Journal of General Microbiology (1993), 139, 827-834.
Printed in Great Britain
827
Transport kinetics, cation inhibition and intracellular location of
accumulated caesium in the green microalga ChZoreZZa salina
SIMONV. A V E R YGEOFFREY
,~
A. CODDand GEOFFREY
M. GADD*
Department of Biological Sciences, University of Dundee, Dundee DDl 4HN, UK
(Received 14 September 1992; revised 16 December 1992; accepted 18 December 1992)
Caesium accumulation by Chlurella salina, from buffer (pH 8.0) supplemented with 50 pM-CsCl and 13'Cs,
continued for approximately 15 h and displayed first-order kinetics, indicating a single rate-limiting transport
process. Efflux of Cs+ from Cs+-loaded cells occurred in two distinct phases: a rapid initial loss, representing
approximately 11% of total cellular Cs+,corresponded to release from the cell surface, whereas a second, slower,
phase of effluxcorresponded to loss from the cytoplasm and vacuole. Analysis of subcellular Cs+compartmentation
revealed that most Cs+ was accumulated into the vacuole of C. salina, with lesser amounts being associated with
the cell surface or located in the cytoplasm. Uptake of Cs+ into the vacuole was correlated with a stoichiometric
exchange for K+. However, no loss of K+ from the cell surface or cytoplasm was evident nor was Cs+ or K+
associated with insoluble intracellular components. Calculated values for the Cs+ flux across the vacuolar
membrane were approximately equal to, or higher than, values for total cellular influx. Cs+ influx obeyed
MichaelisrMenten kinetics over the lower range of external Cs+ concentrations examined (0.01-0.25 mM) and a
single transport system with a K, 0.5 mM was evident. The effects of other monovalent cations on Cs+ influx
implied that K+ and Rb+were competitive, and NHt non-competitive/uncompetitiveinhibitors of Cs+uptake. The
order of inhibition was Rb+ > K+ > NHT. We propose that a single, relatively non-selective, rate-limiting transport
system for Cs+ influx is located on the cytoplasmic membrane of C. salina, while a more permeable vacuolar
membrane facilitates transport of Cs+ into the vacuole.
-
Introduction
Several recent reports of metal-microbe interactions
have concentrated on radionuclides, due to their prevalence in waste effluents from the nuclear industry and in
atomic fallout, e.g. from Chernobyl in 1986 (Burton,
1975; Clark, 1989; Watson et al., 1989; Gadd & White,
1989; Halldin et al., 1990; Avery et al., 1992a; Garnham
et al., 1992). The potential role of micro-organisms in the
mobilization and cycling of Cs+ in the aquatic environment has been described (Avery et al., 1992a) and
previous studies have shown that inhibition of the
growth of cyanobacteria (Avery et al., 1991) and
microalgae (Avery et al., 19926) by Cs+ arises through
replacement of cellular K+.It is now generally considered
that Cs+is accumulated into microbial cells by active K+
transport systems (Bossemeyer et al., 1989; Jones &
*Author for correspondence. Tel. (0382) 23181 ext. 4266; fax (0382)
22318.
t Present address : School of Pure and Applied Biology, University of
Wales College of Cardiff, PO Box 915, Cardiff CF1 3TL, UK.
Gadd, 1990; Avery et al., 1992a), although Cs+ uptake
via the NH,+transport system(s) of the cyanobacterium
Anabaena variabilis has recently been reported (Avery et
application of kinetic models of Cs+
al., 1 9 9 2 ~ )The
.
uptake characteristics and NH,+competition in the latter
study, and to cation transport systems in other microorganisms (Borst-Pauwels, 198 l ) , has provided a more
complete understanding of microbial monovalent cation
uptake. However, detailed kinetic studies of cation
competition with Cs+are still lacking (Avery et al., 1991).
