Photosynth Res (2011) 109:47–57
DOI 10.1007/s11120-010-9608-y
REVIEW
Inorganic carbon transporters of the cyanobacterial CO2
concentrating mechanism
G. Dean Price
Received: 15 September 2010 / Accepted: 14 December 2010 / Published online: 26 February 2011
Ó Springer Science+Business Media B.V. 2011
Abstract Cyanobacteria possess an environmental adap-
tation known as a CO2 concentrating mechanism (CCM)
that evolved to improve photosynthetic performance, par-
ticularly under CO2-limiting conditions. The CCM func-
tions to actively transport dissolved inorganic carbon
species (Ci; HCO3- and CO2) resulting in accumulation of
a pool of HCO3- within the cell that is then utilised to
provide an elevated CO2 concentration around the primary
CO2 fixing enzyme, ribulose bisphosphate carboxylase-
oxygenase (Rubisco). Rubisco is encapsulated in unique
micro-compartments known as carboxysomes and also
provides the location for elevated CO2 levels in the cell.
Five distinct transport systems for active Ci uptake are
known, including two types of Na?-dependent HCO3-
transporters (BicA and SbtA), one traffic ATPase (BCT1)
for HCO3- uptake and two CO2 uptake systems based on
modified NADPH dehydrogenase complexes (NDH-I3 and
NDH-I4). The genes for a number of these transporters are
genetically induced under Ci limitation via transcriptional
regulatory processes. The in-membrane topology structures
of the BicA and SbtA HCO3- transporters are now known
and this may aid in determining processes related to
transporter activation during dark to light transitions or
under severe Ci limitation.
Keywords Cyanobacteria  CO2 concentrating
mechanism  Carboxysomes  CO2-responsive genes 
G. D. Price (&)
Molecular Plant Physiology Cluster, Plant Science Division,
Research School of Biology, Australian National University,
Building #46, Sullivan’s Creek Road, Canberra, ACT 0200,
Australia
e-mail: Dean.Price@anu.edu.au
Photosynthesis  Bicarbonate transporters  CO2 uptake
systems  Genetic regulation
Introduction
In response to ancient changes in atmospheric CO2 and O2
levels, the cyanobacteria (blue-green algae) evolved an
environmental adaptation, known as a CO2 concentrating
mechanism (CCM), which has a significant positive effect
on photosynthetic performance. The CCM functions to
actively transport inorganic carbon species (Ci; HCO3-
and CO2) resulting in conditional accumulation of bicar-
bonate (HCO3-) within the cell. This pool of HCO3- is
thereafter utilised to provide elevated CO2 concentrations
around the primary CO2 fixing enzyme, ribulose bisphos-
phate carboxylase-oxygenase (Rubisco), which is in turn
encapsulated in unique micro-compartments known as
carboxysomes. Cyanobacteria are found in many niches
which arguably have applied evolutionary pressure on the
precise compositional diversity of cyanobacterial CCMs.
This diversity is mostly related to the types of Ci trans-
porters employed by different species. The purpose of this
review is to update discussion of these Ci transporter sys-
tems without attempting to supplant more detailed previous
reviews. Further details can be found in a number of pre-
vious reviews dealing with cyanobacterial and micro-algal
CCMs (Badger et al. 2002, 2006; Badger and Price 2003;
Giordano et al. 2005; Kaplan and Reinhold 1999; Price
et al. 1998, 2002, 2008; Raven et al. 2008).
The group of photosynthetic prokaryotes known as
cyanobacteria occupy an enormous range of ecological
niches that feature the availably at least some free water.
These niches include freshwater (rivers, ponds and lakes),
polar, hot springs, alkaline, estuarine and open ocean,
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saline and symbiotic environments (Badger et al. 2006).
Considering the range of niches occupied, it is interesting
that cyanobacteria fall into two broad CCM groups known
as a- or b-cyanobacteria based primarily on the type of
Rubisco and carboxysomes employed (Badger et al. 2002;
Tabita 1999); b-cyanobacteria, prevalent in freshwater and
coastal areas, utilise Form-1b Rubisco and ccm-type
carboxysomes, whereas a-cyanobacteria utilise Form-1a
Rubisco and cso-type carboxysomes. The abundant and
productive a-cyanobacteria occur in the deep ocean areas
of the world, usually at depths of 100–150 m where they
can often access nutrient upwellings from the deep ocean,
but where light intensity can be as little as 1% of surface
intensity. The species that predominate in these low-light,
low-nutrient environments are relatively slow growers
typically displaying growth rates of about one cell division
per day under optimal conditions, but oceans cover more
the half of the earth’s surface, making these species
abundant enough to be highly productive on a global scale.
It is estimated that some 50% of global primary produc-
tivity occurs annually in the oceans, with oceanic cyano-
bacteria contributing a significant fraction of this
productivity (Field et al. 1998; Liu et al. 1999), possibly as
much as 25% of global productivity. For example, in
oligotrophic oceans located between 40°N and 40°S, pho-
tosynthetic CO2 fixation is dominated by marine a-cyano-
bacteria of the Synechococcus and Prochlorococcus
genera, and together these species have been estimated to
contribute 30–80% of primary production (Liu et al. 1997).
