Plant CellPhysiol. 37(1): 1-7 (1996)
JSPP © 1996
Inorganic Carbon Transport and Fixation in Cells of Anabaena variabilis
Adapted to Mixotrophic Conditions
Mar Nieva and Eduardo Fernandez Valiente
Departamento de Biologi'a, Facultad de Ciencias, Universidad Autdnoma de Madrid, 28049 Madrid, Spain
The cyanobacterium Anabaena variabilis ATCC
29413 grown at low CO2 concentration under mixotrophic
conditions with fructose showed a repression in the ability
to fix inorganic carbon. This repression was not due to a
diminution in the ability to transport external inorganic carbon but could be explained by a decrease of two enzymatic
activities involved in the assimilation of inorganic carbon:
carbonic anhydrase and Rubisco. Carbonic anhydrase
activity was close to 50% lower in mixotrophic than in
autotrophic cells. Moreover growth under mixotrophic conditions reduced Rubisco activity at all dissolved inorganic carbon concentrations assayed (5-60 mM). Maximum
Rubisco activity (Vmn) decreased from 4.7 //mol CO2 mg
protein"1 h~' in autotrophic cells to 2.3 /aaol CO2 mg protein"' h"1 in mixotrophic cells. No significant differences
in Km (Cj) between autotrophic and mixotrophic cells were
however observed. The possible mechanisms involved in
the inhibition of Rubisco are discussed.
Key words: Carbonic anhydrase — Cyanobacteria — Inorganic carbon concentrating mechanism — Mixotrophy —
Photosynthesis — Rubisco.
The most common growing pattern of cyanobacteria
is the photoautotrophic growth. The photosynthetic characteristics of cyanobacteria varied according to the level of
CO2 in the medium. At low (air) level of CO2 the cyanobacteria develop an inorganic carbon (CJ concentrating
mechanism (CCM) which permits to elevate the concentration of CO2 around the active site of the Rubisco, the primary photosynthetic carboxylating enzyme. The results are
an increase in photosynthetic affinity for Q and a higher
photosynthetic efficiency in fixing Q. This mechanism is
not induced in cells growing at high-CO2 concentrations
(for review see Kaplan et al. 1991, Badger and Price 1992).
In addition to photoautotrophic growth, some cyanoAbbreviations: BTP, (l,3-bis[tris(hydroxvmethyl)methylamino]-propane); CA, carbonic anhydrase; CCM, inorganic carbon concentrating mechanism; Cj, inorganic carbon; L-MSO,
L-methionine sulfoximine; RBP, ribulose 1,5-bisphosphate;
Rubisco, ribulosebisphosphate-carboxylase/oxygenase; SIS, sorbitol impermeable-space.
Financial source: Support by Direcci6n General de Investigacibn Cientifica y Tecnica (DGICYT) (PB 90-205).
bacteria are also able to develop a heterotrophic metabolism, utilizing exogenous organic compounds as carbon
source (Rippka et al. 1979). The organic compounds can be
assimilated by cyanobacteria both in the light and in the
dark. Assimilation of organic carbon in the light can be performed with the Cj fixation impaired (photoheterotrophic
conditions) or simultaneously with Q fixation (mixotrophic
conditions).
Organic carbon assimilation under mixotrophic conditions induces changes in both respiratory and photosynthetic metabolism in cyanobacteria. Respect to respiratory metabolism it is known that, in cyanobacteria, carbohydrates
are mainly oxidized by the oxidative pentose phosphate
cycle (Stanier and Cohen-Bazire 1977). The resulting
NADPH is further reoxidized by the respiratory cytochrome electron transport chain which share common intermediates with the photosynthetic chain (Pescheck 1987).
