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Keywords:

  • CopZ;
  • CopA;
  • Metallochaperone;
  • P-type ATPase;
  • Cytochrome oxidase;
  • Atx1

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

The structure of the hypothetical copper-metallochaperone CopZ from Bacillus subtilis and its predicted partner CopA have been studied but their respective contributions to copper export, -import, -sequestration and -supply are unknown. ΔcopA was hypersensitive to copper and contained more copper atoms cell−1 than wild-type. Expression from the copA operator-promoter increased in elevated copper (not other metals), consistent with a role in copper export. A bacterial two-hybrid assay revealed in vivo interaction between CopZ and the N-terminal domain of CopA but not that of a related transporter, YvgW, involved in cadmium-resistance. Activity of copper-requiring cytochrome caa3 oxidase was retained in ΔcopZ and ΔcopA. ΔcopZ was only slightly copper-hypersensitive but ΔcopZcopA was more sensitive than ΔcopA, implying some action of CopZ that is independent of CopA. Significantly, ΔcopZ contained fewer copper atoms cell−1 than wild-type under these conditions. CopZ makes a net contribution to copper sequestration and/or recycling exceeding any donation to CopA for export.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Copper is an essential cofactor for a number of enzymes with roles including electron transfer, oxidase and oxygenase activities, and detoxification of oxygen derived radicals[1]. However, copper can also be toxic in excess due to its binding to adventitious sites and promotion of oxidative damage through the catalysis of free radical formation. It has become apparent that, at least in yeast, efficient homeostatic mechanisms maintain essentially no free copper in the cell cytosol[2] whilst assisting in the delivery of copper to specific intracellular compartments and/or copper-requiring proteins [3–5]. These include copper transporters and copper metallochaperones.

In Saccharomyces cerevisiae the copper metallochaperone Atx1 interacts with and delivers copper to the CPx (or P1)-type ATPase Ccc2 which imports copper into the Golgi-apparatus for insertion into copper enzymes (reviewed in[4]). Atx1-related proteins have also been identified in some prokaryotes, including Atx1 from the cyanobacterium Synechocystis PCC 6803[6] and CopZ from Enterococcus hirae[7]. The latter (EhCopZ hereafter) influences DNA binding by the copper-responsive transcriptional repressor CopY and copper exchange between EhCopZ and EhCopY has been observed in vitro[8]. The cop operon includes copY and copZ along with copA and copB that encode CPx-type ATPases[7] with proposed roles in copper import and export, respectively[9]. Both EhCopA and EhCopB have been suggested as further interactive partners for EhCopZ [4,10], but it remains to be established whether or not EhCopZ interacts with these proteins in vivo.

An Atx1-like protein, designated CopZ, was recently identified in Bacillus subtilis (BsCopZ hereafter) and the solution structures of the apo and a copper(I)-bound form resolved[11]. The structure is similar to related proteins, including EhCopZ[12] and yeast Atx1 [13,14], with typical βαββαβ ferredoxin-like folding. The cytosolic N-terminal region of yeast Ccc2 contains two soluble domains that each adopt a structure similar to Atx1[15] and possess complementary charged surfaces to Atx1 that contribute significantly to interactions between the two proteins[16]. Both proteins possess the motif MX CXX C (where X represents any amino acid) associated with metal binding and the formation of copper-bridged hetero-dimeric species during copper transfer[16]. A potential partner for BsCopZ was identified as the deduced CPx-type ATPase CopA (BsCopA hereafter)[17] encoded adjacent to copZ in the B. subtilis genome (Fig. 1). The N-terminal region of BsCopA (BsCopAN) possesses two putative metal-binding domains and the solution structure of the second, resolved in the apo and copper(I) bound forms, reveals a high degree of similarity to BsCopZ but with complementary charged residues surrounding the MX CXX C metal-binding site that may contribute towards interactions[17].

image

Figure 1. Physical map of the copZcopA region. The copZ and copA genes, corresponding to ORFs yvgY and yvgX respectively in the sequenced B. subtilis genome[18], are shown with the adjacent ORFs yvgZ, yvgW, bcdC and bcdD (shaded rectangles coincide with ORFs); the latter two encoding thiol-disulfide oxidoreductases[19]. The insertion sites of pMUTIN in BFA1116 and BFA1117 (bold arrows), the region of DNA (239 bp) deleted in 168ΔcopZ by introduction of the kanamycin-resistance gene (horizontal line), and the positions of deduced[18] transcriptional terminators (circles) are shown.

