Leptolyngbya Boryana Research Articles

<p><strong><em>Euryhalinema mangrovii gen. nov., sp. nov. </em></strong><strong>and <em>Leptoelongatus litoralis gen. nov., sp. nov.</em> (Leptolyngbyaceae) isolated from an Indian mangrove forest</strong></p>

Molecular data based revision of Leptolyngbya, the largest polyphyletic genus of the family Leptolyngbyaceae (Synechococcales) is imperative. Polyphasic approach to the taxonomic analysis of two (AP9F and AP25) cyanobacteria, tentatively designated positions in the “LPP-group” is described. Cell shapes of AP9F and AP25 were highly elongated whereas the cells of the reference strains (Leptolyngbya boryana and Nodosilinea nodulosa) were occasionally elongated to isodiametrical. Terminal cells of AP9F and AP25 appeared as flattened corners (not rounded), which was different from other Leptolyngbyaceae members. 16S rRNA gene sequences of AP9F (1366 bp) and AP25 (1408 bp) showed 95% and 92% similarities respectively with the non-redundant nucleotide sequences of their closest relatives of the Leptolyngbya genus. Test strains were located in the phylogenetic tree in a clade different from the ones containing the type species. A single operon having both tRNAile and tRNAala genes were present in the ITS regions of AP9F and AP25 compared to two operons in the ITS region of the genera Leptolyngbya and Nodosilinea: one having both tRNAile and tRNAala genes and another lacking both the genes. The secondary structures of the traditionally conservative D-stem region as well as the Box B helix and V3 regions of the ITS operons significantly varied between the test strains and also when compared with the corresponding sequences of L. boryana and N. nodulosa. Molecular phylogenetic and morphological data suggested AP9F and AP25 to be monophyletic taxa for which the names Euryhalinema mangrovii gen. nov., sp. nov. and Leptoelongatus litoralis gen. nov., sp. nov. are proposed respectively.

Antiviral activity of five filamentous cyanobacteria against coxsackievirus B3 and rotavirus.

Coxsackievirus B3 (CVB3) and rotavirus (RV) are pathogens of some chronic human diseases. The aim of this study was to determine in vitro antiviral activity of some cyanobacteria against RV and CVB3 infections. Five cyanobacteria were collected from Egypt, identified, and analyzed biochemically. Then, the inhibition of the cytopathic effect of RV and CVB3 viruses by cyanobacterial extracts was examined. Methanol extract of the cyanobacterial isolates showed high antiviral activity against CVB3 with Therapeutic index (TI) of 50.0, 30.0, 27.6, 16.6, and 20.0 for Leptolyngbya boryana, Arthrospira platensis, Nostoc punctiforme, Oscillatoria sp., and Leptolyngbya sp., respectively. The extracts reduced CVB3 titers comparing to 50% tissue culture infectious doses (TCID50) with values 3.25-5.75 log10 of TCID50. Moreover, extracts of A. platensis, and Oscillatoria sp. exhibited high antiviral activity against RV with TI values of 45 and 42.5, respectively, and a reduction in virus titers by 5.75 log10 and 5.5 log10 of TCID50, respectively. Extracts of L. boryana, Leptolyngbya sp., and N. punctiforme had a moderate to low antiviral activity against RV with TI ranging between 2.8 and 7, respectively, and a reduction in virus titers between 0.5 log10 and 1.5 log10 of TCID50, respectively. This study concluded that extracts of five cyanobacterial isolates possess a potent antiviral effect against CVB3 and RV, making them promising sources of new safe antiviral drugs.

Organization and regulation of cyanobacterial nif gene clusters: implications for nitrogenase expression in plant cells.

