CO2 concentrating mechanisms in cyanobacteria: molecular components,their diversity and evolution

Cyanobacteria have evolved an extremely effective single-cell CO(2) concentrating mechanism (CCM). Recent molecular, biochemical and physiological studies have significantly extended current knowledge about the genes and protein components of this system and how they operate to elevate CO(2) around Rubisco during photosynthesis. The CCM components include at least four modes of active inorganic carbon uptake, including two bicarbonate transporters and two CO(2) uptake systems associated with the operation of specialized NDH-1 complexes. All these uptake systems serve to accumulate HCO(3)(-) in the cytosol of the cell, which is subsequently used by the Rubisco-containing carboxysome protein micro-compartment within the cell to elevate CO(2) around Rubisco. A specialized carbonic anhydrase is also generally present in this compartment. The recent availability of at least nine cyanobacterial genomes has made it possible to begin to undertake comparative genomics of the CCM in cyanobacteria. Analyses have revealed a number of surprising findings. Firstly, cyanobacteria have evolved two types of carboxysomes, correlated with the form of Rubisco present (Form 1A and 1B). Secondly, the two HCO(3)(-) and CO(2) transport systems are distributed variably, with some cyanobacteria (Prochlorococcus marinus species) appearing to lack CO(2) uptake systems entirely. Finally, there are multiple carbonic anhydrases in many cyanobacteria, but, surprisingly, several cyanobacterial genomes appear to lack any identifiable CA genes. A pathway for the evolution of CCM components is suggested.     

Carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii: inorganic carbon transport and CO2 recapture

Many microalgae are capable of acclimating to CO(2) limited environments by operating a CO(2) concentrating mechanism (CCM), which is driven by various energy-coupled inorganic carbon (Ci; CO(2) and HCO(3)(-)) uptake systems. Chlamydomonas reinhardtii (hereafter, Chlamydomonas), a versatile genetic model organism, has been used for several decades to exemplify the active Ci transport in eukaryotic algae, but only recently have many molecular details behind these Ci uptake systems emerged. Recent advances in genetic and molecular approaches, combined with the genome sequencing of Chlamydomonas and several other eukaryotic algae have unraveled some unique characteristics associated with the Ci uptake mechanism and the Ci-recapture system in eukaryotic microalgae. Several good candidate genes for Ci transporters in Chlamydomonas have been identified, and a few specific gene products have been linked with the Ci uptake systems associated with the different acclimation states. This review will focus on the latest studies on characterization of functional components involved in the Ci uptake and the Ci-recapture in Chlamydomonas.     

The environmental plasticity and ecological genomics of the cyanobacterial CO2 concentrating mechanism

Cyanobacteria probably exhibit the widest range of diversity in growth habitats of all photosynthetic organisms. They are found in cold and hot, alkaline and acidic, marine, freshwater, saline, terrestrial, and symbiotic environments. In addition to this, they originated on earth at least 2.5 billion years ago and have evolved through periods of dramatic O2 increases, CO2 declines, and temperature changes. One of the key problems they have faced through evolution and in their current environments is the capture of CO2 and its efficient use by Rubisco in photosynthesis. A central response to this challenge has been the development of a CO2 concentrating mechanism (CCM) that can be adapted to various environmental limitations. There are two primary functional elements of this CCM. Firstly, the containment of Rubisco in carboxysome protein microbodies within the cell (the sites of CO2) elevation), and, secondly, the presence of several inorganic carbon (Ci) transporters that deliver HCO3- intracellularly. Cyanobacteria show both species adaptation and acclimation of this mechanism. Between species, there are differences in the suites of Ci transporters in each genome, the nature of the carboxysome structures and the functional roles of carbonic anhydrases. Within a species, different CCM activities can be induced depending on the Ci availability in the environment. This acclimation is largely based on the induction of multiple Ci transporters with different affinities and specificities for either CO2 or HCO3- as substrates. These features are discussed in relation to our current knowledge of the genomic sequences of a diverse array of cyanobacteria and their ecological environments.     

