Aran Incharoensakdi

Overexpression of a Na+/H+ antiporter confers salt tolerance on a freshwater cyanobacterium, making it capable of growth in sea water

The salt tolerance of a freshwater cyanobacterium, Synechococcus sp. PCC 7942, transformed with genes involved in the synthesis of a Na+/H+ antiporter, betaine, catalase, and a chaperone was examined. Compared with the expression of betaine, catalase, and the chaperone, the expression of the Na+/H+ antiporter gene from a halotolerant cyanobacterium (ApNhaP) drastically improved the salt tolerance of the freshwater cyanobacterium. The Synechococcus cells expressing ApNhaP could grow in BG11 medium containing 0.5 M NaCl as well as in sea water, whereas those expressing betaine, catalase, and the chaperone could not grow under those conditions. The coexpression of ApNhaP with catalase or ApNhaP with catalase and betaine did not further enhance the salt tolerance of Synechococcus cells expressing ApNhaP alone when grown in BG11 medium containing 0.5 M NaCl. Interestingly, the coexpression of ApNhaP with catalase resulted in enhanced salt tolerance of cells grown in sea water. These results demonstrate a key role of sodium ion exclusion by the Na+/H+ antiporter for the salt tolerance of photosynhetic organisms.

The cyanotoxin-microcystins: current overview

The monocyclic heptapeptides microcystins (MCs), are a group of hepatotoxins, produced worldwide by some bloom-forming cyanobacterial species/strains both in marine and freshwater ecosystems. MCs are synthesized non-ribosomally by large multi-enzyme complexes consisting of different modules including polyketide synthases and non-ribosomal peptide synthetases, as well as several tailoring enzymes. More than 85 different variants of MCs have been reported to exist in nature. These are chemically stable, but undergo bio-degradation in natural water reservoirs. Direct or indirect intake of MCs through the food web is assumed to be a highly exposed route in risk assessment of cyanotoxins. MCs are the most commonly found cyanobacterial toxins that cause a major challenge for the production of safe drinking water and pose a serious threat to global public health as well as fundamental ecological processes due to their potential carcinogenicity. Here, we emphasize recent updates on different modes of action of their possible carcinogenicity. Besides the harmful effects on human and animals, MC producing cyanobacteria can also present a harmful effect on growth and development of agriculturally important plants. Overall, this review emphasizes the current understanding of MCs with their occurrence, geographical distribution, accumulation in the aquatic as well as terrestrial ecosystems, biosynthesis, climate-driven changes in their synthesis, stability and current aspects on its degradation, analysis, mode of action and their ecotoxicological effects.

Content and biosynthesis of polyamines in salt and osmotically stressed cells of Synechocystis sp. PCC 6803

The effects of various NaCl and sorbitol concentrations in the growth medium on polyamine content and on two enzymes of the polyamine biosynthesis pathway, arginine decarboxylase (ADC) and S-adenosyl methionine decarboxylase (SAMDC), were investigated in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Synechocystis cells showed no difference in growth rate when the concentration of NaCl was raised up to 550 mM. The growth rate decreased at 300 mM sorbitol, and complete inhibition of growth occurred at concentrations of > or =700 mM sorbitol. Salt stress induced a moderate increase in the total cellular polyamine content, spermine in particular. Osmotic stress caused an apparent increase in the total cellular polyamine content with a marked increase of spermidine induced by 700 mM sorbitol. Importantly, a low level of spermine, which so far has never been detected in cyanobacteria, could be found in Synechocystis sp. PCC 6803. ADC, a key enzyme for putrescine synthesis, was unaffected by salt stress but showed a six-fold increase in enzyme activity upon osmotic stress imposed by 700 mM sorbitol. SAMDC, another important enzyme for spermidine and spermine synthesis, responded to salt and osmotic stresses similarly to the pattern observed for ADC. An analysis by reverse transcription-polymerase chain reaction revealed an increase of ADC mRNA level in cells under salt and osmotic stresses. Most importantly, the increase of ADC mRNA was attributed to its slower turnover rate under both stress conditions. Interestingly, the samdc gene(s) of Synechocystis appear to be unique since comparisons with known gene sequences from other organisms resulted in no homologous sequences identified in the Synechocystis genome.

