High-quality draft genome sequence of pantanalinema sp. GBBB05, a cyanobacterium from cerrado biome

Cyanobacteria comprise one of the oldest and most diverse phyla in the Bacteria domain and are recognized for their importance in the biosphere evolution. Members of this phylum can be found in a wide variety of environments reflecting their photosynthetic ability, adaptability to various environmental conditions, and diversified metabolism. Such characteristics make cyanobacteria one of the preferred targets for research on bioactive compounds and new enzymes (Schirrmeister et al., 2011; Dittmann et al., 2015). Pantanalinema was described as a new genus of the Leptolyngbyaceae cyanobacterial family by a polyphasic approach, which included morphological characteristics, 16S rRNA gene phylogeny, 16S-23S ITS rRNA secondary structures, and physiological characteristics such as adaptability to pH variations (Vaz et al., 2015). This genus has been described only in Brazilian biomes such as the Pantanal and the Amazon, the first isolates being found in a lake. These Pantanalinema isolates were characterized by their ability to grow over a wide pH range (pH 4 to 11) as well as to modify the culture medium pH around neutrality (pH 6 to 7.4). Due to these characteristics, it is thought that this genus can occupy a variety of ecological niches, such as alkaline or slightly acidic water bodies (Vaz et al., 2015; Genuário et al., 2017). Taxonomic classification of Pantanalinema isolates requires the use of molecular markers as this genus is morphologically very similar to the recently described genus Amazoninema, which, in turn, has comparable morphology to other genera of the Leptolyngbyaceae family (Genuário et al., 2018). In this work, we report the genome sequence of a new Pantanalinema strain, named GBBB05, which was isolated from the Brazilian Cerrado biome. This is the first genome assembly for the Pantanalinema genus, which, along with the analyses provided here, is expected to enhance our understanding of this genus’s metabolic potential

Evaluating Eucalyptus leaf colonization by Brasilonema octagenarum (Cyanobacteria, Scytonemataceae) using in planta experiments and genomics.

BACKGROUND: Brasilonema is a cyanobacterial genus found on the surface of mineral substrates and plants such as bromeliads, orchids and eucalyptus. B. octagenarum stands out among cyanobacteria due to causing damage to the leaves of its host in an interaction not yet observed in other cyanobacteria. Previous studies revealed that B. octagenaum UFV-E1 is capable of leading eucalyptus leaves to suffer internal tissue damage and necrosis by unknown mechanisms. This work aimed to investigate the effects of B. octagenarum UFV-E1 inoculation on Eucalyptus urograndis and to uncover molecular mechanisms potentially involved in leaf damage by these cyanobacteria using a comparative genomics approach. RESULTS: Leaves from E. urograndis saplings were exposed for 30 days to B. octagenarum UFV-E1, which was followed by the characterization of its genome and its comparison with the genomes of four other Brasilonema strains isolated from phyllosphere and the surface of mineral substrates. While UFV-E1 inoculation caused an increase in root and stem dry mass of the host plants, the sites colonized by cyanobacteria on leaves presented a significant decrease in pigmentation, showing that the cyanobacterial mats have an effect on leaf cell structure. Genomic analyses revealed that all evaluated Brasilonema genomes harbored genes encoding molecules possibly involved in plant-pathogen interactions, such as hydrolases targeting plant cell walls and proteins similar to known virulence factors from plant pathogens. However, sequences related to the type III secretory system and effectors were not detected, suggesting that, even if any virulence factors could be expressed in contact with their hosts, they would not have the structural means to actively reach plant cytoplasm. CONCLUSIONS: Leaf damage by this species is likely related to the blockage of access to sunlight by the efficient growth of cyanobacterial mats on the phyllosphere, which may hinder the photosynthetic machinery and prevent access to some essential molecules. These results reveal that the presence of cyanobacteria on leaf surfaces is not as universally beneficial as previously thought, since they may not merely provide the products of nitrogen fixation to their hosts in exchange for physical support, but in some cases also hinder regular leaf physiology leading to tissue damage.

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.

Dereplication of Natural Products with Antimicrobial and Anticancer Activity from Brazilian Cyanobacteria.

Cyanobacteria are photosynthetic organisms that produce a large diversity of natural products with interesting bioactivities for biotechnological and pharmaceutical applications. Cyanobacterial extracts exhibit toxicity towards other microorganisms and cancer cells and, therefore, represent a source of potentially novel natural products for drug discovery. We tested 62 cyanobacterial strains isolated from various Brazilian biomes for antileukemic and antimicrobial activities. Extracts from 39 strains induced selective apoptosis in acute myeloid leukemia (AML) cancer cell lines. Five of these extracts also exhibited antifungal and antibacterial activities. Chemical and dereplication analyses revealed the production of nine known natural products. Natural products possibly responsible for the observed bioactivities and five unknown, chemically related chlorinated compounds present only in Brazilian cyanobacteria were illustrated in a molecular network. Our results provide new information on the vast biosynthetic potential of cyanobacteria isolated from Brazilian environments.

Biosynthesis of microcystin hepatotoxins in the cyanobacterial genus Fischerella.

