Two Menkes-type ATPases Supply Copper for Photosynthesis inSynechocystis PCC 6803*

Synechocystis PCC 6803 contains four genes encoding polypeptides with sequence features of CPx-type ATPases, two of which are now designatedpacS and ctaA. We show that CtaA and PacS (but not the related transporters, ZiaA or CoaT) facilitate switching to the use of copper (in plastocyanin) as an alternative to iron (in cytochrome c 6) for the carriage of electrons within the thylakoid lumen. Disruption of pacSreduced copper tolerance but enhanced silver tolerance, andpacS-mediated restoration of copper tolerance was used to select transformants. Disruption of ctaA caused no change in copper tolerance but reduced the amount of copper cell−1. In cultures supplemented with 0.2 μmcopper, photooxidation of cytochrome c 6 (PetJ) was depressed in wild-type cells but remained elevated in bothSynechocystis PCC 6803(ctaA) andSynechocystis PCC 6803(pacS). Conversely, plastocyanin transcripts (petE) were less abundant in both mutants at this [copper]. Synechocystis PCC 6803(ctaA) and Synechocystis PCC 6803(pacS) showed increased iron dependence with impaired growth in deferoxamine mesylate (iron chelator)-containing media. Double mutants also deficient in cytochromec 6, Synechocystis PCC 6803(petJ,ctaA) and Synechocystis PCC 6803(petJ,pacS), were viable, but the former had increased copper dependence with severely impaired growth in the presence of bathocuproinedisulfonic acid (copper chelator). Analogous transporters are likely to supply copper to plastocyanin in chloroplasts.

Cyanobacteria contain internal thylakoid membranes where oxygen-evolving photosynthetic electron transport occurs. Respiratory electron transport occurs in both thylakoid and plasma membranes (1). Thylakoid membranes contain two protein complexes that include photosystems II and I. Within photosynthetically active cells, mobile soluble carriers shuttle electrons between these two complexes. Some cyanobacteria and green algae (see Refs. 2 and 3 and references therein) adapt to copper deficiency by exploiting alternative carriers. In copper-sufficient Synechocystis PCC 6803, electrons transfer between the complexes via copper in plastocyanin (PetE) whereas under copper deficiency heme iron in cytochrome c 6 (PetJ) is used (4). Both PetE and PetJ are located "inside" the thylakoid lumen.
One subset of proteins is imported into cyanobacterial (and plant chloroplast) thylakoids via the Sec system, whereas others are imported via a ⌬pH-dependent pathway (28). The latter transports folded proteins, and its substrates tend to be proteins that require complex cofactors, thereby avoiding separate thylakoid import of the cofactors. Plastocyanin is imported using Sec indicating that a copper delivery system into this compartment is required when Synechocystis PCC 6803 switches from PetJ to PetE. Higher plant chloroplasts rely exclusively on plastocyanin for electron transport between the two photosystems (2), and therefore thylakoid copper import is predicted to be especially important in higher plants.
Copper can impair cell function by associating with adventitious sites or engaging in "elicit" redox chemistry. In recent years, it has become apparent that there are efficient systems to deliver intracellular copper while avoiding adverse interactions en route (5). These include transporters and copper chaperones that target specific intracellular compartments and/or apoproteins (5). How is copper supplied to plastocyanin?
The human copper transporters MNK and WND, aberrant in Menkes and Wilson diseases, respectively, are two (prominent) members of a subgroup of P-type ATPases (6) often termed P 1 - (7) or CPx-type (8). CPx-type ATPases transport larger metal ions, with the founder, CadA from Staphylococcus aureus, exporting Cd 2ϩ (9). Known representatives include the bacterial copper transporters CopA and CopB; CCC2 that transports copper in yeast; ZntA from Escherichia coli that exports zinc but also lead and cadmium; ZiaA, zinc; and CoaT, cobalt (reviewed in Refs. 10 and 11). At present, the metal ion transported and the direction of transport cannot be predicted from the sequence of a CPx-type ATPase. We have previously characterized two of the four ORFs 1 encoding deduced CPx-type ATPases in the genome of Synechocystis PCC 6803 (12), ZiaA (slr0798) (13) and CoaT (slr0797) (14), but ORFs sll1920 and slr1950 remained uncharacterized.