Zinc Biosorption from Aqueous Solution by a Halotolerant Cyanobacterium Aphanothece halophytica

We have investigated the characteristics of zinc biosorption by Aphanothece halophytica. Zinc could be rapidly taken up from aqueous solution by the cells with an equilibrium being reached within 15 min of incubation with 100 mg L−1 ZnCl2. The adsorbed zinc was desorbed by treatment with 10 mM EDTA. The presence of glucose, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and N,N′-dicyclohexylcarbodiimide (DCCD) did not affect the uptake of zinc. The specific uptake of zinc increased at low cell concentration and decreased when cell concentration exceeded 0.2 g L−1. The binding of zinc followed Langmuir isotherm kinetics with a maximum zinc binding capacity of 133 mg g−1 and an apparent zinc binding constant of 28 mg L−1. The presence of an equimolar concentration of Mn2+, Mg2+, Co2+, K+, or Na+ had no effect on zinc biosorption, whereas Ca2+, Hg2+, and Pb2+ showed an inhibitory effect. The biosorption of zinc was low at a pH range from 4 to 6, but increased progressively at pH 6.5 and 7.

Increased H2 production in the cyanobacterium Synechocystis sp. strain PCC 6803 by redirecting the electron supply via genetic engineering of the nitrate assimilation pathway.

The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 contains a single bidirectional NiFe-Hox-hydrogenase, which evolves hydrogen under certain environmental conditions. The nitrate assimilation pathway is a potential competing pathway that may reduce the electron flow to the hydrogenase and thereby limit hydrogen production. To improve H(2) production, the nitrate assimilation pathway was disrupted by genetic engineering to redirect the electron flow towards the Hox-hydrogenase. Mutant strains disrupted in either nitrate reductase (ΔnarB) or nitrite reductase (ΔnirA) or both nitrate reductase and nitrite reductase (ΔnarB:ΔnirA) were constructed and tested for their ability to produce hydrogen. H(2) production and Hox-hydrogenase activities in all the mutant strains were higher than those in wild-type. Highest H(2) production was observed in the ΔnarB:ΔnirA strain. Small changes were observed for Hox-hydrogenase enzyme activities and only minor changes in transcript levels of hoxH and hoxY were not correlated with H(2) production. The results suggest that the high rate of H(2) production observed in the ΔnarB:ΔnirA strain of the cyanobacterium Synechocystis sp. strain PCC 6803 is the result of redirecting the electron supply from the nitrate assimilation pathway, through genetic engineering, towards the Hox-hydrogenase.

Halotolerant cyanobacterium Aphanothece halophytica contains a betaine transporter active at alkaline pH and high salinity.

ABSTRACT Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow in media of up to 3.0 M NaCl and pH 11. This cyanobacterium can synthesize betaine from glycine by three-step methylation using S-adenosylmethionine as a methyl donor. To unveil the mechanism of betaine uptake and efflux in this alkaliphile, we isolated and characterized a betaine transporter. A gene encoding a protein (BetTA. halophytica) that belongs to the betaine-choline-carnitine transporter (BCCT) family was isolated. Although the predicted isoelectric pH of a typical BCCT family transporter, OpuD of Bacillus subtilis, is basic, 9.54, that of BetTA. halophytica is acidic, 4.58. BetTA. halophytica specifically catalyzed the transport of betaine. Choline, γ-aminobutyric acid, betaine aldehyde, sarcosine, dimethylglycine, and amino acids such as proline did not compete for the uptake of betaine by BetTA. halophytica. Sodium markedly enhanced betaine uptake rates, whereas potassium and other cations showed no effect, suggesting that BetTA. halophytica is a Na+-betaine symporter. Betaine uptake activities of BetTA. halophytica were high at alkaline pH values, with the optimum pH around 9.0. Freshwater Synechococcus cells overexpressing BetTA. halophytica showed NaCl-activated betaine uptake activities with enhanced salt tolerance, allowing growth in seawater supplemented with betaine. Kinetic properties of betaine uptake in Synechococcus cells overexpressing BetTA. halophytica were similar to those in A. halophytica cells. These findings indicate that A. halophytica contains a Na+-betaine symporter that contributes to the salt stress tolerance at alkaline pH. BetTA. halophytica is the first identified transporter for compatible solutes in cyanobacteria.

Characterization of UV‐screening compounds, mycosporine‐like amino acids, and scytonemin in the cyanobacterium Lyngbya sp. CU2555

Ultraviolet-screening compounds from the cyanobacterium Lyngbya sp. CU2555 were partially characterized and investigated for their induction by UV radiation, stability under different abiotic factors, and free radical scavenging activity. Based on the high-performance liquid chromatography coupled with diode array detector and ion trap liquid chromatography/mass spectrometry analysis, the compounds were identified as palythine (UVλ max: 319 nm; m/z: 245), asterina (UVλ max: 330 nm; m/z: 289), scytonemin (UVλ max: 384 nm; mw: 544), and reduced scytonemin (UVλ max: 384 nm; m/z: 547). This is the first report for the occurrence of palythine, asterina, and an unknown mycosporine-like amino acids (MAA), M-312 (UVλ max: 312 ± 1 nm), in addition to scytonemin and reduced scytonemin in Lyngbya strains studied so far. Induction of MAAs and scytonemin was significantly more prominent upon exposure to UV-A + UV-B radiation. Both MAAs and scytonemin were highly resistant to some physicochemical factors such as UV-B, heat, and a strong oxidizing agent and exhibited strong antioxidant activity. These results indicate that the studied cyanobacterium may protect itself from deleterious short-wavelength radiation by synthesizing photoprotective compounds in response to harmful UV radiation.

