Microbiology (2012), 158, 398–413
DOI 10.1099/mic.0.054544-0
Low-carbon acclimation in carboxysome-less and
photorespiratory mutants of the cyanobacterium
Synechocystis sp. strain PCC 6803
Claudia Hackenberg,1 Jan Huege,23 Annerose Engelhardt,1
Floyd Wittink,3 Michael Laue,44 Hans C. P. Matthijs,5 Joachim Kopka,2
Hermann Bauwe1 and Martin Hagemann1
Correspondence
Martin Hagemann
[email protected]
1
Universität Rostock, Institut für Biowissenschaften, Pflanzenphysiologie,
Albert-Einstein-Str. 3, D-18059 Rostock, Germany
2
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1,
D-14476 Golm, Germany
3
Microarray Department, Swammerdam Institute for Life Sciences, University of Amsterdam,
Science Park 904, 1098XH Amsterdam, The Netherlands
4
Universität Rostock, Institut für Pathologie, Elektronenmikroskopisches Zentrum,
Strempelstr. 14, D-18055 Rostock, Germany
5
Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam,
Science Park 904, 1098XH Amsterdam, The Netherlands
Received 5 September 2011
Revised
27 October 2011
Accepted 13 November 2011
Using metabolic and transcriptomic phenotyping, we studied acclimation of cyanobacteria to low
inorganic carbon (LC) conditions and the requirements for coordinated alteration of metabolism
and gene expression. To analyse possible metabolic signals for LC sensing and compensating
reactions, the carboxysome-less mutant DccmM and the photorespiratory mutant DglcD1/D2
were compared with wild-type (WT) Synechocystis. Metabolic phenotyping revealed
accumulation of 2-phosphoglycolate (2PG) in DccmM and of glycolate in DglcD1/D2 in LC- but
also in high inorganic carbon (HC)-grown mutant cells. The accumulation of photorespiratory
metabolites provided evidence for the oxygenase activity of RubisCO at HC. The global gene
expression patterns of HC-grown DccmM and DglcD1/D2 showed differential expression of many
genes involved in photosynthesis, high-light stress and N assimilation. In contrast, the transcripts
of LC-specific genes, such as those for inorganic carbon transporters and components of the
carbon-concentrating mechanism (CCM), remained unchanged in HC cells. After a shift to LC,
DglcD1/D2 and WT cells displayed induction of many of the LC-inducible genes, whereas
DccmM lacked similar changes in expression. From the coincidence of the presence of 2PG in
DccmM without CCM induction and of glycolate in DglcD1/D2 with CCM induction, we regard a
direct role for 2PG as a metabolic signal for the induction of CCM during LC acclimation as less
likely. Instead, our data suggest a potential role for glycolate as a signal molecule for enhanced
expression of CCM genes.
3Present address: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Physiology and Cell Biology, Corrensstr. 3, D-06466 Gatersleben,
Germany.
4Present address: Robert Koch Institut, Nordufer 20, D-13353 Berlin, Germany.
Abbreviations: CBB, Calvin–Benson–Bassham; CCM, carbon-concentrating mechanism; Ci, inorganic carbon; HC, high inorganic carbon; HCR,
high-carbon-requiring; LC, low inorganic carbon; 2OG, 2-oxoglutarate; OPP, oxidative pentose phosphate; PEP, phosphoenolpyruvate; 2PG,
2-phosphoglycolate; 3PGA, 3-phosphoglycerate; RbcL, large RubisCO subunit; Ru5P, ribulose 5-phosphate; TCA, tricarboxylic acid; WT, wild-type.
The microarray data discussed in this paper have been submitted to the NCBI Gene Expression Omnibus (GEO) database under accession number
GSE31672.
Two supplementary figures, showing mutant genotypes and growth and global changes in transcriptomic patterns, and three supplementary tables listing
the complete metabolomic and transcriptomic datasets, and the distribution of differentially regulated genes in different metabolic categories in cells of
the WT and DccmM and DglcD1/D2 mutants, are available with the online version of this paper.
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Carboxysomal and photorespiratory Synechocystis mutants
INTRODUCTION
The Calvin–Benson–Bassham (CBB) cycle is the primary
metabolic pathway for CO2 assimilation in photoautotrophic organisms. Covalent binding of CO2 to the acceptor
molecule ribulose 1,5-bisphosphate is catalysed by the
enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase
(RubisCO). This reaction produces two molecules of 3phosphoglycerate (3PGA) that are further metabolized into
carbohydrates. In the presence of O2, RubisCO also shows
oxygenase activity, which generates one molecule of 3PGA
and a second reaction product, 2-phosphoglycolate (2PG),
that cannot be metabolized in the CBB cycle. Furthermore,
2PG and its derivative glycolate inhibit enzymes of the
CBB cycle, leading to lowered CO2 fixation (Kelly & Latzko,
1977; Norman & Colman, 1991). To detoxify 2PG and to
regenerate 3PGA, plants and cyanobacteria possess photorespiratory 2PG metabolism (also referred to as the C2 cycle
or photorespiration; reviewed by Bauwe et al., 2010).
Over geological time, the originally much higher CO2
concentration in the atmosphere decreased (Hetherington
& Raven, 2005). To compensate, cyanobacteria developed
the carbon-concentrating mechanism (CCM), which is also
believed to minimize the competing oxygenase reaction of
RubisCO (reviewed by Badger et al., 2006; Kaplan et al., 2008).
The CCM is composed of (i) specialized transporters for CO2
and HCO^3 , which accumulate inorganic carbon (Ci) inside the
cell, and (ii) carboxysomes, protein-surrounded microcompartments embedded in the cytoplasm containing RubisCO
together with carbonic anhydrase (CA). Synechocystis sp. PCC
6803 (hereafter Synechocystis) possesses two constitutively
expressed low-affinity Ci transporters and three low-carbon
(LC)-inducible high-affinity Ci transporters (SbtA/B, BCT1
and NDH-13; Kaplan et al., 2008). The carboxysomes possess
structural proteins (CcmK and CcmL), which form a shell that
is believed to represent a diffusion barrier for gases (Yeates
et al., 2008). Therefore, carboxysomes allow the enrichment of
CO2 close to RubisCO and possibly prevent inward diffusion
of O2. In most cyanobacteria, such as Synechocystis, many
structural proteins of the carboxysome are encoded by the ccm
operon (ccmKLMNO; Badger et al., 2006). The loss of CcmM
and other structural proteins results in carboxysome-less
mutants. Such mutants are unable to grow under LC conditions and survive only under CO2-enriched conditions,
exhibiting a high-carbon-requiring (HCR) phenotype (Ogawa
et al., 1994; Berry et al., 2005; Woodger et al., 2005).
Recent data show that the CCM does not completely prevent
oxygenation. 2PG is produced in Synechocystis wild-type
(WT) cells even at 5 % CO2 (HC conditions) (Huege et al.,
2011) and is subsequently converted by 2PG metabolism
(Eisenhut et al., 2008a). Cyanobacterial 2PG metabolism
consists of two or three cooperating routes: the plant-like C2
cycle, the bacterial glycerate pathway and, in Synechocystis
and Synechococcus elongatus PCC 7942, the complete
decarboxylation of glyoxylate. The combined loss of these
three pathways as well as the interruption of the glycolate-toglyoxylate conversion by deletion of two redundant glycolate
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dehydrogenases (GlcD, double mutant DglcD1/D2) leads to
an HCR phenotype and growth deficiency similar to CCM
mutants (Eisenhut et al., 2008a).
