Description of Aliinostoc alkaliphilum sp. nov. (Nostocales, Cyanobacteria), a New Bioactive Metabolite-Producing Strain from Salina Verde (Pantanal, Brazil) and Taxonomic Distribution of Bioactive Metabolites in Nostoc and Nostoc-like Genera

Cyanobacteria are a group of oxygenic photosynthetic prokaryotes found in almost all habitats on earth including those characterized as extreme environments. It has been observed that the number of studies dealing with the biodiversity of extremophilic cyanobacteria is limited while studies exploring their bioactive potential are even scarcer. The taxonomy of three Nostoc-like cyanobacterial strains isolated from a shallow lake in Brazil was studied by applying a polyphasic approach. The bioactive potential of the strains was also evaluated using antimicrobial susceptibility testing. The metabolites present in the bioactive HPLC fractions were identified by UPLC/ESI/Q-TOF. Based on our phylogenetic inferences in combination with morphological and ecological information, we describe Aliinostoc alkaliphilum sp. nov., exhibiting antibacterial and antifungal activities. The main bioactive metabolite in all three strains was nocuolin A, which represents the first report of this metabolite in Aliinostoc. Our phylogenetic studies also revealed that many bioactive metabolite-producting strains that are currently assigned to Nostoc belong to other distinct evolutionary lineages. These findings highlight the importance of polyphasic approach studies in both cyanobacterial taxonomy and natural product discovery programs

Genetic and biochemical evidence for redundant pathways leading to mycosporine-like amino acid biosynthesis in the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024

Cyanobacteria have been widely reported to produce a variety of UV-absorbing mycosporine-like amino acids (MAAs). Herein, we reported production of the unusual MAA, mycosporine-glycine-alanine (MGA) in the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 using a newly developed UHPLC-DAD-MS/HRMS (ultra-high performance liquid chromatography-diode array detection-high resolution tandem mass spectrometry) method. MGA had previously been first identified in a red-algae, but S. torques-reginae strain ITEP-024 is the first cyanobacteria to be reported as an MGA producer. Herein, the chemical structure of MGA is fully elucidated from one-dimensional / two-dimensional nuclear magnetic resonance and HRMS data analyses. MAAs are unusually produced constitutively in S. torques-reginae ITEP-024, and this production was further enhanced following UV-irradiance. It has been proposed that MAA biosynthesis proceeds in cyanobacteria from the pentose phosphate pathway intermediate sedoheptulose 7-phosphate. Annotation of a gene cluster encoded in the genome sequence of S. torques-reginae ITEP-024 supports these gene products could catalyse the biosynthesis of MAAs. However, addition of glyphosate to cultures of S. torques-reginae ITEP-024 abolished constitutive and ultra-violet radiation induced production of MGA, shinorine and porphyra-334. This finding supports involvement of the shikimic acid pathway in the biosynthesis of MAAs by this species.Peer reviewe

Genomic and Metabolomic Analyses of Natural Products in Nodularia spumigena Isolated from a Shrimp Culture Pond

The bloom-forming cyanobacterium Nodularia spumigena CENA596 encodes the biosynthetic gene clusters (BGCs) of the known natural products nodularins, spumigins, anabaenopeptins/namalides, aeruginosins, mycosporin-like amino acids, and scytonemin, along with the terpenoid geosmin. Targeted metabolomics confirmed the production of these metabolic compounds, except for the alkaloid scytonemin. Genome mining of N. spumigena CENA596 and its three closely related Nodularia strains—two planktonic strains from the Baltic Sea and one benthic strain from Japanese marine sediment—revealed that the number of BGCs in planktonic strains was higher than in benthic one. Geosmin—a volatile compound with unpleasant taste and odor—was unique to the Brazilian strain CENA596. Automatic annotation of the genomes using subsystems technology revealed a related number of coding sequences and functional roles. Orthologs from the Nodularia genomes are involved in the primary and secondary metabolisms. Phylogenomic analysis of N. spumigena CENA596 based on 120 conserved protein sequences positioned this strain close to the Baltic Nodularia. Phylogeny of the 16S rRNA genes separated the Brazilian CENA596 strain from those of the Baltic Sea, despite their high sequence identities (99% identity, 100% coverage). The comparative analysis among planktic Nodularia strains showed that their genomes were considerably similar despite their geographically distant origin

Convergent evolution of [D-Leucine1] microcystin-LR in taxonomically disparate cyanobacteria