Cyanobacteria, along with diatoms, form the base of the
marine food web and are therefore of critical ecological
and commercial importance.
Evolution of cyanobacterial CCMs
Cyanobacterial progenitors first appeared some 2.7 billion
years ago (Buick 1992) when the O2 level in the atmo-
sphere was low, and CO2 levels were many fold greater
than today (Berner 2006). However, it is near certain that
cyanobacteria have been subjected to periods of rapid
evolutionary change throughout the past 2.7 billion years.
In particular, the marked drop in CO2 levels, and rise in O2
levels, that occurred around 400–350 million years ago
(Berner 1990; Berner 2006), combined with the diffusional
resistance to CO2 transfer in liquid, may have triggered
adaptations to cope with photorespiration and low-effi-
ciency carbon gain. These adaptations would have included
transport mechanisms for active uptake of inorganic carbon
(Ci) and subsequent localised elevation of CO2 around the
primary carboxylating enzyme, ribulose bisphosphate car-
boxylase-oxygenase (Rubisco) and the partitioning of
Rubisco into icosahedral, micro-compartments known as
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Photosynth Res (2011) 109:47–57
carboxysomes (Badger et al. 2002; Badger and Price 2003;
Raven 2003). Collectively, these CCM adaptations func-
tion to raise the concentration of CO2 around Rubisco and
thereby improve the efficiency of CO2 fixation, suppressing
the wasteful oxygenase reaction of Rubisco. In species so
far examined, a basal form of the CCM is obligate for
survival in natural environments.
The widespread occurrence of CCMs in present day
cyanobacteria may have been aided by rapid evolution as
a result of horizontal transfer of large gene sets (Badger
et al. 2002). Another important secondary adaptation that
appears to have accompanied the development of the
CCM is the co-evolution of a Rubisco enzyme that is
adapted for optimal performance under the elevated CO2
conditions produced by the CCM. More precisely, the
evolution of the CCM possibly allowed the retention of a
more ancient form of Rubisco with lower affinities for
both CO2 and O2 than other algal or higher plant coun-
terparts, but with much higher catalytic turnover rates per
unit protein than terrestrial forms of Rubisco (Badger
et al. 1998). The cyanobacterial enzymatic properties of
Rubisco improve the nitrogen use efficiency of photo-
synthesis since the CCM allows Rubisco to operate near
Vmax, thereby requiring a much smaller nitrogen invest-
ment in Rubisco to achieve a particular rate of
photosynthesis.
The acquisition of Ci for photosynthetic CO2-fixation
represents the largest nutrient flux that cyanobacteria
encounter, but ultimately only CO2 can serve as the sub-
strate for Rubisco. A key problem in aquatic environments
is that CO2 availability and supply rate are often severely
limiting for photosynthesis and growth. In large part, this is
because CO2 diffusion in water is 104 times slower than in
air, plus the chemical equilibrium between HCO3- (the
main source of CO2 supply) and CO2 is relatively slow,
especially in the pH range 7 to 8.5. Furthermore, the total
Ci and HCO3- concentrations are sensitive to prevailing
pH, with total Ci and HCO3- being lowest at neutral to
acidic pH. In cyanobacteria, the existence of efficient
active Ci uptake systems and the generation of an elevated
CO2 level within carboxysomes overcame limits that would
normally be posed by poor CO2 availability. Thus, the
cyanobacterial CCM can achieve a saturated rate of CO2
fixation at 10–15 lM exogenous CO2 despite the fact that
cyanobacterial Rubisco enzymes possess a low-affinity for
CO2, with KmCO2 of greater than 150 lM (Badger and
Andrews 1987). Running Rubisco at near substrate satu-
ration also suppresses photorespiration allowing cyano-
bacteria to display better photosynthetic and ecological
competitiveness. It is suggested that the extra metabolic
costs associated with active accumulation of Ci are sig-
nificantly outweighed by the performance advantages
conferred by a CCM (Raven and Lucas 1985).
Photosynth Res (2011) 109:47–57 49

Active accumulation of Ci Genes needed for

β- Carboxysomes:
Cyanobacteria are dependent on active accumulation of Ci Thylakoid
ccmKLMN, ccmO
to achieve a satisfactory rate of CO2 fixation and growth. membrane
RbcLS, CcaA
To date, a total of five different uptake systems have been
identified (see below), including three HCO3- transporters Outer membrane
and two CO2 uptake systems (Fig. 1). Active uptake sys- Plasma membrane
tems vary in maximum flux rate and net affinity for CO2 or (inner)
HCO3-, with some transport activities being constitutively
expressed, while others are genetically inducible by growth Restricted
CO2 leakage Rubisco
under Ci limitation. A majority of cyanobacteria possess CO2
active uptake systems for both CO2 and HCO3-, however, CA
genomic data indicate that Prochlorococcus species (an Internal recycling
of CO2 leakage HCO3-
oceanic clade) only possess gene homologs for HCO3-
uptake (Badger and Price 2003; Badger et al. 2006).
