164, L33259, U00039, and
U04356, respectively).
The activity of copper-ATPases and associated metal ion binding proteins involved in copper homeostasis must be appropriately controlled in response to the copper concentration; hence, tightly regulated systems are required. The two copper-ATPases of E. hirae were shown to be regulated in their expression by the extracellular copper concentration: a low level of expression was observed in 10 µM copper, and strong induction resulted from either limiting or near toxic levels of copper in the media(11) . However, the regulation of the activity and expression of these pumps was so far not understood.
Several copper-regulated bacterial expression systems are known. In Pseudomonas syringae, a two-component regulatory system is required for copper-inducible expression of the plasmid-determined copper resistance operon. It involves two constitutively expressed proteins, CopR and CopS. The current model suggests that the transmembranous CopS senses high levels of free copper ions in the periplasm, phosphorylates CopR, and converts it from an inactive to an active state to induce expression of the cop operon. An effect of copper on the binding of CopR to the promotor could not, however, be demonstrated, and there are no putative copper-binding sequences in the protein. CopR and CopS exhibit sequence similarity to other two-component activator/sensor systems like PhoB/PhoR and OmpR/EnvZ(12, 13) . A similar but less well characterized two-component activator/sensor system exists in E. coli, where PcoR and PcoS regulate plasmid-determined copper resistance and CutR and CutS chromosomally encoded copper resistance, respectively(14) .
The copper-regulated expression system
that has so far been described in most detail is the ACE1 gene
of S. cerevisiae that controls the expression of the CUP1 metallothionein locus. At toxic metal ion concentrations,
the ACE1 protein forms a complex with Cu or, with
lower stability, Ag
, which then binds to a cis-acting copper control sequence upstream of the
transcription initiation site of CUP1 to induce transcription
of the metallothionein gene(15, 16) . A similar
transcription factor, AMT1, was described in Candida glabrata.
AMT1 functions like ACE1 as a trans-activating factor by
binding to a cis-acting element and activating metallothionein
transcription(17) . These and other metalloregulatory proteins
have been called metal-fist transcription factors.
Here, we describe two new E. hirae genes, copY and copZ, that are located upstream of the copAB region that encodes the two copper-ATPases. They control the expression of the two ATPases, by CopY acting as a repressor and CopZ as an activator. CopY represents a novel, bacterial metal-fist type transcription factor.
Figure 1: DNA sequence and protein translation of copY and copZ. The deduced protein translations for copY and copZ are given below the DNA sequence. The first nine amino acids of the previously published sequence (2) of the CopA ATPase are also shown. Ribosome binding sites are double underlined, and relevant restriction sites are indicated above the sequence. The putative promoter regions at the -35 and -10 positions are indicated in italics. Inverted repeats are delineated by arrows above and below the DNA sequence.
Figure 4: Schematic representation of the cop operon and the plasmids used in this study. Only the inserts of E. hirae DNA in the plasmids are shown. cop operon, organization of the cop operon derived from pOA1 and the published sequences for copA and copB(2) . pOA1, pOY1, and pOZ2 are pUC19 derivatives; pCC1, 4, 5, and 6 are pC3 derivatives. Open boxes delineate the open reading frames, the heavy black lines the erythromycin resistance cassettes, the black boxes the promoter/operator region, and the hatched box the part of copY that is not translated in the frameshift-mutated plasmid pCC6. ORFU, open reading frame of unknown function upstream of the cop promoter/operator region P/O. The positions of relevant restriction sites are indicated above the operon and are corresponding in the other sequences. Construction of the plasmids is described under ``Experimental Procedures.''
In normal growth media, which contain about 10 µM total copper, the expression of the two copper-ATPases of E. hirae, CopA and CopB, is strongly down-regulated. However, if the ambient copper concentration is substantially lowered or raised, CopA and CopB are simultaneously induced(11) . To study this regulation, we looked for possible regulatory genes and elements upstream of copAB. We thus cloned the upstream region by chromosome walking. Fig. 1shows the pertinent part of this DNA sequence. It contains two open reading frames, copY and copZ, preceding the copA gene. The derived protein sequences predict CopY and CopZ to be an acidic and a basic protein of 145 and 69 amino acids, respectively. Both genes are preceded by clear ribosome binding sites. A putative promoter/operator region is located immediately upstream of the ribosome binding site of copY. This suggests that the cop operon consists of the four genes copY, copZ, copA, and copB. The -10 promoter region lies within the first half of a 2-fold symmetric inverted repeat encompassing a region of 51 nucleotides. An open reading frame not shown in Fig. 1. starts at nucleotide 79 and encodes a 179-amino acid protein that is unrelated to any known protein. There is no evidence that this gene plays a role in copper metabolism.
