Identification of a homoarginine biosynthetic gene from a microcystin biosynthetic pathway in Fischerella sp. PCC 9339.

Microcystins (MCs) are a family of chemically diverse toxins produced by numerous distantly related cyanobacteria. They are potent inhibitors of eukaryotic protein phosphatases 1 and 2A and are responsible for the toxicosis and death of wild and domestic animals around the world. Microcystins are synthesized on large enzyme complexes comprised of peptide synthetases, polyketide synthases, and additional modifying enzymes. Bioinformatic analysis identified the presence of an additional uncharacterized enzyme in the microcystin (mcy) biosynthetic gene cluster in Fischerella sp. PCC 9339, which we named McyK, that lacked a clearly defined role in the biosynthesis of microcystin. Further bioinformatic analysis suggested that McyK belongs to the inosamine-phosphate amidinotransferase family and could be involved in synthesizing homo amino acids. Quadrupole time-of-flight tandem mass spectrometry (Q-TOFMS/MS) analysis confirmed that Fischerella sp. PCC 9339 produces MC-Leucine2-Homoarginine4(MC-LHar) and [Aspartic acid3]MC-Leucine2-Homoarginine4 ([Asp3]MC-LHar) as the dominant chemical variants. We hypothesized that the McyK enzyme might be involved in the production of microcystin variants containing homoarginine (Har) in the strain. Heterologous expression of a codon-optimized mcyK gene in Escherichia coli confirmed that McyK is responsible for the synthesis of L-Har. These results confirm the production of MC-LHar, a novel microcystin chemical variant [Asp3]MC-LHar, and a new microcystin biosynthetic enzyme involved in supply of the rare homo-amino acid Har to the microcystin biosynthetic pathway in Fischerella sp. PCC 9339. This study provides new insights into the logic underpinning the biosynthesis of microcystin chemical variants and broadens our knowledge of structural diversity of the microcystin family of toxins.

Strain heterogeneity in a non-pathogenic fungus highlights factors contributing to virulence.

Fungal pathogens exhibit extensive strain heterogeneity, including variation in virulence. Whether closely related non-pathogenic species also exhibit strain heterogeneity remains unknown. Here, we comprehensively characterized the pathogenic potentials (i.e., the ability to cause morbidity and mortality) of 16 diverse strains of Aspergillus fischeri, a non-pathogenic close relative of the major pathogen Aspergillus fumigatus. In vitro immune response assays and in vivo virulence assays using a mouse model of pulmonary aspergillosis showed that A. fischeri strains varied widely in their pathogenic potential. Furthermore, pangenome analyses suggest that A. fischeri genomic and phenotypic diversity is even greater. Genomic, transcriptomic, and metabolomic profiling identified several pathways and secondary metabolites associated with variation in virulence. Notably, strain virulence was associated with the simultaneous presence of the secondary metabolites hexadehydroastechrome and gliotoxin. We submit that examining the pathogenic potentials of non-pathogenic close relatives is key for understanding the origins of fungal pathogenicity.

A phylogenetic approach to explore the Aspergillus fumigatus conidial surface-associated proteome and its role in pathogenesis.

Aspergillus fumigatus, an important pulmonary fungal pathogen causing several diseases collectively called aspergillosis, relies on asexual spores (conidia) for initiating host infection. Here, we used a phylogenomic approach to compare proteins in the conidial surface of A. fumigatus, two closely related non-pathogenic species, Aspergillus fischeri and Aspergillus oerlinghausenensis, and the cryptic pathogen Aspergillus lentulus. After identifying 62 proteins uniquely expressed on the A. fumigatus conidial surface, we assessed null mutants for 42 genes encoding conidial proteins. Deletion of 33 of these genes altered susceptibility to macrophage killing, penetration and damage to epithelial cells, and cytokine production. Notably, a gene that encodes glycosylasparaginase, which modulates levels of the host pro-inflammatory cytokine IL-1ß, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that A. fumigatus conidial surface proteins and effectors are important for evasion and modulation of the immune response at the onset of fungal infection.