b-Cyanobacteria from freshwater and estuarine niches face Ci transporters PGA
the greatest extremes in Ci availability, driven by temper- NDH-14 CO2 HCO3-
ature, pH and degree of mixing with air, and accordingly NADPH
these species possess the largest number of discrete trans-
porters (Badger et al. 2006). NDH-13 CO2
NADPH
Regardless of whether CO2 or HCO3- is the substrate
BicA HCO3-
for an uptake system, HCO3- is the species accumulated
Na+
within the cell to ratios as high as 1000-fold relative to total
exogenous Ci levels. Peak internal Ci pools of between 20 SbtA HCO3-
and 40 mM are regularly measured in cyanobacteria Na+
(Kaplan and Reinhold 1999; Price et al. 1998; Sültemeyer ATP
et al. 1995; Woodger et al. 2005b). Furthermore, HCO3- is BCT1 HCO3-
apparently maintained at chemical dis-equilibrium partly CA absent
due to the general absence of carbonic anhydrase (CA)
activity in the cytosol (Price and Badger 1989a; Volokita
et al. 1984) and the CO2 recycling action of the vectorial
CO2 uptake systems (Price et al. 2002). Bicarbonate is the β
preferred species for accumulation since as an ionic form
of Ci it is about 1000-fold less permeable to lipid mem- Fig. 1 A scheme showing a stylised b-cyanobacterium possessing
branes than the uncharged CO2 molecule. Experimental five types of Ci transporters and the b-carboxysome type. Each
support for the view that HCO3- is the accumulated spe- transporter delivers HCO3- to the general cell cytoplasm regardless
of the substrate accepted for uptake. Carbonic anhydrase (CA)
cies came from expressing human CA within the cytoplasm activity is absent for the general cytoplasm and is only present as a
of Synechococcus PCC7942 cells, leading to a high-flux carboxysome-specific form used in the generation of elevated CO2
leakage of CO2 from the cells due to rapid equilibration levels around Rubisco. The ribulose bisphosphate and phosphoglyc-
between HCO3- and CO2 (Price and Badger 1989a). This erate (PGA) are able to transverse the proteinaceous carboxysome
shell. Ci transporters shown in red are usually genetically induced
showed that Ci is normally held in a steady state non- under Ci limitation, whereas blue indicates a constitutive uptake
equilibrium favouring HCO3-, and also that carboxysomes system; purple relates to transporter that can be constitutive or
are the devoted site of CO2 elevation. inducible
An unavoidable feature of active Ci accumulation is the
problem of CO2 leakage. However, a CO2 uptake system carboxysomes (CA assisted) or by spontaneous dehydra-
can aid in the recycling of leaked CO2 before it escapes tion of HCO3-. The experimental evidence for recycling
from the cell (Espie et al. 1991; Fridlyand et al. 1996; by the CO2 pumps is 3-fold. Firstly, a mutant of Syn-
Kaplan and Reinhold 1999; Price and Badger 1989b; Price echococcus PCC7942 lacking both CO2 uptake systems,
et al. 1998, 2002). In fact, CO2 pumps, by default, accept but possessing normal HCO3- uptake, showed substan-
CO2 from any source, whether it originates from outside tially higher rates of spontaneous CO2 efflux from the cell
the cell or is internally generated. Thus, CO2 uptake sys- during steady state photosynthesis (Maeda et al. 2002); this
tems, particularly when located on the thylakoids, are well efflux is not evident in WT cells. Secondly, it has been
placed to intercept CO2 leakage originating from the observed that a high-CO2-requiring (HCR) mutant of

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Synechococcus PCC7942 (mutant #68), with normal Ci
uptake, but extremely low carboxysomal CA activity,
exchanges 18O out of labelled Ci at WT rates (Price and
Badger 1989b; Price et al. 1992); this means that the
carboxysomal CA is not the major 18O-exchange/CA
activity in whole cells (Price et al. 2002). Thirdly, a mutant
lacking active CO2 uptake, but still possessing HCO3-
uptake, showed abnormally low exchange of 18O-label
from labelled Ci compared to WT cells (Maeda et al. 2002)
implicating WT CO2 pumps in active recycling of leaked
CO2 originating from the carboxysomes and cytosol;
effectively, the vectorial hydration of CO2 to HCO3- by
the CO2 pump, followed by dehydration of HCO3- in the
carboxysomes, or cytosol, constitutes a special type of CA
cycle that is missing in mutants unable to take up CO2 (for
details see Price et al. 2002).
The carboxysome micro-compartment
Bicarbonate that is accumulated in the cell is used in the
generation of elevated CO2 levels within the carboxy-
somes (see Fig. 1) which contain most, if not all, of the
Rubisco content of the cell. Carboxysomes range from 90
to 250 nm diameter, depending on the cyanobacterial
species type, and are proteinaceous, polyhedral bodies
containing tightly packed Rubisco molecules (L8S8)
bounded by a thin protein shell of about 3 nm width. As
noted before, there are two types of carboxysomes in
cyanobacteria, each apparently having evolved by parallel
evolution to a convergent evolutionary function (Badger
2003; Badger et al. 2002). Both types of carboxysomes
contain a specific CA for catalytic conversion of HCO3-
to CO2 in the vicinity of Rubisco. In the case of b-
carboxysomes, the CA function is carried out by shell-
associated proteins CcaA/IcfA, or by CcmM in some
strains (Fukuzawa et al. 1992; Pena et al. 2010; So et al.
