LCIB functions as a carbonic anhydrase: evidence from yeast and Arabidopsis carbonic anhydrase knockout mutants.

Chlamydomonas reinhardtii evolved a CO2-concentrating mechanism (CCM) because of the limited CO2 in its natural environment. One critical component of the C. reinhardtii CCM is the limiting CO2 inducible B (LCIB) protein. LCIB is required for acclimation to air levels of CO2. C. reinhardtii cells with a mutated LCIB protein have an 'air-dier' phenotype when grown in low CO2 conditions, meaning they die in air levels of CO2 but can grow in high and very low CO2 conditions. The LCIB protein functions together with its close homolog in C. reinhardtii, limiting CO2 inducible C protein (LCIC), in a hexameric LCIB-LCIC complex. LCIB has been proposed to act as a vectoral carbonic anhydrase (CA) that helps to recapture CO2 that would otherwise leak out of the chloroplast. Although both LCIB and LCIC are structurally similar to ßCAs, their CA activity has not been demonstrated to date. We provide evidence that LCIB is an active CA using a Saccharomyces cerevisiae CA knockout mutant (∆NCE103) and an Arabidopsis thaliana ßCA5 knockout mutant (ßca5). We show that different truncated versions of the LCIB protein complement ∆NCE103, while the full length LCIB protein complements ßca5 plants, so that both the yeast and plant mutants can grow in low CO2 conditions.

Arabidopsis plastid carbonic anhydrase ßCA5 is important for normal plant growth.

Carbonic anhydrases (CAs) are zinc-metalloenzymes that catalyze the interconversion of CO2 and HCO3-. In heterotrophic organisms, CAs provide HCO3- for metabolic pathways requiring a carboxylation step. Arabidopsis (Arabidopsis thaliana) has 14 α- and ß-type CAs, two of which are plastid CAs designated as ßCA1 and ßCA5. To study their physiological properties, we obtained knock-out (KO) lines for ßCA1 (SALK_106570) and ßCA5 (SALK_121932). These mutant lines were confirmed by genomic PCR, RT-PCR, and immunoblotting. While ßca1 KO plants grew normally, growth of ßca5 KO plants was stunted under ambient CO2 conditions of 400 µL L-1; high CO2 conditions (30,000 µL L-1) partially rescued their growth. These results were surprising, as ßCA1 is more abundant than ßCA5 in leaves. However, tissue expression patterns of these genes indicated that ßCA1 is expressed only in shoot tissue, while ßCA5 is expressed throughout the plant. We hypothesize that ßCA5 compensates for loss of ßCA1 but, owing to its expression being limited to leaves, ßCA1 cannot compensate for loss of ßCA5. We also demonstrate that ßCA5 supplies HCO3- required for anaplerotic pathways that take place in plastids, such as fatty acid biosynthesis.

A Rapid Method for Detecting Normal or Modified Plant and Algal Carbonic Anhydrase Activity Using Saccharomyces cerevisiae.

In recent years, researchers have attempted to improve photosynthesis by introducing components from cyanobacterial and algal CO2-concentrating mechanisms (CCMs) into terrestrial C3 plants. For these attempts to succeed, we need to understand the CCM components in more detail, especially carbonic anhydrase (CA) and bicarbonate (HCO3−) transporters. Heterologous complementation systems capable of detecting carbonic anhydrase activity (i.e., catalysis of the pH-dependent interconversion between CO2 and HCO3−) or active HCO3− transport can be of great value in the process of introducing CCM components into terrestrial C3 plants. In this study, we generated a Saccharomyces cerevisiae CA knock-out (ΔNCE103 or ΔCA) that has a high-CO2-dependent phenotype (5% (v/v) CO2 in air). CAs produce HCO3− for anaplerotic pathways in S. cerevisiae; therefore, the unavailability of HCO3− for neutral lipid biosynthesis is a limitation for the growth of ΔCA in ambient levels of CO2 (0.04% (v/v) CO2 in air). ΔCA can be complemented for growth at ambient levels of CO2 by expressing a CA from human red blood cells. ΔCA was also successfully complemented for growth at ambient levels of CO2 through the expression of CAs from Chlamydomonas reinhardtii and Arabidopsis thaliana. The ΔCA strain is also useful for investigating the activity of modified CAs, allowing for quick screening of modified CAs before putting them into the plants. CA activity in the complemented ΔCA strains can be probed using the Wilbur−Anderson assay and by isotope exchange membrane-inlet mass spectrometry (MIMS). Other potential uses for this new ΔCA-based screening system are also discussed.