In addition to kinetic analysis allowing a fuller
understanding of the interactions between ions for a
common uptake mechanism, characterization of the
kinetics of cellular influx and efflux may provide
information relating to the location of rate-determining
ion transport systems and intracellular compartmentation of monovalent cations (Barber, 1978; Baker &
Hall, 1988). Such investigations are particularly pertinent
to Cs+ because of its inhibitory effects on microbial
growth. The selective sequestration of certain potentially
toxic metal ions in the vacuole of eukaryotic algal cells
may represent a cellular detoxification mechanism (Reed
0001-7838 0 1993 SGM
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828
S. V. Avery, G. A. Codd and G. M . Gadd
& Gadd, 1990). Furthermore, the vacuole also acts as a
storage pool for essential ions maintaining cytoplasmic
ionic homeostasis and favourable conditions for metabolic processes (Jones & Gadd, 1990). Differential
extraction techniques have been used to determine solute
levels in different cellular compartments (Huber-Walchli
& Wiemken, 1979; White & Gadd, 1987) and their use
has recently been extended to microalgae. Garnham et
al. (1992) observed higher concentrations of Co2+,Mn2+
and Zn2+ in the vacuoles than in the cytoplasm of the
microalga Chlorella salina, although due to the small
fraction of the cellular volume occupied by the vacuoles
of this organism, total metal levels were higher in the
cytoplasm.
In this study, influx/efflux experiments and differential
extraction techniques have been used to determine the
intracellular localization of Cs+ in C. salina, while kinetic
studies have determined the selectivity of the system(s)
determining Cs+ uptake.
Methods
Organism and culture conditions. Axenic cultures of Chlorella salina
Kufferath CCAP 2 1 1/25 (obtained from Dr G. Russell, Department of
Evolutionary and Environmental Biology, University of Liverpool,
UK) were grown at 22 "C in 100ml MN medium (Waterbury &
Stanier, 1978) of composition (mg 1-') : MgSO,. 7H,O, 37.5 ;
CaCl,. 2H,O, 18-0; NaNO,, 750.0; K,HPO,, 20-0; citric acid, 3.0;
FeSO,. 7H,O, 3.0; EDTA (disodium salt), 0.5; Na,CO,, 20.0; H,BO,,
2.68 ; MnCl, .4H,O, 1.81 ; ZnSO, .7H,O, 0.222 ; Na,MoO, .2H,O, 0.39 ;
CuSO, . 5H,O, 0.079 ; Co(NO,), . 6H,O, 0.049 ; in 75 YO(v/v in distilled
water) filtered seawater (obtained from East Rock, St Andrews,
Scotland; filtered using Whatman no. 1 filter papers). Cultures were
incubated in 250 ml Erlenmeyer flasks with aeration provided by rotary
shaking at 150 cycles min-' and under a photon fluence irradiance
incident on the surface of the flasks of 120 pmol rn-, s-', provided by
white fluorescent light tubes.
Cs+ uptake experiments. Cells from the late exponential growth phase
Cjudged using semi-log growth plots; Avery et al., 1992b) were collected
by centrifugation (1200 g, 10 min), washed thoroughly with 10 mMN-tris(hydroxymethyl)methyl-3-aminopropanes~phonicacid (TAPS)
buffer containing 0.5 M-NaCl, adjusted to pH 8.0 using 1 M-NaOH, and
suspended to a density of approximately 1 x lo6 cells ml-' in 20 ml of
the same buffer. The suspension was then equilibrated for 1 h at 22 "C
and a photon fluence irradiance of 120 pmol mP2s-'. Cs+ uptake was
initiated by the addition of CsCl to the required concentration, with
137Cs(Amersham) added as a tracer to a final activity of between
0.925-7.40 KBq ml-'. Where required, either KC1, RbCl or NH,CI, at
the desired concentration, was added to the cell suspension with the
CsCl and 137Cs.At intervals, 200 p1 samples were removed and
harvested by centrifugation (8000 g, 30 s) through a layer (200 pl)
comprising 40 YO(v/v) Dow Corning 550 silicone oil and 60 YO(v/v)
bis-3,3,5-trimethylhexylphthalate (Fluka) in 500 pl Beckman PRO22
plastic tubes, using an Eppendorf 5412 microcentrifuge. The bottom of
each tube was cut off and placed in 5 ml Ecoscint A scintillation fluid
(National Diagnostics) for 24 h before measuring radioactivity using a
Packard Minaxi Tri-carb 4000 scintillation counter. All glassware was
washed with 1 M-HCI and rinsed thoroughly with distilled deionized
water prior to use.
