Diego Bonaldo Genuário

Microcystin production by a freshwater spring cyanobacterium of the genus Fischerella.

We investigated the production of a hepatotoxic, cyclic heptapeptide, microcystin, by a filamentous branched cyanobacterium belonging to the order Stigonematales, genus Fischerella. The freshwater Fischerella sp. strain CENA161 was isolated from spring water in a small concrete dam in Piracicaba, São Paulo State, Brazil, and identified by combining a morphological description with 16S rRNA gene sequencing and phylogenetic analysis. Microcystin (MCYST) analysis performed using an ELISA assay on cultured cells gave positive results. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis detected 33.6microg MCYST-LR per gram dry weight of cyanobacterial cells. Microcystin profile revealed by quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS) analysis confirmed the production of MCYST-LR. Furthermore, genomic DNA was analyzed by PCR for sequences similar to the ketosynthase (KS) domain of the type I polyketide synthase gene, which is involved in microcystin biosynthesis. This revealed the presence of a KS nucleotide fragment similar to the mcyD and ndaD genes of the microcystin and nodularin synthetase complexes. Phylogenetic analysis grouped the Fischerella KS sequence together with mcyD sequences of the three known microcystin synthetase operon (Microcystis, Planktothrix and Anabaena) and ndaD of the nodularin synthetase operon, with 100% bootstrap support. Our findings demonstrate that Fischerella sp. CENA161 produces MYCST-LR and for the first time identify a nucleotide sequence putatively involved in microcystin synthesis in this genus.

Characterization of a microcystin and detection of microcystin synthetase genes from a Brazilian isolate of Nostoc

A nostocalean nitrogen-fixing cyanobacterium isolated from an eutrophic freshwater reservoir located in Piracicaba, São Paulo, Brazil, was evaluated for the production of hepatotoxic cyclic heptapeptides, microcystins. Morphologically this new cyanobacterium strain appears closest to Nostoc, however, in the phylogenetic analysis of 16S rRNA gene it falls into a highly stable cluster distantly only related to the typical Nostoc cluster. Extracts of Nostoc sp. CENA88 cultured cells, investigated using ELISA assay, gave positive results and the microcystin profile revealed by ESI-Q-TOF/MS/MS analysis confirmed the production of [Dha(7)]MCYST-YR. Further, Nostoc sp. CENA88 genomic DNA was analyzed by PCR for sequences of mcyD, mcyE and mcyG genes of microcystin synthetase (mcy) cluster. The result revealed the presence of mcyD, mcyE and mcyG genes with similarities to those from mcy of Nostoc sp. strains 152 and IO-102-I and other cyanobacterial genera. The phylogenetic tree based on concatenated McyG, McyD and McyE amino acids clustered the sequences according to cyanobacterial genera, with exception of the Nostoc sp. CENA88 sequence, which was placed in a clade distantly related from other Nostoc strains, as previously observed also in the 16S rRNA phylogenetic analysis. The present study describes for the first time a Brazilian Nostoc microcystin producer and also the occurrence of demethyl MCYST-YR variant in this genus. The sequenced Nostoc genes involved in the microcystin synthesis can contribute to a better understanding of the toxigenicity and evolution of this cyanotoxin.

NONHETEROCYTOUS CYANOBACTERIA FROM BRAZILIAN SALINE-ALKALINE LAKES

Saline‐alkaline lakes are extreme environments that limit the establishment and development of life. The Nhecolândia, a subregion of the Pantanal wetland in Brazil, is characterized by the existence of ~500 saline‐alkaline lakes, which support an underexplored and rich diversity of microorganisms. In this study, unicellular and homocytous cyanobacteria from five saline‐alkaline lakes were accessed by culture‐dependent approaches. Morphological evaluation and analyses of near complete sequences (~1400 nt) of the 16S rRNA genes were applied for phylogenetic and taxonomic placement. This polyphasic approach allowed for the determination of the taxonomic position of the isolated strains into the following genera: Cyanobacterium, Geminocystis, Phormidium, Leptolyngbya, Limnothrix, and Nodosilinea. In addition, fourteen Pseudanabaenales and Oscillatoriales representatives of putative novel taxa were found. These sequences fell into five new clades that could correspond to new generic units of the Pseudanabaenaceae and Phormidiaceae families.