Cells adapted to growth on fructose under mixotrophic conditions showed a higher rate of fructose conversion to CO2
(Ferndndez Valiente et al. 1992) and higher value of glucoses-phosphate dehydrogenase activity, the main regulatory enzyme of the oxidative pentose phosphate cycle
(Pelroy et al. 1972, Rozen et al. 1986) than autotrophic
cells. In addition it has been also reported a fructose-induced enhancement in the rate of O2 consumption both in
the dark (Haury and Spiller 1981, Rozen et al. 1986) and in
the light (Fernandez Valiente et al. 1992). These results clearly indicated that growth under mixotrophic conditions enhanced respiratory metabolism in cyanobacteria.
Mixotrophic conditions give also rise to alterations in
the photosynthetic metabolism in cyanobacteria (Haury
and Spiller 1981, Rozen et al. 1988, Fernandez Valiente et
al. 1992, Bloye et al. 1992), green algae (Moroney et al.
1987, Martinez and Orus 1991, Shiraiwa and Umino 1991,
Umino and Shiraiwa 1991) and in vitro cultured plantlets
(Galzy and Compan 1992).
In cyanobacteria a decrease in the rate of net oxygen
evolution by fructose was described in Anabaena variabilis
(Haury and Spiller 1981) and Anabaena azollae (Rozen et
al. 1988). In Synechocystis PCC 6803, it was reported that
addition of glucose induced a decrease in the rate of bicarbonate uptake (Bloye et al. 1992). Likewise, in a previous
paper, we have reported that the photosynthetic affinity for
external Q and the rate of photosynthesis dependent on external inorganic carbon were reduced by fructose in
A. variabilis ATCC 29413 (Fernandez Valiente et al. 1992).
M. Nieva and E. Fernindez Valiente
These data suggest that the CCM could be altered when the
cells were grown under mixotrophic conditions.
In this paper the main steps of the CCM, namely C,
transport and fixation as well as Rubisco and carbonic
anhydrase (CA) activities, have been examined under mixotrophic and photoautotrophic conditions in order to
know the processes involved in the decrease in photosynthesis caused by fructose assimilation in A. variabilis ATCC
29413.
Materials and Methods
Organisms and culture conditions—Photoautotrophic cultures of the cyanobacterium Anabaena variabilis ATCC 29413
were grown under N2-fixing conditions in the nitrogen-free medium reported previously (Mateo et al. 1986). The cultures were
bubbled with sterile air or CO2 enriched (5%) air, depending on
the experiment. Mixotrophic cultures were grown in the same medium supplemented with 10 mM fructose and always bubbled with
low (air) CO2. The cultures were grown at 28°C under constant
light intensity of 90 fiE m~2 s"' provided by 40 W cool white fluorescent tubes. Light intensity was measured with a LiCor LI-1000
Datalogger equipped with a In quantum sensor.
Transport and fixation ofC,—The assays were performed by
using the filtering centrifugation technique (Kaplan et al. 1980).
Prior to the assays the cells were collected by centrifugation at
9,000 x g for 10 min, washed three times with CO2-free 25 mM
HEPES (pH 8). Afterward cells were preincubated in the same
buffer in a closed oxygen electrode chamber to reach the O2 compensation point. This pre-treatment was made to deplete the endogenous pool of CO2 and thus, to overcome a further dilution of
added NaH u CO 3 by endogenous CO2. Samples of cell suspensions were then transferred into microtubes which contained
(from bottom to top): 50 ii\ of 3 M NaOH (alkaline killing solution), 125 ^il oil mixture of 1 : 2 (v/v) dibutyl phthalate with bis
(2-ethylhexyl)phthalate (FLUKA AG, Buchs SG) and 200 /<1 cell
suspension (20 //g Chi ml"'). Incubations were initiated under saturating light by addition of 100/uM NaH14CO3 (final concentration) and the reactions were stopped at different incubation
times (10, 30, 60, 90 and 120 s) by centrifugation of the cells
through the oil layer into the killing solution. Afterwards 15 /il 3
M NaOH were injected into the top layer to minimize the diffusion
of CO2 through the oil layer. The tubes were then quickly frozen
in liquid nitrogen and the bottom, containing the cellular pellet in
the killing solution, cut off and placed in a vial with 700/il 0.1 M
NaOH. Two aliquots of 200 fi\ each were taken from this vial. One
of them was placed into an equal volume of 0.1 M NaOH and assayed for I4C activity by liquid scintillation counting with a LKBWallac 1209 RackBeta (total I4C activity, meaning the transported
Q). The other aliquot was acidified by addition of 200^1 0.5 M
HC1 and left overnight at room temperature for removing acidlabile 14C activity and then measured the remaining radioactivity
(acid-stable 14C activity, showing the fixed Q). The acid-labile 14C
activity (accumulated Q) was estimated from the difference of
radioactivity between alkaline and acidified samples.
