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.

EXPERIMENTAL PROCEDURES

Materials

Cloning and Plasmid Constructions

()

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.''

DNA Sequencing and Analysis

Gene Disruption

Growth Experiments

Protein Expression

RESULTS

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 CXCXCXC. 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.

DISCUSSION

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.

FOOTNOTES

*

This work was supported by Grant 32-37527.93 from the Swiss National Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z46807[GenBank].

§

To whom correspondence should be addressed: Dept. of Clinical Pharmacology, University of Berne, Murtenstrasse 35, 3010 Berne, Switzerland. Tel.: 31-632-3268; Fax: 31-381-4713; solioz{at}ikp.unibe.ch.

()

The abbreviation used is: kb, kilobase.

ACKNOWLEDGEMENTS

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