Cs+ eflux. Cell suspensions were prepared as described above and
incubated in the presence of 50 ~ M - C Slabelled
+
with 137Csto give a final
activity of approximately 3-7 KBq ml-'. After incubation for 15 h, cells
were removed from the loading medium by centrifugation (1200 g,
10 min) and resuspended to the same cell density in 10 mM-TAPS buffer
containing 0.5 M-NaCl, pH 8, and either 20 or 50 p~ non-radioactive
CsCl or 50 ~M-KCI.
Samples were removed at intervals and measured
for radioactivity as described above.
Cellular distribution of Cs+and K+. Cell suspensions were prepared as
described above. Caesium uptake was initiated by the addition of CsC1,
3.7 KBq ml-'), to a final conlabelled with 137Cs(final activity
centration of 50 p ~At. intervals, cells were harvested by centrifugation
(SOOOg, 30 s) of 1 ml of uptake suspension through a layer (0.5 ml)
comprising 40 YO Dow Corning silicone oil and 60 YO bis-3,3,5trimethylhexylphthalate in a 1.5 ml Eppendorf microcentrifuge tube.
After removal of the supernatant, all further operations were carried
out in the microcentrifuge tube. Exchangeable Cs+ at the cell surface
was removed by washing at 4 "C with 3 x 1 ml tracer-free CsCl
solution, at a Cs+ concentration approximating to that which remained
in solution following uptake for the specified incubation time. This was
followed by washing once with 10 mM-Tris/MES buffer containing
0.5 M-NaC1, pH 6 at 0-4 "C. Cells were incubated in the washing
medium for 1 min and separated by microcentrifugation at each step.
All the supernatants were combined and retained. Permeabilization of
the cell membrane was achieved by resuspending the pellet in 1 ml
10 mM-Tris/MES buffer, pH 6, with 0.7 M-sorbitol at 25 "C. DEAEdextran was added to the same buffer (40 pl, 10 mg ml-'), mixed and
the sample incubated for 30 s at 25 "C. Cells were separated by
centrifugation (8000 g, 30 s) and the supernatant removed and retained.
The permeabilized cells were washed with 3 x 0.5 ml 0.7 M-sorbitol in
10 mM-Tris/MES buffer, pH 6, at 0-4 "C, incubating for 1 min at each
wash and separating by centrifugation as described above. Supernatants were retained and combined with that from the permeabilization step. The vacuolar contents were extracted by suspending the
pellet in 60% (v/v) methanol at 0-4 "C for 30 s, centrifuging (SOOOg,
30 s) and removing the supernatant. This was repeated three times and
followed by three further washes in 10 mM-Tris/MES buffer, pH 6, at
W "C, incubating for 1 min and centrifuging as above. The supernatants were retained and combined with those from the methanol
washes. The remaining pellet was suspended in 0 5 ml10 mM-Tris/MES
buffer, pH 6, the suspension removed and the tube washed with a
further 0.5 ml of the same buffer. The suspension and washings were
combined. Radioactivity (137Cs)and K+ (see below) were measured in
each of the four fractions.
-
Measurement of K+. After appropriate dilution with distilled
deionized water, the potassium concentration in each of the fractions
described above was determined using a PYE Unicam SP9 atomic
absorption spectrophotometer, with reference to standard KCl solutions.
Cell counts. Cell numbers were determined using a modified
Fuchs-Rosenthal haemocytometer after appropriate dilution of cell
suspensions with distilled water.
Results
Cs' accumulation
Cs+ uptake by C . salina was examined over 24 h in the
presence of 50 PM-CScl (Fig. 1a). The rate of Cs'
accumulation was highest over the first 2 h [approximately 3.8 nmol Cs+ h-' (lo6cells)-'] but declined there-
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Cs+ transport and cellular location in C. salina
I
5
I
10
Time (h)
829
I
15
Fig. 2. Caesium efflux from C. salina. '37Cs-loadedcells were suspended
to 1 x lo6 ml-' in 10 mM-TAPS buffer containing 0.5 M-NaCl, pH 8,
supplemented with 15 pM-CsCl (O), 50 pM-CsCl ( 0 )or 50 ~M-KCI
(0).
Mean values from three replicate determinations are shown fSEM
where possible.