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Copper-transporting CPx-type ATPases are widespread with representatives described in bacteria, yeast, higher plants and man (reviewed in [20,21]). Other CPx-type ATPases are also known that transport different metal ions including cadmium[22], zinc and lead [23–25], cobalt[26] and silver[27]. These proteins display a high degree of specificity with respect to the metal ions transported but the determinants of metal specificity remain unresolved. Most importantly the metal ion transported or direction of transport cannot be predicted from the sequence of a CPx-type ATPase based upon the current level of understanding. However, similarity of BsCopA to known copper transporters, including CopA (49% identity) and CopB (26%) from E. hirae[9], PacS (45%) and CtaA (35%) from Synechocystis PCC 6803, and CopA (39%) from Escherichia coli[28] encouraged the prediction that BsCopA contributes to copper homeostasis. During the writing of this manuscript Gaballa and Helmann[29] have reported that BsCopA confers copper-resistance and is induced by elevated copper. We attribute similar phenotypes to BsCopA and also detect increased copper accumulation in mutants with disrupted copA supporting a role for BsCopA in copper export.

Here we investigate the role of the putative copper metallochaperone BsCopZ. A bacterial two-hybrid assay shows in vivo interaction between BsCopZ and BsCopAN, but not of a second CPx-type ATPase, YvgW, encoded adjacent to copA (Fig. 1) but with a role in cadmium-resistance[30]. Activity of copper-requiring cytochrome caa3 oxidase at the cytoplasmic membrane is not dependent upon copA or copZ (or yvgW). We show that copZ is required for normal cellular copper content and that copZ alone confers some copper tolerance. This is consistent with a model in which BsCopZ mediates greater internal sequestration of copper in vivo either via accumulation of Cu(I)-BsCopZ or by trafficking to some other ‘advantageous’ site.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Bacterial strains, growth conditions and DNA manipulations

B. subtilis strains 168 or 1A1 (trpC2) (Bacillus Genetic Stock Center), BFA1116 and BFA1117 (http://locus.jouy.inra.fr/cgi-bin/genmic/madbase/progs/madbase.operl) were used. The latter two were generated within the framework of the B. subtilis European consortium and contain insertionally inactivated yvgX (now designated copA) and yvgW, respectively due to integration of pMUTIN[31] into the 168 genome (Fig. 1). B. subtilis strains were grown at 37°C in Luria–Bertani medium (LB), nutrient sporulation medium with phosphate (NSMP)[32] or on tryptose blood agar base (Oxoid) plates. E. coli strains JM101, JM109 (Promega) or BacterioMatch™ (Stratagene) were used. The media were supplemented with antibiotics when appropriate: for B. subtilis, lincomycin (25 μg ml−1), erythromycin (0.3 μg ml−1) or kanamycin (5 μg ml−1) were used; for E. coli, ampicillin (100 μg ml−1) or kanamycin (50 μg ml−1) were used. DNA manipulations were performed as described by Sambrook et al.[33]. Extraction of B. subtilis genomic DNA and transformation of B. subtilis by the Groningen method was performed as described by Bron[34]. All generated plasmid constructs were checked by restriction digestion and DNA sequencing.