Similarity of the deduced products of sll1920 and slr1950 with PacS (15) and CtaA (16), respectively, from Synechococcus PCC 7942, encouraged a prediction that these polypeptides contribute to copper homeostasis. We report that this is correct and designate the Synechocystis genes pacS and ctaA. However, disruption of pacS or ctaA in Synechocystis PCC 6803 confers different phenotypes (in part) to those observed in Synechococcus PCC 7942. PacS is located in thylakoid membranes in Synechococcus PCC 7942 (15), but the direction of copper transport by PacS is unknown. Synechococcus PCC 7942, unlike Synechocystis PCC 6803, does not show copperdependent switching between cytochrome c 6  both pacS and ctaA in Synechocystis PCC 6803 is negative with respect to the photooxidation of cytochrome c 6 and positive toward the accumulation of plastocyanin, petE, transcripts in copper-containing medium. Both of these transporters contribute toward the substitution of copper (in place of iron) for photosynthetic electron transport in the thylakoid, consistent with inward copper transport by both. Deletion of ctaA impairs cellular copper accumulation. Deletion of pacS confers copper sensitivity but silver resistance, which is interpreted in the context of metal ion sequestration within thylakoids. Structural features that confer metal specificity are considered.

EXPERIMENTAL PROCEDURES
Bacterial Strains, DNA Manipulations, and Southern Analyses-Synechocystis PCC 6803 was grown either in liquid BG-11 medium or on medium C plates (17) using previously described conditions (14). BG-11 contains 0.3 M copper. A variant BG-11 (BG-11-C), lacked copper as a microelement but was supplemented with defined [copper]. Growth under iron limitation was analyzed using medium lacking normal [iron] and containing 0.2 M copper (BG-11-FC). Cells were transformed to antibiotic resistance as described by Hagemann and Zuther (18). E. coli strains JM101 or SURE (Stratagene) were grown in Luria-Bertani medium (19). DNA manipulations were performed as described by Sambrook et al. (19). Genomic DNA was isolated from Synechocystis PCC 6803 using a protocol described previously for the isolation of DNA from plant cell cultures but excluding CsCl gradients (20). Aliquots (10 g) of DNA were digested with restriction endonucleases, resolved by agarose gel electrophoresis, transferred to nylon filters, and washed (after probing) to a stringency of 0.5ϫ SSC, 0.1% w/v SDS at 65°C (19).
Insertional Inactivation of pacS-Synechocystis PCC 6803 genomic DNA was used as template for polymerase chain reaction with primers 5Ј-GAAGAATTCAGTAAACCGAAGAGGGGATAG-3Ј and 5Ј-GAAG-GATCCTTGGCCGGGGAATACCCATGCAG-3Ј. The pacS amplification product (2.36 kb) was ligated to pGEM-T (Promega) to create pSTNR-2. A 1.26-kb BamHI fragment of pUK4K (Amersham Pharmacia Biotech) containing a kanamycin resistance gene was incubated with the Klenow fragment of E. coli DNA polymerase I and the "blunt-ended" fragment ligated to a unique MscI site (within sll1920) of pSTNR-2, creating pIN-PACS. Synechocystis PCC 6803 was transformed to kanamycin resistance following incubation with pIN-PACS, transformants selected on solid medium containing 25 g ml Ϫ1 kanamycin prior to growth in liquid medium containing 50 g ml Ϫ1 kanamycin. Interruption of pacS by insertion of the kanamycin resistance gene in all copies of the Synechocystis PCC 6803 chromosome was confirmed by Southern analysis and probing with a 32 P-labeled fragment of pacS and the strain designated Synechocystis PCC 6803(pacS). Subsequently, plasmid pSTNR-2 was used to reintroduce pacS into the chromosome of Synechocystis PCC 6803(pacS), and these transformants were selected on solid medium supplemented with 3 M copper (no kanamycin).
Insertional Inactivation of ctaA-An analogous procedure (to that described for pacS) was used with primers 5Ј-GAAGAATTCCGGTTAA-CAGCAAGGGAGC-3Ј and 5Ј-GAAGGATCCGTATGAAACCCGTCTC-CAAG-3Ј, generating a 2.21-kb ctaA product and subsequently plasmid pSTNR-1. The kanamycin resistance gene was ligated to a unique SmaI site in the pSTNR1 insert to generate plasmid pIN-CTAA. Interruption of ctaA was confirmed by Southern analysis and the strain designated Synechocystis PCC 6803(ctaA).
