Acta Physiol Plant (2010) 32:511–518
DOI 10.1007/s11738-009-0428-7
ORIGINAL PAPER
Characterization of trophic changes and a functional oxidative
pentose phosphate pathway in Synechocystis sp. PCC 6803
Tove Jansén • Dominic Kurian • Wuttinun Raksajit •
Steve York • Michael L. Summers • Pirkko Mäenpää
Received: 13 February 2009 / Revised: 12 September 2009 / Accepted: 16 November 2009 / Published online: 2 December 2009
Ó Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2009
Abstract Cyanobacteria have a tremendous activity to
adapt to environmental changes of their growth conditions. In
this study, Synechocystis sp. PCC 6803 was used as a model
organism to focus on the alternatives of cyanobacterial
energy metabolism. Glucose oxidation in Synechocystis sp.
PCC6803 was studied by inactivation of slr1843, encoding
glucose-6-phosphate dehydrogenase (G6PDH), the first
enzyme of the oxidative pentose phosphate pathway (OPPP).
The resulting zwf strain was not capable of glucose supported
heterotrophic growth. Growth under autotrophy and under
mixotrophy was similar to that of the wild-type strain, even
though oxygen evolution and uptake rates of the mutant were
decreased in the presence of glucose. The organic acids citrate and succinate supported photoheterotrophic growth of
both WT and zwf. Proteome analysis of soluble and membrane fractions allowed identification of four growth condition-dependent proteins, pentose-5-phosphate 3-epimerase
Communicated by H. Gabrys.
T. Jansén P. Mäenpää (&)
Laboratory of Plant Physiology and Molecular Biology,
Department of Biology, University of Turku,
20014 Turku, Finland
e-mail: [email protected]
W. Raksajit
Department of Veterinary Technology, Faculty of Veterinary
Technology, Kasetsart University, 50 Phahon Yothin Rd,
Chatuchak, Bangkok 10900, Thailand
D. Kurian
Henry Wellcome Laboratory for Biogerontology Research,
Newcastle University, Newcastle Upon Tyne NE4 6BE, UK
S. York M. L. Summers
Department of Biology, California State University,
Northridge, CA 91330-8303, USA
(slr1622), inorganic pyrophosphatase (sll0807), hypothetical
protein (slr2032) and ammonium/methylammonium permease (sll0108) revealing details of maintenance of the cellular
carbon/nitrogen/phosphate balance under different modes
of growth.
Keywords Citric acid Glucose G6PDH OPPP Succinic acid zwf
Abbreviations
a-KGDH
a-Ketoglutarate dehydrogenase
BN-PAGE Blue native page
DCBQ
2,6-Dichloro-p-benzoquinone
DCMU
3-(3,4-Dichlorophenyl)-1,1-dimethylurea
G6PDH
Glucose-6-phosphate dehydrogenase
IDH
Isocitrate dehydrogenase
LAHG
Light-activated heterotrophic growth
OPPP
Oxidative pentose phosphate pathway
PPDF
Photosynthetic photon flux density
PPi
Inorganic phosphate
TCA
Tricarboxylic acid
WT
Wild type
zwf
Mutant strain lacking glucose-6-phosphate
dehydrogenase
Introduction
Cyanobacteria, a group of ancient autotrophs, perform
oxygen-evolving photosynthesis similar to that of plants.
Plant chloroplasts have developed from cyanobacteria like
ancestors (Rodrı́guez-Ezpeleta et al. 2005). Cyanobacteria
can face very varying environmental conditions in nature.
Flexible energy metabolism enables optimal adaptation
strategies of many cyanobacterial species. Detailed
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Acta Physiol Plant (2010) 32:511–518
characterization of function and regulation of cyanobacteraial photosynthesis has been performed (DeRuyter and
Fromme 2008), while significances and molecular mechanisms of other pathways of energy metabolism still remain to
be clarified. In this study, cyanobacterium Synechocystis sp.