2.2Generation of a copZ deletion mutant and a copA/copZ double mutant

B. subtilis 1A1 genomic DNA was used as template for PCR with primers 5′-ATGTCTAGACAACCGTTTGGAC-3′ and 5′-CCTGTGAATTCTTTCTATTTTCATCC-3′ and the amplification product, containing 423 bp from immediately upstream of yvgY (now designated copZ), was ligated into the Xba I/Eco RI site of pBluescript (SK), creating pYDS1. A second amplification product generated using primers 5′-ATGACGTCGACAAGTGATTCAAGG-3′ and 5′-GACGGTACCTGTTTCTAAAGCG-3′, containing 427 bp from immediately downstream of copZ, was subsequently ligated into the Sal I/Kpn I site of pYDS1, creating pYDS1S2. A kanamycin-resistance gene, released from pDG780[35] on an Eco RV/Sal I fragment, was ligated into the Eco RV/Sal I site of pYDS1S2, between the copZ flanking sequences, creating pMKNC10. Sca I linearised pMKNC10 (to favour a double crossover recombination event) was used to transform B. subtilis 1A1 to kanamycin-resistance, and deletion of copZ upon integration of the kanamycin-resistance gene was confirmed by PCR. Genomic DNA from B. subtilis 1A1 with disrupted copZ, 1A1ΔcopZ, was then used to transform B. subtilis 168 to kanamycin-resistance and copZ deletion again confirmed by PCR and the resulting strain designated 168ΔcopZ.

To generate mutants lacking both copZ and copA, B. subtilis 168ΔcopZ was transformed to erythromycin- and lincomycin-resistance using genomic DNA from BFA1116 and inactivation of both copZ and copA confirmed by PCR.

2.3Analyses of metal tolerance and copper accumulation

To determine the minimum inhibitory/maximum permissive concentrations of a range of metal ions, cells were grown overnight in LB medium, diluted 1:100 in fresh medium supplemented with ZnSO4, CuSO4, NiSO4, AgNO3, CdCl2 or CoSO4, and growth monitored after ca. 6 h by measuring the absorbance at 600 nm. Subsequent experiments quantified the effects on growth of selected (from the previous experiment) concentrations as a function of time.

To examine copper contents, overnight cultures were diluted 1:100 in LB supplemented with various concentrations of CuSO4 (described in individual experiments) and grown for 4 h. Cells from the resulting cultures, of standardised optical density (A600), were harvested and washed three times with 10 mM Tris–HCl (pH 7.5), 1 mM EDTA and once with Milli-Q H2O. Pelleted cells were dried overnight at 80°C, dissolved in 70% nitric acid, and the metal content measured by atomic absorption spectrophotometry. Metal contents were determined as atoms cell−1 (determined here as a colony-forming unit). Parallel control experiments eliminated any metal contamination from the materials used.

2.4Generation of bacterial two-hybrid constructs containing copAN, yvgWN and copZ

B. subtilis 168 genomic DNA was used as template for PCR with primers 5′-GAATTCCATGGAACAAAAAACATTGC-3′ and 5′-GCTCGAGTCACTTGGCTAC-3′ to amplify copZ, primers 5′-GGATCCATGTTGAGTGAAC-3′ and 5′-GCTCGAGTTACAGTCTCGCCG-3′ to amplify codons 1–163 of copA (copAN) and primers 5′-GGATCCATGAGACTAGTG-3′ and 5′-GCTCGAGTCACATATTGACCATTC-3′ to amplify codons 1–93 of yvgW (yvgWN). All PCR products contained introduced restriction sites suitable for introduction into BacterioMatch™ two-hybrid vectors (Stratagene). The amplification products were ligated to pGEM-T prior to subcloning; copZ into the Not I/Eco RI site of pBT creating pBTCOPZ; and copAN and yvgWN into the Bam HI/Eco RI site of pTRG creating pTRGCOPAN and pTRGYVGWN, respectively.

2.5β-Galactosidase assays

These assays were performed as described previously[36]. B. subtilis cultures were grown overnight in LB medium, diluted 1:100 in fresh medium supplemented with maximum permissive concentrations of Zn(II), Cu(II), Ni(II), Ag(I), Cd(II) or Co(II) and grown at 37°C until OD595 of 0.2–0.5 prior to assay. E. coli cultures (for the two-hybrid assays) were used with an OD595 of 0.6 following 20 h growth at 30°C.