Molecular interaction of nitrate transporter proteins with recombinant glycinebetaine results in efficient nitrate uptake in the cyanobacterium Anabaena PCC 7120.

Nitrate transport in cyanobacteria is mediated by ABC-transporter, which consists of a highly conserved ATP binding cassette (ABC) and a less conserved transmembrane domain (TMD). Under salt stress, recombinant glycinebetaine (GB) not only protected the rate of nitrate transport in transgenic Anabaena PCC 7120, rather stimulated the rate by interacting with the ABC-transporter proteins. In silico analyses revealed that nrtA protein consisted of 427 amino acids, the majority of which were hydrophobic and contained a Tat (twin-arginine translocation) signal profile of 34 amino acids (1-34). The nrtC subunit of 657 amino acids contained two hydrophobic distinct domains; the N-terminal (5-228 amino acids), which was 59% identical to nrtD (the ATP-binding subunit) and the C-terminal (268-591), 28.2% identical to nrtA, suggesting C-terminal as a solute binding domain and N-terminal as ATP binding domain. Subunit nrtD consisted of 277 amino acids and its N-terminal (21-254) was an ATP binding motif. Phylogenetic analysis revealed that nitrate-ABC-transporter proteins are highly conserved among the cyanobacterial species, though variation existed in sequences resulting in several subclades. Nostoc PCC 7120 was very close to Anabaena variabilis ATCC 29413, Anabaena sp. 4-3 and Anabaena sp. CA = ATCC 33047. On the other, Nostoc spp. NIES-3756 and PCC 7524 were often found in the same subclade suggesting more work before referring it to Anabaena PCC 7120 or Nostoc PCC 7120. The molecular interaction of nitrate with nrtA was hydrophilic, while hydrophobic with nrtC and nrtD. GB interaction with nrtACD was hydrophobic and showed higher affinity compared to nitrate.

Mitochondrial carbonic anhydrases are needed for optimal photosynthesis at low CO2 levels in Chlamydomonas.

Chlamydomonas reinhardtii can grow photosynthetically using CO2 or in the dark using acetate as the carbon source. In the light in air, the CO2 concentrating mechanism (CCM) of C. reinhardtii accumulates CO2, enhancing photosynthesis. A combination of carbonic anhydrases (CAs) and bicarbonate transporters in the CCM of C. reinhardtii increases the CO2 concentration at Ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) in the chloroplast pyrenoid. Previously, CAs important to the CCM have been found in the periplasmic space, surrounding the pyrenoid and inside the thylakoid lumen. Two almost identical mitochondrial CAs, CAH4 and CAH5, are also highly expressed when the CCM is made, but their role in the CCM is not understood. Here, we adopted an RNAi approach to reduce the expression of CAH4 and CAH5 to study their possible physiological functions. RNAi mutants with low expression of CAH4 and CAH5 had impaired rates of photosynthesis under ambient levels of CO2 (0.04% CO2 [v/v] in air). These strains were not able to grow at very low CO2 (<0.02% CO2 [v/v] in air), and their ability to accumulate inorganic carbon (Ci = CO2 + HCO3-) was reduced. At low CO2 concentrations, the CCM is needed to both deliver Ci to Rubisco and to minimize the leak of CO2 generated by respiration and photorespiration. We hypothesize that CAH4 and CAH5 in the mitochondria convert the CO2 released from respiration and photorespiration as well as the CO2 leaked from the chloroplast to HCO3- thus "recapturing" this potentially lost CO2.