PCC 6803 (hereafter Synechocystis) was used as a model
organism to focus on alternatives of cyanobacterial energy
metabolism. The Synechocystis is a widely used model
organism in studies of functional and regulatory aspects of
oxygenic photosynthesis. The metabolism of Synechocystis is
primarily phototautotrophic, but Synechocystis is also able to
utilize glucose for photomixotrophic and heterotrophic
growth. This feature is not common to all cyanobacteria
(Rippka 1972) and its biological significance and the genetic
background is undefined (Ikeuchi and Tabata 2001). In cyanobacteria intracellular compartmentalization is limited.
Hence, different metabolic pathways overlap by sharing both
enzymes and reaction intermediates. Tracking and monitoring the carbon flow in glucose catabolism is therefore challenging and remains poorly characterized.
Under heterotrophic growth conditions the oxidative
pentose phosphate pathway (OPPP) has been found to be the
most significant pathway for energy conversion in cyanobacteria (Pelroy and Bassham 1972; Yang et al. 2002). The
primary functions of the OPPP are to generate NADPH for
reductive biosynthesis reactions, and to provide the cell with
ribose-5-phosphate for the synthesis of nucleotides and
nucleic acids. In addition, triose phosphates can be channelled from the OPPP into the lower glycolytic pathway. The
activity of the first enzyme of the OPPP, glucose-6-phosphate
dehydrogenase (G6PDH) encoded by the zwf gene, is controlled by fluctuating ATP and NADPH concentrations as
well as light-induced pH changes in the extra-thylakoidal
environment (Grossman and McGowan 1975). Such sensitivity to the cellular energy level gives G6PDH a central role
in regulating glucose degradation via the OPPP. Glucose
catabolism is completed in the TCA reactions and finally in
the respiratory electron transfer chain coupled to oxidative
phosphorylation. In this study, we have characterized the
significance of the OPPP at the phenotypic, physiological and
proteome levels in Synechocystis. Comparisons of the WT
and zwf inactivation strain with respect to growth, photosynthetic and respiratory activities, as well as both membrane
and soluble proteins were conducted.
strain (zwf) was grown in BG-11 medium (Williams
1988) under photosynthetic photon flux density (PPDF)
of 40 lmol photons m-2 s-1 at 32°C using Philips TLD
30W/865 lamps. The zwf mutant strain lacking glucose6-phosphate dehydrogenase (G6PDH), was constructed
by insertional inactivation, using the primer pair zwf1:
50 -TGG TGC CAG CCA TCT ACC AA-30 and zwf2:
50 -CCA CTC CAA AGG ATG AAC CA-30 to amplify an
internal 1.184 kb fragment of the zwf gene from Synechocystis genomic DNA. The PCR fragment was cloned
into pCR2.1-TOPO (Invitrogen), and inactivated by
insertion of a gentamycin resistance gene cassette into
the unique XmaI site within the coding region of the zwf
gene. The 1.9 kb XmaI gentamycin cassette was obtained
from pHP45Gm, constructed by insertion of a 1.9 kb
BamHI fragment from pRZ1107 (Yin et al. 1988) into
the BamHI site of pHP45 (Prentki and Krisch 1984). The
resulting zwf::Gmr plasmid was transformed into Synechocystis, and colonies surviving gentamycin selection
(10 lg ml-1) were cultured in liquid BG-11 medium
under antibiotic selection. Complete segregation of the
mutation was confirmed by Southern analysis of genomic
DNA blotted to a nylon membrane following the manufacturers’ instructions (PerkinElmer Life Sciences). The
zwf gene probe was generated by random primer biotin
labelling of a PCR fragment produced by the zwfP1/2
primers, and visualized using chemiluminescence
(PerkinElmer Life Sciences). Inactivation of the zwf gene
product was confirmed by the loss of glucose-6-phosphate dehydrogenase enzyme activity, assayed as in
Summers and Meeks (1996). The segregated zwf was
grown in the presence of gentamycin (2 lg ml-1). Both
the effect of glucose and the TCA cycle reaction intermediates succinate and citric acid on growth were
evaluated. The WT strain and zwf strain were grown
photoautotrophically, photomixotrophically (in the presence of 5 mM glucose, succinate or citric acid),
photoheterotrophically (in the presence of 5 mM glucose, succinate or citric acid and 10 lM DCMU), and
heterotrophically (light-activated heterotrophic growth,
LAHG, 5 mM glucose, succinate or citric acid and a
daily 10 min light pulse of 50 lmol photons m-2 s-1).