Abstract\ud \ud \ud \ud Background\ud \ud Many important toxins and antibiotics are produced by non-ribosomal biosynthetic pathways. Microcystins are a chemically diverse family of potent peptide toxins and the end-products of a hybrid NRPS and PKS secondary metabolic pathway. They are produced by a variety of cyanobacteria and are responsible for the poisoning of humans as well as the deaths of wild and domestic animals around the world. The chemical diversity of the microcystin family is attributed to a number of genetic events that have resulted in the diversification of the pathway for microcystin assembly.\ud \ud \ud \ud Results\ud \ud Here, we show that independent evolutionary events affecting the substrate specificity of the microcystin biosynthetic pathway have resulted in convergence on a rare [D-Leu1] microcystin-LR chemical variant. We detected this rare microcystin variant from strains of the distantly related genera Microcystis, Nostoc, and Phormidium. Phylogenetic analysis performed using sequences of the catalytic domains within the mcy gene cluster demonstrated a clear recombination pattern in the adenylation domain phylogenetic tree. We found evidence for conversion of the gene encoding the McyA2 adenylation domain in strains of the genera Nostoc and Phormidium. However, point mutations affecting the substrate-binding sequence motifs of the McyA2 adenylation domain were associated with the change in substrate specificity in two strains of Microcystis. In addition to the main [D-Leu1] microcystin-LR variant, these two strains produced a new microcystin that was identified as [Met1] microcystin-LR.\ud \ud \ud \ud Conclusions\ud \ud Phylogenetic analysis demonstrated that both point mutations and gene conversion result in functional mcy gene clusters that produce the same rare [D-Leu1] variant of microcystin in strains of the genera Microcystis, Nostoc, and Phormidium. Engineering pathways to produce recombinant non-ribosomal peptides could provide new natural products or increase the activity of known compounds. Our results suggest that the replacement of entire adenylation domains could be a more successful strategy to obtain higher specificity in the modification of the non-ribosomal peptides than point mutations.This work was supported by Academy of Finland to KS (118637). TKS was funded by the Helsinki Graduate Program in Biotechnology and Molecular Biology, São Paulo Research Foundation (2009/13455-0), Centre for International Mobility (TM-09-6506) and Finnish Cultural Foundation. UK was funded by the Emil Aaltonen Foundation

Quorum sensing detected by atomic force microscopy imaging of corrals surrounding multicellular arrangement of bacteria

Connectivity of the glycocalyx covering of small communities of Acidithiobacillus ferrooxidans bacteria deposited on hydrophilic mica plates was imaged by atomic force microscopy. When part of the coverage was removed by water rinsing, an insoluble structure formed by corrals surrounding each individual bacterium was observed. A collective ring structure with clustered bacteria (>= 3) was observed, which indicates that the bacteria perceived the neighborhood in order to grow a protective structure that results in smaller production of exopolysaccharides material. The most surprising aspect of these collective corral structures was that they occur at a low bacterial cell density. The deposited layers were also analyzed by confocal Raman microscopy and shown to contain polysaccharides, protein, and glucoronic acid

Biomonitoring genotoxicity and cytotoxicity of Microcystis aeruginosa (Chroococcales, Cyanobacteria) using the Allium cepa test

Water pollution caused by toxic cyanobacteria is a problem worldwide, increasing with eutrophication. Due to its biological significance, genotoxicity should be a focus for biomonitoring pollution owing to the increasing complexity of the toxicological environment in which organisms are exposed. Cyanobacteria produce a large number of bioactive compounds, most of which lack toxicological data. Microcystins comprise a class of potent cyclic heptapeptide toxins produced mainly by Microcystis aeruginosa. Other natural products can also be synthesized by cyanobacteria, such as the protease inhibitor, aeruginosin. The hepatotoxicity of microcystins has been well documented, but information on the genotoxic effects of aeruginosins is relatively scarce. In this study, the genotoxicity and ecotoxicity of methanolic extracts from two strains of M. aeruginosa NPLJ-4, containing high levels of microcystin, and M. aeruginosa NPCD-1, with high levels of aeruginosin, were evaluated. Four endpoints, using plant assays in Allium cepa were applied: rootlet growth inhibition, chromosomal aberrations, mitotic divisions, and micronucleus assays. The microcystin content of M. aeruginosa NPLJ-4 was confirmed through ELISA, while M. aeruginosa NPCD-1 did not produce microcystins. The extracts of M. aeruginosa NPLJ-4 were diluted at 0.01, 0.1, 1 and 10 ppb of microcystins: the same procedure was used to dilute M. aeruginosa NPCD-1 used as a parameter for comparison, and water was used as the control. The results demonstrated that both strains inhibited root growth and induced rootlet abnormalities. The strain rich in aeruginosin was more genotoxic, altering the cell cycle, while microcystins were more mitogenic. These findings indicate the need for future research on non-microcystin producing cyanobacterial strains. Understanding the genotoxicity of M. aeruginosa extracts can help determine a possible link between contamination by aquatic cyanobacteria and high risk of primary liver cancer found in some areas as well as establish water level limits for compounds not yet studied. (C) 2012 Elsevier B.V. All rights reserved.State of Sao Paulo Research Foundation [FAPESP 2009/05474-5, 2010/09867-9]CNPq [308299/2009-4, 151931/2010-0