2002; Yu et al. 1992), whilst in a-carboxysomes the shell
protein CsoS3 (CsoSCA) is required (So et al. 2004).
Modelling indicates that for optimal efficiency carbox-
ysomal CA activity should generate CO2 at a sufficient
rate to match, as closely as possible, the maximal rate of
CO2 fixation (Price and Badger 1989b; Reinhold et al.
1987, 1991). A basic requirement of a CCM is the exis-
tence of an effective mechanism for minimising the
leakage of CO2 from the site of elevation in the carb-
oxysome. In addition to the recycling action of the CO2
pumps, it is envisaged that the hydrophilic shell of the
carboxysome can help impede the transfer of gases such
as CO2 (Reinhold et al. 1987, 1991). Further details about
the gene products involved in forming the two carboxy-
some types can be found in previous reviews (Price et al.
2008; Yeates et al. 2008).
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Photosynth Res (2011) 109:47–57
Ci transporters present in cyanobacteria
There are five types of active Ci uptake so far discovered in
cyanobacteria, and in general, freshwater and estuarine
species have the most Ci transporter types, whereas the
oceanic strains, from environments with remarkably stable
Ci levels, tend to have the fewest. The five Ci uptake
systems are explained in more detail in the subsequent
sections and in Fig. 2. Briefly, the five Ci uptake systems
are
(i) BCT1, an inducible, high-affinity HCO3- transporter
encoded by the cmpABCD operon and belonging to
the traffic ATPase family (Omata et al. 1999).
(ii) SbtA, an inducible, high-affinity Na?-dependent
HCO3- transporter (Shibata et al. 2002) acting as a
probable Na?/HCO3- symporter.
(iii) BicA, a low-affinity, high-flux Na?-dependent
HCO3- transporter belonging to the widespread
SulP/SLC26 family (Price et al. 2004) and also
acting as a probable Na?/HCO3- symporter.
(iv) NDH-I4, a constitutive CO2 uptake system based on a
specialised NADPH dehydrogenase (NDH-I) com-
plex (Maeda et al. 2002; Shibata et al. 2001).
(v) NDH-I3, a second CO2 uptake system based on a
modified NDH-1 complex that is inducible under Ci
limitation and is of higher uptake affinity than NDH-
I4 (Klughammer et al. 1999; Maeda et al. 2002;
Shibata et al. 2001) and has been confirmed to be
located on the thylakoid membrane in Synechocystis
PCC6803 (Prommeenate et al. 2004; Zhang et al.
2004).
The BCT1 bicarbonate transporter
The high-affinity HCO3- transporter, BCT1 (Omata et al.
1999), belongs to the ATP binding cassette (ABC) trans-
porter family, also known as traffic ATPases (Higgins
2001). To date, BCT1 has been physiologically character-
ised in just one cyanobacterium, namely Synechococcus
PCC7942, although close homologs have been detected in
[10 other species (Price et al. 2008). BCT1 is encoded by
the cmpABCD operon and is expressed when cells are
under Ci limitation (Omata et al. 1999). Using gain-of-
function analysis, the transporter displayed a high-photo-
synthetic affinity for HCO3- of around 15 lM, and sup-
ported a medium to low flux rate (Omata et al. 2002). Gene
transcript analyses confirm that the cmpABCD operon is
strongly induced under conditions of relatively severe Ci
limitation (McGinn et al. 2003, 2004; Wang et al. 2004;
Woodger et al. 2003, 2005b). Additionally, high-light
conditions can enhance transcription (McGinn et al. 2004;
Woodger et al. 2003).
Photosynth Res (2011) 109:47–57 51

Fig. 2 Scheme summarising HCO 3- Passive CO2 entry

some of the differences between Porin ? CO2
the five known Ci uptake Outer
membrane
systems (coloured components).
Na+/H+ antiporter EcaA/B
Ancillary components that do CO2
Peptidoglycan
HCO3- HCO3- layer
have, or may have, a secondary
Na+ Na+ HCO3- H+ H+
role in supporting Ci uptake are Na+ Na+ K0.5 (CO2 )
10 μM
shown in grey HCO3-
MnhD1 NdhD4
Plasma
MnhD2 Mnh B B SbtA NdhF4 NDH-14
membrane
MnhB complex NdhB complex
ATP
D ChpX NADPH
NADPH Or Fd
Or Fd H+ CmpC ATP CO2
HCO3-
Cytosol ?
