Viviana R. Lopes

Phylogenetic, chemical and morphological diversity of cyanobacteria from Portuguese temperate estuaries.

Cyanobacteria from estuarine habitats have been poorly studied regarding diversity and potential bioactive compounds production compared with their fresh and marine waters congeners. In this work, 44 cyanobacteria isolates characterised from three Portuguese estuarine environments. Identification was performed based on diacritical morphological features of the isolates (e.g. cell shape, cell size, presence/absence of sheaths) and on 16S rRNA gene sequences phylogenetic analysis. Diversity of produced secondary metabolites was assessed by molecular and analytical tools. The isolates (mostly benthic forms) belonged to: (i) Chroococcales (Cyanobium, Synechocystis and Synechococcus), (ii) Oscillatoriales (Leptolyngbya, Microcoleus, Phormidium and Romeria) and (iii) Nostocales (Nostoc and Nodularia). 19 morphotypes were assigned at the species level, while phylogeny allowed us to distinguish 21 phylotypes spread amongst three distinct large clades. McyA and sxtI gene fragments were detected in some isolates, despite absence of toxins. Simultaneous presence of anabaenopeptins A and D was for the first time identified in Nostoc (LEGE06077). No correlation between morphological/phylogenetic relationships and the secondary-metabolite profile of the isolates was found. This is the first comprehensive study of estuarine cyanobacteria of Portuguese habitats revealing a diverse array of cyanobacteria that constitute an important source of potential bioactive compounds with ecological relevance and/or biomedical application.

The non-protein amino acid β-N-methylamino-L-alanine in Portuguese cyanobacterial isolates.

The tailor made amino acid β-N-methyl-amino-l-alanine (BMAA) is a neurotoxin produced by cyanobacteria. It has been associated with certain forms of progressive neurodegenerative disease, including sporadic Amyotrophic Lateral Sclerosis and Alzheimer’s disease. Some different reports of BMAA in cyanobacterial blooms from lakes, reservoirs, and other water resources have been made by different investigators. We here report the detection of BMAA of both free and protein-bound produced by cyanobacteria, belonging to the Chroococcales, Oscillatoriales and Nostocales ordered. We use a rapid and sensitive HPLC-FD method that utilizes methanol elution and the Waters AQC Tag chemistry. On other hand, we have used three different assay procedures for BMAA extraction from cyanobacteria: Trichloroacetic acid (TCA), Methanol/Acetone and hydrochloric acid (HCl). All assays let successfully detect BMAA in all cyanobacteria samples analyzed. Nevertheless, with TCA and HCl extraction procedures the highest BMAA values, for free as well as protein-bound BMAA were detected. BMAA content could not be related to the taxonomy of the isolates or to their geographical origin, and no correlation between free and protein-bound BMAA concentrations were observed within or between taxonomic groups. These data offer confirmation of the taxonomic and geographic ubiquity of BMAA from naturally occurring populations of cyanobacteria, for the first time reported for estuaries.

Determination of the non protein amino acid β-N-methylamino-L-alanine in estuarine cyanobacteria by capillary electrophoresis

A capillary electrophoretic method for the determination of the amino acid β-N-methylamino-l-alanine (BMAA) was achieved using a fused-silica capillary column (50xa0cmxa0×xa075xa0μm I.D.) filled with 5xa0mM sodium tetraborate solution (pH 9), with an applied voltage of 25xa0kV, at 25xa0°C. The method was then applied in quantifying BMAA in eighteen strains of lyophilized estuarine cyanobacteria, following amino acid extraction using 0.1xa0M trichloroacetic acid and 6xa0M hydrochloric acid, sequentially.

Reversed-phase HPLC/FD method for the quantitative analysis of the neurotoxin BMAA (β-N-methylamino-l-alanine) in cyanobacteria

A method has been developed and optimized in order to detect and quantify the non-protein amino acid β-N-methylamino-L-alanine(BMAA) in cyanobacteria. The novelty of the method is that we have used methanol instead of acetonitrile as the eluent. The method includes extraction with 0.1 M trichloroacetic acid (free BMAA) or protein hydrolysis with 6 M hydrochloric acid (total BMAA), derivatization with AQC (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) and reversed-phase high-performance liquid chromatography analysis with fluorescence detection (HPLC/FD). Detection limits ranged from 0.35 to 0.75 pg injected, while quantification limits ranged from 1.10 to 2.55 pg injected for total and free BMAA hydrolysis, respectively. The linear response range was up to 850 pmol in both methods, embracing three orders of magnitude. The method was successfully applied to a lyophilized estuarine species of Nostoc (LEGE 06077). All previous published methods for BMAA quantification, using HPLC/FD, have reported the usage of acetonitrile. This is the first report using methanol as the mobile phase. Although the elution strength differs with both solvents, the final method proved efficient for the quantification of BMAA in this complex sample. The method resulted effective, low-priced, and simple, being suitable for routine monitoring of BMAA in cyanobacteria.