Thylakoid localized bestrophin-like proteins are essential for the CO2 concentrating mechanism of Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) that helps in successful acclimation to low CO2 conditions. Current models of the CCM postulate that a series of ion transporters bring HCO3- from outside the cell to the thylakoid lumen, where the carbonic anhydrase 3 (CAH3) dehydrates accumulated HCO3- to CO2, raising the CO2 concentration for Ribulose bisphosphate carboxylase/oxygenase (Rubisco). Previously, HCO3- transporters have been identified at both the plasma membrane and the chloroplast envelope, but the transporter thought to be on the thylakoid membrane has not been identified. Three paralogous genes (BST1, BST2, and BST3) belonging to the bestrophin family have been found to be up-regulated in low CO2 conditions, and their expression is controlled by CIA5, a transcription factor that controls many CCM genes. YFP fusions demonstrate that all 3 proteins are located on the thylakoid membrane, and interactome studies indicate that they might associate with chloroplast CCM components. A single mutant defective in BST3 has near-normal growth on low CO2, indicating that the 3 bestrophin-like proteins may have redundant functions. Therefore, an RNA interference (RNAi) approach was adopted to reduce the expression of all 3 genes at once. RNAi mutants with reduced expression of BST1-3 were unable to grow at low CO2 concentrations, exhibited a reduced affinity to inorganic carbon (Ci) compared with the wild-type cells, and showed reduced Ci uptake. We propose that these bestrophin-like proteins are essential components of the CCM that deliver HCO3- accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen.

Physiological responses to salt stress of salt-adapted and directly salt (NaCl and NaCl+Na2SO4 mixture)-stressed cyanobacterium Anabaena fertilissima.

Soil salinity in nature is generally mixed type; however, most of the studies on salt toxicity are performed with NaCl and little is known about sulfur type of salinity (Na2SO4). Present study discerns the physiologic mechanisms responsible for salt tolerance in salt-adapted Anabaena fertilissima, and responses of directly stressed parent cells to NaCl and NaCl+Na2SO4 mixture. NaCl at 500 mM was lethal to the cyanobacterium, whereas salt-adapted cells grew luxuriantly. Salinity impaired gross photosynthesis, electron transport activities, and respiration in parent cells, but not in the salt-adapted cells, except a marginal increase in PSI activity. Despite higher Na+ concentration in the salt mixture, equimolar NaCl appeared more inhibitive to growth. Sucrose and trehalose content and antioxidant activities were maximal in 250 mM NaCl-treated cells, followed by salt mixture and was almost identical in salt-adapted (exposed to 500 mm NaCl) and control cells, except a marginal increase in ascorbate peroxidase activity and an additional fourth superoxide dismutase isoform. Catalase isoform of 63 kDa was induced only in salt-stressed cells. Salinity increased the uptake of intracellular Na+ and Ca2+ and leakage of K+ in parent cells, while cation level in salt-adapted cells was comparable to control. Though there was differential increase in intracellular Ca2+ under different salt treatments, ratio of Ca2+/Na+ remained the same. It is inferred that stepwise increment in the salt concentration enabled the cyanobacterium to undergo priming effect and acquire robust and efficient defense system involving the least energy.

The freshwater cyanobacterium Anabaena doliolum transformed with ApGSMT-DMT exhibited enhanced salt tolerance and protection to nitrogenase activity, but became halophilic.

Glycine betaine (GB) is an important osmolyte synthesized in response to different abiotic stresses, including salinity. The two known pathways of GB synthesis involve: 1) two step oxidation of choline (choline → betaine aldehyde → GB), generally found in plants, microbes and animals; and 2) three step methylation of glycine (glycine → sarcosine → dimethylglycine → GB), mainly found in halophilic archaea, sulphur bacteria and the cyanobacterium Aphanothece (Ap.) halophytica. Here, we transformed a salt-sensitive freshwater diazotrophic filamentous cyanobacterium Anabaena (An.) doliolum with N-methyltransferase genes (ApGSMT-DMT) from Ap. halophytica using the triparental conjugation method. The transformed An. doliolum synthesized and accumulated GB in cells, and showed increased salt tolerance and protection to nitrogenase activity. The salt responsiveness of the transformant was also apparent as GB synthesis increased with increasing concentrations of NaCl in the nutrient solution, and maximal [12.92 µmol (g dry weight)(-1)] in cells growing at 0.5 M NaCl. Therefore, the transformed cyanobacterium has changed its behaviour from preferring freshwater to halophily. This study may have important biotechnological implications for the development of stress tolerant nitrogen-fixing cyanobacteria as biofertilizers for sustainable agriculture.