Insertional Inactivation of petJ-An analogous procedure (to that described for pacS and ctaA) was used but with petJ-specific primers 5Ј-GAAGGATCCGCTGTTAGCTTGCCAAATACTGGG-3Ј and 5Ј-GAA-GAATTTCGAAATGGAGCCCTAGGTATGGTGA-3Ј, generating plasmid pSTNR-3. The chloramphenicol resistance gene was ligated to a unique NheI site (in petJ) to generate plasmid pIN-PETJ. The transporter mutants, Synechocystis PCC 6803(pacS) and Synechocystis PCC 6803(ctaA), were transformed to chloramphenicol resistance following incubation with pIN-PETJ, transformants selected on solid medium containing 7.5 g ml Ϫ1 chloramphenicol (and 50 g ml Ϫ1 kanamycin) prior to growth in liquid medium containing the same concentration of antibiotic. Interruption of petJ was confirmed by Southern analysis and the strains designated Synechocystis PCC 6803(petJ,pacS) and Synechocystis PCC 6803(petJ,ctaA).
Analyses of Metal Tolerance and Accumulation-Logarithmically growing cultures were subcultured on alternate days (to ϳ1 ϫ 10 6 cells ml Ϫ1 ) for a minimum of 7 days (to standardize growth rates). Growth of cultures in metal-supplemented media was examined as previously described (14). Growth under copper limitation was analyzed by adding 300 M bathocuproinedisulfonic acid, 48 h after inoculation with cells derived from cultures of standardized growth rates that had been maintained (at least 7 days) in BG-11-C. To examine effects of iron deprivation, cells were passaged twice through BG-11-FC supplemented with 3 mg ml Ϫ1 ferric ammonium citrate and once in BG-11-FC before inoculation into either BG-11-FC or BG-11-FC supplemented with 10 M deferoxamine mesylate.
To examine metal accumulation, aliquots (30 ml) of logarithmically growing cultures in BG-11-C of standardized optical density (A 540 ) were exposed (2 h) to 0.2, 1, and 2 M copper, and cells were harvested, washed in BG-11-C, and finally resuspended in 1.25 ml of 70% v/v HNO 3 . Metal contents were determined by atomic absorption spectrophotometry. Analyses of cobalt and zinc accumulation used BG-11.
Single Turnover Cytochrome Kinetics-Measurements of cytochromes (f plus c 6 ) and of plastocyanin were made by analyses of flash-induced absorbance changes in the visible region using a protocol developed for use with higher plant thylakoid membranes (21,22). Cells were grown in BG-11-C supplemented with specified [copper]. Cell densities were determined by A 540 , and chlorophyll content was measured as the A 665 of chloroform extracts. Similar chlorophyll contents and cell densities were determined for all genotypes and copper treatments. Cells were diluted in BG-11-C medium to a chlorophyll concentration of 25 g ml Ϫ1 ; required sample volume was 1.5 ml, and pathlength was 1 cm. Saturating actinic flashes were generated using a xenon flashlamp (15-microfarad capacitor at 1000 V; 6-s halfpeak width) filtered with RG625 glass filters. Two lightpipes (1-cm diameter) were used to deliver a train of four flashes at 5 Hz to both sides of the sample cuvette, and the photomultiplier was protected with BG39, OG530, and 580-nm cut-off filters. Transients were recorded sequentially at 542, 554, 563, and 575 nm after dark adaptation for 2 s before each train of flashes. The cycle of four flashes was repeated 20 times, and the transients at each wavelength were averaged. Changes (⌬A) due to cytochrome b 563 at 563 nm, cytochromes f plus c 6 (which have sufficiently similar spectra that are deconvoluted together) at 554 nm, P700 at 542 nm, and plastocyanin at 575 nm were obtained by matrix deconvolution using the matrix of extinction coefficient values obtained for higher plants (21). This deconvolution can be applied to Synechocystis PCC 6803 without the need to dissipate any generated electric field, because there is no equivalent of the carotenoid bandshift, which would otherwise strongly overlap in this region. Each resultant trace represented the absorbance change at one wavelength of a single component. In all experiments, the measuring beam was switched on 50 ms before recording commenced, and during dark periods the photomultiplier was provided with light from a light-emitting diode of intensity equal to that of the measuring beam. In all samples, the size of the transients did not increase on successive flashes, indicating that there was sufficient P700 to cause a full photooxidation of f/c 6 /plastocyanin with a single flash. Hence, only the 20-replicate average of the first flash transient is shown in the figures.