2.6Membrane isolation and assays of cytochrome oxidase activities

For these assays cells were cultured in NSMP. Colony staining for N, N, N′,N′-tetramethyl-p-phenylene diamine (TMPD) oxidation activity was carried out as previously described[37]. Membranes were prepared[38] and cytochrome c oxidase activities measured as described[39] but using a membrane protein concentration of 20 μg ml−1 and reduced cytochrome c (20 μM) from S. cerevisiae (Sigma). Protein concentrations were determined using the BCA method[40] with bovine serum albumin standards.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Disruption of copA causes reduced tolerance to copper and increased cellular copper content

B. subtilis copA encodes an 803-residue protein with sequence features of metal-transporting CPx-type ATPases [41,42] including two MX CXX C metal-binding motifs in the N-terminal region. Disruption of copA in B. subtilis strain BFA1116, due to integration of vector pMUTIN (Fig. 1), was confirmed using PCR (data not shown). Growth of BFA1116 and B. subtilis 168 (wild-type) was tested in multiple liquid cultures supplemented with a range of levels of copper, silver, zinc, cadmium, cobalt and nickel ions to determine maximum permissive concentrations (data not shown). Only resistance to copper appeared to be reduced in BFA1116, with growth inhibited above 0.2 mM copper (inset Fig. 2A). Subsequently, growth was examined as a function of time in response to selected concentrations of copper (Fig. 2A). Unlike wild-type B. subtilis, BFA1116 is unable to grow in LB medium containing 1.5 mM copper.

image

Figure 2. Analysis of ΔcopA. A: Growth of wild-type B. subtilis 168 (open symbols) and BFA1116 (closed symbols) in LB medium supplemented with 0 (squares), or 1.5 mM (triangles) Cu(II). Inset, OD600 cultures (y-axis) against added [Cu(II)] (x-axis) following 6 h growth. B: Copper contents of B. subtilis 168 (open bars) and BFA1116 (closed bars) grown in media supplemented with 0 or 0.15 mM Cu(II). Data points represent the mean values from three separate cultures with standard errors. C: β-Galactosidase activity in BFA1116 grown with no metal supplement or with maximum permissive concentrations of Cu(II) (0.2 mM), Ag(I) (0.5 μM), Zn(II) (0.1 mM), Cd(II) (0.5 μM), Co(II) (0.1 mM) or Ni(II) (0.25 mM). The data points represent the means of three separate assays with standard errors. Similar trends were obtained when the experiment was repeated on two further occasions.

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The total copper content, or copper quota, of both wild-type and BFA1116 cells were determined for cultures grown in normal LB medium or following supplementation with copper at a level non-inhibitory to either strain (0.15 mM). Values were expressed as number of atoms per cell. These values increase by 11-fold in wild-type cells and 18-fold in BFA1116 following copper supplementation, with BFA1116 containing significantly more (three-fold) copper than wild-type cells (Fig. 2B). Disruption of copA promotes copper accumulation consistent with a role in export.

3.2Copper induces copA expression

The pMUTIN vector used to disrupt copA contains a lacZ reporter gene such that, upon integration into the chromosome, transcription of the target gene can be monitored[43]. Expression of copA was therefore examined in BFA1116. Following exposure of cells to biologically significant levels of various metal ions, induction of β-galactosidase activity was only observed in cells exposed to copper (Fig. 2C).

3.3CopZ interacts with CopAN, but not YvgWN, in a bacterial two-hybrid assay

B. subtilis copZ (Fig. 1) encodes a 69-amino acid protein with significant similarity to copper metallochaperones. A likely candidate partner for BsCopZ is BsCopAN[17]. It is now possible to analyse protein–protein interactions within a bacterial (E. coli) cell using the BacterioMatch™ two-hybrid system (Stratagene), and we have used this method previously[6] to reveal interactions between cyanobacterial Atx1 and the N-terminal regions of CtaA and PacS from Synechocystis PCC 6803. Greatly elevated β-galactosidase activity was detected in cells in which BsCopZ and BsCopAN (which included residues preceding the first predicted trans-membrane α-helix of BsCopA) were used as target and bait within this system compared with cells in which one or both partners was/were absent (Fig. 3A). BsCopA can therefore act as an interactive partner for BsCopZ.

image

Figure 3. In a bacterial two-hybrid assay BsCopZ interacts with the amino-terminal domain BsCopAN, but not with YvgWN. A: β-Galactosidase activity in E. coli (BacterioMatch™, Stratagene) containing: the control plasmids pBT and pTRG (−), pBTCOPZ and pTRG (CopZ), pBT and pTRGCOPAN (CopAN) or pBTCOPZ and pBTCOPAN (CopAN/CopZ). B: As panel A but using cells containing pBT and pTRGYVGWN (YvgWN), or pBTCOPZ and pBTYVGWN (YvgWN/CopZ). The data for three independent transformants are shown for cells containing translational fusions of copAN, yvgWN and/or copZ within pBT and pTRG. Data points represent the means of three separate assays for each transformant, with standard errors. Similar trends were obtained when the experiment was repeated on two further occasions.