Decolorization of textile dyes by cyanobacteria

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.State of Sao Paulo Research FoundationState of Sao Paulo Research Foundation [FAPESP 2009/05474-5]Brazilian National Research Council [CNPq 559720/2009-2]Brazilian National Research CouncilCNPq [308299/2009-4, 151931/2010-0]CNPqFAPESP [2010/09867-9, 2006/01671-2, 2010/00321-3]FAPES

Effect of a highly concentrated lipopeptide extract of Bacillus subtilis on fungal and bacterial cells

Lipopeptides produced by Bacillus subtilis are known for their high antifungal activity. The aim of this paper is to show that at high concentration they can damage the surface ultra-structure of bacterial cells. A lipopeptide extract containing iturin and surfactin (5 mg mL-1) was prepared after isolation from B. subtilis (strain OG) by solid phase extraction. Analysis by atomic force microscope (AFM) showed that upon evaporation, lipopeptides form large aggregates (0.1-0.2 mu m2) on the substrates silicon and mica. When the same solution is incubated with fungi and bacteria and the system is allowed to evaporate, dramatic changes are observed on the cells. AFM micrographs show disintegration of the hyphae of Phomopsis phaseoli and the cell walls of Xanthomonas campestris and X. axonopodis. Collapses to fungal and bacterial cells may be a result of formation of pores triggered by micelles and lamellar structures, which are formed above the critical micelar concentration of lipopeptides. As observed for P. phaseoli, the process involves binding, solubilization, and formation of novel structures in which cell wall components are solubilized within lipopeptide vesicles. This is the first report presenting evidences that vesicles of uncharged and negatively charged lipopeptides can alter the morphology of gram-negative bacteria.Fundacao de Pesquisa de Sao Paulo (FAPESP)[96/11193-7]Fundacao de Pesquisa de Sao Paulo (FAPESP)[03/12529-4]Conselho Nacional de Pesquisa (CNPq)FAPESP[2004/16042-5

Prozeßorientierte Organisationsgestaltung - Untersuchung der Kundenauftragsklärung in 25 Unternehmen der Investitionsgüterindustrie zeigt Potentiale

Mangrove ecosystems are tropical environments that are characterized by the interaction between the land and the sea. As such, this ecosystem is vulnerable to oil spills. Here, we show a culture-independent survey of fungal communities that are found in the sediments of the following two mangroves that are located on the coast of Sao Paulo State (Brazil): (1) an oil-spill-affected mangrove and (2) a nearby unaffected mangrove. Samples were collected from each mangrove forest at three distinct locations (transect from sea to land), and the samples were analyzed by quantitative PCR and internal transcribed spacer (ITS)-based PCR-DGGE analysis. The abundance of fungi was found to be higher in the oil-affected mangrove. Visual observation and correspondence analysis (CA) of the ITS-based PCR-DGGE profiles revealed differences in the fungal communities between the sampled areas. Remarkably, the oil-spilled area was quite distinct from the unaffected sampling areas. On the basis of the ITS sequences, fungi that are associated with the Basidiomycota and Ascomycota taxa were most common and belonged primarily to the genera Epicoccum, Nigrospora, and Cladosporium. Moreover, the Nigrospora fungal species were shown to be sensitive to oil, whereas a group that was described as "uncultured Basidiomycota" was found more frequently in oil-contaminated areas. Our results showed an increase in fungal abundance in the oil-polluted mangrove regions, and these data indicated potential fungal candidates for remediation of the oil-affected mangroves