Membrane Isolation and Assays of Cytochrome c Oxidase Activities-Total membranes were prepared according to Norling et al. (24) using cells recovered from 50-ml cultures of A 540 0.3 to 0.6, resuspended in 20 mM potassium phosphate buffer, pH 7.0. Total membrane pellets were resuspended and homogenized in 200 l of potassium phosphate buffer supplemented with 20% v/v glycerol prior to storage at Ϫ80°C. Cytochrome oxidase activities were determined using reduced (1) 3 to 9 M horse heart cytochrome c (type VI) in potassium phosphate buffer and reactions initiated by addition of 10 l of isolated total membranes containing 2 to 10 g of protein. Oxidation of cytochrome c was determined using ⑀ 550 of 29.5 mM Ϫ1 cm Ϫ1 (25).

Mutants of Synechocystis PCC 6803 with a Disrupted pacS
Gene Have Reduced Tolerance to Copper but Enhanced Tolerance to Silver-Mutants, Synechocystis PCC 6803(pacS), were generated by integration of sequences derived from plasmid pIN-PACS, which contains ORF sll1920 interrupted by a kanamycin resistance gene. Growth of Synechocystis PCC 6803(pacS) and wild type was tested in multiple liquid cultures supplemented with a range of levels of cadmium, zinc, cobalt, copper, and silver ions to determine maximum permissive concentrations (data not shown). Only resistance to copper appeared to be reduced in Synechocystis PCC 6803(pacS). Subsequently, growth was examined as a function of time in response to selected concentrations of copper (Fig. 1A). Unlike wild type, Synechocystis PCC 6803(pacS) is unable to grow in BG-11-C medium containing 1 M copper and shows some inhibition of growth in medium containing 0.3 M copper. In BG-11-C medium containing 0.2 M copper, Synechocystis PCC 6803(pacS) has similar growth to wild type (data not shown). Restoration of tolerance to 3 M copper was used as a selectable marker to identify mutants of Synechocystis PCC 6803(pacS) in which pacS had reintegrated into the chromosome by homologous recombination following incubation of cells with the corresponding DNA. The genotypes of Synechocystis PCC 6803(pacS), and cells with reintegrated pacS, were confirmed by Southern analysis; the band of 2.8 kb represents hybridization to pacS on a larger fragment because of the presence of the kanamycin resistance gene within pacS (Fig. 1B). Fig. 1C shows the phenotypes of Synechocystis PCC 6803(pacS), wild type, and cells with pacS reintroduced into the chromosome. There was no significant difference in the copper content of Synechocystis PCC 6803(pacS), compared with wild type, when cells were grown in media containing copper concentrations that allowed equivalent growth (data not shown).
An estimation of the maximum permissive concentration of silver for growth of Synechocystis PCC 6803(pacS) suggested enhanced tolerance (data not shown). Growth of Synechocystis PCC 6803(pacS) was examined as a function of time in response to selected [silver] (Fig. 1A, right panel). Wild type cells were unable to grow in medium containing 0.5 M silver, whereas Synechocystis PCC 6803(pacS) showed only slightly impaired growth. Growth of wild type cells was significantly impaired in 0.3 and 0.4 M silver, concentrations that were not inhibitory to the pacS mutants.