5
10
15
Time (h)
20
25
Fig. 1. (a) Caesium accumulation by C. salina. Cells were suspended to
1 x lo6 ml-' in 10 mM-TAPS buffer containing 0.5 M-NaCl, pH 8,
supplemented with 50 ~M-CSCI.
Mean values from three replicate
determinations are shown kSEM where possible. (b)First-order semi-log
plot of the data shown in (a). C,,, intracellular Cs+ after 24 h; C,,
intracellular Cs+ at the specified time of incubation.
after. The intracellular level of Cs+ was effectively at a
maximum after 15 h incubation, at approximately
23 nmol (lo6cells)-'. The total amount of intracellular
Cs+ increased by only 4 % between 15 and 24 h (Fig.
la). A semi-log plot of the data in Fig. l(a) gave a
straight line (Fig. lb), indicating that Cs+ uptake in C.
salina followed first-order kinetics and probably occurred
into a single intracellular compartment. First-order
uptake kinetics may arise when the tonoplast is much
more permeable than the plasmalemma; under these
conditions the plasmalemma is the rate-limiting membrane.
-
Cs+ e@ux
Cs+ efflux studies were carried out with cells of C. salina
pre-loaded with 13'Cs. Two phases of efflux could be
distinguished and, through extrapolation of the linear
portions of the plot to zero time, the quantity of Cs+
present in the appropriate cellular compartment at the
start of the elution could be determined (Fig. 2). An
initial rapid release of approximately 3-4 nmol Cs+
(lo6cells)-* corresponded to the amount of Cs+ initially
associated with the cell wall. The remaining intracellular
Cs+ [approximately 26-4 nmol ( lo6cells)-'] corresponded
to both the cytoplasmic and vacuolar fractions (rates of
efflux for these fractions could not be distinguished).
Relative amounts of Cs+associated with the cell wall and
cytoplasm plus vacuole were approximately 11.4 % and
88-6%, respectively. Rates of release of intracellular Cs+
were approximately 0.23, 0.29 and 0.35 nmol Cs+ h-'
(lo6cells)-' for cells incubated in the presence of 15 p ~ c s c l , 50 ~ M - K Cand
I 50 pM-cscl, respectively (15 pMCsCl was used as this was representative of the
concentration of Cs+ remaining in solution after incubation of C. salina for 15 h in the presence of 50 p ~ CsCl). Rates of Cs+efflux were slow in comparison to the
initial rates of Cs+ accumulation observed previously
(Fig. la).
Cellular distribution of Cs+ and K+
The intracellular location of Cs+ and K+, over a time
course of Cs+ uptake, was also analysed by cell
fractionation. No Cs+ or K+ was found to be associated
with particulate intracellular material, e.g. organellar
membranes. The greatest degree of Cs+ accumulation
was observed in the vacuolar fraction and this coincided
with a decline in vacuolar K+; the replacement of
vacuolar K+ with Cs' was approximately stoichiometric
and after 15 h incubation approximately 23 nmol Cs+ or
K+ ( lo6 cells)-' had been accumulated or released,
respectively (Fig. 3 a, b). In contrast to the stoichiometric
vacuolar exchange of Cs+ and K+, no release of K+ from
the cell wall or cytoplasm of C. salina was observed,
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830
S. V. Avery, G. A . Codd and G. M . Gadd
30
1989) for Chlorella spp., the following values can be
inserted into the above equation:
I
-4t
40K
@t
30
u
m
-
-
I
Y
I
I
1
I
1
5
10
15
Time (h)
Fig. 3. Subcellular compartmentationof caesium and potassium during
a time-course of caesium uptake in C. salina. Cells were suspended to
1 x lo6 ml-' in 10 mM-TAPS buffer containing 0.5 M-NaCl, pH 8,
supplemented with 50 pM-CsC1. Panels (a)and (b)show (a)intracellular
Cs' (filled symbols) and ( b ) intracellular K+ (open symbols) present in
the vacuole (A, A), cytoplasm (0,
W) and cell wall ( 0 , 0).