CO2
HCO 3- HCO 3- HCO 3- HCO3-
Mnh Complex NADPH ChpY K0.5 (CO2 )
(modified NDH-1) Or Fd 1-2 μM
K0.5 (HCO3-) K0.5 (HCO3-) K0.5 (HCO3-)
NDH-13 NdhD3
~ 15 μ M 90-170 μ M ~ 5 μM Thylakoid
complex NdhF3 membrane
NdhB
Lumen

Three HCO3- transporters H+

Two CO2 uptake systems

The multimeric BCT1 complex is composed of four
different subunits (see Fig. 2) with cmpA coding for the
periplasmic lipoprotein, cmpB coding for the membrane
integral subunit and cmpC/D coding for attached cyto-
plasmic domains. At maturity, CmpA is a 42-kDa mature
peptide that is bound to the periplasmic face of the plasma
membrane where it acts as a mobile binding protein for
HCO3-. The CmpA protein from Synechococcus PCC7942
has been over-expressed in E. coli, purified and shown to
specifically bind HCO3- with a KD of around 5 lM
(Maeda et al. 2000). The crystal structure of CmpA has
also been determined, and unexpectedly it has been found
that the HCO3- binding site also binds Ca2? as a co-factor
(Koropatkin et al. 2007); any potential role for Ca2? in the
transport of HCO3- is unknown. The hydrophobic CmpB
membrane protein probably exists as a dimer within the
membrane, by analogy with other ABC transporters,
allowing the formation of an ion transport channel. Both
CmpC and CmpD are located on the cytoplasmic face of
CmpB, and both possess consensus binding sites for ATP.
However, CmpC has an extra domain not present in CmpD
raising speculation that the domain is involved in allosteric
regulation of the BCT1 transporter since a similar domain
in the analogous NrtC protein of NRT1 has been shown to
have a role in inactivating the transporter when NH4? is
present (Maeda and Omata 1997). Potentially, CmpC could
have a role in dark inactivation of the BCT1 transporter.
Analysis of available genome databases indicates that
BCT1 is generally only present in b-cyanobacteria and
mainly restricted to freshwater or brackish strains (Badger
et al. 2006). Some transition strains of a-cyanobacteria
appear to have gained BCT1 by horizontal gene transfer
(Price et al. 2008) and Rae et al. (CCM7 special issue).
The SbtA bicarbonate transporter
The Na?-dependent SbtA transporter was originally iden-
tified in Synechocystis PCC6803 as a single gene trans-
porter with relatively high affinity for HCO3-, requiring
1 mM Na? for half maximal HCO3- uptake activity
(Shibata et al. 2002). SbtA from PCC6803 has a predicted
size of 40 kDa, but the sbtA gene is generally co-located
with a smaller sbtB gene of unknown function; however, in
Synechococcus PCC7942, inactivation of sbtB does not
eliminate SbtA-dependent HCO3- uptake (B Forster and
GD Price, unpublished). In Synechococcus PCC7002, the
SbtA transporter has been estimated to have a high-pho-
tosynthetic affinity for HCO3- of around 2 lM and was
only capable of supporting a relatively low flux rate (Price
et al. 2004). The SbtA protein has been detected in cyto-
plasmic membranes isolated from Synechocystis PCC6803
with an apparent complex size of around 160 kDa (Zhang
et al. 2004), indicating that SbtA probably exists in the
membrane as a functional tetramer (Fig. 2). This proteomic
study also confirmed that SbtA abundance is dramatically
increased under Ci limitation. A topology map of SbtA
from Synechocystis PCC6803 has been determined (Fig. 3)
showing that the protein has a distinct 5 ? 5 duplicated
structure consisting of 10 transmembrane helices (Price
et al. submitted to CCM7 special issue). At least two
cytoplasmic domains have been identified that could be
involved in allosteric regulation of the transporter.
Strong homologues of SbtA are present in many
b-cyanobacteria (Badger et al. 2002, 2006; Badger and
Price 2003) but appear to be lacking from species such as
Thermosynechococcus, Gloeobacter violaceus and Trich-
odesmium erythraeum (Price et al. 2008). The weak

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Fig. 3 Experimentally
determined topology maps for
the BicA bicarbonate
transporter from Synechococcus
PCC7002 (a) and the SbtA
bicarbonate transporter from
Synechocystis PCC6803 (b).
Transmembrane helices are
shown as coloured cylinders
and the distribution of positively
charged residues are shown as
plus
A T*
homologues of SbtA present in most of the a-cyanobacte-
ria, except some a-Synechococcus species, are probable
HCO3- transporters, but to date there is a lack of physio-
logical data that substantiates this expectation. The kinetic
variability of SbtA transporters from different cyanobac-
teria is still an open question at this point in time.
The BicA bicarbonate transporter
The BicA transporter is another Na?-dependent HCO3-
transporter, unrelated to SbtA. Discovered in the coastal
marine cyanobacterium, Synechococcus PCC7002 (Price
et al. 2004), it has a relatively low-photosynthetic transport
affinity (*38 lM HCO3-) but it is able to support a high-
photosynthetic flux rate. BicA belongs to a large family of
eukaryotic and prokaryotic transporters often annotated as
sulphate transporters in many bacteria (the SulP/SLC26
family), and BicA homologs are represented in most b- and
a-cyanobacteria genomes so far examined (Price et al.
2004; Shelden et al. 2010). Gain-of-function experi-
ments in the freshwater cyanobacterium, Synechococcus
PCC7942, showed that bicA expression alone was suffi-
cient to confer Na?-dependent, HCO3- uptake activity,
requiring 1.7 mM Na? for half maximal HCO3- uptake
activity (Price et al. 2004). A strict Na? requirement
implies that an electrochemical Na? gradient is required to
energise HCO3- uptake via either BicA or SbtA, possibly
as symport processes. In the natural environment, fresh-
water bodies usually possess less than 1 mM Na?, but
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Photosynth Res (2011) 109:47–57
E N
G374
levels can spike or persist above this value, whereas in
brackish and estuarine water bodies Na? levels are usually
much higher. While phosphate and nitrogen levels usually
correlate with the onset cyanobacterial blooms, but it
would be of interest to probe for any correlation with Na?
levels in future studies.
