Cyanobacteria are integral to biogeochemical cycles, influence climate processes, and hold promise for commercial applications. In natural habitats, they form complex consortia with other microorganisms, where interspecies interactions shape their ecological roles. Although in vitro studies of these consortia have significantly advanced our understanding, they often lack the biological replication needed for robust statistical analysis of shared microbiome features and functions. Moreover, the microbiomes of many model cyanobacterial strains, which are central to our understanding of cyanobacterial biology, remain poorly characterized. Here, we expanded on existing in vitro approaches by coculturing five well-characterized model cyanobacterial strains with microorganisms filtered from three distinct freshwater sources, generating 108 stable consortia. Metagenomic analyses revealed that, despite host and inoculum diversity, these consortia converged on a similar set of noncyanobacterial taxa, forming a 25-species core microbiome. The large number of stable consortia in this study enabled statistical validation of both previously observed and newly identified core microbiome functionalities in micronutrient biosynthesis, metabolite transport, and anoxygenic photosynthesis. Furthermore, core species showed significant enrichment of plasmids, and functions encoded on plasmids suggested plasmid-mediated roles in symbiotic interactions. Overall, our findings uncover the potential microbiomes recruited by key model cyanobacteria, demonstrate that laboratory-enriched consortia retain many taxonomic and functional traits observed more broadly in phototroph-heterotroph assemblages, and show that model cyanobacteria can serve as robust hosts for uncovering functional roles underlying cyanobacterial community dynamics.

Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015. Since its conception, MIBiG has been regularly updated to expand data coverage and remain up to date with innovations in natural product research. Here, we describe MIBiG version 4.0, an extensive update to the data repository and the underlying data standard. In a massive community annotation effort, 267 contributors performed 8304 edits, creating 557 new entries and modifying 590 existing entries, resulting in a new total of 3059 curated entries in MIBiG. Particular attention was paid to ensuring high data quality, with automated data validation using a newly developed custom submission portal prototype, paired with a novel peer-reviewing model. MIBiG 4.0 also takes steps towards a rolling release model and a broader involvement of the scientific community. MIBiG 4.0 is accessible online at https://mibig.secondarymetabolites.org/. [GRAPHICS] .

In this study, we used microscopic, spectroscopic, and molecular analysis to characterize endolithic colonization in gypsum (selenites and white crystalline gypsum) from several sites in Sicily. Our results showed that the dominant microorganisms in these environments are cyanobacteria, including: Chroococcidiopsis sp., Gloeocapsopsis pleurocapsoides, Gloeocapsa compacta, and Nostoc sp., as well as orange pigmented green microalgae from the Stephanospherinia clade. Single cell and filament sequencing coupled with 16S rRNA amplicon metagenomic profiling provided new insights into the phylogenetic and taxonomic diversity of the endolithic cyanobacteria. These organisms form differently pigmented zones within the gypsum. Our metagenomic profiling also showed differences in the taxonomic composition of endoliths in different gypsum varieties. Raman spectroscopy revealed that carotenoids were the most common pigments present in the samples. Other pigments such as gloeocapsin and scytonemin were also detected in the near-surface areas, suggesting that they play a significant role in the biology of endoliths in this environment. These pigments can be used as biomarkers for basic taxonomic identification, especially in case of cyanobacteria. The findings of this study provide new insights into the diversity and distribution of phototrophic microorganisms and their pigments in gypsum in Southern Sicily. Furthemore, this study highlights the complex nature of endolithic ecosystems and the effects of gypsum varieties on these communities, providing additional information on the general bioreceptivity of these environments.

Cyanobacteria are central to biogeochemical cycling, climate change, and eutrophication. While they readily develop associations with environmental microorganisms, the question of whether they consistently recruit specific microbiomes remains unresolved. Here, we established in vitro cyanobacterial consortia by inoculating five different cyanobacterial strains with microbiomes from three freshwater environments to determine if similar non-cyanobacterial organisms were recruited into stable cyanobacteria-based communities. Using amplicon and genome-resolved metagenomic methods we determined that stable cyanobacterial communities largely share the same taxa, regardless of the host cyanobacterial species or inoculation source. Specifically, we identified a 25 species core microbiome, which was significantly enriched in functions for micronutrient biosynthesis, metabolite transport/exchange, and anoxygenic photosynthesis. Furthermore, core species were enriched in putative mobile genetic elements, including two large (> 100 kb) circularized and curated plasmids, encoding functions that may support cyanobacteria-heterotroph symbioses. The consistency observed across these systems indicate that cyanobacterial consortia maintained under similar conditions converge to a predictable structure, and support a core microbiome with taxonomic and functional similarity to core microbiomes of other phototroph-heterotroph symbiotic assemblages.### Competing Interest StatementThe authors have declared no competing interest.

