From Biochemistry and Genetics, Medical School,
University of Newcastle, NE2 4HH and § Glynn Research
Laboratory of Bioenergetics, Department of Biology, University College
London, WC1E 6BT, United Kingdom
Received for publication, December 13, 2000, and in revised form, March 6, 2001
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ABSTRACT |
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Synechocystis PCC 6803 contains four genes encoding polypeptides with sequence features of
CPx-type ATPases, two of which are now designated
pacS 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 c6) for the carriage of
electrons within the thylakoid lumen. Disruption of pacS
reduced copper tolerance but enhanced silver tolerance, and
pacS-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 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
c6 (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 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 P1- (7) or
CPx-type (8). CPx-type ATPases transport larger metal ions, with
the founder, CadA from Staphylococcus aureus, exporting
Cd2+ (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
ORFs1 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 copper-dependent switching between
cytochrome c6 and plastocyanin (see Ref. 2).
Here we describe experiments showing that the action of both
pacS and ctaA in Synechocystis PCC
6803 is negative with respect to the photooxidation of cytochrome
c6 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.
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 µM 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 µM 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 µg) of DNA were digested
with restriction endonucleases, resolved by agarose gel electrophoresis, transferred to nylon filters, and washed (after probing) to a stringency of 0.5× 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 5'-GAAGAATTCAGTAAACCGAAGAGGGGATAG-3' and
5'-GAAGGATCCTTGGCCGGGGAATACCCATGCAG-3'. 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 µg ml Insertional Inactivation of ctaA--
An analogous procedure (to
that described for pacS) was used with primers
5'-GAAGAATTCCGGTTAACAGCAAGGGAGC-3' and
5'-GAAGGATCCGTATGAAACCCGTCTCCAAG-3', 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 5'-GAAGGATCCGCTGTTAGCTTGCCAAATACTGGG-3' and
5'-GAAGAATTTCGAAATGGAGCCCTAGGTATGGTGA-3', 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 µg
ml Analyses of Metal Tolerance and
Accumulation--
Logarithmically growing cultures were subcultured on
alternate days (to ~1 × 106 cells
ml
To examine metal accumulation, aliquots (30 ml) of logarithmically
growing cultures in BG-11-C of standardized optical density (A540) were exposed (2 h) to 0.2, 1, and
2 µM copper, and cells were harvested, washed in BG-11-C,
and finally resuspended in 1.25 ml of 70% v/v HNO3. 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 c6) 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 A540, and chlorophyll content was measured as
the A665 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 µg ml RNA Isolation and Northern Analysis--
Total RNA was isolated
from logarithmically growing cultures in BG-11-C medium containing 0.2 µM copper using an established method (23) and treated
with DNase I. Equivalent amounts of total RNA (10 µg) were incubated
(65 °C for 5 min) in 60% v/v formamide, 3.6 mM Tris-HCl
(pH 7.8), 3 mM NaH2PO4, 0.2 mM EDTA resolved on 1.2% w/v formamide-agarose gels (in
the same buffer) and transferred to nylon membranes (Hybond-N; Amersham
Pharmacia Biotech). Blots were hybridized to a 32P-labeled
fragment of petE, generated using primers
5'-TTAACAATCCTCGCTGGCCTTCTGCTGG-3' and
5'-TTACTCAACGACAACTTTGCCTACCAT-3' and of petJ generated using primers
5'-TATTCAACCAAGCTAGCCGAA-3' and 5'-CCGCTTGATCAAGCACGTAGGCCG-3' washed
with 0.5 × SSC, 0.1% w/v SDS at 65 °C.
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
A540 0.3 to 0.6, resuspended in 20 mM potassium phosphate buffer, pH 7.0. Total membrane
pellets were resuspended and homogenized in 200 µl of potassium
phosphate buffer supplemented with 20% v/v glycerol prior to storage
at 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 µM copper and shows some
inhibition of growth in medium containing 0.3 µM copper. In BG-11-C medium containing 0.2 µM copper,
Synechocystis PCC 6803(pacS) has similar growth
to wild type (data not shown). Restoration of tolerance to 3 µM 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 µM 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 µM 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 µM copper, whereas growth of both
cell types was inhibited by 1 µM 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 µM 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 µM 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 µM zinc or 5 or 10 µM cobalt (data
not shown).