Low cellular P-quota and poor metabolic adaptations of the freshwater cyanobacterium Anabaena fertilissima Rao during Pi-limitation.

Anabaena fertilissima is a filamentous freshwater N(2)-fixing cyanobacterium, isolated from a paddy field. Growth of the cyanobacterium was limited by the non-availability of inorganic phosphate (Pi) in the growth medium and was found to be directly related to the cellular P quota, which declined rapidly in Pi-deficient cells. To overcome Pi-deficiency, cells induced both cell-bound and cell-free alkaline phosphatase activities (APase). The activity of cell-bound APase was rapid and 5-6 times higher than that of the cell-free APase activity. Native gel electrophoresis revealed the presence of two APase activity bands for both the cell bound and cell-free APase (Mr ≈42 and 34 kDa). For Pi-deficient cells, APase activity was inversely related to cellular P-quota. In A. fertilissima phosphate uptake was facilitated by single high-affinity phosphate transporter (K ( s ), 4.54 µM; V(max), 4.84 µmol mg protein(-1) min(-1)). Pi-deficiency severely reduced the photosynthetic rate, respiration rate and nitrate uptake, as well as the activities of nitrate reductase, nitrite reductase and nitrogenase enzymes. In photosynthesis, PSII activity was maximally inhibited, followed by PSI and whole chain activities. Transcript levels of five key glycolytic enzymes showed the poor adaptability of the cyanobacterium to switch its metabolic activity to PPi-dependent enzyme variants, which has rather constant cellular concentrations.

Isolation and screening of phlD (+) plant growth promoting rhizobacteria antagonistic to Ralstonia solanacearum.

Tomato (Lycopersicon esculentum) is important widely grown vegetable in India and its productivity is affected by bacterial wilt disease infection caused by Ralstonia solanacearum. To prevent this disease infection a study was conducted to isolate and screen effective plant growth promoting rhizobacteria (PGPR) antagonistic to R. solanacearum. A total 297 antagonistic bacteria were isolated through dual culture inoculation technique, out of which forty-two antagonistic bacteria were found positive for phlD gene by PCR amplification using two primer sets Phl2a:Phl2b and B2BF:BPR4. The genetic diversity of phlD (+) bacteria was studied by amplified 16S rDNA restriction analysis and demonstrated eleven groups at 65% similarity level. Out of these 42 phlD (+) antagonistic isolates, twenty exhibited significantly fair plant growth promoting activities like phosphate solubilization (0.92-5.33%), 25 produced indole acetic acid (1.63-7.78 µg ml(-1)) and few strains show production of antifungal metabolites (HCN and siderophore). The screening of PGPR (phlD (+)) for suppression of bacterial wilt disease in glass house conditions was showed ten isolated phlD (+) bacteria were able to suppress infection of bacterial wilt disease in tomato plant (var. Arka vikas) in the presence R. solanacearum. The PGPR (phlD (+)) isolates s188, s215 and s288 was observed to be effective plant growth promoter as it shows highest dry weight per plant (3.86, 3.85 and 3.69 g plant(-1) respectively). The complete absence of wilt disease symptoms in tomato crop plants was observed by these treatments compared to negative control. Therefore inoculation of tomato plant with phlD (+) isolate s188 and other similar biocontrol agents may prove to be a positive strategy for checking wilt disease and thus improving plant vigor.

Anabaena sp. PCC7120 transformed with glycine methylation genes from Aphanothece halophytica synthesized glycine betaine showing increased tolerance to salt.