Previously, we reported that Synechocystis mutants defective
in enzymes of the photorespiratory 2PG metabolism exhibit
features of LC acclimation already under HC conditions,
indicating a direct or indirect role of photorespiratory metabolites in sensing Ci limitation (Eisenhut et al., 2007, 2008b).
This would fit with an earlier assumption that oxygen and/or
intermediates of 2PG metabolism are involved in the sensing of Ci limitation (Marcus et al., 1983). Alternatively, LC
conditions might be sensed through the declining internal Ci
pool in a process which has also been found to be oxygendependent (Woodger et al., 2005). To examine the possible
role of photorespiratory metabolites in the sensing of and
acclimation to LC environments, we analysed two HCR
mutants of Synechocystis by transcriptional and metabolic
phenotyping, the carboxysome-less mutant DccmM and the
2PG metabolism mutant DglcD1/D2.
METHODS
Strains and culture conditions. The glucose-tolerant strain Synecho-
cystis sp. PCC 6803 served as the WT. Axenic cultures were grown on
Petri dishes at 30 uC under constant illumination of 30 mmol photons
m22 s21 using BG11 medium (Rippka et al., 1979) containing 0.8 % agar
buffered to pH 8.0 with 20 mM TES–KOH. Transformants were selected
on media containing either 10 mg kanamycin ml21 (Km) or 4 mg spectinomycin ml21 (Sp). Subsequently, we used 50 mg Km or 20 mg Sp ml21.
For the physiological and molecular phenotyping, axenic cultures with
an OD750 of approximately 0.8 were grown photoautotrophically in
batch cultures at 29 uC under continuous illumination of 100 mmol
photons m22 s21. Cultures were bubbled with 5 % CO2 in air [defined
as high Ci (HC)] in BG11 medium at pH 8.0, or, alternatively with
ambient air [0.035 % CO2, defined as low Ci (LC)] in BG11 medium at
pH 7.0. Growth was monitored by measuring OD750 (Ultrospec 3000
spectrophotometer, Pharmacia Biotech).
Statistically significant differences in the growth rates or other
parameters between WT and mutant strains with the different
treatments were analysed using Student’s t test.
DNA manipulation and generation of mutants. Total DNA from
Synechocystis was isolated according to Hagemann et al. (1997). All
other DNA techniques were standard methods. The construction of the
Synechocystis double mutant DglcD1/D2 has been previously described
(Eisenhut et al., 2008a). To obtain a ccmM (sll1031) mutant, the coding
sequence was amplified with specific primers (Table 1). A fragment of
1100 bp was excised with StuI and HpaI and replaced by a Kmresistance cartridge excised from pUC4K (Pharmacia) with HincII.
Complete segregation of Synechocystis mutants was checked by PCR
analysis with gene-specific primers (Table 1). Only DNA fragment with
sizes corresponding to the mutated genes were detected with DNA
from mutants, while fragments of WT gene sizes were completely
absent (Supplementary Fig. S1a).
Metabolite profiling. Cells grown in liquid media under standard
HC or LC conditions were harvested by fast filtration in the light and
immediately frozen in liquid N2, as described previously (Schwarz
et al., 2011). Metabolites were determined using a metabolite-targeted
profiling method, and a quantitatively standardized and calibrated
variant of the previously established gas chromatography electron
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Table 1. Strains and primers used in this work
Strain or primer
Strains
Synechocystis sp. PCC 6803
Synechocystis DglcD1/D2
Synechocystis DccmM
Primers
sll0404-fw
sll0404-rev
sll0806-fw
sll0806-rev
sll1031-fw
sll1031-rev
Genotype or sequence (5§A3§)
WT
sll0404 : : Sp/slr0806 : : Km
sll1031 : : Km
Source or reference
Pasteur Culture Collection
Eisenhut et al. (2008a)
This work
GTC AAC CGC CGT TAC CGA T
GCC TAG GTT AAG CCG TTG
GGA GTC GCT CTG GGG ATA ACC
GGT GAG ATA AAA GAT TGG TTG
CCA TCA TCC GCC GTT AAT
ACC GAG ACA AGC TGT TGC
ionization time-of-flight MS (GC–EI–TOF–MS) profiling analysis.
Relative amounts of metabolites were estimated by adding a defined
amount of an internal standard (ribitol and alanine) to the cell
material. The amounts were then normalized to biomass using the
OD750 of each sample (Eisenhut et al., 2008b; Huege et al., 2011).
Electron microscopy. Cells were harvested by centrifugation and
fixed with glutaraldehyde (2.5 %, v/v) in 0.1 M phosphate buffer. Fixed
cells were embedded in low-melting-point agarose, post-fixed with 2 %
osmium tetroxide, dehydrated in ethanol and finally embedded in
EPON resin. Ultrathin sections were analysed with a transmission
electron microscope equipped with a digital charge-coupled device
(CCD) camera (161k). The numbers of carboxysomes in WT and
mutant strains were counted in sections of 50 representative cells
(sectioned approximately in the middle of the cell) from each culture.
Immunoblotting. Proteins were extracted from 20 ml cells. Total
proteins were extracted by sonication in ice-cold 10 mM HEPES buffer
(pH 7.5) containing PMSF. Equal amounts of soluble protein (10 mg)
were separated in 12 % denaturing polyacrylamide gels and subsequently
blotted onto PVDF membranes (Bio-Rad). RubisCO was detected by
an antibody specific for the large RubisCO subunit (RbcL, received
from Professor E. Pistorius, University of Bielefeld) using horseradish
peroxidase-conjugated anti-rabbit IgG (Bio-Rad) as secondary antibody.
RNA isolation and transcriptional phenotyping using DNA
microarrays. Cells from 10 ml of culture were harvested by centrifug-
ation at 3500 g for 5 min at 4 uC, and were immediately frozen at
280 uC. Total RNA was extracted. Labelled cDNA was hybridized to
60-mer oligonucleotide DNA microarrays (Agilent custom design array,
AMADID no. 016989) designed from the complete Synechocystis
chromosomal sequence but lacking the genes found on the plasmid
DNA. Values presented are the means and SDs of three independent
biological replicates. After array normalization, probe signals were
statistically evaluated and those with an adjusted P value below 0.01
were considered reliable. All probes for a single gene were required to
indicate the same regulation, and finally a cut-off value of 2.5-fold up or
0.4-fold down was used to select a reasonable number of regulated genes
(Eisenhut et al., 2007; Hackenberg et al., 2009; Aguirre von Wobeser et al.,
2011).
conditions. As previously shown (Ogawa et al., 1994; Berry
et al., 2005; Eisenhut et al., 2008a), both mutants displayed
the HCR phenotype, i.e. they could not grow under LC
conditions but could be rescued under HC conditions
(Supplementary Fig. S1). Compared with the WT at HC,
cells of DccmM were characterized by an approximately 50 %
lowered growth rate (Supplementary Fig. S1b), while cells of
DglcD1/D2 grew approximately 20 % slower, similar to rates
reported previously (Eisenhut et al., 2008a). Previous studies
with different DccmM mutants of Synechocystis have also
shown compromised growth under HC conditions (Ogawa
et al., 1994; Berry et al., 2005). The reduced growth rate is
in agreement with the lower photosynthetic Vmax of cells of
DccmM and of DglcD1/D2 when grown under HC conditions (Berry et al., 2005; Eisenhut et al., 2008a).