Photooxidation of Cytochrome c6 (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 c6 in
Synechocystis PCC 6803 containing ~4 × 106 copper atoms cell
The kinetics of light-induced change in
Fig. 6 shows that copper
super-supplementation partly complements the Synechocystis
PCC 6803(ctaA) phenotype with respect to photooxidation of
cytochrome c6. In copper-enriched (above 0.2 µM) 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
c6 (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 c6 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 c6 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 µM copper, petE
transcripts were less abundant in Synechocystis PCC
6803(pacS) and Synechocystis PCC
6803(ctaA) than wild type (Fig. 5C). Cytochrome
c6 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 c6, and requires copper (1).
However, cytochrome oxidase activities were not significantly different
from wild type in membranes isolated from either
Synechocystis PCC 6803(pacS) or
Synechocystis PCC 6803(ctaA) grown in 0.2 µM 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).
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 c6 in Synechocystis PCC
6803(ctaA) at 0.2 µM 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 µM copper (Fig.
5C). Impaired copper acquisition in Synechocystis
PCC 6803(ctaA), and impaired ability to switch (at 0.2 µM copper) from use of iron in cytochrome
c6 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 µM copper) (Fig. 7A). More specifically, this predicts enhanced dependence
upon cytochrome c6 (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 c6 at
0.2 µM 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 µM copper (Fig. 5C), and a reduction in growth
in low iron relative to wild type at 0.2 µM 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.
1. In cultures supplemented with 0.2 µM
copper, photooxidation of cytochrome c6 (PetJ)
was depressed in wild-type cells but remained elevated in both
Synechocystis PCC 6803(ctaA) and
Synechocystis 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 cytochrome
c6, 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 kanamycin prior to
growth in liquid medium containing 50 µg ml
1 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
32P-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 µM copper
(no kanamycin).
1 chloramphenicol (and 50 µg ml
1
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).
1) 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 µM 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
1 ferric ammonium
citrate and once in BG-11-FC before inoculation into either BG-11-FC or
BG-11-FC supplemented with 10 µM deferoxamine mesylate.
1; 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-µs 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 b563 at 563 nm, cytochromes f plus c6 (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/c6/plastocyanin with a single
flash. Hence, only the 20-replicate average of the first flash
transient is shown in the figures.
80 °C. Cytochrome oxidase activities were determined using
reduced (1) 3 to 9 µM horse heart cytochrome c
(type VI) in potassium phosphate buffer and reactions initiated by
addition of 10 µl of isolated total membranes containing 2 to 10 µg
of protein. Oxidation of cytochrome c was determined using
550 of 29.5 mM
1
cm
1 (25).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Synechocystis PCC
6803(pacS) has reduced copper but enhanced silver
tolerance. Panel A, growth in BG-11-C medium of wild type
(open symbols) and Synechocystis PCC
6803(pacS) (closed symbols) in the presence of 1 µM (triangles), 0.3 µM
(circles), or no added (squares) copper
(left panel) or 0.5 µM (inverted
triangles), 0.4 µM (circles), 0.3 µM (triangles), or no added
(squares) silver (right panel). All of the data
are means of triplicate determinations with S.D. (some values are too
small to be visible). Equivalent trends have been observed on two
further occasions. Panel B, Southern analysis of
NheI-digested DNA from wild type (lane 1),
Synechocystis PCC 6803(pacS) (lane 2),
and pacS-restored cells (lane 3) electrophoresed
on a 0.8% w/v agarose gel and probed with part of pacS.
Integration of the kanamycin resistance gene into pacS
creates a diagnostic 2.8-kb fragment. Panel C, colonies of
wild type (bottom left), Synechocystis PCC
6803(pacS) (top), and pacS-restored
cells (bottom right) on medium C plates (17)
supplemented with copper (2 µM) or kanamycin
(kan; 50 µg ml 1).
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Fig. 2.