(c)
Tonoplast influx rates for Cs+calculated from the data shown in (a)and
(b), Mean values from three replicate determinations are shown f SEM
where possible.
whereas Cs+ accumulation into these compartments did
occur, to final levels of approximately 5.6 and 8.2 nmol
(lo6cells)-', respectively. Hence the total amount of Cs+
accumulated by C. salina after 15 h exceeded the total
cellular loss of K+ (Fig. 3a, b). The separate estimation
of cytoplasmic and vacuolar ion levels can be employed
to determine their flux across the tonoplast when the
plasmalemma is the rate-limiting membrane, as appears
to be the case here for Cs+ in C. salina. MacRobbie
(1966) showed that in such cases, at time t, when the
concentration of the solute in question is rising simultaneously in both the cytoplasm and vacuole:
where 4t is the tonoplast flux rate, (3, is the product of
vacuolar solute concentration and vacuolar volume, S, is
the cytoplasmic level of solute and S, is the vacuolar level
of solute. By substituting values of nmol Cs+ (lo6cells)-'
in the cytoplasm and in the vacuole, for S, and S,,
respectively, at time t = 1-5h and assuming a vacuolar
volume of 45.5 fl per cell, a value based on data for
cytoplasmic volume (Kirst, 1977) and vacuolar volume
as a percentage of cytoplasmic volume (Raven, 1980,
1
3.34
0.073 mol 1-' x 4.6 x
X
1.5 h
(1.75 - 3.34)
= 4-64 nmol Cs+ h-' ( lo6cells)-'
=
Tonoplast influx rates were also calculated at various
other intervals after the commencement of Cs+ accumulation and were found to increase to a maximal level
of approximately 5.3 nmol Cs+ h-' (lo6 cells)-' after 4 h
(Fig. 3c). Subsequently the rate decreased and, after
incubation for 10 h, the value was approximately
3.7 nmol Cs+ h-' (lo6 cells)-'. The values obtained for
tonoplast flux rate were generally higher than the
maximum total cellular uptake rate estimated previously
[approximately 3.8 nmol Cs+ h-I (lo6 cells)-'] (Fig. 1a),
the latter value presumably being a direct measure of Cs+
transport across the rate-limiting plasmalemma.
Kinetics of Cs+ uptake and the influence of competing
monovalent cations
The rate of Cs+ uptake in C. salina was determined over
a range of CsCl concentrations, from 0.01 to 2 0 - 0 m ~ .
Rates were determined over the initial linear phase of
active uptake (20-120 min) that followed any binding of
Cs+ to the cell surface, and data were fitted to log-log
plots (Reed et al., 1981) due to the wide range of Cs+
uptake rates and Cs+ concentrations examined (Fig. 4a).
The rate of Cs+ uptake was dependent on the external
CsCl concentration supplied. At low concentrations of
CsCl (0-01-0.25 mM), the rate of Cs+ uptake increased
with increasing external concentration and was maximal
at approximately 12.5 nmol Cs' h-' (lo6cells)-'. At
higher concentrations (0.5-20-0 mM-CsCl), no marked
increase in the Cs+ uptake rate was evident and values
approximated to those observed at 0.25 mM-CsC1 (Fig.
4a). When the data were transformed according to
Lineweaver & Burk (1934), a straight line could be fitted
to reciprocal Cs+ uptake rates obtained for cells
incubated in the presence of external concentrations of
0-01-0.25 mM CsCl (Fig. 4b). To give greater emphasis to
the more accurate rate values determined at higher
substrate concentrations, lines were fitted visually. Linear
regression analysis, which gave equal weighting to all
points, indicated similar trends to those described here
although estimated values for the Vmaxand K, differed
slightly. The intercepts on the y - and x-axes gave
estimates of the Vmaxand the K, for Cs+ uptake by C.
salina of 33.3 nmol Cs+ h-' (lo6cells)-' and 0.5 mM,
respectively (Fig. 4 b). At external concentrations of
CsCl> 0.25 mM, uptake rates deviated from classic
Michaelis-Menten kinetics and data did not conform to
Lineweaver-Burk plots.