Comparison of CopY with other proteins in the data base
revealed sequence similarity to the -lactamase repressor proteins
MecI of Staphylococcus epidermis, PenI of Bacillus
licheniformis, and BlaI of Staphylococcus aureus, sharing
32, 30, and 27% identical amino acids, respectively (Fig. 2).
The best studied of these, PenI of B. licheniformis, acts as a
repressor by binding to operator sites between penI and penP to repress the transcription of both genes. The
N-terminal half of PenI appears to be the recognition site for the
operator(26, 27) , whereas the C terminus is
responsible for binding the inducer. In line with such an arrangement
of functional domains, the N terminus of CopY exhibits strong sequence
similarity to the N-terminal DNA binding domains of
-lactamase
repressors, whereas the C terminus of CopY is divergent and contains
the heavy metal binding motif
CXCX
CXC. This would suggest that
CopY functions as a copper-binding repressor.
Figure 2:
Alignment of the deduced amino acid
sequence of CopY with -lactamase repressor proteins. Se
MecI, methicillin resistance regulatory protein MecI of S.
epidermis; Bl PenI, PenI of B. licheniformis(26) ; Sa BlaI, BlaI of S.
aureus(36) . Amino acids identical in CopY and the other
proteins are boxed.
To analyze the
function of CopY, we constructed the gene-disrupted strain
copY by homologous recombination. Expression of CopB in
wild type and mutant
copY was analyzed by Western
blotting with antibodies raised against CopB (we have previously shown
that CopA and CopB are co-regulated; (11) ). Disruption of copY led to constitutive overexpression of CopB, reaching
levels higher than those obtainable by induction of wild type cells
with the known inducers Cu
, Cd
, or
Ag
(Fig. 3A). Plasmid pCC4 (Fig. 4), bearing copY, complemented mutant
copY in trans, whereas the control plasmid pCC5
had no effect. In trans-complemented cells, CopB was not fully
repressed, with levels resembling those of induced wild type cells.
Further induction of these cells with 2 mM CuSO
resulted in only a small additional increase in CopB. This was to
be expected since the presence of large amounts of CopB copper export
ATPase hinders an increase in intracellular copper. Clearly, the
absence of CopY leads to runaway expression; supplementing CopY in trans represses this overexpression, albeit not completely
(see below).
Figure 3:
A, expression of CopB in wild type, mutant
copY, and complemented mutant
copY. Lysates
were prepared from logarithmically growing E. hirae cells, and
1 µl of total extract, corresponding to 3 µg of protein, was
separated on a 10% polyacrylamide gel. Expression of CopB was
visualized by Western blotting as described under ``Experimental
Procedures.'' Where indicated, the cultures were induced with 2
mM CuSO
for 45 min. The arrow identifies
the band corresponding to CopB at a relative molecular weight of 80
kDa. The numbers below the lanes indicate the relative
quantities of CopB as determined by densitometry of the Western blot. Lane 1, wild type; lane 2, induced wild type; lanes 3-6, mutant
copY containing no
plasmid, control plasmid pCC5, copY-bearing plasmid pCC4, or copY-bearing plasmid pCC4 under induced conditions,
respectively. B, growth of wild type, mutant
copY, and complemented mutant
copY in
copper-depleted media. For details see ``Experimental
Procedures.''
, mutant
copY;
, mutant
copY complemented with pCC4 bearing copY;
, mutant
copY supplemented with 250 µM CuSO
;
, wild type.
The overexpression of the cop operon in
copY cells increased the resistance to copper, permitting
growth in 10 mM CuSO
(wild type: 8 mM,
not shown). At the same time,
copY cells had a
copper-dependent phenotype and grew very slowly in copper-depleted
medium (Fig. 3B). Adding 250 µM CuSO
restored growth to wild-type rates, indicating that copper was
the limiting ion. The growth inhibition of
copY by low
copper was fully complemented by supplementing CopY in trans on pCC4. According to our model, CopA serves in the uptake and
CopB in the extrusion of copper, and the two ATPases must thus be
balanced in their activity. Overexpression of the cop operon
apparently upsets this balance and results in hyper-resistance to
copper but also copper deficiency under conditions of limiting copper.
