Adapting from Low to High: An Update to CO2-Concentrating Mechanisms of Cyanobacteria and Microalgae.

The intracellular accumulation of inorganic carbon (Ci) by microalgae and cyanobacteria under ambient atmospheric CO2 levels was first documented in the 80s of the 20th Century. Hence, a third variety of the CO2-concentrating mechanism (CCM), acting in aquatic photoautotrophs with the C3 photosynthetic pathway, was revealed in addition to the then-known schemes of CCM, functioning in CAM and C4 higher plants. Despite the low affinity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of microalgae and cyanobacteria for the CO2 substrate and low CO2/O2 specificity, CCM allows them to perform efficient CO2 fixation in the reductive pentose phosphate (RPP) cycle. CCM is based on the coordinated operation of strategically located carbonic anhydrases and CO2/HCO3- uptake systems. This cooperation enables the intracellular accumulation of HCO3-, which is then employed to generate a high concentration of CO2 molecules in the vicinity of Rubisco's active centers compensating up for the shortcomings of enzyme features. CCM functions as an add-on to the RPP cycle while also acting as an important regulatory link in the interaction of dark and light reactions of photosynthesis. This review summarizes recent advances in the study of CCM molecular and cellular organization in microalgae and cyanobacteria, as well as the fundamental principles of its functioning and regulation.

Putative extracellular α-class carbonic anhydrase, EcaA, of Synechococcus elongatus PCC 7942 is an active enzyme: a sequel to an old story.

Carbonic anhydrase (CA) EcaA of Synechococcus elongatus PCC 7942 was previously characterized as a putative extracellular α-class CA, however, its activity was never verified. Here we show that EcaA possesses specific CA activity, which is inhibited by ethoxyzolamide. An active EcaA was expressed in heterologous bacterial system, which supports the formation of disulfide bonds, as a full-length protein (EcaA+L) and as a mature protein that lacks a leader peptide (EcaA-L). EcaA-L exhibited higher specific activity compared to EcaA+L. The recombinant EcaA, expressed in a bacterial system that does not support optimal disulfide bond formation, exhibited extremely low activity. This activity, however, could be enhanced by the thiol-oxidizing agent, diamide; while a disulfide bond-reducing agent, dithiothreitol, further inactivated the enzyme. Intact E. coli cells that overexpress EcaA+L possess a small amount of processed protein, EcaA-L, whereas the bulk of the full-length protein resides in the cytosol. This may indicate poor recognition of the EcaA leader peptide by protein export systems. S. elongatus possessed a relatively low level of ecaA mRNA, which varied insignificantly in response to changes in CO2 supply. However, the presence of protein in the cells is not obvious. This points to the physiological insignificance of EcaA in S. elongatus, at least under the applied experimental conditions.

The complete genome of a cyanobacterium from a soda lake reveals the presence of the components of CO2-concentrating mechanism.

At present geological epoch, the carbon concentrating mechanism (CCM) of cyanobacteria represents the obligatory tool for adaptation to low content of CO2 in the atmosphere and for the maintenance of sufficient photosynthetic activity. Functional CCM was found in modern cyanobacteria from different ecological niches. However, the presence of such mechanism in species that inhabit soda lakes is not obvious due to high content of inorganic carbon (C i) in the environment. Here we analyze CCM components that have been identified by sequencing of the whole genome of the alkaliphilic cyanobacterium Microcoleus sp. IPPAS B-353. The composition of the CCM components of Microcoleus is similar to that of 'model' ß-cyanobacteria, freshwater and marine Synechococcus or Synechocystis spp. However, CahB1 protein of Microcoleus, which is the homolog of CcaA, the carboxysomal ß-type carbonic anhydrase (CA) of ß-cyanobacteria, appeared to be the only active CA located in cell envelopes. The conservative regions of CcmM, CahG (a homolog of archeal γ-CAs, Cam/CamH), and ChpX of Microcoleus possess single amino acid substitutions that may cause a lack of CA activities. Unlike model cyanobacteria, Microcoleus induces only one BicA-type bicarbonate transporter in response to C i limitation. The differences in the appearance of CCM components and in their characteristics between alkaliphilic Microcoleus and freshwater or marine cyanobacteria are described. The possible reasons for the maintenance of CCM components in cyanobacteria, which permanently live at high concentrations of C i in soda lakes, are discussed.

Genomic Survey and Biochemical Analysis of Recombinant Candidate Cyanobacteriochromes Reveals Enrichment for Near UV/Violet Sensors in the Halotolerant and Alkaliphilic Cyanobacterium Microcoleus IPPAS B353.

Cyanobacteriochromes (CBCRs), which are exclusive to and widespread among cyanobacteria, are photoproteins that sense the entire range of near-UV and visible light. CBCRs are related to the red/far-red phytochromes that utilize linear tetrapyrrole (bilin) chromophores. Best characterized from the unicellular cyanobacterium Synechocystis sp. PCC 6803 and the multicellular heterocyst forming filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Anabaena sp. PCC 7120, CBCRs have been poorly investigated in mat-forming, nonheterocystous cyanobacteria. In this study, we sequenced the genome of one of such species, Microcoleus IPPAS B353 (Microcoleus B353), and identified two phytochromes and seven CBCRs with one or more bilin-binding cGMP-specific phosphodiesterase, adenylyl cyclase and FhlA (GAF) domains. Biochemical and spectroscopic measurements of 23 purified GAF proteins from phycocyanobilin (PCB) producing recombinant Escherichia coli indicated that 13 of these proteins formed near-UV and visible light-absorbing covalent adducts: 10 GAFs contained PCB chromophores, whereas three contained the PCB isomer, phycoviolobilin (PVB). Furthermore, the complement of Microcoleus B353 CBCRs is enriched in near-UV and violet sensors, but lacks red/green and green/red CBCRs that are widely distributed in other cyanobacteria. We hypothesize that enrichment in short wavelength-absorbing CBCRs is critical for acclimation to high-light environments where this organism is found.