Estimation of cellular volume—The results of the transport
and fixation of Q were expressed as a function of the cellular volume, calculated according to Werdan et al. (1972) and Abe et al.
(1987) using also the filtering centrifugation technique. The cells
were incubated with stirring in 3H2O (25^1 ml" 1 ) for 1 min and
then centrifugated through the oil layer. Tritiated water permea-
ble cell space was calculated from the percentage of radioactivity
found in the bottom layer with the cellular pellet respect to the top
layer. To obtain the 14C-sorbitol permeable volume the cells were
incubated with 37 kBq ml" 1 of 14C-sorbitol (11 MBq mmol" 1 ) for
5 s followed by centrifugation and the radioactivity of the bottom
layer respect to the top layer was measured. The volume occupied
by the tritiated water represents the intra- and inter-cellular volume. The volume occupied by the sorbitol is only the intercellular
volume. The cell volume, expressed as SIS (sorbitol impermeablespace), was calculated from the difference between the waterpermeable and sorbitol-permeable volumes.
Enzyme assays—For the enzyme assays the cells were harvested by centrifugation, resuspended in the corresponding buffer
kept on ice and then broken in a French press. Cellular extracts
were properly diluted up to similar protein contents were obtained
in both autotrophic and mixotrophic cultures.
Carbonic anhydrase activity was measured by pH changes in
the crude cell extract resulting from the treatment of French press.
In this case the buffer utilized was 30 mM HEPES-KOH (pH 8.1)
and 1 mM MgSO4. For the assay, 500/d of buffer were preincubated with 500//I of crude cell extract (20/jg Chi ml"1) in a chamber
at constant temperature (1°C) to equilibrate. The reaction was initiated by injection of 1 ml distilled water saturated with CO2 kept
on ice. The pH change rate was monitored with a pH electrode in
the presence or absence of cell extract. Carbonic anhydrase activity was expressed as enzyme units mg protein" 1 . Enzyme units
were calculated from the equation U=(to—t)/t (Yang et al. 1985)
where to and t represent the time needed for the change of one pH
unit without or with cells, respectively. The CA activity was completely inhibited by addition of 10 /iM ethoxyzolamide.
The carboxylase activity of Rubisco was measured in the
supernatant from centrifugation of the cellular extract at 10,000 x
g for 20 min. The extraction buffer utilized in this assay was CO2free 50 mM Bicine (pH 8), 10 mM MgCl2 and 1 mM DTT
(dithiothreitol). 10 mM NaHCO3 was also added for maintaining
the enzyme active (Kaplan et al. 1980, Mouget et al. 1993). For
determining the total Rubisco activity 100 /A of the supernatant
(20 fi% Chi ml" 1 ) was preincubated with 160 /A of the extraction
buffer containing 60 mM NaHCO 3 -NaH l4 CO 3 (2.1 GBq mmol" 1 ,
0.3 mM). The preincubation was performed at 30°C for 15 min.
The reaction was initiated by adding RBP (final concentration 0.6
mM) and it was stopped after 2 minutes by acidification with 0.5
mM acetic acid. The vials were maintained at room temperature
overnight for removing the 14C not fixed. The 14C fixed, showing
the total Rubisco activity, was measured by liquid scintillation
counting. Rubisco activity was expressed as /imol 14Cfixedmg protein" 1 h" 1 .