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Cs+transport and cellular location in C. salina
G -n
3
1.25
1.0
-
0.5
0.25
831
plot, revealed linear inhibition and a Ki for Rb+ of
approximately 0.15 mM. In contrast to K+ and Rb+,
inhibition of Cs+ uptake by NH,+was non-competitive/
uncompetitive (Fig. 5 c). This situation is characterized
by decreases in both the & and Vmaxin the presence of
the inhibitory ion and is intermediate between noncompetitive and uncompetitive inhibition. & values for
Cs+ uptake rates decreased from approximately 0.85 mM
to 0-45 and 0.24 mM in the presence of 0-5 and 2.0 mMNH4C1, respectively, and Vmaxvalues decreased from
approximately 50-0 nmol Cs+ h-' (lo6 cells)-' to 19.2 and
6.9 nmol Cs+ h-' (lo6cells)-' at the same concentrations
of NH4Cl, respectively (Fig. 5c). Secondary plots of the
data obtained from Lineweaver-Burk analysis indicated
that, as with K+ and Rb+, inhibition of Cs+ uptake by
NH; was linear, although in this case a larger K, of
approximately 2.2 mM was calculated.
Discussion
I/[CSCI]( m d )
Fig. 4. Kinetics of caesium uptake by C. salina. Cells were suspended
to 1 x lo6 ml-' in 10 mM-TAPS buffer containing 0-5 M-NaCl, pH 8,
supplemented with the appropriate concentration of CsCl. (a) log-log
plot of Cs+uptake rates against a range of external CsCl concentrations.
Slopes were calculated using the initial (20-120 min) rates. (b)The same
data transformed according to Lineweaver & Burk (1934). Typical
results are shown from at least three experiments.
The effect of externally supplied K+, Rb+ and NH,+on
Cs+ uptake in C. salina was examined over a range of
CsCl concentrations (0.025-0-25mM) at which
Michaelis-Menten kinetics were observed. Cs+ uptake
rates were reduced when KC1 was supplied at external
concentrations of 0.25 and 1.0 mM. Transformation of
the data to a Lineweaver-Burk plot indicated that the
inhibition was of a competitive nature (Fig. 5a). In the
presence of 0.25 mM-KCl the K, increased from 0.5 mM
to approximately 0.7 mM, while the Vmaxremained the
same as for uninhibited Cs+ uptake. At 1.0 mM-KCl the
& was increased further to approximately 2-5 mM,
although in this case an increase in the Vmax,to
approximately 50 nmol Cs+h-' (10-6 cells)-', was also evident (Fig. 5a). A secondary plot (not shown) of the slopes
of the lines obtained from the Lineweaver-Burk analysis,
against KCl concentration, demonstrated that linear
inhibition of Cs+ uptake occurred in the presence of
KCl. The half-maximal inhibitory concentration (Ki),
was approximately 0.39 mM-KCl. Rb+ was also a
competitive inhibitor of Cs+uptake in C. salina (Fig. 5b).
The & increased from approximately 0.50 mM to 0.83
and 2-50 mM in the presence of 0.1 and 0.5 mM-RbCl,
respectively ; the Vmaxremained approximately constant
at 30 nmol Cs+ h-' (lo6cells)-'. A secondary plot, of the
gradients of the lines obtained from the double reciprocal
The results demonstrate that most Cs+ was accumulated
into the vacuole of C. salina, whereas lower amounts
were accumulated in the cytoplasm or associated with
the cell wall. No Cs+ was associated with insoluble
intracellular components, implying that Cs+ is maintained largely in mobile ionic form in C. salina, unlike
Mn, Zn and Co which are mostly associated with
particulate material (Garnham et al., 1992). Other studies
on the intracellular compartmentation of monovalent
cations in microalgae indicate that the distribution of
ions between cellular phases may be species specific. For
example, cells of Chlorella pyrenoidosa acted as a single
compartment for K+, Na+ and C1- (Barber, 1978).
However, Ginzburg (198 1) demonstrated that C1- influx
in Dunaliella parva comprised two phases, and it has
subsequently been suggested that this may also be the
case for K+ and Na+ in Dunaliella tertiolecta (Ehrenfeld
& Cousin, 1982). Hajibagheri et al. (1986) observed a
slight preferential localization of Na+ and C1- in the
vacuole than in the cytoplasm of D. parva, although this
situation was reversed in the case of K+. Analysis of Cs+
uptake data for C. salina revealed first-order kinetics,
which was consistent with uptake of Cs+ into a single
intracellular compartment. Furthermore, it can be
inferred that the tonoplast of C. salina was more
permeable to Cs+ than the plasmalemma as only a single
rate-limiting phase of accumulation could be detected,
presumably located at the plasmalemma (Baker & Hall,
1988). This evidence was further supported by efflux
studies which revealed that, unlike the classic three-phase
loss of ions that is generally observed from plant cells
(Clarkson, 1974), release of Cs+from the cytoplasm of C.
salina could not be distinguished from loss across the
tonoplast. The single rate of efflux from these compart-
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832
S . V. Avery, G. A . Codd and G. M . Gadd
2.0
1.5
1.0
en
n
0.5
.4
8
5
2.0
v
d
1.5
Y
-3
c
3
+m
u
1.0
-2
-1
0
0.5
Q+
2 2.0
-.