CO2-concentrating mechanism in cyanobacterial photosynthesis: organization, physiological role, and evolutionary origin.

The cellular and molecular organization of the CO2-concentrating mechanism (CCM) of cyanobacteria is reviewed. The primary processes of uptake, translocation, and accumulation of inorganic carbon (Ci) near the active site of carbon assimilation by the enzyme ribulose-1,5-bisphosphate carboxylase in the C3 cycle in cyanobacteria are described as one of the specialized forms of CO2 concentration which occurs in some photoautotrophic cells. The existence of this form of CO2 concentration expands our understanding of photosynthetic Ci assimilation. The means of supplying Ci to the C3 cycle in cyanobacteria is not by simple diffusion into the cell, but it is the result of coordinated functions of high-affinity systems for the uptake of CO2 and bicarbonate, as well as intracellular CO2/HCO3 (-) interconversions by carbonic anhydrases. These biochemical events are under genetic control, and they serve to maintain cellular homeostasis and adaptation to CO2 limitation. Here we describe the organization of the CCM in cyanobacteria with a special focus on the CCM of relict halo- and alkaliphilic cyanobacteria of soda lakes. We also assess the role of the CCM at the levels of the organism, the biosphere, and evolution.

Identification and functional role of the carbonic anhydrase Cah3 in thylakoid membranes of pyrenoid of Chlamydomonas reinhardtii.

The distribution of the luminal carbonic anhydrase Cah3 associated with thylakoid membranes in the chloroplast and pyrenoid was studied in wild-type cells of Chlamydomonas reinhardtii and in its cia3 mutant deficient in the activity of the Cah3 protein. In addition, the effect of CO(2) concentration on fatty acid composition of photosynthetic membranes was examined in wild-type cells and in the cia3 mutant. In the cia3 mutant, the rate of growth was lower as compared to wild-type, especially in the cells grown at 0.03% CO(2). This might indicate a participation of thylakoid Cah3 in the CO(2)-concentrating mechanism (CCM) of chloroplast and reflect the dysfunction of the CCM in the cia3 mutant. In both strains, a decrease in the CO(2) concentration from 2% to 0.03% caused an increase in the content of polyunsaturated fatty acids in membrane lipids. At the same time, in the cia3 mutant, the increase in the majority of polyunsaturated fatty acids was less pronounced as compared to wild-type cells, whereas the amount of 16:4ω3 did not increase at all. Immunoelectron microscopy demonstrated that luminal Cah3 is mostly located in the thylakoid membranes that pass through the pyrenoid. In the cells of CCM-mutant, cia3, the Cah3 protein was much less abundant, and it was evenly distributed throughout the pyrenoid matrix. The results support our hypothesis that CO(2) might be generated from HCO(3)(-) by Cah3 in the thylakoid lumen with the following CO(2) diffusion into the pyrenoid, where the CO(2) fixing Rubisco is located. This ensures the maintenance of active photosynthesis under CO(2)-limiting conditions, and, as a result, the active growth of cells. The relationships between the induction of CCM and restructuring of the photosynthetic membranes, as well as the involvement of the Cah3 of the pyrenoid in these events, are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Extracellular ß-class carbonic anhydrase of the alkaliphilic cyanobacterium Microcoleus chthonoplastes.

The gene for ß-class carbonic anhydrase (CA), which was designated as cahB1, was cloned from the genomic library of the alkaliphilic cyanobacterium Microcoleus chthonoplastes. The product of the cahB1 gene was expressed in Escherichia coli. The protein revealed high specific activity of CA, which was inhibited with ethoxyzolamide. The maximum activity of the recombinant CA was detected at alkaline pH (∼9.0) and its minimum - at neutral pH (∼7.0). Western blotting analysis with the antibodies raised against the recombinant CahB1 protein revealed its localization in cell envelopes of M. chthonoplastes. Immunocytochemical localization of the CahB1 in cells confirmed its extracellular location. The newly characterized CahB1 of Microcoleus was similar in amino acid and nucleotide sequences to well known ß-CAs of Synechococcus sp. PCC 7942 (IcfA) and Synechocystis sp. PCC 6803 (CcaA), although those CAs were attributed to the carboxysomal shells of cyanobacteria. Previously we have reported ß-class CA which was associated with PS II of alkaliphilic cyanobacteria. Here we first report extracellular localization of ß-class CA and provide a scheme for its possible involvement in the maintenance of a balance between external sources of inorganic carbon and photosynthesis in extreme environments of soda lakes.

The thylakoid carbonic anhydrase associated with photosystem II is the component of inorganic carbon accumulating system in cells of halo- and alkaliphilic cyanobacterium Rhabdoderma lineare.

The organization of carbonic anhydrase (CA) system in halo- and alkaliphilic cyanobacterium Rhabdoderma lineare was studied by Western blot analysis and immunocytochemical electron microscopy. The presence of putative extracellular alpha-CA of 60 kDa in the glycocalyx, forming a tight sheath around the cell, and of two intracellular beta-CA is reported. We show for the first time that the beta-CA of 60 kDa is expressed constitutively and associated with polypeptides of photosystem II (beta-CA-PS II). Another soluble beta-CA of 25 kDa was induced in low-bicarbonate medium. Induction of synthesis of the latter beta-CA was accompanied by an increase in the intracellular pool of inorganic carbon, which suggests an important role of this enzyme in the functioning of a CO(2)-concentrating mechanism.