Enhanced accumulation of glycogen, lipids and polyhydroxybutyrate under optimal nutrients and light intensities in the cyanobacterium Synechocystis sp. PCC 6803.

Glycogen (GL) and lipids (LP) are efficient biofuel substrates, whereas polyhydroxybutyrate (PHB) is a potent biodegradable plastic. This study aimed to increase accumulation of these three compounds in Synechocystis sp. PCC 6803.

Inactivation of uptake hydrogenase leads to enhanced and sustained hydrogen production with high nitrogenase activity under high light exposure in the cyanobacterium Anabaena siamensis TISTR 8012

BackgroundBiohydrogen from cyanobacteria has attracted public interest due to its potential as a renewable energy carrier produced from solar energy and water. Anabaena siamensis TISTR 8012, a novel strain isolated from rice paddy field in Thailand, has been identified as a promising cyanobacterial strain for use as a high-yield hydrogen producer attributed to the activities of two enzymes, nitrogenase and bidirectional hydrogenase. One main obstacle for high hydrogen production by A. siamensis is a light-driven hydrogen consumption catalyzed by the uptake hydrogenase. To overcome this and in order to enhance the potential for nitrogenase based hydrogen production, we engineered a hydrogen uptake deficient strain by interrupting hupS encoding the small subunit of the uptake hydrogenase.ResultsAn engineered strain lacking a functional uptake hydrogenase (∆hupS) produced about 4-folds more hydrogen than the wild type strain. Moreover, the ∆hupS strain showed long term, sustained hydrogen production under light exposure with 2–3 folds higher nitrogenase activity compared to the wild type. In addition, HupS inactivation had no major effects on cell growth and heterocyst differentiation. Gene expression analysis using RT-PCR indicates that electrons and ATP molecules required for hydrogen production in the ∆hupS strain may be obtained from the electron transport chain associated with the photosynthetic oxidation of water in the vegetative cells. The ∆hupS strain was found to compete well with the wild type up to 50 h in a mixed culture, thereafter the wild type started to grow on the relative expense of the ∆hupS strain.ConclusionsInactivation of hupS is an effective strategy for improving biohydrogen production, in rates and specifically in total yield, in nitrogen-fixing cultures of the cyanobacterium Anabaena siamensis TISTR 8012.

Halotolerant Cyanobacterium Aphanothece halophytica Contains NapA-Type Na+/H+ Antiporters with Novel Ion Specificity That Are Involved in Salt Tolerance at Alkaline pH

ABSTRACT Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow at NaCl concentrations up to 3.0 M and at pH values up to 11. The genome sequence revealed that the cyanobacterium Synechocystis sp. strain PCC 6803 contains five putative Na+/H+ antiporters, two of which are homologous to NhaP of Pseudomonas aeruginosa and three of which are homologous to NapA of Enterococcus hirae. The physiological and functional properties of NapA-type antiporters are largely unknown. One of NapA-type antiporters in Synechocystis sp. strain PCC 6803 has been proposed to be essential for the survival of this organism. In this study, we examined the isolation and characterization of the homologous gene in Aphanothece halophytica. Two genes encoding polypeptides of the same size, designated Ap-napA1-1 and Ap-napA1-2, were isolated. Ap-NapA1-1 exhibited a higher level of homology to the Synechocystis ortholog (Syn-NapA1) than Ap-NapA1-2 exhibited. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 complemented the salt-sensitive phenotypes of an Escherichia coli mutant and exhibited strongly pH-dependent Na+/H+ and Li+/H+ exchange activities (the highest activities were at alkaline pH), although the activities of Ap-NapA1-2 were significantly lower than the activities of the other polypeptides. Only one these polypeptides, Ap-NapA1-2, complemented a K+ uptake-deficient E. coli mutant and exhibited K+ uptake activity. Mutagenesis experiments suggested the importance of Glu129, Asp225, and Asp226 in the putative transmembrane segment and Glu142 in the loop region for the activity. Overexpression of Ap-NapA1-1 in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942 enhanced the salt tolerance of cells, especially at alkaline pH. These findings indicate that A. halophytica has two NapA1-type antiporters which exhibit different ion specificities and play an important role in salt tolerance at alkaline pH.