Glucose 6-phosphate dehydrogenase was determined spectrophotometrically following the rate of NADPH formation at 340
nm (Karni et al. 1984). The buffer used in this assay contains
20 mM BTP (pH 6.5), 10 mM MgCl2 and 2.5 mM glucose-6phosphate. One milliliter of reaction mixture contained 10 mM
MgCl2, 0.5 mM NADP + , 10 mM glucose-6-phosphate and around
100 fig protein from the supernatant resulting from centrifugation. Enzymatic activity was expressed as nmol NADP + reduced
mg protein" 1 min" 1 .
Protein content was estimated according to Bradford (1976).
Chlorophyll concentration was determined in cold methanol according to the spectrophotometric method of Marker (1972).
Results and Discussion
Utilization of inorganic carbon—We have previously
Q assimilation in mixotrophic cells
30
60
90
120
30
60
90
120
30
60
90
120
time (s)
Fig. 1 Time courses of transport (A),fixation(B) and accumulation (C) of inorganic carbon in autotrophic (O) and mixotrophic (•)
cells of A. variabilis. SIS was 0.21 and 0.60 n\ftg Chi"1 in autotrophic and mixotrophic cells, respectively. Experimental conditions
are described in Materials and Methods.
reported in A. variabilis ATCC 29413 that fructose reduced
the rate of photosynthesis dependent on external Q, increased the concentration of Q required to reach the halfmaximum rates of photosynthesis [KU2(C{)] and decreased
the photosynthetic KmM (Fernandez Valiente et al. 1992).
These results could be explained by a decrease in the ability
to transport the external Q and/or by a decrease in the
efficiency in fixing the transported Q. To examine these
possibilities we undertook a study of the ability to transport, fix and accumulate the external Q in autotrophic and
mixotrophic cells.
In Fig. 1 results were expressed as a function of cell volume (SIS) in order to calculate internal concentration of Q.
In this way it was possible to compare the external and internal concentration in autotrophic and mixotrophic cells.
Cells grown under autotrophic conditions exhibited, apparently, a higher ability to transport and tofixQ than mixotrophic cells (Fig. 1 A, B respectively). At 60 s the amount
of I4C accumulated into the cells was around 0.7 mM [//mol
(ml SIS)"1] for autotrophic cultures (7 times higher than
the initial external Q concentration) and 0.2 mM [fimol (ml
SIS)"1] for mixotrophic cultures (2 times higher than the initial external C, concentration) (Fig. 1C). Thus, autotrophic
cells accumulated Q more than two times than mixotrophic
cells. Nevertheless these differences decreased along the experimental time due to the increase in the ability to fix the
accumulated Q, which is higher in the autotrophic cells
(Fig. IB).
However, it is important to point out that, as we described in Table 1, SIS value per cell is almost three times
higher in mixotrophic cells. Thus, the decrease in the internal concentration of inorganic carbon showed by mixotrophic cells could be due to an increase in cell volume
instead of a decrease in the ability to transport inorganic
carbon.
Since autotrophic and mixotrophic cultures showed
the same value of chlorophyll content per cell (Table 1), it
seems more appropiate to express transport and fixation activities on chlorophyll basis. Results of transport, fixation
and accumulation of Q as a function of chlorophyll are
shown in Fig. 2. The amount of Q transported by cells
(acid-labile 14C plus acid-stable MC) increased gradually
with time in both types of cultures. Although the transported carbon was slightly higher in autotrophic cells the
differences with respect to mixotrophic cells were not significant (Fig. 2A). These results contrast with those reported in
Synechocystis PCC 6803 (Bloye et al. 1992) which indicated
a decrease in the rate of bicarbonate uptake, expressed on
chlorophyll basis, in the presence of glucose. Whether
these differences are due to the different experimental
method or reflect differences between the two sugars or the
two strains, remains to be established.