Physiological characterization and light response of the CO2-concentrating mechanism in the filamentous cyanobacterium Leptolyngbya sp. CPCC 696

We studied the interactions of the CO(2)-concentrating mechanism and variable light in the filamentous cyanobacterium Leptolyngbya sp. CPCC 696 acclimated to low light (15 μmol m(-2) s(-1) PPFD) and low inorganic carbon (50 μM Ci). Mass spectrometric and polarographic analysis revealed that mediated CO(2) uptake along with both active Na(+)-independent and Na(+)-dependent HCO(3)(-) transport, likely through Na(+)/HCO(3)(-) symport, were employed to concentrate Ci internally. Combined transport of CO(2) and HCO(3)(-) required about 30 kJ mol(-1) of energy from photosynthetic electron transport to support an intracellular Ci accumulation 550-fold greater than the external Ci. Initially, Leptolyngbya rapidly induced oxygen evolution and Ci transport to reach 40-50% of maximum values by 50 μmol m(-2) s(-1) PPFD. Thereafter, photosynthesis and Ci transport increased gradually to saturation around 1,800 μmol m(-2) s(-1) PPFD. Leptolyngbya showed a low intrinsic susceptibility to photoinhibition of oxygen evolution up to PPFD of 3,000 μmol m(-2) s(-1). Intracellular Ci accumulation showed a lag under low light but then peaked at about 500 μmol photons m(-2) s(-1) and remained high thereafter. Ci influx was accompanied by a simultaneous, light-dependent, outward flux of CO(2) and by internal CO(2)/HCO(3)(-) cycling. The high-affinity and high-capacity CCM of Leptolyngbya responded dynamically to fluctuating PPFD and used excitation energy in excess of the needs of CO(2) fixation by increasing Ci transport, accumulation and Ci cycling. This capacity may allow Leptolyngbya to tolerate periodic exposure to excess high light by consuming electron equivalents and keeping PSII open.     

Genes essential to sodium-dependent bicarbonate transport in cyanobacteria: function and phylogenetic analysis

The cyanobacterium Synechocystis sp. strain PCC 6803 possesses two CO(2) uptake systems and two HCO(3)(-) transporters. We transformed a mutant impaired in CO(2) uptake and in cmpA-D encoding a HCO(3)(-)transporter with a transposon inactivation library, and we recovered mutants unable to take up HCO(3)(-) and grow in low CO(2) at pH 9.0. They are all tagged within slr1512 (designated sbtA). We show that SbtA-mediated transport is induced by low CO(2), requires Na(+), and plays the major role in HCO(3)(-) uptake in Synechocystis. Inactivation of slr1509 (homologous to ntpJ encoding a Na(+)/K(+)-translocating protein) abolished the ability of cells to grow at [Na(+)] higher than 100 mm and severely depressed the activity of the SbtA-mediated HCO(3)(-) transport. We propose that the SbtA-mediated HCO(3)(-) transport is driven by DeltamuNa(+) across the plasma membrane, which is disrupted by inactivating ntpJ. Phylogenetic analyses indicated that two types of sbtA exist in various cyanobacterial strains, all of which possess ntpJ. The sbtA gene is the first one identified as essential to Na(+)-dependent HCO(3)(-) transport in photosynthetic organisms and may play a crucial role in carbon acquisition when CO(2) supply is limited, or in Prochlorococcus strains that do not possess CO(2) uptake systems or Cmp-dependent HCO(3)(-) transport.     

Carbon concentration mechanisms in photosynthetic microorganisms

Unicellular green algae and cyanobacteria have mechanism to actively concentrate dissolved inorganic carbon into the cells, only if they are grown with air levels of CO2. The carbon concentration mechanisms are commonly known as "CCM" or "DIC-pumps". The DIC-pumps are environmental adaptation that function to actively transport and accumulate inorganic carbon (HCO3- and CO2; Ci) within the cell and then uses this Ci pool to actively increase the concentration of CO2 at the site of ribulose bisphosphate carboxylase-oxygenase (Rubisco), the primary CO2-fixing enzyme. The current working model for dissolved inorganic carbon concentration mechanism in unicellular green algae includes several isoforms of carbonic anhydrase (CA), and ATPase driven active transporters at the plasmalemma and at the inner chloroplast envelopes. In the past fifteen years, significant progress has been made in isolating and characterizing the various isoforms of carbonic anhydrase at the biochemical and molecular level. However, we have an inadequate understanding of active transporters that are located on the plasmalemma and at the chloroplast envelopes. In this mini-review we focus on certain aspects of the induction, function and significance of the dissolved inorganic carbon concentration mechanisms in aquatic photosynthetic microorganisms.     