The gain-of-function approach allowed two other BicA
transporters to be characterised, including one from the
ecologically important oceanic strain, Synechococcus
WH8102 and the other was from Synechocystis PCC6803.
Including BicA-7002, the three BicA transporters showed
transport affinities for 14C-HCO3- uptake that ranged from
74 to 353 lM, revealing an interesting trade-off for Km
verses Vmax (Fig. 4). However, the collection of kinetic
data from a greater diversity of BicA transporters would be
necessary to complete this analysis. In Synechococcus
PCC7002, BicA expression is highly inducible under Ci
limitation and is lowly expressed in high-CO2 grown cells
(Price et al. 2004; Woodger et al. 2007). In contrast, the
BicA gene in Synechocystis PCC6803 is constitutively
expressed (Wang et al. 2004).
The predicted bicA gene product from Synechococcus
PCC7002 is *60 kDa and recent topology mapping indi-
cates that it possess 12 transmembrane helices (see Fig. 3)
with both the C- and N-termini inside the cell (Shelden
et al. 2010). The prominent, hydrophilic C-terminus known
as the STAS (sulphate transporter anti-sigma factor-like
domain) domain is known to be a regulatory domain in
some mammalian homologues (Ko et al. 2004) and
Photosynth Res (2011) 109:47–57
1400
ptake rate
1200
h-1)
1000
Vmax, Grross 14 C-HCO 3 - up
( mmol.mg Chl -1.h
800
600
400
200
0
0 100
Fig. 4 Data replotted from Price et al. (2004) based on the gain-of-
function analysis of BicA bicarbonate transporters from Synechocystis
PCC6803, Synechococcus PCC7002 and Synechococcus WH8102
based on 14C-HCO3- uptake. The plot shows Km(HCO3-) data
against the Vmax for gross HCO3- uptake
Arabidopsis sulphate transporters (Shibagaki and Gross-
man 2004), and this is also likely for BicA. The cyto-
plasmic loop between helices 8 and 9 is another domain
likely to be involved in allosteric regulation (Shelden et al.
2010).
CO2 uptake systems based on specialised NDH-1
complexes
Two types of CO2 uptake systems have been identified in
cyanobacteria. They are not true transporters since the
uptake process involves passive entry of CO2 into the cell
followed by energised, vectorial conversion to HCO3-.
Both systems are based on modified, plastoquinone oxi-
doreductase NADPH dehydrogenase (NDH-1) respiratory
complexes that are related to complex-I from mitochon-
dria. The basal system, NDH-14, is constitutively expres-
sed, whilst NDH-13 is inducible under Ci limitation,
displaying uptake affinities of around 10 and 1–2 lM,
respectively (Klughammer et al. 1999; Ohkawa et al.
2000a; Price et al. 2002). A set of NDH-1 genes common
to both respiratory and CO2 uptake systems are present as
single copies in the genome, namely ndhAIGE, ndhB,
ndhCJK, ndhH and ndhL. However, most b-cyanobacteria
possess multiple copies of ndhD and ndhF, with some of
these being specific for CO2 uptake systems. Thus, in
Synechococcus PCC7002, the ndhF3-ndhD3-chpY-orf133
gene cluster encodes specific components necessary for
inducible, high-affinity CO2 uptake (Klughammer et al.
1999; Sültemeyer et al. 1997). In contrast, NdhD4 and
NdhF4 are specific subunits required for constitutive CO2
53
uptake (Maeda et al. 2002; Ohkawa et al. 2000b; Shibata
PCC6803
et al. 2001).
Two unique proteins are involved in CO2 uptake activity
PCC7002 by modified NDH-1 complexes, and these are the CO2
hydration proteins, ChpX (NDH-I4 complex) and ChpY
(NDH-I3 complex) in Synechococcus PCC7942 and
PCC7002 (Maeda et al. 2002; Woodger et al. 2007); also
known in Synechocystis PCC6803 as CupB and CupA
(Shibata et al. 2001), respectively. Proteomic studies in
WH8102 Synechocystis PCC6803 have confirmed the presence of
NdhF3, NdhD3, ChpY, sll1735/orf133 (Herranen et al.
pH 9.3 assay, 2004; Prommeenate et al. 2004; Zhang et al. 2004) as being
Silicone oil centriguation-filtration present in thylakoid membranes and part of a sub-complex
200 300 400 that is induced under Ci limitation. Unfortunately, such
Km (HCO 3- ), μ M studies have so far failed to detect the constitutively
expressed NdhF4, NdhD4, ChpX proteins in thylakoid or
plasma membranes. It is possible that the NDH-I4 complex
is of low abundance or is located on the plasma membrane
and is easily lost during cell fractionation. Inhibitor studies
with CO2 uptake mutants of Synechococcus PCC7942
showed a differential sensitivity to DCMU, with the NDH-
I4 system being largely unaffected, whereas the thylakoid-
based NDH-I3 system was inhibited (Maeda et al. 2002).