The photosynthetic fixation of Q (acid-stable I4C) increased with time under both growth conditions. The differences in the ability to fix C, expressed as a function of chlorophyll in autotrophic and mixotrophic cells were lower
than those expressed as a function of SIS (Fig. IB). However, the diminution in fixed Q observed in mixotrophic
cells were also significant when data were expressed as a
function of chlorophyll (Fig. 2A). Both types of cells showTable 1 Effect of mixotrophic conditions on cell volume
and on chlorophyll and protein content per cell in Anabaena variabilis
Culture
conditions
SIS cell
Chi cell"1
protcell'
Autotrophic
3.05 x 10"8
1.45 x 10"'
6.45 xlO" 6
Mixotrophic
8.70 xlO" 8
1.45 x 10~7
7.81 x 10"6
Cells were grown under autotrophic or mixotrophic conditions for
48 h. Data are mean of 3 experiments.
M. Nieva and E. Fernandez Valiente
o
60
o
e
30
c
60
90
120
30
60
90
120
time (s)
Fig. 2 Time courses of transport (A), fixation (B) and accumulation (C) of inorganic carbon as a function of chlorophyll in
autotrophic (O) and mixotrophic (•) cells of A. variabilis. Experimental conditions are described in Materials and Methods.
ed a slow rate of 14C fixation during the first 60 s and afterwards the rate increased. At the longest experimental period (120 s) the amount of 14C fixed was almost 40% lower in
mixotrophic cells.
The time courses of the accumulation of Q (acid-labile
I4
C) inside the cells are showed in Fig. 2C. In autotrophic
cells, the pattern of accumulation is the characteristic of
low CO2-grown cells (Kaplan et al. 1980): the concentration of internal Q increased rapidly for the first 60 s and decreased afterwards concurrently with the raise in the rate of
I4
C fixation (Fig.2B). However in mixotrophic cells the
lower Cj fixation leads to a gradual increase in internal pool
of C; over the experimental period. The differences in the
amount of Q accumulated after 90 s were more obvious
when results were expressed on chlorophyll basis (Fig. 2C).
In summary, mixotrophic cells showed a lower rate of
I4
C fixation but not of transport of Q (Fig. 2A, B). This
fact indicated a lower efficiency in assimilating the transported Cj in cells grown with fructose.
It is thought that the assimilatory step is performed in
the carboxysomes, where most of the two main enzymatic
activities involved in the Q fixation: carbonic anhydrase
(CA) and Rubisco (Badger and Price 1989, McKay et al.
1993, Price et al. 1992) are located. Therefore next objective of our study was to measure the activity of these enTable 2 Carbonic anhydrase activity in A. variabilis
Culture conditions
CA activity
(units mg protein"')
Autotrophic (low CO2)
8.5±1.8
Autotrophic (high CO2)
3.9±0.5
Mixotrophic (low CO2)
4.4±0.9
Cells were grown for 48 h under autotrophic conditions at low
(0.02% v/v) or high (5% v/v) CO2 concentration and mixotrophic
conditions. Cellular extracts contained 0.8 mg protein ml"1 in all
culture conditions. Values are means of five experiments±SD.
zymes.
Enzyme activities—CA activity plays a crucial role in
the Cj assimilation since this enzyme catalyzes the conversion of HCOf into CO2 in the vicinity of Rubisco in the
carboxysome (Kaplan et al. 1991, Badger and Price 1992).
As a consequence of the induction of the CCM, the activity
of the enzyme increases greatly when cells are transferred from high to low-CO2 concentrations (Shiraiwa and
Miyachi 1985, Bedu and Joset 1991).
The results presented in Table 2 indicate that the level
of CA activity of mixotrophic cells was similar to that
shown by autotrophic high-CO2 cells, namely around a
50% lower than that of autotrophic low-CO2 cells. These
data indicate that the induction of CA activity, which takes
place in cells grown at low concentrations of CO2, was suppressed by the presence of fructose (mixotrophic cells).
Similar effect has been reported in the green alga Chlorella
vulgaris l l h by glucose (Shiraiwa and Umino 1991) and in
Chlorella regularis by various metabolizable organic carbon sources (Umino and Shiraiwa 1991).
Not much is known about the CA activity regulation.