Recent progresses on the genetic basis of the regulation of CO2 acquisition systems in response to CO2 concentration

Marine diatoms, the major primary producer in ocean environment, are known to take up both CO(2) and HCO(3)(-) in seawater and efficiently concentrate them intracellularly, which enable diatom cells to perform high-affinity photosynthesis under limiting CO(2). However, mechanisms so far proposed for the inorganic carbon acquisition in marine diatoms are significantly diverse despite that physiological studies on this aspect have been done with only limited number of species. There are two major hypotheses about this; that is, they take up and concentrate both CO(2) and HCO(3)(-) as inorganic forms, and efficiently supply CO(2) to Rubisco by an aid of carbonic anhydrases (biophysical CO(2)-concentrating mechanism: CCM); and as the other hypothesis, biochemical conversion of HCO(3)(-) into C(4) compounds may play a major role to supply concentrated CO(2) to Rubisco. At moment however, physiological evidence for these hypotheses were not related well to molecular level evidence. In this study, recent progresses in molecular studies on diatom-carbon-metabolism genes were related to the physiological aspects of carbon acquisition. Furthermore, we discussed the mechanisms regulating CO(2) acquisition systems in response to changes in pCO(2). Recent findings about the participation of cAMP in the signaling pathway of CO(2) concentration strongly suggested the occurrences of mammalian-type-signaling pathways in diatoms to respond to changes in pCO(2). In fact, there were considerable numbers of putative adenylyl cyclases, which may take part in the processes of CO(2) signal capturing.     

Involvement of the cynABDS operon and the CO2-concentrating mechanism in the light-dependent transport and metabolism of cyanate by cyanobacteria

The cyanobacteria Synechococcus elongatus strain PCC7942 and Synechococcus sp. strain UTEX625 decomposed exogenously supplied cyanate (NCO-) to CO2 and NH3 through the action of a cytosolic cyanase which required HCO3- as a second substrate. The ability to metabolize NCO- relied on three essential elements: proteins encoded by the cynABDS operon, the biophysical activity of the CO2-concentrating mechanism (CCM), and light. Inactivation of cynS, encoding cyanase, and cynA yielded mutants unable to decompose cyanate. Furthermore, loss of CynA, the periplasmic binding protein of a multicomponent ABC-type transporter, resulted in loss of active cyanate transport. Competition experiments revealed that native transport systems for CO2, HCO3-, NO3-, NO2-, Cl-, PO4(2-), and SO4(2-) did not contribute to the cellular flux of NCO- and that CynABD did not contribute to the flux of these nutrients, implicating CynABD as a novel primary active NCO- transporter. In the S. elongatus strain PCC7942 DeltachpX DeltachpY mutant that is defective in the full expression of the CCM, mass spectrometry revealed that the cellular rate of cyanate decomposition depended upon the size of the internal inorganic carbon (Ci) (HCO3- + CO2) pool. Unlike wild-type cells, the rate of NCO- decomposition by the DeltachpX DeltachpY mutant was severely depressed at low external Ci concentrations, indicating that the CCM was essential in providing HCO3- for cyanase under typical growth conditions. Light was required to activate and/or energize the active transport of both NCO- and Ci. Putative cynABDS operons were identified in the genomes of diverse Proteobacteria, suggesting that CynABDS-mediated cyanate metabolism is not restricted to cyanobacteria.     

Mitochondrial-driven bicarbonate transport supports photosynthesis in a marine microalga