A Plastic Biosynthetic Pathway for the Production of Structurally Distinct Microbial Sunscreens.

Mycosporine-like amino acids (MAAs) are small, colorless, and water-soluble secondary metabolites. They have high molar extinction coefficients and a unique UV radiation absorption mechanism that make them effective sunscreens. Here we report the discovery of two structurally distinct MAAs from the lichen symbiont strain Nostoc sp. UHCC 0926. We identified these MAAs as aplysiapalythine E (C23H38N2O15) and tricore B (C34H53N4O15) using a combination of high-resolution liquid chromatography-mass spectrometry (HR-LCMS) analysis and nuclear magnetic resonance (NMR) spectroscopy. We obtained a 8.3 Mb complete genome sequence of Nostoc sp. UHCC 0926 to gain insights into the genetic basis for the biosynthesis of these two structural distinct MAAs. We identified MAA biosynthetic genes encoded in three separate locations of the genome. The organization of biosynthetic enzymes in Nostoc sp. UHCC 0926 necessitates a branched biosynthetic pathway to produce two structurally distinct MAAs. We detected the presence of such discontiguous MAA biosynthetic gene clusters in 12% of the publicly available complete cyanobacterial genomes. Bioinformatic analysis of public MAA biosynthetic gene clusters suggests that they are subject to rapid evolutionary processes resulting in highly plastic biosynthetic pathways that are responsible for the chemical diversity in this family of microbial sunscreens.

A cylindrospermopsin-producing cyanobacterium isolated from a microbial mat in the Baltic Sea.

Toxic benthic mats of cyanobacteria are associated with water quality problems and animal poisonings around the world. A strain of the filamentous cyanobacterial genus Kamptonema was isolated from a water bloom in the Baltic Sea four decades ago and later shown to produce cylindrospermopsins. However, the exact habitat of this strain remains unclear and cylindrospermopsins have not yet been reported from water blooms in the Baltic Sea. Here, we report the isolation of Kamptonema sp. UHCC 0994 from a benthic microbial mat collected in shallow water on the coast of Helsinki. We obtained draft genome sequences for the Kamptonema spp. PCC 7926 and UHCC 0994 strains that were isolated from the Baltic Sea. These genomes were 90-96% similar to previously studied Kamptonema sp. PCC 6506 and Kamptonema formosum PCC 6407, which were isolated from benthic and North American freshwater environments, respectively. The genomes of all four Kamptonema strains encode complete cylindrospermopsin biosynthetic gene clusters. We detected the production of cylindrospermopsin and 7-epi-cylindrospermopsin in the four Kamptonema strains using high-resolution liquid chromatography mass spectrometry. The four strains encode genes for producing gas vesicles distributed in two to three different regions of their genomes. Kamptonema spp. UHCC 0994 and PCC 7926 have both retained the ability to regulate their buoyancy when grown in liquid culture. Together this suggests that these toxic cyanobacteria may exhibit a tychoplanktic lifestyle in the Baltic Sea. This study suggests that microbial mats containing cyanobacteria could be a source of environmental toxins in the Baltic Sea.

Direct pathway cloning and expression of the radiosumin biosynthetic gene cluster.

Radiosumins are a structurally diverse family of low molecular weight natural products that are produced by cyanobacteria and exhibit potent serine protease inhibition. Members of this family are dipeptides characterized by the presence of two similar non-proteinogenic amino acids. Here we used a comparative bioinformatic analysis to identify radiosumin biosynthetic gene clusters from the genomes of 13 filamentous cyanobacteria. We used direct pathway cloning to capture and express the entire 16.8 kb radiosumin biosynthetic gene cluster from Dolichospermum planctonicum UHCC 0167 in Escherichia coli. Bioinformatic analysis demonstrates that radiosumins represent a new group of chorismate-derived non-aromatic secondary metabolites. High-resolution liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy and chemical degradation analysis revealed that cyanobacteria produce a cocktail of novel radiosumins. We report the chemical structure of radiosumin D, an N-methyl dipeptide, containing a special Aayp (2-amino-3-(4-amino-2-cyclohexen-1-ylidene) propionic acid) with R configuration that differs from radiosumin A-C, an N-Me derivative of Aayp (Amyp) and two acetyl groups. Radiosumin C inhibits all three human trypsin isoforms at micromolar concentrations with preference for trypsin-1 and -3 (IC50 values from 1.7 µM to >7.2 µM). These results provide a biosynthetic logic to explore the genetic and chemical diversity of the radiosumin family and suggest that these natural products may be a source of drug leads for selective human serine proteases inhibitors.