For over 50 years scientists have considered the possibility of engineering a plant with nitrogen fixation capability, freeing farmers from their dependence on nitrogen fertilizers. With the development of the tools of synthetic biology, more progress has been made toward this goal in the last 5 years than in the previous five decades. Most of the effort has focused on nitrogenase genes from Klebsiella oxytoca, which has complex gene regulation. There may be advantages in using nitrogenase genes from cyanobacteria, which comprise large polycistronic gene clusters that may be easier to manipulate and eventually express in a plant. The fact that some diatoms have a cyanobacterial nitrogen fixing organelle further supports the idea that a cyanobacterial nitrogenase gene cluster may function in a newly-engineered, cyanobacterial-based plant organelle, a nitroplast. This review describes recent attempts to express the nif genes from Anabaena variabilis ATCC 29413, Leptolyngbya boryana dg5 and Cyanothece sp. ATCC 51142 in heterologous cyanobacteria in the context of the organization of the nitrogenase genes and their regulation by the transcription factor CnfR via its highly conserved binding sites.

Accessory Proteins of the Nitrogenase Assembly, NifW, NifX/NafY, and NifZ, Are Essential for Diazotrophic Growth in the Nonheterocystous Cyanobacterium Leptolyngbya boryana.

Since nitrogenase is extremely vulnerable to oxygen, aerobic or micro-aerobic nitrogen-fixing organisms need to create anaerobic microenvironments in the cells for diazotrophic growth, which would be one of the major barriers to express active nitrogenase in plants in efforts to create nitrogen-fixing plants. Numerous cyanobacteria are able to fix nitrogen with nitrogenase by coping with the endogenous oxygen production by photosynthesis. Understanding of the molecular mechanisms enabling to the coexistence of nitrogen fixation and photosynthesis in nonheterocystous cyanobacteria could offer valuable insights for the transfer of nitrogen fixation capacity into plants. We previously identified the cnfR gene encoding the master regulator for the nitrogen fixation (nif) gene cluster in the genome of a nonheterocystous cyanobacterium Leptolyngbya boryana, in addition to initial characterization of the nif gene cluster. Here we isolated nine mutants, in which the nif and nif-related genes were individually knocked out in L. boryana to investigate the individual functions of (1) accessory proteins (NifW, NifX/NafY, and NifZ) in the biosynthesis of nitrogenase metallocenters, (2) serine acetyltransferase (NifP) in cysteine supply for iron-sulfur clusters, (3) pyruvate formate lyase in anaerobic metabolism, and (4) NifT and HesAB proteins. ΔnifW, ΔnifXnafY, and ΔnifZ exhibited the most severe phenotype characterized by low nitrogenase activity (<10%) and loss of diazotrophic growth ability. The phenotypes of ΔnifX, ΔnafY, and ΔnifXnafY suggested that the functions of the homologous proteins NifX and NafY partially overlap. ΔnifP exhibited significantly slower diazotrophic growth than the wild type, with lower nitrogenase activity (22%). The other four mutants (ΔpflB, ΔnifT, ΔhesA, and ΔhesB) grew diazotrophically similar to the wild type. Western blot analysis revealed a high correlation between nitrogenase activity and NifD contents, suggesting that NifD is more susceptible to proteolytic degradation than NifK in L. boryana. The phenotype of the mutants lacking the accessory proteins was more severe than that observed in heterotrophic bacteria such as Azotobacter vinelandii, which suggests that the functions of NifW, NifX/NafY, and NifZ are critical for diazotrophic growth of oxygenic photosynthetic cells. L. boryana provides a promising model for studying the molecular mechanisms that produce active nitrogenase, to facilitate the creation of nitrogen-fixing plants.

High diversity of thermophilic cyanobacteria in Rupite hot spring identified by microscopy, cultivation, single-cell PCR and amplicon sequencing.

Genotypic and morphological diversity of cyanobacteria in the Rupite hot spring (Bulgaria) was investigated by means of optical microscopy, cultivation, single-cell PCR, and 16S rRNA gene amplicon sequencing. Altogether, 34 sites were investigated along the 71-39°C temperature gradient. Analysis of samples from eight representative sites shown that Illumina, optical microscopy, and Roche 454 identified 72, 45 and 19% respective occurrences of all cumulatively present taxa. Optical microscopy failed to detect species of minor occurrence; whereas, amplicon sequencing technologies suffered from failed primer annealing and the presence of species with extensive extracellular polysaccharides production. Amplicon sequencing of the 16S rRNA gene V5-V6 region performed by Illumina identified the cyanobacteria most reliably to the generic level. Nevertheless, only the combined use of optical microscopy, cultivation and sequencing methods allowed for reliable estimate of the cyanobacterial diversity. Here, we show that Rupite hot-spring system hosts one of the richest cyanobacterial flora reported from a single site above 50°C. Chlorogloeopsis sp. was the most abundant at the highest temperature (68°C), followed by Leptolyngbya boryana, Thermoleptolyngbya albertanoae, Synechococcus bigranulatus, Oculatella sp., and Desertifilum sp. thriving above 60°C, while Leptolyngbya geysericola, Geitlerinema splendidum, and Cyanobacterium aponinum were found above 50°C.