Copper tolerance is unaltered in
Synechocystis PCC 6803(ctaA).
Panel A, Southern analysis of NheI-digested DNA
from wild type (lane 1) and Synechocystis PCC
6803(ctaA) (lane 2) electrophoresed on a 0.8%
w/v agarose gel and probed with part of ctaA. Integration of
the kanamycin resistance gene into ctaA creates a diagnostic
2.3-kb fragment. Panel B, growth (in BG-11) of wild type
(closed symbols) and Synechocystis PCC
6803(ctaA) (open symbols) supplemented with 2 µM (triangles), 1 µM
(circles), or no additional (squares) copper. All
of the data are means of triplicate determinations with S.D. Equivalent
trends have been observed on two further occasions.
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Fig. 3.
Synechocystis PCC
6803(ctaA) accumulates less copper.
Cell-associated copper in Synechocystis PCC
6803(ctaA) (filled columns) and wild type cells
(open columns). Cells were grown in BG-11-C medium and then
exposed to the indicated copper concentrations for 2 h prior to
harvesting, acid digestion, and determination of copper content by atom
absorption spectroscopy.
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Fig. 4.
Kinetics and magnitude of photooxidation of
cytochrome c6 in Synechocystis
PCC 6803 grown in different [copper]. Panel A, a
diagrammatic representation of electron flow within the thylakoid lumen
in Synechocystis PCC 6803. Upon light (h )
activation of photosystems (PS) I and II, electrons are
donated to a membrane complex that includes PS I from a reduced
electron carrier located within the thylakoid lumen. The carrier is
subsequently re-reduced by the cytochrome
b6f complex (Cyt
b6f), associated with PS II, and must shuttle between
the sites of electron acquisition and donation. In copper sufficiency
the carrier is plastocyanin-encoded by petE, whereas under
copper limitation this is substituted with cytochrome
c6 encoded by petJ. Both carriers can
also donate electrons to cytochrome c oxidase (Cyt c
Ox.). Panel B, the kinetics of light-induced absorbance
change, deconvoluted at 554 nm for cytochrome
c6, plus f (
A) in
response to a 6-µs pulse of actinic light (coincident with the drop
in
A) in cells grown in BG-11-C with different added
[copper] (0, 0.2, 0.3, and 1 µM). The subsequent rise
in
A is because of re-reduction of cytochrome
c6 by the cytochrome
b6f complex. Panel C, the
magnitude of the decrease in
A at each [copper] and
hence the relative amount of photooxidation of cytochrome
c6. The residual change in
A in 1 µM copper is the contribution of cytochrome
f.
1 grown in 0.3 µM 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
additional contribution at 554 nm because of transient oxidation of
cytochrome c6 (4) (at rest the carrier is
reduced). Growth of Synechocystis PCC 6803(pacS)
is inhibited by 0.3 µM copper (Fig. 1A)
precluding the use of this [copper] in subsequent experiments. Growth
is not inhibited at 0.2 µM copper. Photooxidation of
cytochrome c6 (plus cytochrome f) has
been compared in BG-11-C containing 0, 0.2, 0.3, and 1 µM
copper (Fig. 4B). The transient decrease in
A
at 554 nm is less in 0.2 µM compared with no added copper
and similar to that observed in 0.3 µM copper. This
implies that plastocyanin replaces cytochrome c6
in 0.2 µM copper (Fig. 4C). Deconvolution at
575 nm (for plastocyanin) revealed a small (consistent with the lesser
575 for plastocyanin compared with
554
for cytochrome c6) change upon exposure to a
pulse of actinic light in cells grown in 0.2 µM copper
that was not evident in cells grown without added copper (data not shown).
A deconvoluted at
554 nm was compared in Synechocystis PCC
6803(pacS), Synechocystis PCC
6803(ctaA), and wild type cells grown in 0.2 µM 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
c6 replaces that of plastocyanin in the
mutants. Photooxidation of cytochrome
c6 was unaltered in Synechocystis PCC
6803(ziaA) or Synechocystis PCC
6803(coaT) grown in BG-11-C containing 0.2 µM
copper (Fig. 5B).