The number of carboxysomes in HC-acclimated and LCshifted cells was analysed using electron microscopy. As
reported before, cells of DccmM are free of carboxysomes
(Fig. 1a, b; Berry et al., 2005). In contrast, cells of DglcD1/D2
possessed slightly more carboxysomes per cell compared
with WT cells under HC conditions (Fig. 1a, b). An increase
in carboxysome number is a typical feature of LC-grown
WT cells. The finding of enhanced carboxysome numbers in
HC-acclimated cells of photorespiratory mutants, DglcD1
and DgvcT in previous work, led to the hypothesis that
photorespiratory intermediates might act as a signal for
some aspects of CCM induction (Eisenhut et al., 2007). Cells
were shifted to LC conditions for 24 h. This shift increased
the carboxysome number in WT cells and to a greater extent
in DglcD1/D2 cells (Fig. 1b). Carboxysomes did not appear
in LC-shifted cells of DccmM. In accordance with the
increased carboxysome number, WT cells contained a
higher amount of RubisCO after the shift to LC (Fig. 1c).
A similar increase in RubisCO content was observed in both
mutant strains at LC. The DglcD1/D2 mutant accumulated
more RubisCO than DccmM (Fig. 1c).
RESULTS AND DISCUSSION
Characterization of strains
Metabolic phenotyping of mutants DccmM and
DglcD1/D2 compared with WT
The growth of the mutants and WT was analysed after
cultivation under HC (5 % CO2) and LC (0.035 % CO2)
Steady-state metabolite levels of intermediates of the
primary C and N metabolism (Fig. 2) of the WT and
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Carboxysomal and photorespiratory Synechocystis mutants
Fig. 1. Analysis of carboxysomes and RubisCO in the Synechocystis mutants DccmM and DglcD1/D2. (a) Representative
electron micrographs of WT, DccmM and DglcD1/D2 cells are shown. Cells were grown under standard HC or for 24 h under
LC conditions. Carboxysomes (C), polyphosphate bodies (P) and glycogen granules (G) are marked with arrows. (b)
Carboxysome number per cell is shown (means and confidence intervals). Statistically supported differences in the
carboxysome number compared with the WT grown under HC conditions are indicated (*P,0.01). (c) Immunoblotting analyses
with protein extracts to determine the amount of RubisCO. Cells were cultivated under HC or for 24 h under LC conditions.
Samples (10 mg per lane) of total soluble protein were applied to an SDS–PAGE gel (upper panel to show equal loading). RbcL
was detected by a specific antibody (aRbcL) via chemiluminescence.
mutants under different Ci conditions were measured
(Supplementary Table S1 displays the complete metabolome dataset). For comparison, all data are displayed as
relative increases/decreases (fold changes) compared with
WT levels under HC conditions (set to 1).
HC conditions. Cells of the DccmM strain were characterized
by 1.8-fold increased 2PG levels compared with the WT
(Fig. 3). The 2PG accumulation in this carboxysome-less
mutant can be explained by the unshielded, direct exposure
of RubisCO to O2 in the cytoplasm of the cell, which
evidently increases oxygenase activity. In contrast to the
higher 2PG content, the photorespiratory intermediates
glycine, serine and glycerate dropped in DccmM to about
50 % of the WT level. Accumulated 2PG is known to
exhibit an inhibitory effect on CBB cycle enzymes (Kelly
& Latzko, 1977), which could explain the lowered growth
and photosynthesis of DccmM cells despite the HC environment. In addition, the CBB cycle intermediate ribulose
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5-phosphate (Ru5P) was below background in WT but
clearly observed in HC-grown cells of DccmM, whereas the
3PGA content of DccmM remained similar to that of the WT
at HC. Furthermore, DccmM cells contained lower levels of
glutamate, citrate, sucrose and 6-phosphogluconate under
HC conditions (Figs 3 and 4). The observed changes are
unlikely to be explained by changes in enzyme amounts,
because no significant changes in the expression of the
corresponding genes were detected by transcriptomics (see
below). These findings suggest that 2PG interferes directly
with actual enzyme catalysis, and thus might not be restricted
in its effects to CBB cycle steps but might possibly also
affect other enzymes of primary metabolism. Additionally,
enzymes of the CBB cycle, e.g. Ru5P kinase, are activated
via reduction and inactivated under oxidative conditions
(Dietz & Pfannschmidt, 2011). Since HC-grown cells of the
DccmM strain showed increased transcript levels for many
genes involved in the oxidative stress response (see Table 2),
it can be assumed that the cytoplasm of DccmM cells is
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C. Hackenberg and others
Fig. 2. Scheme of central carbon and nitrogen metabolism in Synechocystis. The sites of mutation in DccmM and DglcD1/D2
are indicated.
more oxidized than in WT cells. Therefore, redox-sensitive
enzymes might be negatively affected in their activity in
this mutant. The redox regulation of Ru5P kinase is also
mediated by its redox-dependent binding to or release from
the small peptide CP12 (Wedel & Soll, 1998; Tamoi et al.,
2005).
As expected from the genetic lesion, the double mutant
DglcD1/D2 accumulated glycolate under HC conditions
(three times more than WT), while 2PG and Ru5P were not
changed. The block in photorespiratory 2PG metabolism
(Fig. 2) that gives rise to the glycolate accumulation is also
illustrated by the decreased levels of the downstream metabolites glycine, serine and glycerate in HC-grown DglcD1/D2
cells. Also, sucrose and glutamate levels decreased, as
observed in DccmM, whereas other metabolites remained
at levels similar to those in the WT (Figs 3 and 4).
From the metabolomics data with HC cells, we conclude that
glycolate accumulation in cells of mutant DglcD1/D2 hampers
general cell metabolism less than does 2PG accumulation in
mutant DccmM. This conclusion nicely coincides with the
order of growth rates at HC: growth of WT.growth of
DglcD1/D2.growth of DccmM (Supplementary Fig. S1).
402
LC conditions. 2PG accumulated in WT cells 24 h after the
shift to LC (Fig. 3), illustrating an increased proportion
of the oxygenase relative to the carboxylase reaction of
RubisCO (Huege et al., 2011). Interestingly, in the same
sample, the amount of the CBB cycle intermediate Ru5P was
90-fold higher. This finding could be taken as an indication
that the CBB cycle enzyme Ru5P kinase is inhibited by the
action of 2PG and/or inhibited by oxidation of the protein
in association with CP12 (Tamoi et al., 2005). The other
metabolites of the photorespiratory 2PG metabolism remained
unchanged (glycolate, glycerate) or decreased (glycine, serine)
in WT cells.
The most severe metabolic changes were observed in the
carboxysome-less mutant after LC shift. In DccmM, 2PG and
Ru5P, both already elevated at HC, increased to even higher
levels 3 h after the transfer to LC (Fig. 3). Probably, 2PG
phosphatase represents a rate-limiting step in 2PG metabolism under conditions strongly favouring RubisCO
oxygenation, i.e. after sudden transition from HC to LC in
WT cells, and particularly in the absence of functional
carboxysomes, as in DccmM. The increase in Ru5P and 2PG
is accompanied by decreased levels of the primary CO2
fixation product 3PGA and the photorespiratory C2 cycle
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Carboxysomal and photorespiratory Synechocystis mutants
Fig. 3. Metabolic phenotyping in the Synechocystis mutants DccmM and DglcD1/D2. Fold
changes of photorespiratory and CBB metabolites in cells of the WT and DccmM and
DglcD1/D2 mutants. Cells were grown under
HC conditions and shifted for 3 or 24 h to LC
conditions. n.d., Not detected. Statistically
supported differences compared with the WT
grown at HC are indicated (one asterisk,
P,0.05; two asterisks, P,0.01).
intermediates serine, glycine and glycerate in cells of DccmM
under LC conditions.