Mutants of Synechocystis PCC 6803 with a Disrupted ctaA Gene Have Unaltered Metal Tolerance but Reduced Accumulation of Copper-Mutants with disrupted ctaA were generated by integration of sequences derived from plasmid pIN-CTAA, which contains ORF slr1950 interrupted by a kanamycin resistance gene. Southern analysis confirmed integration of the antibiotic resistance gene into ctaA on all copies of the chromosome ( Fig. 2A). Growth of Synechocystis PCC 6803(ctaA) and wild type was tested in multiple liquid cultures supplemented with a range of concentrations of cadmium, zinc, cobalt, copper, and silver ions to determine maximum permissive concentrations. Tolerance to all metals appeared unaltered (data not shown). Subsequently, growth was examined as a function of time in response to selected concentrations of copper (Fig. 2B). Neither cell line grew in BG-11 medium supplemented with 2 M copper, whereas growth of both cell types was inhibited by 1 M copper to a similar extent. In contrast, disruption of ctaA from Synechococcus PCC 7942 resulted in increased tolerance to copper ions compared with wild type (16).
Cultures of Synechocystis PCC 6803(ctaA) and wild type cells were exposed for 2 h to BG-11-C medium containing 0.2, 1, or 2 M copper, and significantly less copper was detected in the mutant at the two higher metal concentrations (Fig. 3). The total copper content of the mutants after this "short exposure" to 0.2 M copper was not significantly different from wild type. The cobalt and zinc contents of Synechocystis PCC 6803(ctaA) did not differ from wild type cells following 2 h of exposure to either 8 or 16 M zinc or 5 or 10 M cobalt (data not shown).
Photooxidation of Cytochrome c 6 (PetJ) in Copper-containing Medium Is Greater in Cells Disrupted in ctaA or pacS-The observation that in some media Synechocystis PCC 6803(ctaA) contains less copper than wild type (Fig. 3) suggests a role for CtaA in copper import. Does CtaA supply copper for PetE, and what influence, if any, does PacS have on the substitution of PetE for PetJ (Fig. 4A)?
Previous workers detected no cytochrome c 6 in Synechocystis PCC 6803 containing ϳ4 ϫ 10 6 copper atoms cell Ϫ1 grown in 0.3 M copper (4). Under these conditions, the decrease in ⌬A (absorbance deconvoluted at a defined wavelength for a defined component) at 554 nm upon exposure to a pulse of actinic light is attributable to cytochrome f alone, whereas in the absence of added copper this change becomes greater because of an addi-tional contribution at 554 nm because of transient oxidation of cytochrome c 6 (4) (at rest the carrier is reduced). Growth of Synechocystis PCC 6803(pacS) is inhibited by 0.3 M copper (Fig. 1A) precluding the use of this [copper] in subsequent experiments. Growth is not inhibited at 0.2 M copper. Photooxidation of cytochrome c 6 (plus cytochrome f) has been compared in BG-11-C containing 0, 0.2, 0.3, and 1 M copper (Fig.  4B). The transient decrease in ⌬A at 554 nm is less in 0.2 M compared with no added copper and similar to that observed in 0.3 M copper. This implies that plastocyanin replaces cytochrome c 6 in 0.2 M copper (Fig. 4C). Deconvolution at 575 nm (for plastocyanin) revealed a small (consistent with the lesser ⑀ 575 for plastocyanin compared with ⑀ 554 for cytochrome c 6 ) change upon exposure to a pulse of actinic light in cells grown in 0.2 M copper that was not evident in cells grown without added copper (data not shown).
The kinetics of light-induced change in ⌬A deconvoluted at 554 nm was compared in Synechocystis PCC 6803(pacS), Synechocystis PCC 6803(ctaA), and wild type cells grown in 0.2 M copper (Fig. 5). Both of the mutants show a greater change in ⌬A at 554 nm than wild type cells exposed to equivalent exogenous [copper] (Fig. 5) and indeed a larger change in ⌬A at 554 nm than observed in wild type cells grown in the absence of added copper (Fig. 4C). No light-induced change in ⌬A deconvoluted at 575 nm (for plastocyanin) was detected in the mutants (data not shown). Hence these data overall show that usage of cytochrome c 6 replaces that of plastocyanin in the mutants. Photooxidation of cytochrome c 6 was unaltered in Synechocystis PCC 6803(ziaA) or Synechocystis PCC 6803(coaT) grown in BG-11-C containing 0.2 M copper (Fig. 5B). Fig. 6 shows that copper super-supplementation partly complements the Synechocystis PCC 6803(ctaA) phenotype with respect to photooxidation of cytochrome c 6 . In copper-enriched (above 0.2 M) medium it is presumed that some copper is acquired via other (perhaps nonspecific) metal importers, partly restoring the use of plastocyanin.