CopZ encodes a protein of 69 amino acids, which contains
the conserved heavy metal binding motif GMXCXXC (Fig. 5). This motif is repeated six times in the polar
N-terminal regions of the putative human Menkes and Wilson
copper-ATPases and once in CopA of E. hirae(28) and
PacS of Synechococcus ((9) , Fig. 5). The same
motif is also found in the mercuric reductase MerA of Serratia
marcescens, which reduces Hg to
Hg
(29) , and in the periplasmic mercury binding
proteins MerP of Serratia marcescens(30) and MerP of Shigella flexneri(31) . CopZ is most similar to CopP
of H. pylori (41% identity), a protein of unknown function,
encoded downstream of a putative copper-ATPase gene
(GenBank
/EMBL Data Bank accession number L33259).
Figure 5: Protein sequence alignment of CopZ with related proteins involved in heavy metal ion metabolism. Eh CopZ, CopZ of E. hirae; Hp CopP, putative copper binding protein of H. pylori; Menkes, copper-ATPase encoded by the Menkes gene; Eh CopA, copper-ATPase of E. hirae; Sy PacS, copper-ATPase of Synechococcus; Sm MerP, periplasmic components of plasmid-determined mercury resistance system from S. marcescens, Sm MerA, mercuric reductase of Serratia marcescens. Regions of sequence identity between these proteins and CopZ are boxed. The sequences were aligned with the program Pileup of the Genetics Computer Group(21) .
To
illuminate the role of CopZ in metal ion homeostasis, we also
constructed the corresponding null mutant copZ.
Disruption of copZ prevented induction of CopB (Fig. 6A), suggesting that CopZ is an activator. Neither
addition of copper or silver ions nor chelation of copper with o-phenanthroline or 8hydroxyquinoline could stimulate the
expression of the cop operon in
copZ cells.
Transformation of this mutant with pCC6 that contains a functional copZ gene (copY was inactivated by a frameshift
mutation introduced at the AsnI site) reactivated CopB
expression. However, wild type levels of expression could not be
obtained in the complemented system (see below).
Figure 6:
A, expression of CopB in wild type, mutant
copZ, and complemented mutant
copZ. 5
µl of total extracts, corresponding to 15 µg of protein, were
analyzed for expression of CopB as described in the legend to Fig. 3. Lane 1, induced wild type; lane 2,
uninduced wild type; lanes 3-7, mutant
copZ containing no plasmid, control plasmid pCC5, copZ-bearing plasmid pCC6, plasmid pCC6 under copper induced
conditions, or plasmid pCC6 with induction by 500 µMo-phenanthroline, respectively. B, growth of
wild type, mutant
copZ, and complemented mutant
copZ in media containing 750 µM CuSO
.
, mutant
copZ;
,
mutant
copZ complemented with pCC6;
, wild type.
The level of CopB expression in lane 1 is underestimated in
this experiment because of signal
saturation.
Fig. 6B shows that mutant copZ cells were very
copper-sensitive and ceased to grow 1 h after adding 750 µM CuSO
to the medium. Supplying CopZ in trans on plasmid pCC6 restored growth under these conditions. Whereas
wild type cells grow in media containing up to 8 mM CuSO
, mutant
copZ complemented with pCC6
did not acquire full wild type resistance and did not grow at copper
concentrations above 1 mM.
There could be several reasons why both, copY- and copZ-disrupted cells could only be partially complemented in trans. According to our model, CopY and CopZ form a two-component regulatory system with a repressor and an activator component (see ``Discussion''). The balance of the two proteins will be crucial in this regulation. With one component on a plasmid, the system may become imbalanced, be it by altered expression levels of the proteins or by a gene-dosage effect of the cop promoter/operator region that is also present on the plasmids used for complementation. In addition, the erythromycin cassette introduced into the regulatory genes in the null mutants may disturb the regulation.
Taken together, our results show that the E. hirae cop operon consists of the four genes copY, copZ, copA, and copB, preceded by an operator/promotor region. CopY and CopZ possess heavy metal ion binding domains and regulate the expression of the two copper-ATPases, CopA and CopB. CopY and CopZ act in trans, with CopY functioning as a repressor and CopZ as an activator protein.