Exploring the Relationship between Biosynthetic Gene Clusters and Constitutive Production of Mycosporine-like Amino Acids in Brazilian Cyanobacteria.

Cyanobacteria are oxygenic phototrophic prokaryotes that have evolved to produce ultraviolet-screening mycosporine-like amino acids (MAAs) to lessen harmful effects from obligatory exposure to solar UV radiation. The cyanobacterial MAA biosynthetic cluster is formed by a gene encoding 2-epi-5-epi-valiolone synthase (EVS) located immediately upstream from an O-methyltransferase (OMT) encoding gene, which together biosynthesize the expected MAA precursor 4-deoxygadusol. Accordingly, these genes are typically absent in non-producers. In this study, the relationship between gene cluster architecture and constitutive production of MAAs was evaluated in cyanobacteria isolated from various Brazilian biomes. Constitutive production of MAAs was only detected in strains where genes formed a co-linear cluster. Expectedly, this production was enhanced upon exposure of the strains to UV irradiance and by using distinct culture media. Constitutive production of MAAs was not detected in all other strains and, unexpectedly, production could not be induced by exposure to UV irradiation or changing growth media. Other photoprotection strategies which might be employed by these MAA non-producing strains are discussed. The evolutionary and ecological significance of gene order conservation warrants closer experimentation, which may provide a first insight into regulatory interactions of genes encoding enzymes for MAA biosynthesis.

Bacterial Communities Along Environmental Gradients in Tropical Soda Lakes.

Soda lake environments are known to be variable and can have distinct differences according to geographical location. In this study, we investigated the effects of different environmental conditions of six adjacent soda lakes in the Pantanal biome (Mato Grosso do Sul state, Brazil) on bacterial communities and their functioning using a metagenomic approach combined with flow cytometry and chemical analyses. Ordination analysis using flow cytometry and water chemistry data from two sampling periods (wet and dry) clustered soda lakes into three different profiles: eutrophic turbid (ET), oligotrophic turbid (OT), and clear vegetated oligotrophic (CVO). Analysis of bacterial community composition and functioning corroborated this ordination; the exception was one ET lake, which was similar to one OT lake during the wet season, indicating drastic shifts between seasons. Microbial abundance and diversity increased during the dry period, along with a considerable number of limnological variables, all indicative of a strong effect of the precipitation-evaporation balance in these systems. Cyanobacteria were associated with high electric conductivity, pH, and nutrient availability, whereas Actinobacteria, Alphaproteobacteria, and Betaproteobacteria were correlated with landscape morphology variability (surface water, surface perimeter, and lake volume) and with lower salinity and pH levels. Stress response metabolism was enhanced in OT and ET lakes and underrepresented in CVO lakes. The microbiome dataset of this study can serve as a baseline for restoring impacted soda lakes. Altogether, the results of this study demonstrate the sensitivity of tropical soda lakes to climate change, as slight changes in hydrological regimes might produce drastic shifts in community diversity.

Biosynthesis of Guanitoxin Enables Global Environmental Detection in Freshwater Cyanobacteria.