This would be consistent with NDH-I4 being located on the
plasma membrane and therefore not in close association
with Photosystem-II. In Fig. 2, NDH-I4 is shown as being
located on the plasma membrane, but a location on the
thylakoid is possible, subject to further analysis. Single
particle analysis of NDH-I3 complexes isolated from Syn-
echocystis PCC6803 indicates that the complex assumes a
flattened U-shape, with the bottom of the ‘‘U’’ being
membrane intrinsic, whereas CupA (ChpY) is believed to
be located on one of the cytoplasmic-facing lobes (Folea
et al. 2008).
It has been suggested that the ChpX and ChpY poly-
peptides may direct the vectorial conversion of CO2 to
HCO3-, linked to electron transport and proton transloca-
tion functions associated with the NDH-1 complex (Maeda
et al. 2002; Price et al. 2002). As noted above, proteomic
data indicates that NDH-I3 complexes are restricted to
the thylakoid membrane thus linking them directly to
the photosynthetic electron transport chain. A model of the
operation of such an NDH-13/4 CO2-uptake complex has
been presented previously (Badger and Price 2003; Price
et al. 2002) based on the generation a nucleophilic Zn-OH-
species within the ChpX or ChpY proteins of specialised
NDH-1 complexes at the expense of NADPH or reduced
ferredoxin as an energy source. At present, there has been
no further advancement or further confirmation of this
model.
Analysis of 70? cyanobacterial genome databases
confirms significant diversity of the two CO2 uptake sys-
tems in the a- and b-cyanobacteria, as previously reported
123
54
(Badger and Price 2003; Badger et al. 2006; Price et al.
2008). In general, the b-cyanobacteria usually possess
genes for both CO2 uptake systems, whereas the oceanic
a-Synechococcus possess only the low-affinity CO2 uptake
system; the observation that Prochlorococcus species lack
genes for any known active CO2 uptake systems is a fea-
ture of all Prochlorococcus genomes so far examined. A
recent update on the presence of genes encoding CO2
uptake systems in cyanobacteria can be found in Rae et al.
(CCM7 special issue).
Ancillary systems supporting Ci uptake
MnhD1 and MnhD2 (also known as NdhD5, D6, respec-
tively) are polypeptides in yet another specialised NDH-1
complex, the Mnh complex, that is thought to be involved
in Na?/H? antiport in bacteria (Hiramatsu et al. 1998) or as
a primary Na? extrusion pump involved in enhancing the
generation of an electrochemical Na? gradient (Fig. 2). An
Mnh-like operon is known to be low-Ci-responsive in
Synechocystis PCC6803 (Wang et al. 2004) and could
potentially aid in Na?-dependent energization of HCO3-
uptake due to SbtA and BicA. Interestingly, an Mnh
cluster, including mnhD1 and mnhD2, is expressed as part
of the bicA operon in Synechococcus PCC7002 and was
found to be responsible for improving the efficiency of
HCO3--dependent photosynthesis under Ci limitation
(Woodger et al. 2007). The Na?/H? antiporter, NhaS3, is
also part of this operon and could help generate a Na?
gradient for BicA.
Another ancillary component, the PxcA H? extrusion
pump, has been shown to be needed for homeostatic H?
extrusion associated with the initial phase of CO2 uptake
(Sonoda et al. 1998). PxcA is widespread in cyanobacteria
but only at protein sequence identities of 30–50%. Two
other ancillary components with possible subtle roles in Ci
uptake are the periplasmic-located CAs, EcaA and EcaB,
and the possible HCO3- porin, PorB. The two periplasmic
CA forms are not widespread in cyanobacteria, and use of
insertional mutants indicates that have very little effect on
CO2 uptake (So et al. 1998). Likewise, PorB has been
suggested to be a HCO3- porin in the outer membrane but
has little effect on Ci uptake when inactivated (Woodger
et al. 2007).
Genetic regulation of Ci transporters
All cyanobacteria examined to date display a basal form of
the CCM when grown under high-CO2 conditions, indi-
cating that many of genes coding for carboxysome proteins
and low-affinity Ci transporters are under constitutive
123
Photosynth Res (2011) 109:47–57
expression with limited plasticity (Kaplan and Reinhold
1999; Price et al. 1998, 2002). This basal form of the CCM
is essential for survival since mutants lacking CO2 uptake
systems (Woodger et al. 2005b), or lacking functional
carboxysomes (Friedberg et al. 1989; Price and Badger
1989b), possess a HCR phenotype, necessitating 2–3%
CO2 for normal rates of CO2 fixation. Importantly, when
subjected to Ci-limited conditions, b-cyanobacteria, in
particular, are able to up regulate the expression of genes
required for high-affinity Ci transporters such as NDH-I3,
SbtA, BCT1, and also the low-affinity transporter, BicA.
Cells can reach full physiological induction within
60–90 min (Woodger et al. 2003), but transcripts for CO2-
responsive genes can respond to Ci limitation in \15 min
(McGinn et al. 2003; Woodger et al. 2003, 2005a, b, 2007).