Functional expression of an oxygen-labile nitrogenase in an oxygenic photosynthetic organism

Transfer of nitrogen fixation ability to plants, especially crops, is a promising approach to mitigate dependence on chemical nitrogen fertilizer and alleviate environmental pollution caused by nitrogen fertilizer run-off. However, the need to transfer a large number of nitrogen fixation (nif) genes and the extreme vulnerability of nitrogenase to oxygen constitute major obstacles for transfer of nitrogen-fixing ability to plants. Here we demonstrate functional expression of a cyanobacterial nitrogenase in the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803). A 20.8-kb chromosomal fragment containing 25 nif and nif-related genes of the diazotrophic cyanobacterium Leptolyngbya boryana was integrated into a neutral genome site of Synechocystis 6803 by five-step homologous recombination together with the cnfR gene encoding the transcriptional activator of the nif genes to isolate CN1. In addition, two other transformants CN2 and CN3 carrying additional one and four genes, respectively, were isolated from CN1. Low but significant nitrogenase activity was detected in all transformants. This is the first example of nitrogenase activity detected in non-diazotrophic photosynthetic organisms. These strains provide valuable platforms to investigate unknown factors that enable nitrogen-fixing growth of non-diazotrophic photosynthetic organisms, including plants.

In vivo transposon tagging in the nonheterocystous nitrogen-fixing cyanobacterium Leptolyngbya boryana.

Nitrogenase is an oxygen-vulnerable metalloenzyme that catalyzes nitrogen fixation. It largely remains unknown how nitrogenase coexists with oxygenic photosynthesis in nonheterocystous cyanobacteria, since there have been no appropriate model cyanobacteria so far. Here, we demonstrate in vivo transposon tagging in the nonheterocystous cyanobacterium Leptolyngbya boryana as a forward genetics approach. By conjugative transfer, a mini-Tn5-derived vector, pKUT-Tn5-Sm/Sp, was transferred from Escherichia coli to L. boryana cells. Of 1839 streptomycin-resistant colonies, we isolated three mutants showing aberrant diazotrophic growth. Genome resequencing identified the insertion sites of the transposon in the mutants. This in vivo transposon tagging mutagenesis of L. boryana provides a promising system to investigate molecular mechanisms to resolve the Oxygen Paradox between nitrogen fixation and oxygenic photosynthesis in cyanobacteria.

The Effect of Two Amino acid Residue Substitutions via RNA Editing on Dark-operative Protochlorophyllide Oxidoreductase in the Black Pine Chloroplasts

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a key enzyme to produce chlorophyll in the dark. Among photosynthetic eukaryotes, all three subunits chlL, chlN, and chlB are encoded by plastid genomes. In some gymnosperms, two codons of chlB mRNA are changed by RNA editing to codons encoding evolutionarily conserved amino acid residues. However, the effect of these substitutions on DPOR activity remains unknown. We first prepared cyanobacterial ChlB variants with amino acid substitution(s) to mimic ChlB translated from pre-edited mRNA. Their activities were evaluated by measuring chlorophyll content of dark-grown transformants of a chlB-lacking mutant of the cyanobacterium Leptolyngbya boryana that was complemented with pre-edited mimic chlB variants. The chlorophyll content of the transformant cells expressing the ChlB variant from the fully pre-edited mRNA was only one-fourth of the control cells. Co-purification experiments of ChlB with Strep-ChlN suggested that a stable complex with ChlN is greatly impaired in the substituted ChlB variant. We then confirmed that RNA editing efficiency was markedly greater in the dark than in the light in cotyledons of the black pine Pinus thunbergii. These results indicate that RNA editing on chlB mRNA is important to maintain appropriate DPOR activity in black pine chloroplasts.