The CA induction mechanism has been mainly characterized in periplasmic CA in Chlamydomonas reinhardtii,
which is the main CA activity in Chlamydomonas (Husic
1991, Coleman et al. 1991). It has been described in this organism that periplasmic CA is synthesized "de novo" in the
cytoplasm when the CO2 concentration decreases and this
induction is dependent on photosynthesis and light (for
review see Badger and Price 1992). In Chlorella regularis it
has been reported a suppression of CA biosynthesis by glucose and/or its metabolites, by inhibitors of glycolate metabolism and by L-MSO (blocking glutamine synthase)
(Umino and Shiraiwa 1991), but the nature of the signal
that induces the synthesis is unknown at the moment.
Although the Q transport was not clearly inhibited in
mixotrophic cells, the inhibition of CA activity resembles
the behaviour of cells grown under high CO 2 concentration, in which CCM and consequently the CA activity are
C( assimilation in mixotrophic cells
not induced. This alteration in mixotrophic cells could be
related to the higher levels of internal Q due to their higher
respiration rate (Fernandez Valiente et al. 1992). However
the regulation of the processes involved in adaptation to
low-CO2 conditions remains unclear yet and more work
should be performed in this sense.
Another enzyme playing a key role in the photosynthetic carbon fixation is Rubisco. In A. variabilis it has been
reported that Rubisco is the most important carboxylating
enzyme and its activity is 10 times higher than PEPcarboxylase activity (Kaplan et al. 1980). Our results of total
Rubisco activity as a function of different concentrations
of HCOf are shown in Figure 3. The mixotrophic cells
present lower activity than autotrophic cells at all the
HCOf concentrations tested. The Kmax for total Rubisco activity in autotrophic cells was 4.7 ^/mol CO2 mg prot" 1 h" 1
and in mixotrophic cells 2.3 ^mol CO2 mgprot" 1 h" 1 . As
protein content per cell is slightly higher in mixotrophic
cells (Table 1) we calculated the Rubisco activity based on
cellular protein content. Results were 3 x 10~8 and 1.8 x
10~8 fimo\ CO2 mg prot per cell"1 h" 1 for autotrophic and
mixotrophic cells respectively, and also showed a significant decrease in Rubisco activity under mixotrophic conditions. The Km (Cj) values calculated from the double reciprocal plot (Fig. 3B) were 40 mM and 24 mM for
autotrophic and mixotrophic cells respectively. The differences in Km were not statistically significant. Thus, mixotrophic cells seem to show a similar affinity for Q than
autotrophic cells.
It has been described that the Rubisco activity is not
altered by the concentration of CO2 at which cells were
grown (Kaplan et al. 1980) and so, the inhibition of
Rubisco activity might be an effect not related to the alteration of the CCM observed in mixotrophic cells. Moreover,
we have seen that this inhibition is independent on the substrate concentration presents in the assay: HCOf (Fig. 3)
and RBP (data not shown). This fact together with the
similar values for Km (Q) suggest that mixotrophic cells
1
1
1
1
B1 4
1
'
'
45
1 2
30
*/
/
10
P
/
/
/
/
/i
/
/
08
06
1.5
04
0 /
1
,
20
40
[HCO3"] m
,
1
1
1
<
- 0 10-0 05 0 00 0 05 0 10 0 15 0.20
1/[HCOS"J mil
Fig. 3 (A) Total activity of Rubisco in autotrophic (o) and mixotrophic (•) cells of A. variabilis as a function of Q in the assay
medium.
(B) Double reciprocal plot of Rubisco activity versus
HCOf concentration.
might posses a lower quantity of active enzyme.