Phylogeny of culturable cyanobacteria from Brazilian mangroves

The cyanobacterial community from Brazilian mangrove ecosystems was examined using a culture-dependent method. Fifty cyanobacterial strains were isolated from soil, water and periphytic samples collected from Cardoso Island and Bertioga mangroves using specific cyanobacterial culture media. Unicellular, homocytous and heterocytous morphotypes were recovered, representing five orders, seven families and eight genera (Synechococcus, Cyanobium, Cyanobacterium, Chlorogloea, Leptolyngbya, Phormidium, Nostoc and Microchaete). All of these novel mangrove strains had their 16S rRNA gene sequenced and BLAST analysis revealed sequence identities ranging from 92.5 to 99.7% when they were compared with other strains available in GenBank. The results showed a high variability of the 16S rRNA gene sequences among the genotypes that was not associated with the morphologies observed. Phylogenetic analyses showed several branches formed exclusively by some of these novel 16S rRNA gene sequences. BLAST and phylogeny analyses allowed for the identification of Nodosilinea and Oxynema strains, genera already known to exhibit poor morphological diacritic traits. In addition, several Nostoc and Leptolyngbya morphotypes of the mangrove strains may represent new generic entities, as they were distantly affiliated with true genera clades. The presence of non-ribosomal peptide synthetase, polyketide synthase, microcystin and saxitoxin genes were detected in 20.5%, 100%, 37.5% and 33.3%, respectively, of the 44 tested isolates. A total of 134 organic extracts obtained from 44 strains were tested against microorganisms, and 26% of the extracts showed some antimicrobial activity. This is the first polyphasic study of cultured cyanobacteria from Brazilian mangrove ecosystems using morphological, genetic and biological approaches.

Characterization of freshwater benthic biofilm-forming Hydrocoryne (Cyanobacteria) isolates from Antarctica

The aims of this work were to study cyanobacterial isolates resembling the genus Hydrocoryne using a combination of morphology and phylogeny of 16S rRNA and nifH sequences and to investigate genes involved in cyanotoxin and protease inhibitor production. Four new cyanobacterial strains, isolated from biofilm samples collected from King George Island, Antarctica, were studied. In terms of morphology, these new strains share traits similar to true Anabaena morphotypes (benthic ones), whereas phylogenetic analysis of their 16S rRNA gene sequences grouped them with the sequence of the type species Hydrocoryne spongiosa (H. Schwabe ex Bornet and Flahault 1886–1888), but not with sequences of the type species from the genus Anabaena. This cluster is the sister group of Anabaena morphotypes isolated only from the Gulf of Finland. In addition, this cluster is related to two other clusters formed by sequences of Anabaena isolated from different sites. Partial nifH genes were sequenced from two strains and the phylogenetic tree revealed that the Antarctic nifH sequences clustered with sequences from Anabaena. Furthermore, two strains were tested, using PCR with specific primers, for the presence of genes involved in cyanotoxins (microcystin and saxitoxin) and protease inhibitor (aeruginosin, and cyanopeptolin). Only cyanopeptolin was amplified using PCR. These four Hydrocoryne strains are the first to be isolated and sequenced from Antarctica, which improves our knowledge on this poorly defined cyanobacterial genus.

Heterocytous cyanobacteria of the Ulu Peninsula, James Ross Island, Antarctica

The Antarctic cyanobacterial microflora is one of the main components of the diversity of freshwater phototrophic communities in coastal areas. It is little known according to the modern taxonomic criteria (polyphasic approach). Populations of heterocytous cyanobacteria from Ulu Peninsula, the northern part of James Ross Island, NW Weddell Sea, Antarctica, were therefore reviewed. The identified morphospecies were compared with specimens from other localities in maritime Antarctica, especially from the South Shetland Islands. Ecological demands, morphological variations and, if possible, their phylogenetic positions (based on 16S rRNA gene sequencing) of registered populations were analyzed. Altogether, 11 species of heterocytous cyanoprokaryotes were recognized and studied in detail. These species were dominant in the characteristic habitats, and four novel species were described. All have a restricted endemic Antarctic distribution according to both morphology and 16S rRNA gene analyses. Three species of the genus Calothrix, one species of the genus Dichothrix (Rivulariaceae), and four species from the family Tolypotrichaceae were recognized and documented. Few species from this family belong to the recently recognized and revised genera Dactylothamnos and Hassallia, based on molecular analyses. Nodularia quadrata and two species, taxonomically classified to the genus Hydrocoryne (Nostocaceae), were studied. The complex of the genus Nostoc (especially of N. commune) exists as numerous morpho- and ecotypes, and it is diverse phylogenetically, morphologically and ecologically, and will be analyzed in special studies. Our study is important for the exact identification of cyanobacterial microflora in Antarctica, which plays a dominant role in the colonization of deglaciated areas.