Identification of a cis-acting element in nitrogen fixation genes recognized by CnfR in the nonheterocystous nitrogen-fixing cyanobacterium Leptolyngbya boryana.

The filamentous cyanobacterium Leptolyngbya boryana has the ability to fix nitrogen without any heterocysts under microoxic conditions. Previously, we identified the cnfR gene for a master transcriptional activator for nitrogen fixation (nif) genes in a 50-kb gene cluster containing nif and nif-related genes in L. boryana. We showed that CnfR activates the transcription of nif genes in response to low oxygen conditions, which allows the oxygen-vulnerable enzyme nitrogenase to function. However, the regulatory mechanism that underlies regulation by CnfR remains unknown. In this study, we identified a conserved cis-acting element that is recognized by CnfR. We established a reporter system in the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 using luciferase genes (luxAB). Reporter analysis was performed with a series of truncated and modified upstream regulatory regions of nifB and nifP. The cis-element can be divided into nine motifs I-IX, and it is located 76 bp upstream of the transcriptional start sites of nifB and nifP. Six motifs of them are essential for transcriptional activation by CnfR. This cis-acting element is conserved in the upstream regions of nif genes in all diazotrophic cyanobacteria, including Anabaena and Cyanothece, thereby suggesting that the transcriptional regulation by CnfR is widespread in nitrogen-fixing cyanobacteria.

Sucrose in bloom-forming cyanobacteria: loss and gain of genes involved in its biosynthesis.

Bloom-forming cyanobacteria are widely distributed in freshwater ecosystems. To cope with salinity fluctuations, cyanobacteria synthesize compatible solutes, such as sucrose, to maintain the intracellular osmotic balance. The screening of cyanobacterial genomes revealed that homologues to sucrose metabolism-related genes only occur in few bloom-forming strains, mostly belonging to Nostocales and Stigonematales orders. Remarkably, among Chroococcales and Oscillatoriales strains, homologues were only found in M. aeruginosa PCC 7806 and Leptolyngbya boryana PCC 6306, suggesting a massive loss of sucrose metabolism in bloom-forming strains of these orders. After a complete functional characterization of sucrose genes in M. aeruginosa PCC 7806, we showed that sucrose metabolism depends on the expression of a gene cluster that defines a transcriptional unit, unique among all sucrose-containing cyanobacteria. It was also demonstrated that the expression of the encoding genes of sucrose-related proteins is stimulated by salt. In view of its ancestral origin in cyanobacteria, the fact that most bloom-forming strains lack sucrose metabolism indicates that the genes involved might have been lost during evolution. However, in a particular strain, like M. aeruginosa PCC 7806, sucrose synthesis genes were probably regained by horizontal gene transfer, which could be hypothesized as a response to salinity fluctuations.

Comparison of the terrestrial cyanobacterium Leptolyngbya sp. NIES-2104 and the freshwater Leptolyngbya boryana PCC 6306 genomes.

The cyanobacterial genus Leptolyngbya is widely distributed throughout terrestrial environments and freshwater. Because environmental factors, such as oxygen level, available water content, and light intensity, vary between soil surface and water bodies, terrestrial Leptolyngbya should have genomic differences with freshwater species to adapt to a land habitat. To study the genomic features of Leptolyngbya species, we determined the complete genome sequence of the terrestrial strain Leptolyngbya sp. NIES-2104 and compared it with that of the near-complete sequence of the freshwater Leptolyngbya boryana PCC 6306. The greatest differences between these two strains were the presence or absence of a nitrogen fixation gene cluster for anaerobic nitrogen fixation and several genes for tetrapyrrole synthesis, which can operate under micro-oxic conditions. These differences might reflect differences in oxygen levels where these strains live. Both strains have the genes for trehalose biosynthesis, but only Leptolyngbya sp. NIES-2104 has genetic capacity to produce a mycosporine-like amino acid, mycosporine-glycine. Mycosporine-glycine has an antioxidant action, which may contribute to adaptation to terrestrial conditions. These features of the genomes yielded additional insights into the classification and physiological characteristics of these strains.