Planktonic and benthic cyanobacteria of European brackish waters: a perspective on estuaries and brackish seas

Cyanobacteria are photosynthetic organisms found in aquatic and terrestrial biotopes and ecosystems throughout the world. Marine and freshwater cyanobacteria have been extensively studied due to the toxic hazards that some of them create and biotechnological interest. In contrast, the cyanobacteria of brackish waters have been less studied despite being able to form toxic blooms like their freshwater and marine counterparts. We review the occurrence, diversity and toxicity of cyanobacteria species in the brackish waters of Europe, mainly estuaries and brackish environments of the Atlantic Ocean and the Mediterranean and Baltic Seas. The dominant cyanobacteria belong mainly to the planktonic genera Nodularia, Aphanizomenon, Microcystis and Anabaena, but also the benthic forms of Anabaena and Phormidium. Most genera from brackish waters are reported to be hepatotoxin producers (microcystin and nodularin). However, anatoxin-a and other bioactive compounds (e.g. apoptogens) can also be found and are produced exclusively by benthic forms. Nodularin is the best-characterized brackish-water cyanotoxin, being primarily produced by N. spumigena. Data are presented on cyanotoxin production, accumulation, and potential food chain transfer, with a particular focus on nodularin, which induces multiple effects on food-chain dynamics. Microcystins and anatoxin-a are also considered. Potential risks to animals and humans from cyanotoxin exposure are discussed, although reports of animal poisoning by brackish-water toxic cyanobacteria are scarce and no cases of human intoxication are yet known.

Cytotoxicity in L929 fibroblasts and inhibition of herpes simplex virus type 1 Kupka by estuarine cyanobacteria extracts

The cyanobacteria are known to be a rich source of metabolites with a variety of biological activities in different biological systems. In the present work, the bioactivity of aqueous and organic (methanolic and hexane) crude extracts of cyanobacteria isolated from estuarine ecosystems was studied using different bioassays. The assessment of DNA damage on the SOS gene repair region of mutant PQ37 strain of Escherichia coli was performed. Antiviral activity was evaluated against influenza virus, HRV-2, CVB3 and HSV-1 viruses using crystal violet dye uptake on HeLa, MDCK and GMK cell lines. Cytotoxicity evaluation was performed with L929 fibroblasts by MTT assay. Of a total of 18 cyanobacterial isolates studied, only the crude methanolic extract of LEGE 06078 proved to be genotoxic (IF > 1.5) in a dose-dependent manner and other four were putative candidates to induce DNA damage. Furthermore, the crude aqueous extract of LEGE 07085 showed anti- herpes type 1 activity (IC50 = 174.10 μg dry extract mL(-1)) while not presenting any cytotoxic activity against GMK cell lines. Of the 54 cyanobacterial extracts tested, only the crude methanolic and hexane ones showed impair on metabolic activity of L929 fibroblasts after long exposure (48-72 h). The inhibition of HSV-1 and the strong cytotoxicity against L929 cells observed emphasizes the importance of evaluating the impact of those estuarine cyanobacteria on aquatic ecosystem and on human health. The data also point out their potential application in HSV-1 treatment and pharmacological interest.

The conifer biomarkers dehydroabietic and abietic acids are widespread in Cyanobacteria

Terpenes, a large family of natural products with important applications, are commonly associated with plants and fungi. The diterpenoids dehydroabietic and abietic acids are defense metabolites abundant in resin, and are used as biomarkers for conifer plants. We report here for the first time that the two diterpenoid acids are produced by members of several genera of cyanobacteria. Dehydroabietic acid was isolated from two cyanobacterial strains and its identity was confirmed spectroscopically. One or both of the diterpenoids were detected in the cells of phylogenetically diverse cyanobacteria belonging to four cyanobacterial ‘botanical orders’, from marine, estuarine and inland environments. Dehydroabietic acid was additionally found in culture supernatants. We investigated the natural role of the two resin acids in cyanobacteria using ecologically-relevant bioassays and found that the compounds inhibited the growth of a small coccoid cyanobacterium. The unexpected discovery of dehydroabietic and abietic acids in a wide range of cyanobacteria has implications for their use as plant biomarkers.