Photosynthetic, nitrogen-fixing Anabaena strains play an important role in the carbon and nitrogen cycles in tropical paddy fields although they are salt sensitive. Improvement in salt tolerance of Anabaena cells by expressing glycine betaine-synthesizing genes is an interesting subject. Due to the absence of choline in cyanobacteria, choline-oxidizing enzyme could not be used for the synthesis of glycine betaine. Here, the genes encoding glycine-sarcosine and dimethylglycine methyltransferases (ApGSMT-DMT) from a halotolerant cyanobacterium Aphanothece halophytica were expressed in Anabaena sp. strain PCC7120. The ApGSMT-DMT-expressing Anabaena cells were capable of synthesizing glycine betaine without the addition of any substance. The accumulation level of glycine betaine in Anabaena increased with rise of salt concentration. The transformed cells exhibited an improved growth and more tolerance to salinity than the control cells. The present work provides a prospect to engineer a nitrogen-fixing cyanobacterium having enhanced tolerance to stress by manipulating de novo synthesis of glycine betaine.

An alkaline phosphatase/phosphodiesterase, PhoD, induced by salt stress and secreted out of the cells of Aphanothece halophytica, a halotolerant cyanobacterium.

Alkaline phosphatases (APases) are important enzymes in organophosphate utilization. Three prokaryotic APase gene families, PhoA, PhoX, and PhoD, are known; however, their functional characterization in cyanobacteria largely remains to be clarified. In this study, we cloned the phoD gene from a halotolerant cyanobacterium, Aphanothece halophytica (phoD(Ap)). The deduced protein, PhoD(Ap), contains Tat consensus motifs and a peptidase cleavage site at the N terminus. The PhoD(Ap) enzyme was activated by Ca(2+) and exhibited APase and phosphodiesterase (APDase) activities. Subcellular localization experiments revealed the secretion and processing of PhoD(Ap) in a transformed cyanobacterium. Expression of the phoD(Ap) gene in A. halophytica cells was upregulated not only by phosphorus (P) starvation but also under salt stress conditions. Our results suggest that A. halophytica cells possess a PhoD that participates in the assimilation of P under salinity stress.

Cyanobacteria: an emerging source for drug discovery.

The c group of Gram-negative gliding bacteria, has a long history of cosmopolitan occurrence. It has great biodiversity despite the absence of sexual reproduction. This wide biodiversity may be reflected in the wide spectrum of its secondary metabolites. These cyanobacterial secondary metabolites are biosynthesized by a variety of routes, notably by non-ribosomal peptide synthetase or polyketide synthetase systems, and show a wide range of biological activities including anticancer, antibacterial, antiviral and protease inhibition activities. This high degree of chemical diversity in cyanobacterial secondary metabolites may thus constitute a prolific source of new entities leading to the development of new pharmaceuticals.

Microcystin producing cyanobacterium Nostoc sp. BHU001 from a pond in India.

The cyanobacterium Nostoc sp. BHU001 is a new isolate from a pond in India. The cyanobacterium produces more than ten peptides including five microcystin (MC) variants, MC-LR, -WR, -AR, -LA and methylated MC-LR, and a new peptide similar to cyanopeptolin. Total MC content determined by ELISA was 25.2 microg g(-1) dry wt of the cyanobacterium, dominated by MC-LR (54%). This is the first report of MC producing Nostoc strain from India.

Enrichment of sugar content in melon fruits by hydrogen peroxide treatment.

Since sweetness is one of the most important qualities of many fruits, and since sugars are translocated from leaves to fruits, the present study investigates photosynthetic activity, activity of sugar metabolizing enzymes, sugar content in leaves and fruits and endogenous levels of hydrogen peroxide in leaves of melon plants treated with various dilutions of hydrogen peroxide, a nonspecific signaling molecule in abiotic stress. For this purpose, 4-month-old melon plants were treated with various concentrations (<50mM) of hydrogen peroxide by applying 300 mL per day to the soil of potted plants. The treatments resulted in increased fructose, glucose, sucrose and starch in the leaves and fruits. The most effective concentration of hydrogen peroxide was 20mM. During the day, soluble sugars in leaves were highest at 12:00 h and starch at 15:00 h. Furthermore, the peroxide treatment increased the photosynthetic activity and the activities of chloroplastic and cytosolic fructose-1,6-bisphosphatase, sucrose phosphate synthase and invertases. Thus, our data show that exogenous hydrogen peroxide, applied to the soil, can increase the soluble sugar content of melon fruits.