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Fig. 5.
Disruption of pacS or
ctaA increases the magnitude of photooxidation of
cytochrome c6 and reduces petE
transcript abundance. Panel A, the kinetics of
light-induced absorbance change deconvoluted at 554 nm
( A) in response to a 6-µs pulse of actinic light in
wild type cells and each of the individual transporter
(pacS, ctaA, coaT, and
ziaA) mutants grown in BG-11-C supplemented with 0.2 µM copper. Panel B, the magnitude of the
decrease in
A deconvoluted at 554 nm in each of the
mutants and hence the relative amount of photooxidation of cytochrome
c6. Equivalent trends have been observed on two
further occasions. Panel C, Northern analysis showing
plastocyanin (petE) (upper panel) and cytochrome
c6 (petJ) (lower panel),
transcript abundance in Synechocystis PCC
6803(ctaA) (lane 1), Synechocystis PCC
6803(pacS) (lane 2), and wild type (lane
3) cells grown in BG-11-C containing 0.2 µM copper.
DNase-treated RNA was resolved on a 1.2% w/v formamide-agarose gel and
probed with petE.
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Fig. 6.
Copper super-supplementation decreases
photooxidation of cytochrome c6 in cells
disrupted in ctaA. The magnitude of the decrease
in A deconvoluted at 554 nm in Synechocystis
PCC 6803(ctaA), and hence the relative amount of
photooxidation of cytochrome c6, in cells
cultured in media containing greater than 0.2 µM copper
is shown.
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Fig. 7.
Growth of Synechocystis PCC
6803(pacS) and Synechocystis PCC
6803(ctaA) in low iron and of
petJ/transporter double mutants in low copper.
Panel A, growth in BG-11-FC of wild type
(squares), Synechocystis PCC
6803(pacS) (triangles), and
Synechocystis PCC 6803(ctaA) (circles)
in media with no added iron (left panel) or supplemented
with the iron chelator deferoxamine mesylate (right panel). Panel
B, Southern analysis of HincII-digested DNA from
Synechocystis PCC 6803(petJ,ctaA) (lane
1), Synechocystis PCC 6803(petJ, pacS)
(lane 2), and wild type (lane 3) electrophoresed
on a 0.8% w/v agarose gel and probed with part of petJ.
Integration of the 0.8-kb chloramphenicol acetyl transferase gene into
petJ creates a 1.9-kb fragment containing part of
petJ. Panel C, growth in BG-11-C of Synechocystis
PCC 6803(pacS) (open triangles),
Synechocystis PCC 6803(petJ,pacS) (closed
triangles), Synechocystis PCC 6803(ctaA)
(open circles), and Synechocystis PCC
6803(petJ,ctaA) (closed circles); media were
supplemented with the copper chelator bathocuproinedisulfonic acid
after 48 h (indicated by an arrow). Data are the means
of triplicate determinations with S.D. Equivalent trends have been
observed on two further occasions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
A model for the action of each of the four
related CPx-type transporters in Synechocystis PCC
6803. Arrows indicate the direction of transport.
Metals for which homeostasis is known to be altered following deletion
of the respective transporter are shown. The model locates PacS within
thylakoid membranes supplying copper for plastocyanin, PetE, in the
thylakoid lumen. Shaded ovals represent a single
amino-terminal metal-associated motif (CXXC), and a
non-shaded oval (ZiaA) represents an additional
HXH motif.
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 transport 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.
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ACKNOWLEDGEMENTS |
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We thank C. Mullineaux and J. Cavet for advice.
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FOOTNOTES |
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* This work was supported in part by the Plant and Microbial Sciences Committee at Biotechnology and Biological Sciences Research Council (BBSRC). S. T. was supported by a BBSRC committee studentship.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 191-222-7695; Fax: 191-222-7424; E-mail: n.j.robinson@newcastle.ac.uk.
Published, JBC Papers in Press, March 22, 2001, DOI 10.1074/jbc.M011243200
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ABBREVIATIONS |
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The abbreviations used are: ORF(s), open reading frame(s); kb, kilobase.
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REFERENCES |
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