0
*
1.5
1.0
0.5
-10
0
10
20
30
l/[CsCl] (mM-1)
40
Fig. 5. Lineweaver-Burk plots of cation inhibition of Cs' uptake by C.
salina. Cells were suspended to 1 x lo6 ml-' in 10 mM-TAPS buffer
containing 0.5 M-NaCl, pH 8. Initial rates of Cs+ uptake from 0.025 to
0.25 mM-CsC1 were determined in the presence of: (a) 0 (O),
0.25 ( 0 )
and 1.0 (0)
mM-KC1; (b) 0 (O),
0.1 ( 0 )and 0-5 (0)
mM-RbC1; ( c ) 0
(O),0.5 ( 0 ) and 2-0 (0)
mM-NH,Cl. Enlarged portions of the
interaction areas of the x- and y-axes are shown in the insets. Typical
results are shown from at least three experiments.
ments was lower than the rate of accumulation observed
previously, implying that net uptake of Cs+(K+)occurred
under the incubation conditions specified. Transport
systems dealing with K+/Na+ or K+/H+ exchange in
Chlorella spp., in addition to those determining K+/K+
exchange, have been widely reported in the literature
(Schaedle & Jacobsen, 1965; Paschinger & Vanicek,
1974; Tromballa, 1981; Ehrenfeld & Cousin, 1984) and
the operation of such mechanisms would explain the
present observations. As a consequence, it is probable
that the rates of efflux of Cs+ may serve as a direct
measurement of the rate of Cs+/Cs+(K') exchange. The
initial rapid release of Cs+ during efflux corresponded to
levels of the ion initially associated with the cell wall and
represented 11.4% of the total cellular Cs+. This is in
agreement with the results of Ahmad & Hellebust (1984),
who estimated that the total bound fraction of monovalent cations in the halotolerant microalga Chlorella
autotrophica represented 10-5YO of the total cellular
content.
A more accurate determination of the cellular localization of Cs+ in C. salina was obtained by fractionation
of subcellular compartments. Results provided by this
technique indicated that cytoplasmic and vacuolar levels
of Cs+ in C. salina at equilibrium were approximately
22.3 and 62.5% of the total cellular Cs+ content,
respectively. The remaining 15.2YO accounted for Cs+
bound to the cell wall and was similar to the 11.4%
estimated by efflux studies. These results correlate well
with those obtained by similar fractionation techniques
with yeast, where Cs+ was accumulated mostly into the
vacuole (Perkins & Gadd, 1991). The proportion of
vacuolar Cs+ reported here is particularly high considering that in ' non-vacuolate' cells like Chlorella spp.,
where small vacuoles are distributed throughout the
cytoplasm (Kirst, 1977), the total vacuolar volume may
represent < 10% of the total cell volume (Raven, 1980,
1989). The diminutive size of the vacuolar fraction of C.
autotrophica may have led Ahmad & Hellebust (1984) to
the assumption that all unbound monovalent cations
were present in the cytoplasm of this micro-organism. It
is unlikely that the accumulation of Cs+ into vacuoles
represents a specific detoxification mechanism. Cs+ is
taken up via K+ transport systems and apparently
distributes itself accordingly ; a stoichiometric exchange
of vacuolar K+ for Cs+ was evident. It should be noted,
however, that the toxicity of Cs+ to various yeast strains
has been shown to be species specific and the extent of
toxicity appears to correlate with an increased cytoplasmic content of this ion (Perkins & Gadd, 1991). The
separate estimation of cytoplasmic and vacuolar ion
levels in C. salina permitted the determination of
tonoplast influx rates for Cs' according to MacRobbie
(1966). The calculated rates of Cs+ transport across the
tonoplast were generally higher than total cellular influx,
further supporting the conclusion that Cs+ is more
permeant through the tonoplast than the plasmalemma
of C. salina. These results are therefore in agreement with
the model proposed by Hajibagheri et al. (1986) for D.
parva. These workers suggested that the cytoplasmic
membrane was restrictive to monovalent cation fluxes,
whereas the tonoplast was more permeable.