The growth rates of the WT and zwf cells were monitored spectrophotometrically at an optical density of
730 nm for 2 weeks.
Materials and methods
Oxygen evolution and uptake measurements in vivo
Strains, mutant construction, culture conditions and
estimation of growth rate
For each measurement, aliquots of cell cultures containing 10 or 100 lg chlorophyll for photosynthesis and
respiration measurements, respectively, were centrifuged
and the pellets suspended in 1 ml of fresh BG-11 medium. The chlorophyll concentration was determined as
The glucose-tolerant strain of Synechocystis sp. PCC
6803 referred here as wild type (WT) and the zwf mutant
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Acta Physiol Plant (2010) 32:511–518
described (Bennet and Bogorad 1973). Oxygen evolution
was measured under saturating white light with a Clarktype oxygen electrode (Hansatech) at 32°C using the
tungsten halogen bulb (Type 6958, 24V, 250W G6.35,
Philips) of a slide projector as the light source. PSII
activity measurements contained 0.25 mM DCBQ (2,6dichloro-p-benzoquinone) plus 0.25 mM ferricyanide,
and for the measurement of the photosynthetic capacity,
0.25 mM sodium bicarbonate was used as electron
acceptor. Samples were prepared from photoautotrophic
and photomixotrophic conditions as well as LAHG
adapted cultures.
Separation and identification of membrane and soluble
proteins
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Results
Inactivation of the Synechocystis zwf gene
In order to construct the zwf insertional mutant, a gentamycin resistance cassette was inserted into the zwf open
reading frame (slr1843) as illustrated in Fig. 1a. Successful
transformation and disruption of zwf within the chromosome was confirmed by Southern blotting (Fig. 1b), demonstrating that a fully segregated zwf inactivation mutant
was obtained. Functional inactivation of zwf by the antibiotic cassette was confirmed by loss of G6PDH enzymatic
activity in the zwf mutant strain (data not shown).
Growth of the wild type and zwf strains under various
conditions
Thylakoid-enriched membrane fractions were prepared
from WT and zwf cells grown photoautotrophically,
photomixotrophically and photomixotrophically followed
by a 48 h dark incubation. Separation of membrane
protein complexes was performed in the first dimension
by blue native page (BN-PAGE) and in the second
dimension by conventional SDS-PAGE essentially as
described (Herranen et al. 2004). The soluble proteomes
of WT and zwf cells grown photoautotrophically and
photomixotrophically were also subjected to 2D gel
protein analysis using isoelectric focusing in the first
dimension (pH 4–7) and conventional SDS-PAGE in the
second dimension essentially as described previously
(Kurian et al. 2006). All gels were silver-stained and the
gel images were captured with a Fluorchem 8000 system
(Alpha Innotech Corporation). For identification, excised
protein spots from gels were subjected to treatment as
described previously (Kurian et al. 2006). Both MaldiTOF and nanoLC-MS/MS were employed in the protein
identifications.
To address the role of the OPPP in the survival and growth
of Synechocystis under different modes of growth, WT and
zwf strains were cultured under photoautotrophic, photomixotrophic, photoheterotrophic and LAHG conditions.
The growth rates of WT and zwf strains were identical to
each other under both photoautotrophic and photomixotrophic growth conditions (Fig. 2a). Under mixotrophy
only glucose promoted growth while succinate and citric
acid left the growth rate unchanged (data not shown).
Under glucose-induced photoheterotrophic growth, when
linear photosynthetic electron flow was inhibited by the
addition of DCMU, only WT grew, whereas the zwf culture
became bleached and the optical density decreased within
few days (Fig. 2b). When the external carbon source
(glucose) was exchanged to an organic acid (succinic or
citric acid), both WT and zwf were able of photoheterotrophic growth (Fig. 2c). Here, WT grew faster than zwf.