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Adjacent to copA in the B. subtilis genome is a second gene, yvgW, for a deduced metal-transporting CPx-type ATPase (Fig. 1). In contrast to BsCopA, YvgW contains only a single MX CXX C motif in its N-terminal region and was shown[30] to have a role in cadmium, but not copper, resistance. We investigated whether or not BsCopZ could also interact with YvgWN (the N-terminal region of YvgW). Fig. 3B shows no detectable increase in β-galactosidase activity when BsCopZ and YvgWN (which included residues preceding the first predicted trans-membrane α-helix of YvgW) were used within the bacterial two-hybrid system compared with cells in which one or both partners was/were absent.

3.4Deletion of copZ causes a slight reduction in copper tolerance

The demonstrated in vivo interaction between BsCopZ and BsCopAN (Fig. 3A) suggests that BsCopZ may also have a role in copper homeostasis. To test this, a copZ-deficient mutant of B. subtilis strain 1A1 was obtained following chromosomal integration of pMKNC10, which contains sequences from immediately upstream and downstream of the copZ coding region separated by a kanamycin-resistance gene. PCR analyses confirmed integration via a double homologous recombination event at the copZ locus (data not shown) and the resulting strain was designated 1A1ΔcopZ. A copZ deletion mutant of B. subtilis strain 168 was subsequently generated using genomic DNA from 1A1ΔcopZ to transform B. subtilis 168 to kanamycin-resistance. Deletion of copZ was again confirmed by PCR and the resulting strain designated 168ΔcopZ. Growth of B. subtilis 168 (wild-type) and 168ΔcopZ was tested in multiple liquid cultures supplemented with a range of levels of copper. Resistance to copper appeared to be slightly reduced in 168ΔcopZ compared to wild-type cells, with a greater inhibition of growth observed in medium containing ≥1 mM copper (inset of Fig. 4A). Subsequently, growth was examined as a function of time in LB medium with or without 1.5 mM copper added (Fig. 4A). Growth of 168ΔcopZ was significantly more inhibited than growth of wild-type cells in medium containing 1.5 mM copper, revealing a contribution of copZ to copper tolerance.

image

Figure 4. Mutants deficient in copZ have reduced copper content and a slight reduction in copper tolerance. A: Growth of wild-type B. subtilis 168 (open symbols) and 168ΔcopZ (dark-grey symbols) in LB medium supplemented with 0 (squares) or 1.5 mM (triangles) Cu(II). Inset, OD600 cultures (y-axis) against added [Cu(II)] (x-axis) following 6 h growth. B: Copper contents of B. subtilis 168 (open bars) and 168ΔcopZ (dark-grey bars) grown in media supplemented with 0 or 0.4 mM Cu(II). C: Growth of BFA1116 (closed symbols) and 168ΔcopZcopA (light-grey symbols) in LB medium supplemented with 0 (squares) or 0.2 mM (triangles) Cu(II). Inset, OD600 cultures (y-axis) against added [Cu(II)] (x-axis) following 7 h growth. Data points represent the mean values from three separate cultures with standard errors. Similar trends were obtained when the experiments were repeated on two further occasions.

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3.5copZ enhances cellular copper content

BsCopZ binds copper(I) in vitro [11,44]. Two naive models are that copZ-mediated resistance to elevated exogenous copper results from (i) enhanced export via donation to BsCopA or (ii) enhanced intracellular sequestration either directly by BsCopZ or by donation to other ‘non-adventitious’ copper sites. To test this the copper quotas of B. subtilis 168 (wild-type) and 168ΔcopZ were examined following growth in normal LB medium or in LB medium supplemented with a level of copper non-inhibitory to either strain (0.4 mM). Values increased by 40-fold and 22-fold for wild-type and 168ΔcopZ, respectively, as exogenous copper levels increased (Fig. 4B). Most notably, cells containing functional copZ had significantly, 2.6-fold, more cellular copper than 168ΔcopZ cells. The reduced copper content in 168ΔcopZ implies that BsCopZ binds and sequesters copper in vivo, thereby increasing endogenous copper levels.