At the lower external Cs+ concentrations examined
(< 0-25 mM-CsCl), Cs+ uptake by C. salina obeyed
Michaelis-Menten kinetics, as is the case for Cs+ uptake
by higher plants (Shaw & Bell, 1989), and a single Cs'
transport system of apparent K, 0.5 mM was evident.
Deviations from Michaelis-Menten kinetics, similar to
those reported here at higher external Cs+ concentrations, have been observed previously for other metal
ions and micro-organisms and were attributed to a
decrease in the cellular surface potential (Borst-Pauwels,
1981). Although no kinetic data are available for K+
transport in C. salina, two Rb+ (K+) uptake systems are
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-
Cs+ transport and cellular location in C . salina
known to occur in C. pyrenoidosa, with K, values of
0-038 and 6.45 mM (Kannan, 1971), and in the cyanobacterium Anabaena variabilis, with Km values of 0.04
and 4.5 mM (Reed et al., 1981). It should be noted that
the presence of two K+ uptake systems has also been
reported in yeast, although their operation/detection
may depend on incubation conditions (see Jones &
Gadd, 1990). In some cases a single system is evident
with an apparent K, of 0.5 mM (Armstrong & Rothstein,
1964). Should multiple K+ transport systems occur in C.
salina, it is possible that a similar situation to that
reported for A. variabilis is manifest and Cs+ is
apparently only accumulated by one of the systems
(Avery et al., 1992~).Models of ion transport by dual
mechanisms in algal/plant cells have proposed that the
systems are either both located on the plasmalemma
(Kannan, 1971), or on separate membranes, e.g. the
plasmalemma and the tonoplast (Laties, 1969). The
single system detected here is presumably located on the
plasma membrane as this is the rate-limiting membrane.
Competition experiments indicated that Cs+, K+ and
Rb+ were taken up via a common transport system. It
should be stressed that, in certain cases, increases in the
Km in the presence of an inhibitor do not always
represent unequivocal evidence of competitive inhibition
(Jongbloed et al., 1991). However, the interpretation is
probably justified here in view of the mutual transport
mechanisms for Cs', K+ and Rb+ that are known to
occur in other micro-organisms and the similar physicochemical behaviour normally displayed by these ions
(Bossemeyer et al., 1989;Jones & Gadd, 1990; Hughes &
Poole, 1991; Avery et al., 1992a). The non-competitive/
uncompetitive inhibition of Cs+ uptake by NH,+ in C.
salina differed from that observed in A . variabilis, where
inhibition by NH,+ was competitive/non-competitive
(Avery et al., 1992~).The present study suggests that
although NH,+has the potential to reduce Cs+ uptake by
C. salina (by binding to both the carrier and the Cs+carrier complex), these two ions are transported via
different systems in C . salina, as was demonstrated in
fungi (Jongbloed et al., 1991). The order of inhibition of
Cs+ uptake reported here was Rb+ > K+ > NH,+.This is
surprising as K+ generally has a higher affinity than Rb+
for microbial monovalent cation transport systems
(Kannan, 1971; Reed et al., 1981; Jones & Gadd, 1990).
Furthermore, due to the larger ionic radius of Rb+, K+ is
the stronger Lewis acid and is considered to interact
more readily with both organic and inorganic ligands
(Hughes & Poole, 1991). It is likely that due to the low
levels of Rb+ in the natural environment, C. salina has
never been subjected to the selective pressure to develop
a transport mechanism that discriminates as effectively
against Rb+ as it would other, more prevalent monovalent cations, like Na+. It seems likely that for C. salina,
833
in common with Synechocystis PCC 6803 (Avery et al.,
1991), K+may not represent as great a restriction for Cs+
uptake as has previously been indicated (Plato &
Denovan, 1974).
S. V. A. gratefully acknowledges the receipt of a NERC postgraduate
research studentship for the work described.
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