Allergenicity of airborne cyanobacteria Phormidium fragile and Nostoc muscorum.

In recent times, airborne microorganisms and their constituents have become prominent safety and health concern. Ongoing climatic changes coupled with unwarranted human activities have significantly deteriorated the ambient air quality. In certain environments, airborne algae contribute significantly to the total biological load of the atmosphere, hitherto dominated by bacteria and fungi. Present study was aimed to investigate the allergenic potency of two frequent viable algal forms i.e., Phormidium fragile and Nostoc muscorum found in the atmosphere of Varanasi City, India. To test the allergenic potency, crude extracts of these strains were subjected to intra-dermal allergy test and subsequent leukocyte counts, which revealed their allergenic nature. Both the species varied in their allergenic potency. N. muscorum appeared to be more allergenic than P. fragile. However, when the allergens were mixed in equal amounts, the severity of allergenicity increased significantly. A limited pattern of cross-reactivity between the species was also evident.

Growth and cellular ion content of a salt-sensitive symbiotic system Azolla pinnata-Anabaena azollae under NaCl stress.

Salinity, at a concentration of 10 mM NaCl affected the growth of Azolla pinnata-Anabaena azollae association and became lethal at 40 mM. Plants exposed up to 30 mM NaCl exhibited longer roots than the control, especially during the beginning of incubation. Average root number in plants exposed to 10 and 20 mM NaCl remained almost the same as in control. A further rise in NaCl concentration to 30 mM reduced the root number, and roots shed off at 40 mM NaCl. Presence of NaCl in the nutrient solution increased the cellular Na+ of the intact association exhibiting differential accumulation by individual partners, while it reduced the cellular Ca2+ level. However, cellular K+ content did not show significant change. Cellular Na+ based on fresh weight of respective individual partners (host tissues and cyanobiont) remained higher in the host tissues than the cyanobiont, while reverse was true for K+ and Ca2+ contents. The contribution of A. azollae in the total cellular ion content of the association was a little because of meagre contribution of the cyanobiont mass (19-21%). High salt sensitivity of Azolla-Anabaena complex is due to an inability of the association to maintain low Na+ and high Ca2+ cellular level.

Diversity and seasonal variation of viable algal particles in the atmosphere of a subtropical city in India.

To characterize the airborne algal diversity in a populous subtropical urban environment, sampling was done at a height of 2.5m, the normal human breathing zone. Results indicated that airborne algae are the permanent constituent of Varanasi city atmosphere. The nature, composition, and relative ratio of constituting groups differed among sampling sites. Cyanobacteria, possibly due to their broad ecological distribution, dominate the fluctuating climates of subtropical regions such as Varanasi. The majority of the airborne algae were of local origin, indicating short-distance transport of the algae. Soilborne algae constituted the bulk of aeroalgal flora. This might be due to their ability to withstand the dehydrating effect of the atmosphere. Composition of the aeroalgal community also exhibited seasonal variation along with the change in climatic condition of the area. Thus, the physiological ability of an algal group to tolerate different types of abiotic stresses and the climatic conditions of the area appeared to be the two major factors responsible for regulating the structure of the aeroalgal community in the air.

Phosphate metabolism in the cyanobacterium Anabaena doliolum under salt stress.

In the present study, we have investigated the effects of NaCl concentrations on the growth and phosphate metabolism of an Anabaena doliolum strain isolated from a paddy field, in order to determine the possible effects of salinization. Growth rate, chlorophyll content, and protein content decreased with increasing salt concentration in the growth medium, while carbohydrate concentration increased. Phosphate content and phosphate uptake rate decreased. There was an increase in total alkaline phosphatase activity, with an approximately 7-fold increase in extracellular activity compensating for an approximately 3-fold decrease in cell-bound activity. NaCl effects on protein and chlorophyll concentrations were greater in P-deficient medium, while presence or absence of P in the medium had little effect on cellular carbohydrate concentrations. It is concluded that growth in high salt likely leads to reduced phosphate uptake in A. doliolum.