Harmful cyanobacterial blooms (cyanoHABs) cause recurrent toxic events in global watersheds. Although public health agencies monitor the causal toxins of most cyanoHABs and scientists in the field continue developing precise detection and prediction tools, the potent anticholinesterase neurotoxin, guanitoxin, is not presently environmentally monitored. This is largely due to its incompatibility with widely employed analytical methods and instability in the environment, despite guanitoxin being among the most lethal cyanotoxins. Here, we describe the guanitoxin biosynthesis gene cluster and its rigorously characterized nine-step metabolic pathway from l-arginine in the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024. Through environmental sequencing data sets, guanitoxin (gnt) biosynthetic genes are repeatedly detected and expressed in municipal freshwater bodies that have undergone past toxic events. Knowledge of the genetic basis of guanitoxin biosynthesis now allows for environmental, biosynthetic gene monitoring to establish the global scope of this neurotoxic organophosphate.

Mucilaginibacter sp. Strain Metal(loid) and Antibiotic Resistance Isolated from Estuarine Soil Contaminated Mine Tailing from the Fundão Dam.

In 2015 a mine dam with Mn-Fe-rich tailings collapsed releasing million tons of sediments over an estuary, in the Southwest of Brazil. The tailings have a high concentration of metals that contaminated soil until the present day. The high contaminant concentrations possibly caused a selection for microorganisms able to strive in such harsh conditions. Here, we isolated metal(loid) and anti-biotic resistance bacteria from the contaminated estuarine soil. After 16S rDNA sequencing to identify the strains, we selected the Mucilaginibacter sp. strain for a whole-genome sequence due to the bioprospective potential of the genus and the high resistance profile. We obtained a complete genome and a genome-guided characterization. Our finding suggests that the 21p strain is possibly a new species of the genus. The species presented genes for resistance for metals (i.e., As, Zn, Co, Cd, and Mn) beyond resistance and cross-resistance for antibiotics (i.e., quinolone, aminoglycoside, ß-lactamase, sulphonamide, tetracycline). The Mucilaginibacter sp. 21p description as new species should be further explored, as their extracellular polymeric substances and the potential of this strain as bioremediation and as a growth promoter in high met-al(loid) contaminated soil.

Trends in Free-access Genomic Data Accelerate Advances in Cyanobacteria Taxonomy.

Free access databases of DNA sequences containing microbial genetic information have changed the way scientists look at the microbial world. Currently, the NCBI database includes about 516 distinct search results for Cyanobacterial genomes distributed in a taxonomy based on a polyphasic approach. While their classification and taxonomic relationships are widely used as is, recent proposals to alter their grouping include further exploring the relationship between Cyanobacteria and Melainabacteria. Nowadays, most cyanobacteria still are named under the Botanical Code; however, there is a proposal made by the Genome Taxonomy Database (GTDB) to harmonize cyanobacteria nomenclature with the other bacteria, an initiative to standardize microbial taxonomy based on genome phylogeny, in order to contribute to an overall better phylogenetic resolution of microbiota. Furthermore, the assembly level of the genomes and their geographical origin demonstrates some trends of cyanobacteria genomics on the scientific community, such as low availability of complete genomes and underexplored sampling locations. By describing how available cyanobacterial genomes from free-access databases fit within different taxonomic classifications, this mini-review provides a holistic view of the current knowledge of cyanobacteria and indicates some steps towards improving our efforts to create a more cohesive and inclusive classifying system, which can be greatly improved by using large-scale sequencing and metagenomic techniques.

Amazonocrinis nigriterrae gen. nov., sp. nov., Atlanticothrix silvestris gen. nov., sp. nov. and Dendronalium phyllosphericum gen. nov., sp. nov., nostocacean cyanobacteria from Brazilian environments.