The photosynthetic affinity for Ci rises significantly under
induction; for instance, in Synechococcus PCC7942 the
K0.5(Ci) drops from around 170 lM to as low as 10–15 lM
(Woodger et al. 2003).
The expression of CO2-responsive genes in b-cyano-
bacteria is controlled by one or two LysR-type transcrip-
tion regulators known as CcmR (NdhR) or CmpR, acting as
activators or suppressors of transcription (Omata et al.
2001; Price et al. 2008; Wang et al. 2004; Woodger et al.
2007). The precise signal transduction pathway through
CcmR or CmpR is still unknown; however, evidence
indicates that expression of CO2-responsive genes corre-
lates inversely to the size of the internal Ci pool during
illumination (Woodger et al. 2003, 2005a, b). One possi-
bility is that CcmR itself may be able to respond directly to
the HCO3- accumulation level in the cell (Woodger et al.
2005b, 2007; Zhang et al. 2007); alternatively, a signal
transduction pathway may transduce a signal to CcmR or
CmpR. For instance, the FtsH protease can play an enig-
matic role in activation of CcmR in Synechocystis
PCC6803 (Zhang et al. 2007).
It has also been suggested that increases in photorespi-
ratory intermediates, arising from the wasteful oxygenase
reaction of Rubisco, could be involved in sensing Ci lim-
itation (see Kaplan and Reinhold 1999). Indeed, it has been
established that Synechocystis PCC6803 has plant-like and
bacterial-type cycles for dealing with metabolism of pho-
torespiratory phosphoglycolate (Eisenhut et al. 2008) and
although mutants display unaltered expression of low-CO2-
responsive transporter genes there are some minor genes
that change due to higher phosphoglycolate/glycolate lev-
els in the cell (Eisenhut et al. 2007). Mutants defective in
all three glycolate turnover pathways (Eisenhut et al. 2008)
exhibit significantly increased glycolate levels at high CO2,
although the percentage photorespiratory flux via Rubisco,
relative to carboxylation, remains unknown. Such mutants
will only survive at high CO2, and although photosynthetic
Vmax is nearly half that of WT cells at high CO2, the affinity
Photosynth Res (2011) 109:47–57
for Ci is not abnormally induced at high CO2 as would be
expected if phosphoglycolate is an inducer molecule for
CO2 responsive transporter genes. Recent in vitro DNA-
binding studies with CmpR indicate that binding to the
cmpABCD promoter of Synechococcus PCC7942 can be
enhanced by low lM concentrations of phosphoglycolate
(Nishimura et al. 2008), but since binding can occur
without phosphoglycolate it is concluded that it may act as
a co-inducer.
It is known that light is a prerequisite signal for
expression of CO2-responsive genes (McGinn et al. 2003,
2005), and although this could involve redox or phyto-
chrome signals, the mechanism is unknown. However,
light can also enhance the transcriptional response to Ci
limitation by enhancing the expression of CO2-responsive
genes (McGinn et al. 2004; Woodger et al. 2003). In
addition to light, there have been suggestions that the
control of genes involved in Ci and nitrate uptake may be
co-ordinated via the involvement of the PII (glnB) protein
(Hisbergues et al. 1999; Osanai and Tanaka 2007), but firm
evidence of this interaction is still lacking.
Allosteric or post-translational regulation of Ci
transport systems
A major gap in our picture of the regulation of Ci uptake
processes relates to the nature of the mechanisms con-
trolling post-translational or allosteric activation of Ci
transporters. Cyanobacterial Ci transporters are inactive in
the dark, possibly to prevent futile cycling, but upon
illumination uptake systems are quickly activated, with
CO2 uptake being very rapid (\5 s) and HCO3- uptake
activated within 20–30 s. There is some evidence that
activation of HCO3- transporters might involve a redox
signal (Kaplan et al. 1987), however, activation due to a
protein kinase-mediated phosphorylation of transporters is
also likely. For instance, phosphorylation via protein
phosphokinases of the Ser/Thr class has been implicated
in rapid induction of latent HCO3- uptake capacity in
Synechococcus PCC7942 and PCC7002 species subjected
to severe Ci limitation (Sültemeyer et al. 1998a, b). One
rationale for determination of in-membrane topology
maps for SbtA and BicA was to identify the putative
regulatory domains on the cytoplasmic faces of these
transporters that could be involved in activation or
deactivation (Fig. 3). Conserved residues in these regions
are currently being studied by site-directed mutagenesis.
This type of information is needed towards a concerted
effort of introducing cyanobacterial Ci transporter into the
chloroplasts of C3 crop plants for the purpose of
improving photosynthetic productivity (Parry et al. 2011;
Price et al. 2008).
55
Concluding remarks
Over the past decade, the CCM field has amassed a large
body of information about the types, diversity and regu-
lation of cyanobacterial transporters used for the active
uptake of CO2 and HCO3- to support photosynthetic CO2
fixation. However, there is still much to discover yet on the
molecular structure of each transporter, its substrate
stoichiometry, its mechanism of energization and its traf-
ficking/folding into the membrane and genetic and post-
translational controls in regulating cyanobacteria Ci
transporters.
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