Transformation of the Cyanobacterium Leptolyngbya boryana by Electroporation

Leptolyngbya boryana (L. boryana) (formerly Plectonema boryanum) is a versatile, filamentous cyanobacterium that has the ability to fix nitrogen under microoxic conditions and to grow heterotrophically with glucose in the dark, providing an excellent system to investigate photosynthesis, nitrogen fixation, and their regulatory mechanisms. While L. boryana is not naturally transformable different from the unicellular cyanobacterium Synechocystis sp. PCC 6803, it can be transformed by electroporation. Here we describe the transformation of L. boryana by electroporation to isolate mutants in which a targeted gene is disrupted.

Loss of cytochrome cM stimulates cyanobacterial heterotrophic growth in the dark.

Although cyanobacteria are photoautotrophs, they have the capability for heterotrophic metabolism that enables them to survive in their natural habitat. However, cyanobacterial species that grow heterotrophically in the dark are rare. It remains largely unknown how cyanobacteria regulate heterotrophic activity. The cyanobacterium Leptolyngbya boryana grows heterotrophically with glucose in the dark. A dark-adapted variant dg5 isolated from the wild type (WT) exhibits enhanced heterotrophic growth in the dark. We sequenced the genomes of dg5 and the WT to identify the mutation(s) of dg5. The WT genome consists of a circular chromosome (6,176,364 bp), a circular plasmid pLBA (77,793 bp) and two linear plasmids pLBX (504,942 bp) and pLBY (44,369 bp). Genome comparison revealed three mutation sites. Phenotype analysis of mutants isolated from the WT by introducing these mutations individually revealed that the relevant mutation is a single adenine insertion causing a frameshift of cytM encoding Cyt c(M). The respiratory oxygen consumption of the cytM-lacking mutant grown in the dark was significantly higher than that of the WT. We isolated a cytM-lacking mutant, ΔcytM, from another cyanobacterium Synechocystis sp. PCC 6803, and ΔcytM grew in the dark with a doubling time of 33 h in contrast to no growth of the WT. The respiratory oxygen consumption of ΔcytM grown in the dark was about 2-fold higher than that of the WT. These results suggest a suppressive role(s) for Cyt cM in regulation of heterotrophic activity.

Tolerance of soil algae and cyanobacteria to drought stress.

Tolerance to drought stress in soil crust microorganisms is essential for exploiting suitable organisms for restoring soil. In this study, the responses to drought stress of two drought-tolerant species, a green alga and a cyanobacterium, were compared with those of two non-tolerant green algae. In response to drought stress, induced by treatment with polyethylene glycol, the intracellular proline levels increased and were associated with increases in malondialdehye, pigment contents, and enzyme activities such as superoxide dismutase (SOD) and peroxidase (POD). Our results suggest that tolerance to drought stress could be indicated by the intracellular levels of proline, SOD, and carotenoids. This study provides insights into the drought physiology of the photosynthetic microorganisms and suggests that Leptolyngbya boryana and Chlorella vulgaris are suitable pioneer organisms for soil restoration.

Polyphasic characterization of benthic cyanobacterial diversity from biofilms of the Guadarrama river (Spain): morphological, molecular, and ecological approaches(1).