The photorespiratory mutant DglcD1/D2 accumulated
much more glycolate but exhibited a 3PGA level similar to
the WT when shifted to LC. The amount of 2PG and Ru5P
in cells of DglcD1/D2 increased after transfer to LC to levels
similar to those in the WT. Compared with LC-shifted WT
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cells, DglcD1/D2 also contained more glycine and serine at
3 h after LC shifts, while glycerate and 2PG contents were
similar (Fig. 3). The increase of serine in DglcD1/D2 clearly
shows that in addition to 2PG metabolism, which is blocked
at the glycolate-to-glyoxylate step in this mutant, further
pathways exist for serine biosynthesis in Synechocystis, as
discussed by Knoop et al. (2010).
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C. Hackenberg and others
Fig. 4. Metabolic phenotyping in the Synechocystis mutants DccmM and DglcD1/D2. Fold
changes in key metabolites of the central C
and N metabolism in cells of the WT, DccmM
and DglcD1/D2 mutants. Cells were grown
under HC conditions and shifted for 3 or
24 h to LC conditions. n.d., Not detected.
Statistically supported differences compared
with the WT grown at HC are indicated (one
asterisk, P,0.05; two asterisks, P,0.01).
Interesting differences were also observed for metabolites
of the associated central C and N metabolism in WT cells.
Phosphoenolpyruvate (PEP) levels increased in Synechocystis
WT (Fig. 4) and Synechococcus elongatus PCC 7942 after shift
to LC (Eisenhut et al., 2008b; Huege et al., 2011; Schwarz
et al., 2011). In contrast, sucrose, malate, glutamine and
glutamate decreased (Figs 3 and 4). These findings indicate
that carbon is mostly exported from the CBB cycle to
404
glycolysis, while carbon storage through sucrose as well as
N assimilation through glutamine synthetase/glutamineoxoglutarate aminotransferase (GS-GOGAT) is decreased in
WT cells after LC shifts. The latter is of direct interest for
balanced N/C coupling.
In accordance with the strongly decreased levels of 3PGA in
DccmM the PEP pool also significantly decreased after the
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Carboxysomal and photorespiratory Synechocystis mutants
shift to LC in this mutant (Figs 3 and 4). The lowered CO2
fixation and export of carbon to glycolysis also explain
the lowered levels of the tricarboxylic acid cycle (TCA)
intermediates citrate and 2-oxoglutarate (2OG), and that
of glutamine. Interestingly, the intermediates malate (Fig.
4) and fumarate (Supplementary Table S1) in the other
branch of the incomplete TCA cycle (Fig. 2) were much
higher in DccmM cells than in WT 24 h after LC shift. The
malate accumulation could be taken as indicating that in
DccmM the carboxylation via PEP carboxylase increases.
The increased C4 carboxylation could help in compensating for the lowered RubisCO carboxylation activity in
the absence of carboxysomes. However, expression of the
PEP carboxylase gene did not change accordingly (see
Supplementary Table S2). It should be mentioned that,
inside the cytoplasm of the DccmM, RubisCO is exposed
to a high concentration of HCO^3 , the substrate of PEPcarboxylase, while RubisCO depends on the CA-mediated
back-conversion of HCO^3 to CO2. Indications of increased carboxylation via PEP carboxylase have also been
obtained by 13C-labelling experiments with photorespiratory mutants (DglcD1, DgcvT) after HC-to-LC shifts
(Huege et al., 2011).
Overall transcriptional changes in DccmM and
DglcD1/D2
The mutant DccmM also behaved differently from the other
strains regarding the internal amounts of several amino
acids. For example, the small amount of arginine at HC
increased considerably after LC shifts in this mutant, while
it remained at low levels in WT and DglcD1/D2 (Fig. 4).
Arginine originates from aspartate, which is generated
from oxaloacetate and glutamate with the release of
fumarate and 2OG (Fig. 2). The arginine precursor
oxaloacetate is the direct HCO^3 fixation product of PEPcarboxylase and seems to be increased in DccmM cells.
Thus, the decreasing levels of glutamine and glutamate
indicate enhanced transamination of oxaloacetate to
aspartate, increasing the arginine levels in this mutant.
Under LC conditions, DccmM showed fewer changes (69
genes with lower and 90 genes with higher transcript levels)
compared with WT (99 genes with lower and 116 genes with
higher transcript levels) and with DglcD1/D2 (103 genes with
lower and 124 genes with higher transcript levels). Genes
with increased transcript levels varied among the strains,
e.g. many more genes for photosynthesis/respiration and
regulatory proteins increased in DglcD1/D2 than in WT or
DccmM (Supplementary Fig. S2, Supplementary Table S3).
For the WT and mutant DglcD1/D2, the transcriptional
changes were also analysed at 3 h after LC shift. A
remarkably low number of genes changed in mRNA level
in DglcD1/D2 (eight genes with lower and 13 genes with
higher transcript levels) compared with WT shortly after the
shift to LC.
Despite accumulating glycolate, the photorespiratory
mutant DglcD1/D2 showed only slight changes in metabolite levels after the shift to LC conditions. Most metabolites
remained at the levels observed under HC conditions
(Fig. 4, Supplementary Table S1). One exception was
6-phosphogluconate, an intermediate of the oxidative
pentose phosphate (OPP) pathway. It accumulated after
3 h in DglcD1/D2 and remained enhanced after 24 h at LC
(Fig. 4), in a similar manner to the glycolate levels of these
cells. This finding indicates continuing glycogen catabolism
under LC conditions, possibly in combination with
inhibition of 6-phosphogluconate dehydrogenase by glycolate. The rather small amount of glycogen granules in
DglcD1/D2 cells, already at HC compared with WT cells,
supports this notion (Fig. 1a). The lowered glycogen could
be interpreted as an adaptation to lowered CO2 fixation.
Instead of glycogen storage, the pool of low-molecularmass organic carbon precursors for biosynthesis seems to
be increased at HC in DglcD1/D2, which is also characteristic for LC-shifted WT cells.
http://mic.sgmjournals.org
We next compared the metabolic phenotyping results
with data from DNA microarray analyses (Supplementary
Table S2 contains the complete transcriptome dataset). The
question was whether the changed levels of photorespiratory intermediates in HC-grown cells of the HCR mutants
DccmM and DglcD1/D2 corresponded to changes in the
mRNA levels of the genes that are suggested to be
specifically regulated by HC-to-LC shifts, or whether other
potentially compensating responses needed consideration.
Under HC conditions, we found 81 genes with decreased
and 88 genes with increased mRNA levels in DccmM, while
DglcD1/D2 showed approximately double the number of
transcriptional changes (143 genes with lower and 136
genes with higher transcript levels compared with WT at
HC; Supplementary Fig. S2, Supplementary Table S3).
Generally, mostly genes for hypothetical and unknown
proteins were differentially regulated in the mutants
compared with the WT, although many genes for
photosynthetic proteins and transport processes were
affected too.