Microcystins (MCs) are serine/threonine phosphatase inhibitors synthesized by several members of the phylum Cyanobacteria. Mining the draft genome sequence of the nostocalean MC-producing Fischerella sp. strain CENA161 led to the identification of three contigs containing mcy genes. Subsequent PCR and Sanger sequencing allowed the assembling of its complete biosynthetic mcy gene cluster with 55,016 bases in length. The cluster encoding ten genes (mcyA-J) with a central bidirectional promoter was organized in a similar manner as found in other genera of nostocalean cyanobacteria. However, the nucleotide sequence of the mcy gene cluster of Fischerella sp. CENA161 showed significant differences from all the other MC-producing cyanobacterial genera, sharing only 85.2 to 74.1% identities. Potential MC variants produced by Fischerella sp. CENA161 were predicted by the analysis of the adenylation domain binding pockets and further investigated by LC-MS/MS analysis. To our knowledge, this study presents the first complete mcy cluster characterization from a strain of the genus Fischerella, providing new insight into the distribution and evolution of MCs in the phylum Cyanobacteria.

Production of High Amounts of Hepatotoxin Nodularin and New Protease Inhibitors Pseudospumigins by the Brazilian Benthic Nostoc sp. CENA543.

Nostoc is a cyanobacterial genus, common in soils and a prolific producer of natural products. This research project aimed to explore and characterize Brazilian cyanobacteria for new bioactive compounds. Here we report the production of hepatotoxins and new protease inhibitors from benthic Nostoc sp. CENA543 isolated from a small, shallow, saline-alkaline lake in the Nhecolândia, Pantanal wetland area in Brazil. Nostoc sp. CENA543 produces exceptionally high amounts of nodularin-R. This is the first free-living Nostoc that produces nodularin at comparable levels as the toxic, bloom-forming, Nodularia spumigena. We also characterized pseudospumigins A-F, which are a novel family of linear tetrapeptides. Pseudospumigins are structurally related to linear tetrapeptide spumigins and aeruginosins both present in N. spumigena but differ in respect to their diagnostic amino acid, which is Ile/Leu/Val in pseudospumigins, Pro/mPro in spumigins, and Choi in aeruginosins. The pseudospumigin gene cluster is more similar to the spumigin biosynthetic gene cluster than the aeruginosin gene cluster. Pseudospumigin A inhibited trypsin (IC50 4.5 µM after 1 h) in a similar manner as spumigin E from N. spumigena but was almost two orders of magnitude less potent. This study identifies another location and environment where the hepatotoxic nodularin has the potential to cause the death of eukaryotic organisms.

Genetic Organization of Anabaenopeptin and Spumigin Biosynthetic Gene Clusters in the Cyanobacterium Sphaerospermopsis torques-reginae ITEP-024.

Cyanobacteria produce a broad range of natural products, many of which are potent protease inhibitors. Biosynthetic gene clusters encoding the production of novel protease inhibitors belonging to the spumigin and anabaenopeptin family of nonribosomal peptides were identified in the genome of the bloom-forming cyanobacterium Sphaerospermopsis torques-reginae ITEP-024. The genetic architecture and gene organization of both nonribosomal peptide biosynthetic clusters were compared in parallel with their chemical structure variations obtained by liquid chromatography (LC-MS/MS). The spumigin (spu) and anabaenopeptin (apt) gene clusters are colocated in the genomes of S. torques-reginae ITEP-024 and Nodularia spumigena CCY9414 and separated by a 12 kb region containing genes encoding a patatin-like phospholipase, l-homophenylalanine (l-Hph) biosynthetic enzymes, and four hypothetical proteins. hphABCD gene cluster encoding the production of l-Hph was linked to all eight apt gene clusters investigated here. We suggest that while the HphABCD enzymes are an integral part of the anabaenopeptin biosynthetic pathway, they provide substrates for the biosynthesis of both anabaenopeptins and spumigins. The organization of the spu and apt suggests a plausible model for the biosynthesis of the 4-(4-hydroxyphenyl)-2-acid (Hpoba) precursor of spumigin variants in S. torques-reginae ITEP-024 based on the acceptable substrates of HphABCD enzymes.

Antifungal compounds from cyanobacteria.

Cyanobacteria are photosynthetic prokaryotes found in a range of environments. They are infamous for the production of toxins, as well as bioactive compounds, which exhibit anticancer, antimicrobial and protease inhibition activities. Cyanobacteria produce a broad range of antifungals belonging to structural classes, such as peptides, polyketides and alkaloids. Here, we tested cyanobacteria from a wide variety of environments for antifungal activity. The potent antifungal macrolide scytophycin was detected in Anabaena sp. HAN21/1, Anabaena cf. cylindrica PH133, Nostoc sp. HAN11/1 and Scytonema sp. HAN3/2. To our knowledge, this is the first description of Anabaena strains that produce scytophycins. We detected antifungal glycolipopeptide hassallidin production in Anabaena spp. BIR JV1 and HAN7/1 and in Nostoc spp. 6sf Calc and CENA 219. These strains were isolated from brackish and freshwater samples collected in Brazil, the Czech Republic and Finland. In addition, three cyanobacterial strains, Fischerella sp. CENA 298, Scytonema hofmanni PCC 7110 and Nostoc sp. N107.3, produced unidentified antifungal compounds that warrant further characterization. Interestingly, all of the strains shown to produce antifungal compounds in this study belong to Nostocales or Stigonematales cyanobacterial orders.