A. G. Markelova

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 β-class carbonic anhydrase of the alkaliphilic cyanobacterium Microcoleus chthonoplastes

The gene for β-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 β-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 β-class CA which was associated with PS II of alkaliphilic cyanobacteria. Here we first report extracellular localization of β-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.

Distribution and functional role of carbonic anhydrase Cah3 associated with thylakoid membranes in the chloroplast and pyrenoid of Chlamydomonas reinhardtii

Localization of lumenal carbonic anhydrase Cah3 in thylakoid membranes of Chlamydomonas reinhardtii was studied using wild-type algae and photosynthetic mutants with different composition of chlorophyll-protein complexes in the photosystems. In addition, the photosynthetic characteristics of wild-type C. reinhardtii and cia3 mutants lacking the activity of carbonic anhydrase Cah3 were examined. Western blot analysis revealed the lack of cross reaction with antibodies to Cah3 in the mutant lacking the photosystem II (PSII) reaction center, in contrast to the mutant deficient in light-harvesting complex of PSII. These data show that the lumenal Cah3 is associated with polypeptides on the donor side of PSII reaction center. Using immunoelectron microscopy and antibodies to Cah3 from C. reinhardtii, we showed for the first time that the major part of thylakoid Cah3 is localized in the pyrenoid where the bulk of Rubisco is located. The rate of photosynthetic oxygen evolution and PSII photochemical efficiency were lower in C. reinhardtii cia3 mutant than in the wild type, especially in the cells grown at limiting CO2 concentrations. These observations show that Cah3 takes part in CO2-concentrating mechanism of the chloroplast. The results support our hypothesis [1, 2] that the carboxylation reaction in microalgae proceeds in the pyrenoid, a specific Rubisco-containing part of the chloroplast, which acquires CO2 from the lumen of intrapyrenoid thylakoids. We discuss significance of the pyrenoid as an autonomous metabolic microcompartment, in which Cah3 plays a key role in the production and concentration of CO2 for Rubisco. These functions may promote the photosynthetic efficiency owing to the effective CO2 supply for the Calvin cycle.

A Comparison of Cytochemical Methods for the Rapid Evaluation of Microalgal Viability

We compared various rapid methods for the evaluation of cell viability of 30 algal strains from 15 genera using dyes for both light and fluorescence microscopy. Algal strains demonstrated considerable staining specificity. Staining with fluorescein diacetate helps distinguish between living and dead cells and also predicts the physiological state of the unicellular alga.

The effect of nitrogen starvation on the ultrastructure and pigment composition of chloroplasts in the acidothermophilic microalga Galdieria sulphuraria

We studied the effect of nitrogen starvation on growth indices, vitality, ultrastructure, and the photosynthetic apparatus of unique acidothermophilic microalga Galdieria sulphuraria (Galdieri) Merola. Long-term nitrogen starvation ceased G. sulphuraria growth and cell division. During the first days of starvation, phycobiliproteins degraded first, then the content of chlorophyll and carotenoids decreased to trace amounts, chloroplast reduced, cell wall became thinner, and storage compounds accumulated. However, the cells were alive. A comparison with the effects of nitrogen starvation on other photosynthesizing organisms showed that suppression of cell division, reduction of the photosynthetic apparatus to some minimum, and accumulation of storage compounds are a universal response to this stress.

Carbonic anhydrase of the alkaliphilic cyanobacterium Microcoleus chthonoplastes

The activity of carbonic anhydrase (CA) was studied in different cell fractions of the alkaliphilic cyanobacterium Microcoleus chthonoplastes. The activity of this enzyme was found in the soluble and membrane protein fractions, as well as in intact cells and in a thick glycocalyx layer enclosing the cyanobacterium cells. The localization of CA in glycocalyx of M. chthonoplastes was shown by western blot analysis and by immunoelectron microscopy studies with antibodies to the thylakoid CA from Chlamydomonas reinhardtii (Cah3). At least one of the CA forms occurring in M. chthonoplastes CA was shown to be an α-type enzyme. A possible mechanism of the involvement of the glycocalyx CA in calcification of cyanobacteria is discussed.

Capacity of Spirulina platensis to accumulate manganese and its distribution in cell

Effects of manganese salt (MnCl2) on growth of Spirulina platensis and capacity of the cyanobacteria to accumulate the metal in various cell components were studied. S. platensis cells were shown to tolerate high concentrations of manganese and preserve, although strongly suppressed, the capacity to grow in the medium containing 5.1 mM MnCl2. The concentrations of manganese that did not inhibit growth considerably altered cell ultrastructure and changed the protein profile. The accumulation of manganese in S. platensis cells was proportional to the period of culturing and manganese concentration in the medium, reaching a plateau at about 2.5 mM. A threshold intracellular concentration of this metal is estimated as 28 ± 3 μmol/g dry wt. The fractionation of the manganese-enriched biomass demonstrated that the major portion of intracellular manganese (over 90%) was found in the total protein fraction. The chromatographic separation of the soluble protein fraction showed that manganese was incorporated into proteins with molecular weight of 5 to 15 kD. Dry biomass adsorbed manganese cations; this evidence seems to indicate a considerable contribution of biosorption to manganese accumulation by S. platensis cells.

Specific features of the system of carbonic anhydrases of alkaliphilic cyanobacteria

This review brings together our recent data on carbonic anhydrases of representatives of alkaliphilic cyanobacteria inhabiting soda lakes, which are considered as the relicts of the ancient terrestrial microbiota. The modern information about cyanobacterial carbonic anhydrases is based mainly on the study of model strains, such as Synechocystis and Synechococcus. Our results are compared with literature data. The role of carbonic anhydrases in the assimilation of inorganic carbon by cyanobacteria of soda lakes is discussed in terms of evolution of the CO2-concentrating mechanism.

Compartmentation of α- and β-Carbonic Anhydrases in Cells of Halo- and Alkalophilic Cyanobacteria Rhabdoderma lineare

The organization of carbonic anhydrase (CA) system in halo- and alkalophilic cyanobacteria Rhabdoderma lineare was studied by Western blot analysis and immunocytochemical electron microscopy. The presence of extracellular α-CA (60 kD) in the glycocalyx, forming a tight sheath around the cell, and of two intracellular β-CA is reported. One β-CA (60 kD) is associated with polypeptides of photosystem II (PSII) and is a constitutive enzyme. Another β-carbonic anhydrase (25 kD) was induced by low content of bicarbonate in the culture medium; this inducible CA was found in the fraction of total soluble proteins. The expressed synthesis of inducible β-CA was accompanied by the increase in the intracellular pool of inorganic carbon, which suggests an important role of this enzyme in the functioning of CO2-concentrating mechanism.