Specific transcriptional differences between
DccmM, DglcD1/D2 and WT at HC
Contradicting our expectations, we did not find changed
transcript levels for Ci transporters or CCM components in
the 2PG-accumulating DccmM cells at HC, i.e. none of the
affected gene products in this mutant is involved in the
specific acclimation to LC (Table 3). Instead, many genes
encoding proteins involved in the stress response, metal
homeostasis and decreased nitrogen utilization displayed
changed mRNA levels in DccmM (Table 2). The altered
amounts of mRNAs for stress protein genes (Fig. 5)
indicate that DccmM suffers from oxidative stress, which is
likely caused by impaired CO2 fixation, the main sink for
reduced redox equivalents produced by the photosynthetic
light reaction. Interestingly, transcripts of genes flv2 and
flv4 (encoding proteins thought to protect photosystem II;
Zhang et al., 2009), and psbA2 and psbA3, were increased in
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C. Hackenberg and others
Table 2. Transcript changes of selected genes in the DccmM and DglcD1/D2 mutants grown under HC conditions
The relative expression of genes is given for cells grown under HC (5 % CO2) and LC (0.035 % CO2) conditions. Data were obtained by DNA
microarrays and represent fold ratios of transcript levels in mutants exposed to HC compared with the WT grown under HC conditions. Transcript
levels in strains exposed to LC were compared with the respective strain grown under HC conditions. Genes were considered differentially regulated
when the fold change was higher than 2.5-fold and below 0.4-fold (numbers shown in bold type).
ORF
Gene
Annotation
Fold change
DccmM HC/
DglcD1/D2
HC/WT HC
WT LC/
WT HC
DccmM LC/
DccmM HC
4.17
5.11
3.30
2.87
7.75
6.69
7.15
3.61
2.99
8.89
6.11
6.45
16.01
13.78
2.55
1.71
0.71
1.36
1.30
2.07
3.24
1.42
1.12
1.16
2.97
4.81
1.36
1.94
4.18
1.55
1.53
0.93
0.17
0.10
0.20
0.28
0.27
0.09
0.35
0.61
2.59
2.15
1.81
7.14
3.64
1.36
2.26
3.02
1.18
0.29
2.25
1.40
0.82
1.12
4.87
3.67
2.84
2.67
1.35
0.80
0.36
0.92
1.57
1.40
0.81
1.33
0.42
0.47
0.96
0.49
35.39
23.45
2.34
0.89
1.16
1.73
34.96
21.27
4.78
1.67
1.08
2.08
6.69
15.27
1.93
WT HC
Stress proteins
ssl2542
hliA
ssr2595
hliB
ssl1633
hliC
sll0306
sigB
sll1514
hspA
slr1204
htrA
slr1291
ndhD2
sll0856
sigH
slr0233
slr1675
trxM
hypA1
slr1641
ssr2061
sll0430
sll0755
clpB1
grx
htpG
tpx
Flavoproteins
sll0219
sll0217
sll0550
Metal
homeostasis
sll0792
slr0797
slr0798
sll1878
Regulatory
proteins
slr1738
sll0790
slr1285
slr0083
sll0789
sll1626
ssl0707
Nitrogen
metabolism
ssl0452
ssl0453
ssl1911
flv2
flv4
flv3
Flavoprotein 2
Flavoprotein 4
Flavoprotein 3
ziaR
coaT
ziaA
futC
Zinc-responsive repressor ZiaR
Cobalt transporter CoaT
Zinc exporter ZiaA
Iron(III)-transport ATP-binding protein
3.45
6.19
4.74
2.90
2.60
0.70
1.23
0.84
1.08
0.63
0.88
0.75
1.04
3.18
0.71
0.46
0.35
1.03
0.41
0.98
perR
hik31
hik34
crhR
copR
lexA
glnB
Transcription regulator Fur family
Two-component sensor histidine kinase
Two-component sensor histidine kinase
DeaD RNA helicase light
Rre34 two-component response regulator
LexA repressor
Nitrogen regulatory protein P-II
1.37
1.80
1.27
2.15
1.96
0.85
0.47
7.45
3.75
3.57
2.90
2.82
0.33
0.35
1.08
0.54
1.05
1.38
0.84
1.53
1.15
1.43
1.43
1.55
0.78
1.97
0.98
1.27
0.31
0.18
0.31
0.40
0.22
2.85
3.46
nblA1
nblA2
gifA
Phycobilisome degradation protein NblA1
Phycobilisome degradation protein NblA2
Glutamine synthetase inactivating factor
IF7
Glutamine synthetase inactivating factor
IF17
Threonine synthase
Ammonium/methylammonium permease
Gutamate-ammonia ligase
12.29
4.59
6.28
12.91
5.44
2.24
5.66
3.01
8.88
5.05
5.04
2.42
0.26
0.34
0.53
10.10
20.33
31.66
8.38
0.26
3.84
0.20
0.24
4.77
0.12
0.92
2.73
0.24
0.24
0.68
0.67
0.22
0.51
1.59
1.13
sll1515
gifB
sll1688
sll0108
slr1756
thrC
amt1
glnA
406
High-light-inducible polypeptide HliA
High-light-inducible polypeptide HliB
High-light-inducible polypeptide HliC
RpoD group 2 RNA polymerase sigma factor
16.6 kDa small heat-shock protein
Protease
NADH dehydrogenase subunit 4
RpoE group 3 RNA polymerase sigma
factor
Thioredoxin M1
Hydrogenase expression/formation
protein
ClpB protein
Glutaredoxin
Heat-shock protein HtpG
2-Cys peroxiredoxin thioredoxin
peroxidase, ycf42
DglcD1/D2 LC/
DglcD1/D2 HC
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Microbiology 158
Carboxysomal and photorespiratory Synechocystis mutants
Table 2. cont.
ORF
Gene
Annotation
Fold change
DccmM HC/
WT HC
slr0898
sll1450
nirA
nrtA
slr0447
urtA
sll1502
Carbon
metabolism
ssl3364
slr0953
slr1124
slr1875
sll1356
DglcD1/D2
HC/WT HC
WT LC/
WT HC
DccmM LC/
DccmM HC
DglcD1/D2 LC/
DglcD1/D2 HC
0.15
0.09
1.12
0.59
0.19
0.10
0.57
0.35
0.85
0.77
0.24
0.54
0.28
0.34
1.07
gltB
Ferredoxin-nitrite reductase
Nitrate/nitrite transport system substratebinding protein
Substrate-binding protein of ABC
transporter
NADH-dependent glutamate synthase
1.13
0.40
1.43
0.64
2.50
cp12
spsB
gpmB
glgX
glgP
CP12 polypeptide
Sucrose-phosphate phosphatase
Phosphoglycerate mutase
Isoamylase
Glycogen phosphorylase GlgP
1.90
1.60
0.39
0.43
1.19
3.52
3.06
0.52
0.38
0.69
1.14
0.90
0.41
0.73
1.86
1.50
0.91
0.58
0.49
0.57
0.64
0.40
1.26
3.13
2.88
the DccmM mutant at HC. Probably, photosystem II is
particularly affected by the absence of carboxysomes.
Increased transcript levels for these genes have been
reported previously for LC-shifted (Wang et al., 2004;
Eisenhut et al., 2007) as well as high-light-, salt- or H2O2treated, and iron-limited Synechocystis WT cells (Hihara
et al., 2001; Los et al., 2008). In our experiments, many of
these transcripts also increased after LC shifts in WT cells.
Their mutation (e.g. flv2; Zhang et al., 2009) did not,
however, result in an HCR phenotype, ruling out an
essential and specific function of the respective proteins in
LC acclimation.