Plastocyanin (petE) Transcripts Are Less Abundant, and Cytochrome c 6 (petJ) Transcripts Are More Abundant, in Cells Deficient in Functional ctaA or pacS-It has previously been shown (4) that a decline in photooxidation of cytochrome c 6 in copper-containing medium corresponds with a decline in abundance of the PetJ polypeptide and petJ transcript. The abundance of plastocyanin transcripts and polypeptides shows a reciprocal response, increasing in copper (2,4). The data in Fig.  5B imply more electron flow through cytochrome c 6 in Synechocystis PCC 6803(pacS) and Synechocystis PCC 6803(ctaA) and hence predict less plastocyanin and less petE transcripts. In cells grown in BG-11-C plus 0.2 M copper, petE transcripts were less abundant in Synechocystis PCC 6803(pacS) and Synechocystis PCC 6803(ctaA) than wild type (Fig. 5C). Cytochrome c 6 and petJ transcripts were more abundant in Synechocystis PCC 6803(pacS) and Synechocystis PCC 6803(ctaA) than wild type at this [copper] (Fig. 5C).
Disruption of ctaA or pacS Increases Sensitivity to Low Iron-It was speculated that a greater dependence upon PetJ rather than PetE for photosynthetic electron transport in Synechocystis PCC 6803(pacS) and Synechocystis PCC 6803(ctaA) may confer a greater dependence on iron. Iron deficiency, generated by subculture either in BG-11-FC media with no added iron or by addition of the iron chelator deferoxamine mesylate, slowed the growth of all cell lines (compare y axis on Fig. 7A with Fig. 1A and Fig. 2B). Fig. 7A shows that Synechocystis PCC 6803(pacS) and Synechocystis PCC 6803(ctaA) are more sensitive to low iron than wild type cells; this was observed using both types of low iron media and in two further replicates (not shown) of each of these experiments.
Disruption of ctaA Increases Dependence upon petJ and Reduces Cytochrome Oxidase Activity in Low Copper-Despite the observed enhanced dependence upon iron, it was possible to obtain, on copper-replete medium, double mutants (petJ,ctaA and petJ,pacS) in which petJ was insertionally inactivated on all copies of the Synechocystis PCC 6803 chromosome (Fig. 7B). However, Synechocystis PCC 6803(petJ,ctaA) was sensitive to copper depletion, with (slightly) reduced growth on BG-11-C (data not shown) and severely impaired growth in the presence of the copper chelator bathocuproinedisulfonic acid (Fig. 7C).
Copper is implicated in respiratory electron transport, as well as photosynthetic electron transport. In Synechocystis PCC 6803, cytochrome c oxidase resides at both the plasma and thylakoid membranes, can accept electrons from plastocyanin, as well as from cytochrome c 6 , and requires copper (1). However, cytochrome oxidase activities were not significantly different from wild type in membranes isolated from either Syn-echocystis PCC 6803(pacS) or Synechocystis PCC 6803(ctaA) grown in 0.2 M copper (data not shown). When cells were grown in BG-11-C medium, cytochrome oxidase activities were lower (mean 58%) in membranes from Synechocystis PCC 6803(ctaA) compared with wild type in each of eight independent experiments. It is noted that a related copper transporter has recently been implicated in the biogenesis of cytochrome c oxidase in Rhodobacter capsulatus (26).

DISCUSSION
Several lines of evidence support roles for PacS and CtaA in the delivery of copper for photosynthetic electron transport in Synechocystis PCC 6803. Plastocyanin is the electron carrier in the thylakoid lumen of higher plant chloroplasts (2), organelles that share close common ancestors with cyanobacteria (27). It is speculated that equivalent copper transporters act in chloroplasts.