Heterocyte-forming cyanobacteria from Brazilian saline-alkaline lakes

Studies investigating the diversity of cyanobacteria from tropical environments are scarce, especially those devoted to the isolation and molecular characterization of the isolated strains. Among the Brazilian biomes, Pantanal has mainly been examined through microscopic observation of environmental samples, resulting in lists of morphotypes without any genetic information. Recently, two studies were conducted evaluating the morphologic and genetic diversity of cultured non-heterocytous cyanobacteria in this biome, which resulted in the separation and description of two novel genera. In order to complement the diversity of cultured cyanobacteria from saline-alkaline lakes in Pantanal, the present study is dedicated to the examination of cultured nitrogen-fixing heterocytous cyanobacteria from this extreme and underexplored environment. A total of fourteen cyanobacterial strains were isolated. According to morphological examination they belong to the order Nostocales and to the subsections IV.I and IV.II, according to the International Code of Nomenclature for Algae, Fungi and Plants and the Bergeys Manual of Systematic Bacteriology, respectively. Phylogenetic evaluation of their 16S rRNA gene sequences resulted in the formation of five clusters. Among them, one is clearly related to the genus Anabaenopsis whilst the remaining clusters may represent new genetic lineages. These novel sequences aid in the delimitation of problematic groups, especially those containing sequences belonging to mixed genera. The application of both morphologic and phylogenetic studies has proven to be an important tool in resolving problematic groups in cyanobacteria systematics. This strategy is essential in order to detect novel cyanobacteria genera from other tropical environments.

Decolorization of textile dyes by cyanobacteria

Cianobacterias estao amplamente distribuidas no ambiente e podem ser uma alternativa eficaz e economica para a remocao de corantes de efluentes de industrias texteis. O presente trabalho investigou o potencial de seis linhagens de cianobacterias de descolorir onze tipos de corantes texteis. A absorcao maxima de cada corante foi verificada usando um espectrofotometro. Espectrometria de massas foi utilizada na verificacao da degradacao e possivel remocao de corantes pelas cianobacterias. Os resultados mostraram que todas as linhagens avaliadas foram capazes de remover indigo, amarelo palanil, amarelo indantreno, azul indantreno, azul dispersol, vermelho indantreno e vermelho dispersol em mais de 50%. O isolado brasileiro Phormidium sp. CENA135 foi capaz de descolorir e remover completamente o indigo azul BANN 30. Este estudo confirmou a capacidade das cianobacterias de descolorir e, possivelmente, degradar corantes texteis estruturalmente diferentes, sugerindo a possibilidade de sua aplicacao em estudos de biorremediacao. Cyanobacteria are widely distributed in the environment and may be an effective and economic alternative for removing dyes from textile industry effluents. The present work investigated the potential of six cyanobacterial strains in decolorizing eleven types of textile dyes. The maximum absorbance of each dye was verified using a spectrophotometer. Mass spectrometry was used to verify the removal and possible degradation of dyes by the cyanobacteria. The results showed that all of the evaluated cyanobacteria were able to remove indigo, palanil yellow, indanthrene yellow, indanthrene blue, dispersol blue, indanthrene red and dispersol red by more than 50%. The Brazilian isolate Phormidium sp. CENA135 was able to decolorize and completely remove indigo blue BANN 30. This study confirmed the capacity of cyanobacteria to decolorize and possibly to structurally degrade different textile dyes, suggesting the possibility of their application in bioremediation studies.

Cyanobacterial community and microcystin production in a recreational reservoir with constant Microcystis blooms

Cyanobacterial blooms are increasing worldwide favored by eutrophic conditions of aquatic ecosystems associated with climatic perturbations. Generally, inland lentic systems are more susceptible to the development of harmful blooms. In the Salto Grande Reservoir (Brazil), Microcystis is the most common bloom-forming genus along with a wide range of co-occurring and less-known cyanobacteria taxa. The cyanobacterial community and microcystin production were studied in Salto Grande Reservoir applying biological, toxicological, and molecular approaches. Thirteen cyanobacterial strains belonging to eight genera were isolated and taxonomically investigated based on morphological traits and phylogenetic analyses of their 16S rRNA gene sequence. The morphotypes identified were, in general, in agreement with their phylogeny. The presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) was investigated using PCR gene amplification, which were detected in 76.9 and 84.4% of the strains, respectively. Positive enzyme-linked immunosorbent assays (ELISA) reactions for microcystins were obtained only from the strain Leptolyngbya sp. CENA129. ELISA and high-performance liquid chromatography (HPLC) analyses of the environmental water samples showed the highest microcystin concentration during the dry and rainy seasons, respectively. This study highlights that microcystin production must be suspected in benthic forms as well as in genera that are morphologically similar but belonging to other evolutionary lineages.