Some authors have proposed CA activity (because of
its involvement in early steps of Q metabolism) as the regulatory key of the process of CO 2 fixation (Bedu and Joset
1991). If this were the case the inhibition of Rubisco observed in mixotrophic cells could be a consequence of the
decrease in CA activity shown previously (Table 2). However, this possibility would be rejected if we take into account the experimental conditions. We have seen that
Rubisco activity is inhibited in mixotrophic cells even at the
maximal C, concentration assayed, 60 mM. At the experimental pH (8.0), at equilibrium, a 2% of Cj must be
present as CO2 and thus there would be 1.2 mM of CO 2 in
the Rubisco assay. If we calculate the affinity of Rubisco
for CO2 in the Rubisco assays, the value obtained is around
0.5 mM (approximately the same for both autotrophic and
mixotrophic cells), which is in the range to those reported
for other cyanobacteria (Badger 1980, Bedu and Joset
1991). As this affinity value is much lower than CO2 concentration it seems evident that the inhibition of Rubisco activity observed in mixotrophic cells would not be related to
the inhibition in the CA activity presented in these cells.
The explanation for the inhibitory effect of fructose on
Rubisco activity could concern to the Rubisco regulation.
It is known that before catalysis the enzyme must be activated via the carbamylation at a specific Lys residue at the active site on the large subunit and afterwards the carbamylated enzyme is stabilized by divalent cations (Tabita
1987, Li and Tabita 1994). Rubisco activation is catalyzed
by another protein, Rubisco activase (Li and Tabita 1994)
which has been also proposed to catalyze the removal of
some inhibitory compounds maximizing Rubisco activity
in vivo. Several phosphorylated metabolites, such as RBP,
2-carboxyarabinitol monophosphate (CA1P) or 6-phosphogluconate have been described as important inhibitors to
Rubisco activity under certain conditions (Li and Tabita
1994). In that way, 6-phosphogluconate is one of the most
effective in regulating both the partially active and the fully
activated enzyme in the cell (Tabita 1987) and it is known
that this effect is mediated by the small subunit of the
Rubisco (Li and Tabita 1994).
The 6-phosphogluconate is one of the products of the
glucose-6-P-dehydrogenase activity, which plays a key role
in the oxidative pentose phosphate pathway, the main respiratory pathway in cyanobacteria (Stanier and CohenBazire 1977). Our results from glucose-6-P-dehydrogenase
activity were in agreement with previous data which indicated an enhancement of respiratory metabolism in mixotrophic cells of A. variabilis (Fernandez Valiente et al.
1992). Glucose-6-P-dehydrogenase activity was 201 nmol
NADP + reduced mg prot" 1 min" 1 in mixotrophic cells and
70 nmol NADP + reduced mg prot" 1 min" 1 in autotrophic
cells. The increase in this enzymatic activity under mixotrophic conditions could rise the level of 6-P-gluconate in-
M. Nieva and E. Fernandez Valiente
hibiting the Rubisco activity although to estimate the quantity of this compound would be necessary to validate this
hypothesis.
Other possibility involving the metabolic regulation of
photosynthetic genes expression by sugars could also explain the inhibition of Rubisco activity in mixotrophic
cells. This kind of regulation has been described in protoplasts of maize by different sugars and acetate (Sheen 1990)
and in Chlorella using an analog of glucose (2-deoxy-D-glucose, 3-O-methylglucose) (Semenenko et al. 1992).
Recently a co-ordinated regulation of de Q and glucose assimilation has been proposed in Synechocystis
PCC6803 since several enzymatic steps are common to the
assimilation of both sources of carbon (Beuf et al. 1994).
The authors have identified a glucose-induced protein involved in the Q assimilation but the role of this protein is
not known yet. However this kind of regulation would
point out to a much more complex regulation system under
mixotrophic conditions, which could clarify the relationship between organic and inorganic carbon assimilation.
In summary our results show that the presence of an
organic carbon source (fructose) during growth produces
in A. variabilis a decrease in the photosynthetic activity
depending on the external Q (Fernandez Valiente et al.
1992). This fact seems to be due to two different factors: (i)
the alteration in the efficiency to supply the transported Q
to Rubisco, evidenced by a lower CA activity; (ii) the
diminution in the Rubisco activity itself by metabolic
regulation.
We thank Dr Antonio Quesada and Arsenio Villarejo for
their useful criticisms and corrections on the manuscript.
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(Received November 8, 1994; Accepted October 12, 1995)