3.6copZ and copA are additive with respect to copper tolerance

To test for additivity with respect to copper tolerance, a double mutant with both copA and copZ disrupted was generated by transforming 168ΔcopZ to erythromycin and lincomycin-resistance using genomic DNA from BFA1116 (ΔcopA). Disruption of copA by pMUTIN and retention of the copZ deletion were confirmed using PCR (data not shown) and the resulting strain designated 168ΔcopZ/ΔcopA. Copper-resistance appeared to be slightly reduced in 168ΔcopZcopA compared to BFA1116 (inset Fig. 4C). Supplementation of the medium with 0.2 mM copper caused a greater reduction in the growth of 168ΔcopZcopA compared to that of BFA1116 (Fig. 4C). While the interaction between BsCopZ and BsCopAN provides support for BsCopZ acting in conjunction with BsCopA, BsCopZ alone also provides some copper-resistance.

3.7Cytochrome caa3 oxidase activity is unaffected in mutants with disrupted copA, copZ or yvgW

Disruption of Atx1 and the copper transporters CtaA (cellular import) and PacS (thylakoid import) in Synechocystis PCC 6803 results in phenotypes associated with impaired copper supply to plastocyanin and cytochrome c oxidase at the thylakoid compartment [6,45]. B. subtilis of course lacks an internal thylakoid compartment and contains a copper-requiring caa3-type cytochrome oxidase at the cytoplasmic membrane[39]. To examine whether or not the related proteins in B. subtilis have a role in the supply of copper to this enzyme, cytochrome caa3 oxidase activity was examined in 168ΔcopZ and BFA1116 (ΔcopA). Due to the location of yvgW, adjacent to copA, activity was also examined in BFA1117 in which yvgW is disrupted (confirmed by PCR, data not shown). Staining of colonies with the cytochrome caa3-specific substrate TMPD, showed that the mutants have the same positive TMPD-oxidation activity as wild-type cells (data not shown) revealing that cytochrome caa3 oxidase activity was retained. The level of cytochrome oxidase activity in isolated membranes was subsequently determined and activity was found to be similar in membranes isolated from BFA1116, 168ΔcopZ, BFA1117 and wild-type cells (Table 1). These values correlate well with the level of activity (0.15 μmol min−1 mg protein−1) previously reported for B. subtilis strain 3G18[39]. In contrast, ΔctaCD mutants which lack two of the structural genes for cytochrome caa3 oxidase[39], have only 3% of the cytochrome oxidase activity detected in wild-type cells (data not shown).

Table 1. 
  1. Cytochrome oxidase activity (normalised for cytochrome c concentration) in membrane preparations of cells grown in NSMP. Data values are the mean (with standard errors) of at least three separate assays performed using membranes from two separate preparations.

StrainCytochrome oxidase activity (μmol min−1 mg protein−1)
B. subtilis 1680.168 (±0.011)
BFA11160.196 (±0.012)
168ΔcopZ0.215 (±0.022)
BFA11170.229 (±0.027)

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Evidence that BsCopA is involved in copper export includes: (i) disruption of copA caused increased cytosolic copper levels (Fig. 2B), (ii) disruption of copA caused a reduction (five-fold) in tolerance to elevated copper while normal tolerance to other metals was retained (Fig. 2A), and (iii) copA expression was substantially increased by elevated copper but not by other metals (silver, zinc, cadmium, nickel and cobalt) at maximum permissive concentrations (Fig. 2C). The observed in vivo interaction between BsCopZ and BsCopAN (Fig. 3A) suggests that BsCopZ may also contribute to copper export, while the absence of any detectable interaction between BsCopZ and YvgWN (Fig. 3B) illustrates the specificity of BsCopZ towards the copper transporter. From the structures of BsCopZ and BsCopA, a mechanism of copper transfer and adduct formation similar to that described[16] for eukaryotic Atx1 and Ccc2 has been proposed[17].