When the WT and zwf cells were introduced to LAHG
conditions, only WT grew (Fig. 2b). In contrast to the
Fig. 1 Illustration depicting insertional inactivation of the zwf gene
a, and autoradiograph of Southern blot confirmation of the segregated
mutant. b Southern confirmation shows probe hybridized to a 4 kb
HindIII fragment of the wild-type strain (WT), and to a 5.9 kb
fragment of the zwf mutant (zwf) in addition to a common 0.85 kb
fragment of zwf. H, HindIII; X, XmaI; sizes indicated in kilobases
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514
A
Acta Physiol Plant (2010) 32:511–518
3
In vivo oxygen evolution and uptake rates under
different growth conditions
w tPM
w tPA
2.5
zw fPA
OD730
2
To investigate whether different growth modes and elimination of the OPPP by zwf inactivation affects respiration,
PSII activity or the photosynthetic capacity, oxygen uptake
and evolution rates were determined for both the WT and
zwf strains grown under photoautotrophy or photomixotrophy (Table 1) and oxygen uptake rates for WT cell
grown under LAHG. The oxygen evolution rates indicating
PSII activity and photosynthetic capacity were similar in
WT and zwf cells grown photoautotrophically. In contrast,
zwf cells exhibited reduced PSII activity (62%) and total
photosynthetic capacity (48%) when compared to WT cells
grown photomixotrophically. Oxygen uptake activities
were similar in both strains studied under photoautotrophy,
but under glucose-induced photomixotrophy the WT strain
was capable of increasing the oxygen uptake rate over 4fold compared to a modest 1.3-fold increase observed for
the zwf strain. When WT cells were grown under LAHG
for 14 days, oxygen uptake increased by more than an
order of magnitude (49 ± 4) compared to photoautotrophic
conditions. The zwf strain being unable to grow under
LAHG conditions, oxygen uptake measurements were not
performed for this strain.
zw fPM
1.5
1
0.5
0
0
2
4
6
8
10
12
14
10
12
14
Time (days)
B
1.6
w tPH
1.4
w tLAHG
1.2
zw fPH
OD730
zw fLAHG
1
0.8
0.6
0.4
0.2
0
0
2
4
6
8
Time (days)
C
0.25
Proteome analysis of soluble and thylakoid-enriched
membrane proteins
w tS
w tC
To clarify if increased soluble enzymes would be present in
the WT strain to account for increased respiratory capacity,
whole cell soluble proteins from photoautotrophically and
photomixotrophically grown WT and zwf cells were compared using IEF-PAGE analysis. Inspection of the gel
images revealed no detectable differences between the
WT and zwf proteomes (data not shown). However, comparison between growth modes allowed identification of
three proteins that were down-regulated under glucoseinduced photomixotrophic growth in both strains (Fig. 3).
zw fS
0.2
OD730
zw fC
0.15
0.1
0.05
0
0
2
4
6
8
10
12
14
Time (days)
Fig. 2 Growth curves of wild type Synechocystis sp. PCC 6803 cells
and zwf mutant. a Growth of WT and zwf under photoautotrophic
(PA) and glucose-induced photomixotrophic (PM) conditions. b
Growth of WT and zwf under glucose-induced photoheterotrophic
(PH) and light-activated heterotrophic (LAHG) conditions. c Growth
of WT and zwf under organic acid (citric acid-C; succinic acid-S)
induced photoheterotrophy. Representative results from one experiment are presented (n = 3)
bleaching observed for zwf under photoheterotrophic conditions, the zwf cultures remained green under LAHG.
Organic acids were unable to support growth of either WT
or zwf under LAHG conditions (data not shown).