Additionally, transcripts for many proteins involved in
metal homeostasis increased in DccmM (e.g. ziaR, coaT)
that were not induced by LC in WT (Table 2) but are
regulated by limiting trace metal concentrations (Garcı́aDomı́nguez et al., 2000). This observation further supports
the notion that the carboxysome-less mutant suffers from
oxidative stress, which is known to affect metal centres in
protein complexes (e.g. Imlay, 2006).
Transcripts for subunits of phycobilisomes, photosystems,
the cytochrome b6f complex and ATP synthase were lower in
HC-grown cells of DccmM (Supplementary Fig. S2, Supplementary Table S3). The downregulation of antenna size and
photosystem content observed in DccmM cells usually
occurs as part of the acclimation of Synechocystis WT
towards high-light stress to prevent the production of
reactive oxygen species (ROS; Hihara et al., 2001). It has
also been proposed that the CCM itself may serve as a
mechanism to dissipate excess light energy, which explains
the elevated transcript levels of carboxysomal genes under
high-light stress (Hihara et al., 2001; Tchernov et al., 2003).
Thus, the downregulation of photosynthetic processes in the
DccmM mutant can be interpreted as an effort to limit the
synthesis of ROS when CO2 fixation and the CCM are not
properly functioning as energy sinks.
http://mic.sgmjournals.org
Another group of genes strongly affected under HC conditions by mutation of ccmM encode proteins involved in N
assimilation (Fig. 5, Table 2). Correspondingly, increased
transcript levels of nblA1/2 and gifA/B, which encode proteins
involved in phycobilisome degradation and the downregulation of GS, respectively, were found. Since similar changes
occur in cyanobacterial WT cells only after HC-to-LC shift
(Eisenhut et al., 2007; Schwarz et al., 2011), the mutant
displays a partial phenocopy of LC-treated WT cells under HC
conditions. In many regards, our dataset on the diminished
N assimilation after LC shifts corresponds with recently
published data on transcriptomic changes of Synechocystis
under nitrate limitation based on the same DNA microarray
platform (Aguirre von Wobeser et al., 2011).
Despite distinct changes in the metabolome of HC-grown
DccmM cells compared with WT cells, we did not find
significantly altered mRNA contents for proteins of the
CBB, OPP and TCA cycles, 2PG metabolism or glycolysis
that explained or correlated with these changes. Therefore,
the metabolite changes do not result from transcriptional
regulation of enzyme abundances. Instead, they are probably due to the regulation of key enzyme activities, e.g.
by direct inhibition or activation via 2PG and/or other
metabolites as well as redox changes to redox-sensitive
enzymes. Only a few genes for enzymes of central carbon
metabolism showed decreased transcript levels. In DccmM,
one phosphoglycerate mutase (gpmB, reversibly converting
3PGA to 2PGA) involved in the shunt to PEP exhibited
decreased mRNA, similar to LC-treated WT cells (Eisenhut
et al., 2007). Thus, transcriptional control of the gpmB gene
could be involved in the observed parallel changes of 3PGA
and PEP pool sizes at different Ci levels in WT cells
(Eisenhut et al., 2008b, Huege et al., 2011).
The number of genes with changed transcript amounts
under HC conditions in DglcD1/D2 relative to WT was
similar or even higher than in DccmM. As discussed above,
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C. Hackenberg and others
Table 3. Transcript changes of selected genes in cells after shifts from HC to LC conditions for 24 h
The relative expression of genes is given for cells grown under HC (5 % CO2) and LC (0.035 % CO2) conditions. Fold ratios of transcript levels in
mutants exposed to HC were compared with the WT grown under HC conditions, and transcript levels in strains exposed to LC were compared
with the respective strain grown under HC conditions. Genes were considered differentially regulated when the fold change was higher than 2.5-fold
and below 0.4-fold (numbers shown in bold type).
ORF
Gene
Annotation
Fold change
DccmM HC/
Ci transporter
slr0040
cmpA
slr0041
slr0042
slr0043
cmpB
slr0044
cmpD
slr1512
sbtA
slr1513
sbtB
sll1732
ndhF3
sll1733
ndhD3
sll1734
cupA
sll0026
ndhF4
sll0027
ndhD4
slr1302
cupB
sll0834
bicA
Regulatory
proteins
sll0030
sll1594
sll0822
Photosynthesis
slr1835
slr1834
slr1311
sll1867
sll0849
cmpC
DglcD1/D2
HC/WT HC
WT LC/
WT HC
DccmM LC/ DglcD1/D2 LC/
DccmM HC DglcD1/D2 HC
1.08
1.06
23.24
6.69
20.63
0.78
0.98
1.03
0.99
1.05
1.08
17.27
12.57
6.04
3.06
3.67
2.22
16.25
6.01
8.94
1.27
1.08
6.77
1.97
10.23
1.88
0.65
27.24
2.07
11.95
1.32
0.47
12.52
1.71
5.59
1.62
0.64
12.88
1.34
5.14
1.90
0.83
12.85
0.77
12.21
2.10
0.79
15.02
0.71
10.76
0.64
0.49
0.82
0.76
1.25
0.61
0.65
0.80
0.65
0.98
0.91
1.02
0.59
1.15
0.64
0.79
0.68
0.74
0.72
0.84
cmpR
ndhR
abrB
Cmp operon transcriptional regulator
NdhR operon transcriptional regulator
AbrB-like protein
0.97
1.50
0.52
0.89
1.14
0.66
1.98
3.54
1.14
1.67
2.51
0.44
2.19
1.25
2.50
psaA
psaB
psbA2
psbA3
psbD1
P700 apoprotein subunit Ia
P700 apoprotein subunit Ib
Photosystem II D1 protein
Photosystem II D1 protein
Photosystem II reaction centre D2
protein
Photosystem II reaction centre D2
protein
Cytochrome b559 b subunit
Photosystem II PsbJ protein
Photosystem II PsbT protein (ycf8)
0.49
0.34
2.44
2.69
1.02
0.22
0.18
0.97
1.12
0.68
4.47
3.91
11.75
12.17
3.81
1.05
0.88
1.84
1.79
1.20
12.25
15.35
0.74
0.56
0.94
1.37
0.78
7.02
1.34
1.11
0.34
0.30
1.07
0.50
0.35
0.48
2.69
2.53
3.79
0.76
0.72
0.93
6.35
7.42
5.14
slr0927
psbD2
sll0819
sml0008
smr0001
psbF
psbJ
psbT
408
Bicarbonate transport system substratebinding protein
Bicarbonate transport system permease
Probable porin; major outer membrane
Bicarbonate transport system ATPbinding protein
Bicarbonate transport system ATPbinding protein
Sodium-dependent bicarbonate
transporter
Periplasmatic protein; function
unknown
NADH dehydrogenase subunit 5
(involved in low-CO2-inducible highaffinity CO2 uptake)
NADH dehydrogenase subunit 4
(involved in low-CO2-inducible highaffinity CO2 uptake)
Protein involved in low-CO2-inducible
high-affinity CO2 uptake
NADH dehydrogenase subunit 5
(involved in constitutive, low-affinity
CO2 uptake)
NADH dehydrogenase subunit 4
(involved in constitutive, low-affinity
CO2 uptake)
Protein involved in constitutive, lowaffinity CO2 uptake
Low-affinity Na+-dependent HCO^3
transporter of the SulP family
WT HC
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Carboxysomal and photorespiratory Synechocystis mutants
Table 3. cont.