BsCopZ binds copper(I) in vitro [11,44] and increased copper accumulation in wild-type cells compared to 168ΔcopZ, at non-inhibitory copper levels (Fig. 4B), supports the assertion that BsCopZ binds copper in vivo. Cyanobacterial Atx1 interacts with the cellular copper importer CtaA but can also acquire copper from other locations and an attractive proposition[6] is that Atx1 contributes to recycling endogenous copper. No specific copper import proteins have so far been described for B. subtilis. However, it is tempting to speculate that BsCopZ contributes to endogenous copper levels by effectively scavenging copper from importers or weak cytosolic sites, such as degraded metallo-proteins or adventitious copper-binding sites, and sequestering copper either directly or by donation to advantageous copper sites. The latter could include copper requiring apo-proteins or a specific copper sequestering macromolecule.

Cyanobacterial Atx1 and PacS supply copper for cytochrome caa3 oxidase at the thylakoid compartment and activity of this enzyme is reduced in Δatx1 or ΔpacS mutants[6]. We investigated whether or not BsCopZ and BsCopA have an analogous role in B. subtilis, although with BsCopA transporting copper ions across the cytoplasmic membrane rather than into the thylakoid compartment. Cytochrome caa3 oxidase activity was retained in ΔcopA and ΔcopZ (and ΔyvgW) mutants, with similar levels of activity being detected in membranes from wild-type and the mutant cells (Table 1). Our data therefore do not support a role for BsCopA and BsCopZ in the supply of copper to cytochrome caa3 oxidase. Cytochrome c oxidase activity in yeast requires the action of Sco1, at the inner mitochondrial membrane, in addition to the copper metallochaperone Cox17[46]. Sco1 is proposed to accept copper(I) from Cox17 for subsequent insertion into the CuA site of cytochrome caa3 oxidase[47]. A homologue of Sco1, YpmQ, has been identified in B. subtilis and shown to be required for the activity of cytochrome caa3 oxidase, but not of menaquinol oxidase with only a CuB site[48]. It remains to be established whether or not a protein functionally analogous (but different in sequence) to yeast Cox17 therefore delivers copper to YpmQ in B. subtilis for subsequent incorporation into cytochrome caa3 oxidase.

BsCopZ confers some copper-resistance (Fig. 4A). In vivo interaction with BsCopAN (Fig. 3A) is suggestive of copper donation from BsCopZ to BsCopAN for export. However, additivity with respect to copper tolerance (Fig. 4C) supports, at least some, independent action of BsCopZ and our data (Fig. 4B) are consistent with a role (direct or indirect) in intracellular sequestration (Fig. 5). While it is apparent that copZ causes some additional accumulation of copper it is proposed that this is less than the total amount of copper sequestered, BsCopZ thereby causing some reduction in the ‘available toxic pool’ of copper. At non-inhibitory copper levels BsCopZ may encounter, and donate copper to, apo-proteins at a higher frequency than to apo-BsCopAN. However, at higher copper concentrations the probability of encountering holo-proteins will be greater and interactions with apo-BsCopAN may be enhanced by the increased expression (Fig. 2C) of the transporter under these conditions. With the emergence of a class of copper-metallochaperones that interact with CPx-type ATPases it is now necessary to establish what, if any, the effect is of a metallochaperone on copper transport.

image

Figure 5. The role of CopZ in B. subtilis. In the model black arrows represent the pathways for copper, ovals represent metal-binding domains and CO represents cytochrome caa3 oxidase. The data shown in (B) imply that, at 0.4 mM exogenous copper, BsCopZ makes a greater contribution to sequestration/storage and/or recycling of endogenous copper than to BsCopA-mediated export. Copper-sensitivity of 168ΔcopZ implies that removal of copper from adventitious sites and/or donation to BsCopA for export provides some resistance.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

We thank Nigel Robinson for helpful discussions and valuable input into the preparation of this manuscript, Claes von Wachenfeldt and Lars Hederstedt for kindly providing the ΔctaCD strain and Zoltán Prágai for advice regarding the genetic manipulation of B. subtilis. This work was supported by The Royal Society (J.S.C./N.L.B.), D.S.R. is supported by a Luccock Studentship from the University of Newcastle and M.A.K. by a studentship from the BBSRC.

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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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