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Table 1 Oxygen-evolving and uptake activity of Synechocystis WT
and zwf strains in vivo
WT PA
ZWF PA
WT PM
ZWF PM
PSII
309 ± 13
298 ± 12
345 ± 49
213 ± 23
PS capacity
288 ± 5
332 ± 28
282 ± 23
135 ± 22
O2 uptake
3.5 ± 0
3.6 ± 0
15.0 ± 2
4.7 ± 0
Photosystem II activity (PSII) was measured in the presence of
DCBQ; photosynthetic capacity (PS capacity) was measured in the
presence of sodium bicarbonate; Oxygen uptake (O2 uptake) was
measured in the dark. Values in lmol O2 (mg Ch-1 h-1) and represent means ± standard error from three independent experiments. PA
photoautotrophy, PM glucose-induced photomixotrophy
Acta Physiol Plant (2010) 32:511–518
Fig. 3 IEF analysis on narrow range strips (pH 4–7) of the soluble
WT and zwf proteome. a Total image under photoautotrophy,
b enlarged section of the boxed area, photoautotrophy, c enlarged
section of the boxed area, photomixotrophy. Spot 1, slr1622; spot 2,
sll0807; spot 3, slr2032
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The identified proteins were inorganic pyrophosphatase
(spot 1, slr1622, Ppa), pentose-5-phosphate epimerase (spot
2, sll0807) and a hypothetical protein (spot 3, slr2032).
To test the hypothesis that the observed differences in
oxygen evolution and uptake under different modes of
growth and between strains were related to protein changes
in the photosynthetic membranes, thylakoid-enriched
membrane preparations were subjected to BN-PAGE
analysis (Fig. 4). The gel images corresponded well to
earlier published images (Kurian et al. 2006; Zhang et al.
2004). In autotrophic and photomixotrophic conditions,
there were no discernable differences neither between the
WT and zwf strains nor the growth modes (Fig. 4a). Under
LAHG conditions (14 days of incubation) the WT strain
lacked membrane complexes associated with inorganic
carbon concentration (SbtA and NDH-S) and NDH-M and
NDH-L complexes associated with cyclic electron transfer
and respiration, respectively, were present in equal quantities. A novel LAHG-induced protein, not reflected in gels
made from photoautotrophy or photomixotrophy, was
identified as ammonium/methyl-ammonium permease
(sll0108, amt1) (Fig. 4b). To evaluate the role of OPPP in
acclimation to LAHG conditions, we transferred photomixotrophically grown WT and zwf cells to LAHG conditions for 48 h. Analysis of the membranes showed no
alterations from photomixotrophically grown expression
patterns in WT membranes, but zwf membranes showed a
marked decrease in the quantity of all of the membrane
Fig. 4 BN-PAGE analysis of thylakoid-enriched membrane fractions. a Image representing photoautotrophy and photomixotrophy, b image
representing LAHG conditions (WT), Amt1 (sll108) encircled, c image representing mixotrophy plus 48 h dark (zwf)
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complexes listed above (Fig. 4c). A decrease in the optical
density of mutant cultures was also observed after 48 h
incubation in LAHG conditions (data not shown).
Discussion
The group of cyanobacteria is spread to extremely varying
environmental conditions on the Earth. Many cyanobacterial species additionally have a wide adaptive capacity
when facing changes in their environment. Environmental
changes induce signalling cascades that form molecular
basis of adaptive response. They take place via accurate
regulation of different sets of genes (Murata and Suzuki
2006). Due to the easy cultivation of cyanobacteria under
laboratory conditions, their high adaptive capacity and the
possibility to genetic manipulation, there are high hopes to
their utilization as producers of oxygen, bio-energy or
human food supplements, both under normal and extreme
conditions. One problem before these future plans become
reality is our limited knowledge of cyanobacterial molecular metabolism, the knowledge of which forms the basis
of all applications.
In the present work, we utilized the model organism
Synechocystis for further characterization of cyanobacterial
energy metabolisms. The relative significance of the OPPP
compared to other pathways participating in glucose oxidation remains undetermined. Comparison of growth rates
under different growth modes (Fig. 2) as well as monitoring acclimation of the WT and the zwf strains to LAHG
(Fig. 4c) clearly demonstrates that G6PDH, and thus the
OPPP, is obligatory for both glucose supported photoheterotrophy and LAHG in Synechocystis. Recent evidence
suggests that the glycolytic pathway is used during mixotrophic and dark heterotrophic growth (Yang et al.