Evidence that CtaA is involved in (i) copper import and (ii) copper supply for plastocyanin includes a reduction in the copper content of Synechocystis PCC 6803(ctaA) compared with wild type (Fig. 3), an increase relative to wild type in photooxidation of cytochrome c 6 in Synechocystis PCC 6803(ctaA) at 0.2 M copper (Fig. 5, A and B), a decrease relative to wild type in the abundance of petE transcripts, and an increase in petJ transcripts in Synechocystis PCC 6803(ctaA) at 0.2 M copper (Fig. 5C). Impaired copper acquisition in Synechocystis PCC 6803(ctaA), and impaired ability to switch (at 0.2 M copper) from use of iron in cytochrome c 6 to copper in plastocyanin, predicts increased iron dependence of Synechocystis PCC 6803(ctaA). This is consistent with an observed reduction in growth of this genotype in low iron (at 0.2 M copper) (Fig. 7A). More specifically, this predicts enhanced dependence upon cytochrome c 6 (PetJ), and Fig. 7C shows that growth of Synechocystis PCC 6803(petJ,ctaA) is severely inhibited by the copper chelator bathocuproinedisulfonic acid under conditions in which Synechocystis PCC 6803(ctaA) continues to grow.
Copper sensitivity of Synechocystis PCC 6803(pacS) (Fig. 1) indicates that PacS is also involved in copper transport. Two CPx-type ATPases, CopA and CopB, have been described in Enterococcus hirae, with CopA involved in copper import and CopB conferring resistance via export (8). If PacS were analogous to CopB it would either act negatively or have no effect upon switching to plastocyanin in the presence of copper. However, in common with Synechocystis PCC 6803(ctaA), Synechocystis PCC 6803(pacS) also shows an increase in photooxidation of cytochrome c 6 at 0.2 M copper (Fig. 5, A and B), a decrease in the abundance of petE transcripts coincident with a (less marked) increase in petJ transcripts at 0.2 M copper (Fig.  5C), and a reduction in growth in low iron relative to wild type at 0.2 M copper (Fig. 7A). The requirement for two transporters for efficient switching to plastocyanin is consistent with (i) copper traversing two membranes before holoplastocyanin is made in the thylakoid lumen, (ii) inward transport by both PacS and CtaA, and (iii) location of PacS within thylakoid membranes (Fig. 8). Why does PacS confer resistance to copper? Copper may promote fewer adverse interactions within the thylakoid than elsewhere in the cell because of sequestration by plastocyanin and/or an abundance of anti-oxidant systems in this photosynthetic compartment. Silver resistance of Synechocystis PCC 6803(pacS) may be caused by less silver binding to, and inhibiting of, thylakoid proteins. Deletion of pacS in Synechococcus PCC 7942 conferred the opposite phenotype, silver sensitivity (15). This could relate to differences in the expression of plastocyanin, or differences in the direction of transport by PacS, in the two cyanobacteria. The decline in petE transcript levels in Synechocystis PCC 6803(pacS) suggests that transcriptional switching also requires copper to reach the thylakoid.
The presence of multiple CPx-type ATPases in a single organism but differing in metal specificity provides an opportunity to identify structural elements that discriminate between metals. The E. coli zinc exporter (ZntA) is known to also trans-port lead (11), and deletion of ZiaA confers lead (in addition to zinc) sensitivity, a phenotype not detected in any of the other CPx-type ATPase mutants of Synechocystis PCC 6803 (data not shown). As illustrated in Fig. 8, the known metal preferences are CoaT, cobalt; ZiaA, zinc and lead; PacS, copper and silver; and CtaA, copper. Both PacS and CtaA contain an extended motif CPCALGLATP surrounding the sequence (CPC) thought to associate with metals during transport. This sequence is conserved in the majority of the CPx-type ATPases that have been assigned a role in the transport of copper. In ZiaA, it is replaced with the motif CPCALVISTP, and a similar extended motif is present in other zinc and cadmium transporters.
The amino-terminal cytoplasmic domains of most CPx-type ATPases contain the sequence GMXCXXC (where X is any residue), sometimes repeated (11). Both PacS and CtaA contain a single copy of this motif, but this is absent from CoaT, whereas in ZiaA it is associated with a second putative metal binding region containing repeated HXH motifs (Fig. 8). To what extent (and how) do differences in these regions influence metal discrimination? By analogy to the interaction between CCC2 and ATX1 in yeast (5), it is speculated that metal donors, metallochaperones, deliver (or acquire) metals to (from) these amino-terminal domains, at least of PacS and CtaA. There is now a quest for ORFs encoding metallochaperones.