The CO(2)-concentrating mechanism (CCM) of the marine eustigmatophycean microalga Nannochloropsis gaditana consists of an active HCO(3)(-) transport system and an internal carbonic anhydrase to facilitate accumulation and conversion of HCO(3)(-) to CO(2) for photosynthetic fixation. Aqueous inlet mass spectrometry revealed that a portion of the CO(2) generated within the cells leaked to the medium, resulting in a significant rise in the extracellular CO(2) concentration to a level above its chemical equilibrium that was diagnostic for active HCO(3)(-) transport. The transient rise in extracellular CO(2) occurred in the light and the dark and was resolved from concurrent respiratory CO(2) efflux using H(13)CO(3)(-) stable isotope techniques. H(13)CO(3)(-) pump-(13)CO(2) leak activity of the CCM was unaffected by 10 microM 3(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of chloroplast linear electron transport, although photosynthetic O(2) evolution was reduced by 90%. However, low concentrations of cyanide, azide, and rotenone along with anoxia significantly reduced or abolished (13)CO(2) efflux in the dark and light. These results indicate that H(13)CO(3)(-) transport was supported by mitochondrial energy production in contrast to other algae and cyanobacteria in which it is supported by photosynthetic electron transport. This is the first report of a direct role for mitochondria in the energization and functioning of the CCM in a photosynthetic organism.     

The Chlamydomonas reinhardtii cia3 mutant lacking a thylakoid lumen-localized carbonic anhydrase is limited by CO2 supply to rubisco and not photosystem II function in vivo

The Chlamydomonas reinhardtii cia3 mutant has a phenotype indicating that it requires high-CO(2) levels for effective photosynthesis and growth. It was initially proposed that this mutant was defective in a carbonic anhydrase (CA) that was a key component of the photosynthetic CO(2)-concentrating mechanism (CCM). However, more recent identification of the genetic lesion as a defect in a lumenal CA associated with photosystem II (PSII) has raised questions about the role of this CA in either the CCM or PSII function. To resolve the role of this lumenal CA, we re-examined the physiology of the cia3 mutant. We confirmed and extended previous gas exchange analyses by using membrane-inlet mass spectrometry to monitor(16)O(2),(18)O(2), and CO(2) fluxes in vivo. The results demonstrate that PSII electron transport is not limited in the cia3 mutant at low inorganic carbon (Ci). We also measured metabolite pools sizes and showed that the RuBP pool does not fall to abnormally low levels at low Ci as might be expected by a photosynthetic electron transport or ATP generation limitation. Overall, the results demonstrate that under low Ci conditions, the mutant lacks the ability to supply Rubisco with adequate CO(2) for effective CO(2) fixation and is not limited directly by any aspect of PSII function. We conclude that the thylakoid CA is primarily required for the proper functioning of the CCM at low Ci by providing an ample supply of CO(2) for Rubisco.     

The cyanobacterial bicarbonate transporter BicA: its physiological role and the implications of structural similarities with human SLC26 transporters

The cyanobacterial Na+-dependent HCO3- transporter BicA is a member of the ubiquitous and important SulP/SLC26 family of anion transporters found in eukaryotes and prokaryotes. BicA is an important component of the cyanobacterial CO2 concentrating mechanism, an adaptation that contributes to cyanobacteria being able to achieve an estimated 25% of global primary productivity, largely in the oceans. The human SLC26 members are involved in a range of key cellular functions involving a diverse range of anion transport activities including Cl-/HCO3-, I-/HCO3-, and SO42-/HCO3- exchange; mutations in SLC26 members are known to be associated with debilitating diseases such as Pendred syndrome, chondrodysplasias, and congenital chloride diarrhoea. We have recently experimentally determined the membrane topology of BicA using the phoA-lacZ reporter system and here consider some of the extrapolated implications for topology of the human SLC26 family and the Sultr plant sulphate transporters.     

Properties of mutants of Synechocystis sp. strain PCC 6803 lacking inorganic carbon sequestration systems

A mutant (Delta5) of Synechocystis sp. strain PCC 6803 constructed by inactivating five inorganic carbon sequestration systems did not take up CO(2) or HCO(3)(-) and was unable to grow in air with or without glucose. The Delta4 mutant in which BicA is the only active inorganic carbon sequestration system showed low activity of HCO(3)(-) uptake and grew under these conditions but more slowly than the wild-type strain. The Delta5 mutant required 1.7% CO(2) to attain half the maximal growth rate. Electron transport activity of the mutants was strongly inhibited under high light intensities, with the Delta5 mutant more susceptible to high light than the Delta4 mutant. The results implicated the significance of carbon sequestration in dissipating excess light energy.     