ORF
Gene
Annotation
Fold change
DccmM HC/
sll1194
psbU
sll0519
sll0522
sll0521
slr0261
sll0520
slr1281
slr1280
slr0343
RubisCO
slr0009
ndhA
ndhE
ndhG
ndhH
ndhI
ndhJ
ndhK
petD
rbcL
slr0011
slr0012
rbcX
rbcS
Photosystem II 12 kDa extrinsic
protein
NADH dehydrogenase subunit 1
NADH dehydrogenase subunit 4L
NADH dehydrogenase subunit 6
NADH dehydrogenase subunit 7
NADH dehydrogenase subunit NdhI
NADH dehydrogenase subunit I
NADH dehydrogenase subunit NdhK
Cytochrome b6f complex subunit 4
Ribulose bisphosphate carboxylase
large subunit
Possible RubisCO chaperonin
Ribulose bisphosphate carboxylase
small subunit
DglcD1/D2 cells also accumulate mRNAs for many stress
proteins to higher levels than WT cells. The defect in 2PG
metabolism of DglcD1/D2 probably accounts for this, since
it has recently been demonstrated that photorespiration
is a high-light protective mechanism that dissipates excess
energy and prevents photoinhibition in cyanobacteria
(Hackenberg et al., 2009). Interestingly, the genes for
Flv2 and Flv4 and those related to metal homeostasis
remained unchanged (Fig. 5, Table 2). High similarities
exist in the changes of genes for proteins involved in N
assimilation; however, the expression of some genes with
decreased transcription in DccmM did not change in
DglcD1/D2 (e.g. the nrt/mer operon, urtA/B). This suggests
a relatively higher excess of N over C in DccmM with
respect to DglcD1/D2, corresponding to the greater growth
decrease of the carboxysome-less mutant under HC
conditions (Supplementary Fig. S1). Compared with WT
and DccmM, many genes for potential regulatory proteins,
among them Hik34 (a repressor of heat-shock proteins;
Suzuki et al., 2005) and PerR (the repressor of oxidative
stress response; Li et al., 2004), showed higher transcript
levels in HC-grown cells of DglcD1/D2 (Table 2). The many
changes in the expression of genes for transcriptional
regulators could well be related to the larger number of
changes in gene expression observed in mutant DglcD1/D2
at HC (Supplementary Fig. S2, Supplementary Table S3).
Specific transcriptional changes in DccmM,
DglcD1/D2 and WT after LC shift
LC-treated WT cells showed the expected changes, i.e. genes
for Ci transporters such as SbtA, BCT1 (cmpA-D) and NDH13 (sll1732–1736), regulators (ndhR, cmpR), photosystem I
and, to a lesser degree, photosystem II were characterized by
http://mic.sgmjournals.org
WT HC
DglcD1/D2
HC/WT HC
WT LC/
WT HC
DccmM LC/ DglcD1/D2 LC/
DccmM HC DglcD1/D2 HC
1.09
0.54
3.29
1.44
2.31
1.62
1.65
2.09
2.54
2.56
2.19
2.24
0.96
0.62
0.69
0.84
0.85
0.93
0.72
0.98
0.54
3.41
3.04
3.29
2.68
5.69
3.26
3.61
2.53
0.86
0.57
0.60
1.04
0.61
0.69
0.84
1.43
1.76
2.92
2.01
2.03
3.75
3.73
2.95
2.13
1.17
0.22
1.26
0.31
5.09
1.34
1.02
0.30
0.25
1.28
1.83
0.25
0.36
6.69
7.88
coordinately increased mRNA contents (Tables 2 and 3).
Additionally, proteins involved in N assimilation showed
lowered transcript levels, while genes involved in the
decrease of N assimilation (e.g. nblA1/2, gifA/B) showed
higher levels, as reported previously (Wang et al., 2004;
Eisenhut et al., 2007; Schwarz et al., 2011). Genes for
proteins involved in translation were found to be globally
downregulated in all investigated strains (Supplementary
Fig. S2, Supplementary Table S3), indicating a general
acclimation to lowered growth rates due to decreased CO2
fixation.
The gene induction pattern of LC-shifted WT cells was
almost completely absent from LC-shifted cells of the 2PGaccumulating mutant DccmM (Tables 2 and 3). This
mutant lacks the typical increase of Ci transporters after LC
shift, and these genes are also not already expressed at
higher levels under HC conditions. Only a few genes, e.g.
cmpAB and sbtA, increased their mRNA levels, but to a
much lower extent than in WT cells. A similar change has
been observed in the mutant DccmM of Synechococcus
elongatus PCC 7942, but, in contrast to our results, the
induction of cmpA and sbtA was suppressed, while chpY
(cupA) transcripts accumulated at least during short LC
incubations (Woodger et al., 2005). The studies with
Synechococcus elongatus PCC 7942 indicate that the
induction of the CCM is mainly sensed via the twofold
larger internal Ci pool. However, the Ci-dependent
stimulation of the CCM is only found in the presence of
oxygen, i.e. conditions favouring RubisCO oxygenase
activity (Woodger et al., 2005). In Synechocystis, the
expression of cmpA has been shown to be less responsive
to Ci levels than the expression of sbtA and ndhF3 (McGinn
et al., 2004). Interestingly, BCT1 is induced by the positive
regulator CmpR, whose binding to the promoter of the
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409
C. Hackenberg and others
Fig. 5. Transcriptomic phenotyping in the
Synechocystis mutants DccmM and DglcD1/
D2. Fold changes in mRNA levels of selected
genes in cells of the WT, DccmM and DglcD1/
D2 mutants. The relative expression of genes
was calculated by dividing the expression level
of a defined gene by the corresponding level in
WT cells grown under HC conditions (WT HC
was set to 1). Data were obtained using a DNA
microarray comprising probes for all chromosomal genes of Synechocystis sp. PCC
6803. Cells were grown under HC conditions
and shifted for 3 or 24 h to LC conditions. n.i.,
Not investigated (3 h LC for DccmM).
410
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Microbiology 158
Carboxysomal and photorespiratory Synechocystis mutants
cmpABC operon is promoted by 2PG (Nishimura et al.,
2008). However, despite 2PG already being at elevated
levels in DccmM cells at HC (Fig. 3), the cmpAB mRNA
levels did not change (Table 3). Our results suggest that the
major signal that triggers LC acclimation is not 2PG alone.
Instead, additional signals are necessary, for example
changes in the Ci pool size, as proposed elsewhere (e.g.
Woodger et al., 2005), which may regulate transcriptional
factors such as NdhR and AbrB (Wang et al., 2004;
Lieman-Hurwitz et al., 2009).
In contrast to DccmM, DglcD1/D2 showed signs of LCinduced acclimation similar to the WT strain, but this
acclimation was incomplete and delayed. When cells of
DglcD1/D2 were shifted for only 3 h to LC, almost no
changes in gene expression occurred; however, after 24 h
some of the typical LC-induced genes such as sbtA, cmpA
and ndhF3 showed increased mRNA levels (Table 3). The
delay can be explained by the finding that part of the LC
acclimation occurred already in HC-grown cells of DglcD1/
D2, as discussed above. Interesting differences among
potential regulators became apparent. The transcript level
of cmpR increased slightly after 24 h LC, while the level of
ndhR remained at the level observed under HC conditions
(Table 3). A third regulator, the AbrB-like protein Sll0822,
has also been shown to be involved in LC regulation
(Lieman-Hurwitz et al., 2009). The mRNA level of abrB
was 2.5-fold elevated after LC shift only in cells of DglcD1/
D2 (Fig. 5), suggesting a possible activating role for the
transcription of sbtA and ndhF3. However, similar to NdhR
(Figge et al., 2001), AbrB has been described as a negative
regulator of the expression of Ci transporter genes under
HC conditions (Lieman-Hurwitz et al., 2009), although it is
not known whether it also represses its own transcription
under LC conditions. Our results suggest that the accumulating glycolate possibly acts as a signal in DglcD1/D2,
stimulating the transcription of abrB by inactivating AbrB
binding to promoters, e.g. for Ci transporter genes.