2002; Knowles and Plaxton 2003). However, our results
show that under heterotrophy the glycolytic pathway
cannot compensate for the loss of a functional OPPP in
Synechocystis.
In the presence of glucose the Synechocystis zwf strain
had a phenotype resembling the E. coli zwf mutant, namely,
growth properties identical to those of the parent strain. In
the E. coli zwf strain, loss of the OPPP was compensated
for by increased carbon flow through glycolysis to the TCA
cycle, where isocitrate dehydrogenase and malic enzyme
were responsible for NADPH production. In addition, the
glycolytic intermediates glyceraldehyde-3-phosphate and
fructose-6-phosphate were channelled to the non-oxidative
part of the OPPP for the synthesis of erythrose-4-phosphate
and ribose-5-phosphate (Zhao et al. 2004). This could be
the case also for Synechocystis, but the Calvin cycle, producing 5-carbon sugars under autotrophic and mixotrophic
conditions, makes the carbon flow more complex in
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Acta Physiol Plant (2010) 32:511–518
cyanobacteria. Interestingly, organic acids were observed
to support growth of both WT and zwf under photoheterotropy, the lack of a functional OPPP in zwf reduced
though the growth rate. We hypothesize that the provided
organic acids are converted to some extent to glucose
through TCA reactions and gluconeogenesis and subsequently oxidized through glycolysis, OPPP and TCA
reactions as described for E. coli. The significance of the
OPPP, evidenced by the different growth curves of WT and
zwf, in this process remains yet undetermined. Growth
supported by citric acid further indicates that a fully
functional TCA cycle functions in Synechocystis as has
been speculated earlier (Cooley and Vermaas 2001). Citric
acid oxidized sequentially by aconitase and IDH results in
a-ketoglutarate, the substrate for the cryptic cyanobacterial
a-KGDH. In the absence of photosynthesis and other
external reduced carbon sources, the cellular metabolism
relies primarily on the TCA cycle for reductant and carbon
skeletons. If not for a complete TCA cycle being able to
convert a-ketoglutarate to succinyl-CoA, surviving and
growth would be impossible.
As suggested by similar growth rates between the
strains, identical soluble proteomes were found for WT and
zwf strains grown autotrophically or mixotrophically.
However, three growth condition-dependent proteins were
present in both proteomes that were down-regulated under
mixotrophic growth conditions (Fig. 3). Two of the identified proteins are known to be involved in cellular
metabolism (slr1622, pentose-5-phosphate 3-epimerase and
sll0807, inorganic pyrophosphatase, Ppa), more specifically
in the sugar-phosphate interconversion common to the
OPPP and Calvin cycle, and intracellular (PPi)-recycling,
respectively (Gomez-Garcia et al. 2003). The decrease in
the amount of pentose-5-phosphate 3-epimerase may
indicate that exogenous glucose enters the Calvin cycle as
fructose-6-phosphate, resulting in the production of excess
amounts of pentose phosphates. Since twice the amount of
xylulose-5-phosphate is synthesized during the Calvin
cycle relative to ribose-5-phosphate, less epimerase would
be required for maximal conversion of xylulose-5-phosphate to ribulose-5-phosphate, especially during mixotrophic conditions when exogenously added glucose can
contribute additional carbon skeletons to the Calvin cycle
intermediates. Down-regulation of Ppa (sll0807) hints
towards a decrease in functional anabolic reactions under
mixotrophic growth. The decreased level of Ppa under
mixotrophic growth might also reflect the regulatory role of
PPi on photosynthesis (Forti and Meyer 1969). The third
identified protein is a hypothetical protein (slr2032)
showing similarity to algal Ycf23 and its metabolic role
under autotrophic growth conditions remains elusive.