Bioactivity of Benthic and Picoplanktonic Estuarine Cyanobacteria on Growth of Photoautotrophs: Inhibition versus Stimulation

Understanding potential biochemical interactions and effects among cyanobacteria and other organisms is one of the main keys to a better knowledge of microbial population structuring and dynamics. In this study, the effects of cyanobacteria from benthos and plankton of estuaries on other cyanobacteria and green algae growth were evaluated. To understand how the estuarine cyanobacteria might influence the dynamics of phytoplankton, experiments were carried out with the freshwater species Microcystis aeruginosa and Chlorella sp., and the marine Synechocystis salina and Nannochloropsis sp. exposed to aqueous and organic (70% methanol) crude extracts of cyanobacteria for 96 h. The most pronounced effect observed was the growth stimulation. Growth inhibition was also observed for S. salina and M. aeruginosa target-species at the highest and lowest concentrations of cyanobacterial extracts. The methanolic crude extract of Phormidium cf. chalybeum LEGE06078 was effective against S. salina growth in a concentration-dependent manner after 96 h-exposure. All of the cyanobacterial isolates showed some bioactivity on the target-species growth, i.e., inhibitory or stimulating effects. These results indicate that the analyzed cyanobacterial isolates can potentially contribute to blooms’ proliferation of other cyanobacteria and to the abnormal growth of green algae disturbing the dynamic of estuarine phytoplankton communities. Since estuaries are transitional ecosystems, the benthic and picoplanktonic estuarine cyanobacteria can change both freshwater and marine phytoplankton succession, competition and bloom formation. Furthermore, a potential biotechnological application of these isolates as a tool to control cyanobacteria and microalgae proliferation can be feasible. This work is the first on the subject of growth responses of photoautotrophs to cyanobacteria from Atlantic estuarine environments.

Morphological, toxicological and molecular characterization of a benthic Nodularia isolated from Atlantic estuarine environments

A polyphasic study of a benthic Nodularia isolate (LEGE06071) from an Atlantic environment, specifically salt pans, was performed. LEGE06071 resembled both type strains of Nodularia sphaerocarpa and Nodularia harveyana, while ACOI00729 (purchased isolate) was identified as N. sphaerocarpa. The length and width of vegetative cells varied from 3.10 to 3.15 microm and from 3.71 to 4.25 microm, respectively, while heterocyts were 3.91-4.89 microm long and 4.20-4.74 microm wide. None of the isolates had aerotopes. The sequencing of the 16S rRNA gene from the two Nodularia isolates indicated that they belonged neither to Nodularia spumigena nor N. harveyana. Nodularin and other cyanotoxin synthesis-associated genes could not be detected, nor could nodularin production be detected by ELISA. However, MALDI-TOF analysis of LEGE06071 revealed the presence of other compounds, namely, glycolipids. Hence, toxicological screenings against Artemia nauplii, Escherichia coli and Salmonella typhimurium were performed. Toxic effects could only be observed against Artemia, with 48 h-LC(50) values for the aqueous and crude extract of methanol of 53.21 mg ml(-1) and 17.81 mg ml(-1), respectively. This study presents the first evidence of a non-nodularin-producing Nodularia isolate in Atlantic salt pan ecosystems and its potential ecotoxicity against Artemia sp.

Cyanobacterial diversity held in microbial biological resource centers as a biotechnological asset: the case study of the newly established LEGE culture collection

Cyanobacteria are a well-known source of bioproducts which renders culturable strains a valuable resource for biotechnology purposes. We describe here the establishment of a cyanobacterial culture collection (CC) and present the first version of the strain catalog and its online database (http://lege.ciimar.up.pt/). The LEGE CC holds 386 strains, mainly collected in coastal (48%), estuarine (11%), and fresh (34%) water bodies, for the most part from Portugal (84%). By following the most recent taxonomic classification, LEGE CC strains were classified into at least 46 genera from six orders (41% belong to the Synechococcales), several of them are unique among the phylogenetic diversity of the cyanobacteria. For all strains, primary data were obtained and secondary data were surveyed and reviewed, which can be reached through the strain sheets either in the catalog or in the online database. An overview on the notable biodiversity of LEGE CC strains is showcased, including a searchable phylogenetic tree and images for all strains. With this work, 80% of the LEGE CC strains have now their 16S rRNA gene sequences deposited in GenBank. Also, based in primary data, it is demonstrated that several LEGE CC strains are a promising source of extracellular polymeric substances (EPS). Through a review of previously published data, it is exposed that LEGE CC strains have the potential or actual capacity to produce a variety of biotechnologically interesting compounds, including common cyanotoxins or unprecedented bioactive molecules. Phylogenetic diversity of LEGE CC strains does not entirely reflect chemodiversity. Further bioprospecting should, therefore, account for strain specificity of the valuable cyanobacterial holdings of LEGE CC.