The cyanobacterial genus Nostoc is an important contributor to carbon and nitrogen bioavailability in terrestrial ecosystems and a frequent partner in symbiotic relationships with non-diazotrophic organisms. However, since this currently is a polyphyletic genus, the diversity of Nostoc-like cyanobacteria is considerably underestimated at this moment. While reviewing the phylogenetic placement of previously isolated Nostoc-like cyanobacteria originating from Brazilian Amazon, Caatinga and Atlantic forest samples, we detected 17 strains isolated from soil, freshwater, rock and tree surfaces presenting patterns that diverged significantly from related strains when ecological, morphological, molecular and genomic traits were also considered. These observations led to the identification of the evaluated strains as representative of three novel nostocacean genera and species: Amazonocrinis nigriterrae gen. nov., sp. nov.; Atlanticothrix silvestris gen. nov., sp. nov.; and Dendronalium phyllosphericum gen. nov., sp. nov., which are herein described according to the rules of the International Code of Nomenclature for algae, fungi and plants. This finding highlights the great importance of tropical and equatorial South American ecosystems for harbouring an unknown microbial diversity in the face of the anthropogenic threats with which they increasingly struggle.

Phylogenomic analysis of Anabaenopsis elenkinii (Nostocales, Cyanobacteria).

The saline-alkaline lakes (soda lakes) are the habitat of the haloalkaliphilic cyanobacterium Anabaenopsis elenkinii, the type species of this genus. To obtain robust phylogeny of this type species, we have generated whole-genome sequencing of the bloom-forming Anabaenopsis elenkinii strain CCIBt3563 isolated from a Brazilian soda lake. This strain presents the typical morphology of A. elenkinii with short and curved trichomes with apical heterocytes established after separation of paired intercalary heterocytes and also regarding to cell dimensions. Its genome size is 4 495 068 bp, with a G+C content of 41.98 %, a total of 3932 potential protein coding genes and four 16S rRNA genes. Phylogenomic tree inferred by RAxML based on the alignment of 120 conserved proteins using GTDB-Tk grouped A. elenkinii CCIBt3563 together with other genera of the family Aphanizomenonaceae. However, the only previous available genome of Anabaenopsis circularis NIES-21 was distantly positioned within a clade of Desikacharya strains, a genus from the family Nostocaceae. Furthermore, average nucleotide identity values from 86-98 % were obtained among NIES-21 and Desikacharya genomes, while this value was 76.04 % between NIES-21 and the CCIBt3563 genome. These findings were also corroborated by the phylogenetic tree of 16S rRNA gene sequences, which also showed a strongly supported subcluster of A. elenkinii strains from Brazilian, Mexican and Kenyan soda lakes. This study presents the phylogenomics and genome-scale analyses of an Anabaenopsis elenkinii strain, improving molecular basis for demarcation of this species and framework for the classification of cyanobacteria based on the polyphasic approach.

Effect of ultraviolet radiation on the metabolomic profiles of potentially toxic cyanobacteria.

Interactions between climate change and ultraviolet radiation (UVR) have a substantial impact on aquatic ecosystems, especially on photosynthetic organisms. To counteract the damaging effects of UVR, cyanobacteria developed adaptive strategies such as the biosynthesis of secondary metabolites. This study aimed to evaluate the effects of UVR on the metabolomic profiles of potentially toxic cyanobacteria. Twelve strains were irradiated with ultraviolet A and ultraviolet B radiation and parabolic aluminized reflector lamps for 3 days, followed by liquid chromatography-tandem mass spectometry (LC-MS/MS) analysis to assess changes in metabolomic profiles. Matrices were used to generate principal component analysis biplots, and molecular networks were obtained using the Global Natural Products platform. Most strains showed significant changes in their metabolomic profiles after UVR exposure. On average, 7% of MS features were shown to be exclusive to metabolomic profiles before UVR exposure, while 9% were unique to metabolomic profiles after UVR exposure. The identified compounds included aeruginosins, spumigins, cyanopeptolins, microginins, namalides, pseudospumigins, anabaenopeptins, mycosporine-like amino acids, nodularins and microcystins. Data showed that cyanobacteria display broad metabolic plasticity upon UVR exposure, including the synthesis and differential expression of a variety of secondary metabolites. This could result in a competitive advantage, supporting cyanobacterial blooms under various UVR light exposures.

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.