Characterisation of Cyanobacterial Bicarbonate Transporters in E. coli Shows that SbtA Homologs Are Functional in This Heterologous Expression System

Cyanobacterial HCO₃ ^- transporters BCT1, SbtA and BicA are important components of cyanobacterial CO₂-concentration mechanisms. They also show potential in applications aimed at improving photosynthetic rates and yield when expressed in the chloroplasts of C3 crop species. The present study investigated the feasibility of using Escherichia coli to assess function of a range of SbtA and BicA transporters in a heterologous expression system, ultimately for selection of transporters suitable for chloroplast expression. Here, we demonstrate that six β-forms of SbtA are active in E. coli, although other tested bicarbonate transporters were inactive. The sbtA clones were derived from Synechococcus sp. WH5701, Cyanobium sp. PCC7001, Cyanobium sp. PCC6307, Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803, and Synechococcus sp. PCC7002. The six SbtA homologs varied in bicarbonate uptake kinetics and sodium requirements in E. coli. In particular, SbtA from PCC7001 showed the lowest uptake affinity and highest flux rate and was capable of increasing the internal inorganic carbon pool by more than 8 mM relative to controls lacking transporters. Importantly, we were able to show that the SbtB protein (encoded by a companion gene near sbtA) binds to SbtA and suppresses bicarbonate uptake function of SbtA in E. coli, suggesting a role in post-translational regulation of SbtA, possibly as an inhibitor in the dark. This study established E. coli as a heterologous expression and analysis system for HCO₃ ^- transporters from cyanobacteria, and identified several SbtA transporters as useful for expression in the chloroplast inner envelope membranes of higher plants.     

Inorganic carbon acquisition in red tide dinoflagellates

Carbon acquisition was investigated in three marine bloom-forming dinollagellates-Prorocentrum minimum, Heterocapsa triquetra and Ceratium lineatum. In vivo activities of extracellular and intracellular carbonic anhydrase (CA), photosynthetic O2 evolution, CO2 and HCO3- uptake rates were measured by membrane inlet mass spectrometry (MIMS) in cells acclimated to low pH (8.0) and high pH (8.5 or 9.1). A second approach used short-term 14C-disequilibrium incubations to estimate the carbon source utilized by the cells. All three species showed negligible extracellular CA (eCA) activity in cells acclimated to low pH and only slightly higher activity when acclimated to high pH. Intracellular CA (iCA) activity was present in all three species, but it increased only in P. minimum with increasing pH. Half-saturation concentrations (K1/2) for photosynthetic O2 evolution were low compared to ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) kinetics. Moreover, apparent affinities for inorganic carbon (Ci) increased with increasing pH in the acclimation, indicating the operation of an efficient CO2 concentration mechanism (CCM) in these dinoflagellates. Rates of CO2 uptake were comparably low and could not support the observed rates of photosynthesis. Consequently, rates of HCO3- uptake were high in the investigated species, contributing more than 80% of the photosynthetic carbon fixation. The affinity for HCO3- and maximum uptake rates increased under higher pH. The strong preference for HCO3- was also confirmed by the 14C-disequilibrium technique. Modes of carbon acquisition were consistent with the 13C-fractionation pattern observed and indicated a strong species-specific difference in leakage. These results suggest that photosynthesis in marine dinoflagellates is not limited by Ci even at high pH, which may occur during red tides in coastal waters.     

Microalgal carbon-dioxide-concentrating mechanisms: Chlamydomonas inorganic carbon transporters

Aquatic photosynthetic micro-organisms have adapted to the variable and often-limiting availability of CO(2), and inorganic carbon (Ci) in general, by development of inducible CO(2)-concentrating mechanisms (CCMs) that allow them to optimize carbon acquisition. Both microalgal and cyanobacterial CCMs function to facilitate CO(2) assimilation when Ci is limiting via active Ci uptake systems to increase internal Ci accumulation and carbonic anhydrase activity to provide elevated internal CO(2) concentrations through the dehydration of accumulated bicarbonate. These CCMs have been studied over several decades, and details of the cyanobacterial CCM function have emerged over recent years. However, significant advances in understanding of the microalgal CCM have been more recent. With the aid of mutational approaches and the availability of multiple microalgal genome sequences, an integrated picture of the functional components of the microalgal CCMs is emerging, together with the molecular details regarding the function and regulation of the CCM. This review will focus on the recent advances in identifying and characterizing the Ci transport components of the microalgal CCM, especially in the model organism Chlamydomonas reinhardtii Dangeard.