The occurrence and environmental factors responsible for the distribution of benthic cyanobacteria in running waters remain largely unexplored in comparison with those of other aquatic ecosystems. In this study, combined data of ecological characteristics, molecular analysis (based on 16S rRNA gene), and direct microscopic inspection of environmental samples were analyzed in parallel with the morphological characterization of the isolated strains to investigate benthic cyanobacterial diversity in the Guadarrama river (Spain). A total of 17 species were identified that belonged to the genera Aphanocapsa, Pleurocapsa, Chroococcus, Chamaesiphon, Cyanobium, Pseudan-abaena, Leptolyngbya, Phormidium, Nostoc, and Tolypothrix. Phenotypic features were associated with the results of 16S rRNA gene sequencing, complementing existing morphological and genetic databases. A decrease in the cyanobacterial diversity was observed along a pollution gradient in the river. Water quality differed among the sampling sites, and variation in nutrient content was the principal difference among locations. These characteristics were closely associated with an upstream-downstream eutrophic gradient. Canonical correspondence analysis distinguished three groups of species with respect to the eutrophication gradient. The first group (Tolypothrix cf. tenuis, Nostoc punctiforme, Nostoc piscinale, Chamaesiphon investiens, Chroococcus minor, Leptolyngbya nostocorum, and Leptolyngbya tenuis) was characteristic of waters with low levels of nutrients. The second group (Cyanobium sp., Chamaesiphon polymorphus, Leptolyngbya boryana, Phormidium autumnale, Phormidium sp., and Aphanocapsa cf. rivularis) was characteristic of polluted waters, its members appearing mainly in great abundance under eutrophic-hypertrophic conditions. The third group of species (Pseudanabaena catenata, Aphanocapsa muscicola, and Nostoc carneum) was present at upstream and downstream sites.

A new structural insight into differential interaction of cyanobacterial and plant ferredoxins with nitrite reductase as revealed by NMR and X-ray crystallographic studies

Ferredoxin (Fd), which plays a pivotal role in photosynthesis as an electron carrier, forms a transient complex with various Fd-dependent enzymes, such as nitrite reductase (NiR), to achieve efficient intermolecular electron transfer. We studied the protein-protein interaction of Fd and NiR by NMR spectroscopy and determined three acidic regions of Fd to be major sites for the interaction with NiR, indicating that the complex is stabilized through electrostatic interaction. During this study, we found Fds from higher plant and cyanobacterium, in spite of their high structural similarities including the above acidic regions, differ remarkably in the interaction with cyanobacterial NiR. In activity assay of NiR, K(m) value for maize Fd (74.6 µM) was 9.6 times larger than that for Leptolyngbya boryana Fd (7.8 µM). The two Fds also showed a similar difference in binding assay to NiR-immobilized resin. Comparative site-specific mutagenesis of two Fds revealed that their discriminative ability for the interaction with NiR is attributed mainly to non-charged residues in the peripheral region of [2Fe-2S] cluster. These non-charged residues are conserved separately between Fds of plant and cyanobacterial origins. Our data highlight that intermolecular force(s) other than electrostatic attraction is(are) also crucial for the molecular interaction between Fd and partner enzyme.

Crystallization and preliminary X-ray studies of an electron-transfer complex of ferredoxin and ferredoxin-dependent glutamate synthase from the cyanobacterium Leptolyngbya boryana.

Ferredoxin (Fd) dependent glutamate synthase (Fd-GOGAT) is a key enzyme involved in nitrogen assimilation that catalyzes the two-electron reductive conversion of Gln and 2-oxoglutarate to two molecules of Glu. Fd serves as an electron donor for Fd-GOGAT and the two proteins form a transient electron-transfer complex. In this study, these two proteins were cocrystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed at 2.65 Å resolution. The crystals belonged to space group P4(3), with unit-cell parameters a = b = 84.95, c = 476.31 Å.

Functional Evaluation of a Nitrogenase-Like Protochlorophyllide Reductase Encoded by the Chloroplast DNA of Physcomitrella patens in the Cyanobacterium Leptolyngbya boryana

Dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) is a nitrogenase-like enzyme consisting of the two components, L-protein (a ChlL dimer) and NB-protein (a ChlN-ChlB heterotetramer), to catalyze Pchlide reduction in Chl biosynthesis. While nitrogenase is distributed only among certain prokaryotes, the probable structural genes for DPOR are encoded by chloroplast DNA in lower plants. Here we show functional evaluation of DPOR encoded by chloroplast DNA in a moss Physcomitrella patens by the complementation analysis of the cyanobacterium Leptolyngbya boryana and the heterologous reconstitution of the moss L-protein and the cyanobacterial NB-protein. Two shuttle vectors to overexpress chlL and chlN-chlB from P. patens were introduced into the cyanobacterial chlL- and chlB-lacking mutants, respectively. Both transformants restored the ability to perform Chl biosynthesis in the dark, indicating that the chloroplast-encoded DPOR components form an active complex with the cyanobacterial components. The L-protein of P. patens was purified from the cyanobacterial transformant, and DPOR activity was reconstituted in a heterologous combination with the cyanobacterial NB-protein. The specific activity of the L-protein from P. patens was determined to be 118 nmol min(-1) mg (-1), which is even higher than that of the cyanobacterial L-protein (76 nmol min(-1) mg (-1)). Upon exposure to air, the activity of the L-protein from P. patens decayed with a half-life of 30 s, which was eight times faster than that of the cyanobacterial L-protein (240 s). These results suggested that the chloroplast-encoded L-protein functions as efficiently as the cyanobacterial L-protein but is more oxygen labile than the cyanobacterial L-protein.

Nitrate Assimilatory Genes and Their Transcriptional Regulation in a Unicellular Red Alga Cyanidioschyzon merolae: Genetic Evidence for Nitrite Reduction by a Sulfite Reductase-Like Enzyme

Cyanidioschyzon merolae is a unicellular red alga living in acid hot springs, which is able to grow on ammonium, as well as nitrate as sole nitrogen source. Based on the complete genome sequence, proteins for nitrate utilization, nitrate transporter (NRT) and nitrate reductase (NR), were predicted to be encoded by the neighboring nuclear genes CMG018C and CMG019C, respectively, but no typical nitrite reductase (NiR) gene was found by similarity searches. On the other hand, two candidate genes for sulfite reductase (SiR) were found, one of which (CMG021C) is located next to the above-noted nitrate-related genes. Given that transcripts of CMG018C, CMG019C and CMG021C accumulate in nitrate-containing media, but are repressed by ammonium, and that SiR and NiR are structurally related enzymes, we hypothesized that the CMG021C gene product functions as an NiR in C. merolae. To test this hypothesis, we developed a method for targeted gene disruption in C. merolae. In support of our hypothesis, we found that a CMG021G null mutant in comparison with the parental strain showed decreased cell growth in nitrate-containing but not in ammonium-containing media. Furthermore, expression of CMG021C in the nirA mutant of a cyanobacterium, Leptolyngbya boryana (formerly Plectonema boryanum), could genetically complement the NiR defect. Immunofluorescent analysis indicated the localization of CMG021C in chloroplasts, and hence we propose an overall scheme for nitrate assimilation in C. merolae.

Microcystin production in epiphytic cyanobacteria on submerged macrophytes

Cyanotoxins have been largely studied in planktonic and benthic cyanobacteria, but microcystin (MCYST) production in epiphytic cyanobacteria has not been reported yet. The present study reports for the first time the MCYST production in epiphytic cyanobacteria on submerged macrophytes. During this study, four common submerged macrophytes in eutrophic pond in Saudi Arabia were surveyed for the presence of toxic epiphytic cyanobacteria. The results showed that chlorophyll-a and total biovolume of epiphytic cyanobacteria differed significantly among submerged plants with highest values obtained in Stratiotes aloides and lowest in Elodea canadensis. Epiphytic materials collected from Ceratophyllum demersum and S. aloides had higher species diversities than materials collected from E. canadensis and Myriophyllum verticillatum. The cyanobacteria, Merismopedia tenuissima and Leptolyngbya boryana were recorded with a high abundance in epiphytic materials collected from all submerged macrohpytes. Based on Enzyme-linked immunosorbent assay (ELISA), these two species were found to produce MCYSTs (MCYSTs) with concentrations of 1438 and 630 μg g −1 dry weight, respectively. HPLC analysis of the methanolic extracts of the two species showed that M. tenuissima extract contained MCYST-RR and -LR/demethyl LR plus 3 minor unidentified MCYSTs, while L. boryana extract contained MCYST-YR, -LR/demethyl LR, and 2 minor unidentified MCYSTs. This study suggests that epiphytic species should be considered during monitoring of toxic cyanobacteria in water sources.