The impaired CO2 fixation of DccmM presumably leads
to an increasing excess of N over C after the shift from HC
to LC. In support of this, the level of 2OG decreased
and several amino acids accumulated markedly (Fig. 4).
Accordingly, genes encoding proteins involved in N
assimilation are further decreased (Table 2). In contrast
to the WT, genes encoding photosynthesis-related proteins
did not show altered mRNA levels, i.e. their expression
remained at the decreased level observed at HC. The usual
acclimation towards LC, i.e. upregulation of photosynthesis and the CCM (Eisenhut et al., 2007), was missing in
DccmM.
Interestingly, DglcD1/D2 responded differently from WT
and DccmM to LC shifts. The mRNA levels of proteins
involved in N assimilation were not further diminished,
while genes involved in the decrease in N assimilation (e.g.
gifA/B) showed significantly lower levels (Fig. 5, Table 2).
These results indicate that DglcD1/D2 does not exhibit
nitrogen excess over carbon after the shift to LC, which is
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typical for LC-shifted WT cells. The higher levels of, e.g.
2OG, 6-phosphogluconate and rather small glycogen
granules in DglcD1/D2 cells are also indicative of
mobilization of organic carbon to compensate for the
lower CO2 fixation and rebalance metabolic activity. This
assumption is supported by the increased mRNA levels of
two glycogen-degrading enzymes (glgP and glgX) and the
decreased level of sucrose phosphate phosphatase (spsB),
an enzyme involved in the synthesis of sucrose. A similar
observation has been made by Osanai et al. (2006) and
Aguirre von Wobeser et al. (2011) while studying acclimation to N limitation by transcriptomics.
In addition, the mRNA levels of the rbcLXS operon were
significantly elevated only in DglcD1/D2 (Fig. 5, Table 3),
while the protein amount of RubisCO increased in all
strains (Fig. 1c). In Synechocystis WT, the protein stability
of RubisCO and carboxysomal shell proteins has been
shown to increase during acclimation to LC, causing higher
protein amounts and carboxysome numbers, despite rather
small changes in the corresponding mRNA levels (Eisenhut
et al., 2007). The increase in RubisCO content in all strains
can be interpreted as a measure to compensate for the
decreased CO2 fixation. However, the underlying regulatory mechanisms seem to be distinct, since only DglcD1/D2
shows regulation at the mRNA level. In cells of WT and
DglcD1/D2, the carboxysome number increased in accordance with increased RubisCO content, although to a higher
extent in the mutant strain (Fig. 1b). These findings
support the assumption that glycolate, which accumulates
in DglcD1/D2, rather than 2PG, might act as a metabolic
signal in addition to the internal Ci level for the regulation
of at least part of the increased CCM activity at LC.
Conclusions
The two HCR mutants are characterized by specific changes
at the metabolic level. These changes probably result from
direct influences on the activity of several enzymes, e.g. the
direct inhibitory action of 2PG on CBB enzymes (Kelly &
Latzko, 1977) and the redox-based changes of enzyme
activities (Tamoi et al., 2005; Dietz & Pfannschmidt, 2011).
This conclusion is based on the observation that the
expression of genes for key enzymes of central metabolism
is unaltered in the two mutants. However, the mRNA levels
of several stress-regulated genes were elevated, and those of
genes encoding photosynthesis components and proteins for
N assimilation were decreased in the two mutants under HC
conditions. These genes show similar expression changes
in WT cells only after a shift to LC conditions. These
observations indicate that both HCR mutants suffer from
high-light and oxidative stress, and an excess of N over C
caused by impaired CO2 fixation, in combination with
defects in the CCM or photorespiration, representing major
electron sinks in the WT.
The carboxysome-less mutant DccmM showed a significant
increase in 2PG content and a decrease in the 3PGA level
under our conditions, a clear sign of increased oxygenase
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C. Hackenberg and others
and decreased carboxylase activity of RubisCO. Such a
behaviour was expected, because without the carboxysome, i.e. with a defective CCM, CO2 fixation is directly
dependent on the amount and biochemical properties of
RubisCO. In cyanobacteria, this enzyme is characterized by
a low CO2 affinity and a low specificity factor (Badger,
1980; Tcherkez et al., 2006). Initially, we expected that at
least the defective CCM might be compensated by an
increased expression of LC-induced genes. It has been
shown that the transcriptional factor CmpR binds the
RubisCO oxygenase reaction product 2PG, which promotes its in vitro binding to the target promoter of the cmp
operon (Nishimura et al., 2008). This and older findings
(e.g. Marcus et al., 1983) gave rise to the hypothesis that
2PG could serve as the metabolic signal for coordinated
changes in gene expression after HC-to-LC shifts. Our
observation of an unchanged cmpABC expression in 2PGaccumulating cells of the DccmM mutant at HC indicates
that 2PG is not the only signal for transcriptional activation
of this operon.
The other mutant, DglcD1/D2, accumulated glycolate
already under HC conditions, and further increased
glycolate levels rather than 2PG after the HC-to-LC shift,
because of the blockade of photorespiratory 2PG metabolism at the glycolate oxidation step (Eisenhut et al., 2008a).
In contrast to the carboxysome-less mutant DccmM, the
photorespiratory mutant DglcD1/D2 displayed acclimation
to Ci limitation to a higher extent than the WT, e.g. the
expression of photosynthetic genes and RubisCO, and
carboxysome number, increased in the mutant to higher
levels than in the WT. The accumulated glycolate could
therefore act as a signal involved in the acclimation to Cilimiting conditions.
Bauwe, H., Hagemann, M. & Fernie, A. R. (2010). Photorespiration:
players, partners and origin. Trends Plant Sci 15, 330–336.
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Dietz, K.-J. & Pfannschmidt, T. (2011). Novel regulators in photosyn-
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B. W., Bauwe, H., Matthijs, H. C. P. & Hagemann, M. (2007). Long-
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ACKNOWLEDGEMENTS
The help of Dr Martijs Jonker (Microarray Department, University of
Amsterdam, The Netherlands) during DNA microarray data evaluation, and the gift of the anti-RubisCO antibody by Professor E.
Pistorius (University of Bielefeld, Germany) are highly appreciated.
The excellent technical assistance of Klaudia Michl and Ute Schulz is
gratefully acknowledged. The work was supported by a grant from the
Deutsche Forschungsgemeinschaft (DFG) to M. H.
Hetherington, A. M. & Raven, J. A. (2005). The biology of carbon
dioxide. Curr Biol 15, R406–R410.
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Huege, J., Goetze, J., Schwarz, D., Bauwe, H., Hagemann, M. &
Kopka, J. (2011). Modulation of the major paths of carbon in
photorespiratory mutants of Synechocystis. PLoS ONE 6, e16278.
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