The regulation of the cop operon of E. hirae is unique in that both low and high copper concentrations induce expression, with maximal repression observed in 10 µM extracellular copper. We identified two genes, copY and copZ, that are involved in this mechanism. Both of the proteins encoded by these genes contain metal binding motifs and could thus be modulated in their activity by copper.
The N terminus of
CopY shares sequence identity with lactamase repressors in the
N-terminal but not the C-terminal half. This is in line with the
proposed domain structure of these proteins, with the N terminus
interacting with the operator and the C terminus with the inducer. In
crystallographic studies of several repressors, it became apparent that
the DNA binding region consists of a helix-turn-helix motif, located in
the N-terminal part of the protein(32) . There is evidence that
this motif occurs in a large number of repressors that form the
Cro/LysR family of repressors(33) . From the crystal structure
of the bacteriophage 434 repressor, it became apparent that the side
chains of the Gln-Gln-29 pair can only be properly matched by the
nucleotide sequence ACA that seems to be invariant in all operators (34) . In CopY, there is an analogous Gln-Gln-31 pair that
could establish the interaction with the ACA triplet present in the cop operator. This suggests that CopY is a repressor similar
to the 434 repressor.
The C-terminal domain of CopY encompasses a
putative copper binding site with the consensus sequence
CXCXCXC. This sequence is also
found in many metallothioneins and in the C-terminal half of the yeast
protein MAC1. This protein is required for basal level expression of a
component of plasma membrane
Cu
/Fe
-reductase and for induction
of cytosolic catalase(35) . Copper and DNA binding domains
similar to those of MAC1 are also found in ACE1 and AMT1 of S.
cerevisiae. These three yeast transcription factors are believed
to interact with heavy metal ions and have correspondingly been dubbed
metal-fist transcription factors.
Our data support the proposal that CopY is a copper-regulated repressor and appears to be the first representative of a prokaryotic metal-fist transcription factor. Final proof for this concept will, of course, require DNA and copper binding studies paired with mutational analysis. Work along these lines is in progress.
As discussed above, the presumed copper binding motif GMXCXXC of CopZ occurs in most of the proposed copper-ATPases and is even repeated up to six times in some of them. In addition, a single region with sequence similarity over the entire length of CopZ (39% identical residues) can be found in the N termini of the Menkes and Wilson ATPases and, more degeneratedly, also in the other copper-ATPases. This suggests that CopZ is a structural element that forms a building block in the N termini of copper-ATPases. The role of this structural element is, in all likelihood, copper binding.
Based on our data, we propose the model for the regulation of the cop operon outlined in Fig. 7: CopY functions as a
repressor for transcription of the cop operon by binding to
the DNA. An inverted repeat just preceding the copY gene is a
probable binding site. Inverted repeats are a common scheme in
operators and a nearly identical sequence precedes the -lactamase
gene blaZ and functions as an operator in Staphylococcus
aureus(36) . Similar promoter-operator regions were also
found in the
-lactamase operon of B. licheniformis,
upstream of the
-lactamase gene penP and preceding the
-lactamase repressor gene penI(26) . CopZ acts as
an antirepressor and is required for activation of the cop operon. The unusual activation of the cop operon by both
high and low ambient copper, could be explained as follows: under
copper limiting conditions both CopY and CopZ have no copper bound and
are free in the cytoplasm, allowing expression of the cop operon. When the cytoplasmic copper concentration is in the
physiological range, CopY binds copper and represses transcription by
binding to the operator. At cytoplasmic copper concentrations
approaching toxic levels, CopZ also binds copper and acts as an
antirepressor by binding to CopY and releasing it from the operator,
hence activating transcription of the cop operon. Clearly,
further experiments are required to substantiate this model of
bipartite regulation of the cop operon by CopY and CopZ, and
corresponding work is in progress.
Figure 7: Model of the regulation of the cop operon in E. hirae. The black bar indicates the promotor/operator region containing the inverted repeats. Under physiological copper conditions, CopY complexes copper and acts as a repressor of the cop operon. When excessive copper is present, CopZ also complexes copper and functions as an activator, possibly by antagonizing CopY. Under copper-limiting conditions, CopY has no copper bound and thus does not act as repressor.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z46807[GenBank].