In cyanobacterial respiration the membrane bound
NDH-dehydrogenases extract electrons from NAD(P)H
Acta Physiol Plant (2010) 32:511–518
and shuttle them to the respiratory electron transfer chain.
In addition to the NDH complexes, the importance of
succinyl dehydrogenases (SDH) to the respiration of Synechocystis has been reported (Cooley and Vermaas 2001).
We observed an increased oxygen uptake rate in cells
grown for 14 days under LAHG conditions and assumed
that this was an indication of increased respiratory activity.
We therefore analyzed WT membranes from LAHG conditions by BN-PAGE to identify possible changes in the
membrane proteome. Although we could not point out any
newly synthesized complexes associated with respiration,
NDH-M (medium size) and NDH-L (large size) complexes
were present in equal quantities. This is in contrast to levels
expressed under autotrophic and mixotrophic conditions,
where NDH-M complexes are more abundant than NDH-L
complexes. Recently, the Synechocystis NDH-complexes
have been assigned cellular roles (Herranen et al. 2004;
Zhang et al. 2004) and the NDH-L complex was reported to
be of respiratory significance, thus increased dark respiration can be explained by increased levels of NDH-L
complexes.
Under LAHG, photosynthetic light reactions are absent
and CO2 assimilation by the Calvin cycle ceases. In line
with these events, SbtA and NDH-S complexes associated
with the uptake of inorganic carbon are absent under
LAHG (Fig. 4b), which is probably due to the requirement
for photosynthetic electron transfer for the expression of
these complexes (Herranen et al. 2004). We were, however, able to identify one LAHG-inducible protein
(Fig. 4b), the ammonium/methylammonium permease
(sll0108, Amt1). The Amt1 transporter is preferentially
expressed in nitrogen-starved cells (Montesinos et al.
1998), and responds to the cellular carbon/nitrogen balance
in Synechocystis (Vásquez-Bermúdez et al. 2002). The
presence of Amt1 under LAHG conditions is either
required for recovery of ammonium that diffuses out of the
cell, or a response to nitrogen limitation. Nitrate assimilation is coupled to photosynthesis that provides the
reducing power for the conversion of nitrate to nitrite
(Flores et al. 2005) and therefore the latter alternative, i.e.
the ammonium specific Amt1 substituting for inefficient
nitrate uptake under LAHG, seems more likely.
Respiration is claimed to be inactive at growth light
intensities (Cogne et al. 2003) and the activity of G6PDH
down-regulated by the photosynthetic products ATP and
NADPH (Grossman and McGowan 1975). Still we
observed that mixotrophically grown WT cells had three
times higher oxygen uptake rates compared to the zwf cells
(Table 1). The difference in oxygen uptake rates between
the WT and zwf strain under photomixotrophic conditions
can only be explained by the lack of the OPPP in the zwf
inactivation strain, since oxygen uptake rates under autotrophic conditions were comparable between the strains.
517
Enzymes involved in the OPPP were up-regulated on
enzymatic and protein level under LAHG (Kurian et al.
2006). Furthermore, genes encoding G6PDH and transaldolase have been shown to be transcriptionally induced in
as little as 4 h after addition of an exogenous carbon source
in Nostoc punctiforme (Summers et al. 1995) and this may
be a universal phenomenon in cyanobacteria resulting in
enhanced potential for this metabolic pathway. It appears
that under mixotrophic growth, the wild-type strain senses
the presence of glucose and makes necessary adaptations to
use exogenous glucose immediately should light become
limiting. The major increase in oxygen uptake of fully
LAHG-adapted WT cells can be explained by a functionally up-regulated OPPP, resulting in elevated substrate
levels for the respiratory chain, in conjunction with
increases in NDH-L mentioned above.
Acknowledgments This work was supported by the Academy of
Finland to P.M. (203352), by NSF grant and NIH grants to M.L.S
(MCB0093327, GM48680, GM63787) and by the Finnish Cultural
Foundation to T.J. We thank Wim Vermaas for providing us with the
glucose tolerant Synechocystis sp. strain PCC 6803 and Richard J.
Debus for pHP45Gm.
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