Post-mining lakes are anthropogenic water bodies increasing globally due to mining suppression and recultivation. These lakes have ecological and socioeconomic significance, providing freshwater sources, wildlife habitats, and recreation potential. However, research on primary producers in these lakes is limited, with most studies focusing on phytoplankton while neglecting algal mats and meadows. If developed in high biomass, algal mats and macroalgal meadows play a crucial role in aquatic ecosystems. They contribute to the trophic structure, nutrient fluxes, primary production and to the purification of entire waterbodies. Yet, there is a lack of knowledge regarding the nutrient acquisition and the contribution of algal mats and meadows to total primary production and phosphorus (P) cycling, especially in oligotrophic lakes, including post-mining lakes. Our team investigates the benthic algal primary producers - algal mats and meadows in the littoral zone of tree newly established post-mining lakes located in the Czech Republic (Europe). Our main objectives are to describe the quality and quantity of primary producers, quantify the contribution of algal mats and meadows to the primary production and phosphorus cycling in the oligotrophic post-mining lakes. We have studied them throughout all seasons with a special focus on the generally neglected winter/spring season. Our sampling strategy encompasses three gradients of variability along which the composition and ecological role of benthic algae change: spatial variability along the depth gradient within each lake, i temporal variability throughout the seasons within each lake and successional variability between the lakes of different age. spatial variability along the depth gradient within each lake, i temporal variability throughout the seasons within each lake and successional variability between the lakes of different age. The methodological approach consists of measuring the limnological and chemical parameters of lakes, taxonomic determination, biomass estimation and C:N:P stoichiometry of algal biomass along the whole gradient of the euphotic zone (0-15m). The in situ measurement of primary production of benthic algae from various depths using the oxygen meter Fibox 3 (PreSens, Germany) with a combination of laboratory experiments was employed too (Čapková 2022). We measure the kinetic parameters of P uptake of algal mats and meadows using 33 P-labelled orthophosphates under controlled conditions similar to the nature (Konopáčová 2021). According to our survey, algal mats and meadows exhibited high productivity in the entire littoral zone. Algal mats dominate the depth up to 3m Fig. 1 and are highly diverse, with more than 400 identified taxa (Bešta 2022). Macroalgal meadows formed by the genus Chara together with the genus Vaucheria , are key taxa occupying the deeper zone (3-15m) Fig. 2. The distribution of benthic algae in lakes is influenced by depth gradients, substrate quality, seasonality and age of lakes foundation, resulting in complex spatio-temporal patterns. Specific emphasis is placed on the fate of P, which is the limiting nutrient in investigated lakes. High C:P molar ratios in microalgal mats indicate strong P deficiency in studied lakes. Over the vegetation season, microalgal biomass doubled, while the P content in biomass dropped to 60% of the values from the start of the vegetation season. The maximum uptake velocity and specific P uptake affinity decreased by an order of magnitude from April to October, and P uptake affinity was measured for comparison in plankton too. Based on our results, we proposed a possible mechanism underlying a stable coexistence of planktonic and benthic primary producers, with plankton prospering primarily in summer and autumn and algal mats and meadows in winter and spring seasons. By integrating data collected during five years of investigation, our study aims to provide a comprehensive understanding of the ecological roles of primary producers in post-mining lakes.

Hydric recultivation—flooding of abandoned mining pits—creates a completely new, underexplored habitat for a wide range of aquatic organisms. Periphyton, dominated by algae and cyanobacteria, is frequently a key component of newly established aquatic ecosystems. Periphyton and its response to abiotic factors were studied in the littoral zone of three post-mining lakes with different ages of foundation situated in the Czech Republic. The microbial diversity of phototrophs as a major component of periphyton is largely unknown in such localities. The studied habitat proved to harbour a huge periphytic diversity—25% of diatom species found in the respective watershed (~ 5500 km2) inhabited exlusively the studied lakes. Species composition of phototrophic microorganisms varied significantly (Permutational Multivariate Analysis of Variance) among the studied lakes, seasons, and sampling years. However, the sampling depths and sampling site of the studied lake have not shown a significant impact on the diversity, indicating the homogeneous composition of the littoral periphyton within a particular lake and growing season. The seasonal dynamics of periphyton were unique for each lake, documenting three distinct successional patterns. The proportion of diatoms in the periphytic community decreases with the higher trophic state and flooding age of the post-mining lakes. Cyanobacteria and mobile diatom forms prevailed later in the growing season, suggesting that they could utilise nutrients released from the accumulated periphyton biomass. Calcium ions were one of the best correlates of species data among other abiotic variables tested, offering the intriguing question of the role of calcium in the formation of periphytic mats for future research.

Vacuolar myelinopathy is a fatal neurological disease that was initially discovered during a mysterious mass mortality of bald eagles in Arkansas in the United States. The cause of this wildlife disease has eluded scientists for decades while its occurrence has continued to spread throughout freshwater reservoirs in the southeastern United States. Recent studies have demonstrated that vacuolar myelinopathy is induced by consumption of the epiphytic cyanobacterial species Aetokthonos hydrillicola growing on aquatic vegetation, primarily the invasive Hydrilla verticillata Here, we describe the identification, biosynthetic gene cluster, and biological activity of aetokthonotoxin, a pentabrominated biindole alkaloid that is produced by the cyanobacterium A. hydrillicola We identify this cyanobacterial neurotoxin as the causal agent of vacuolar myelinopathy and discuss environmental factors-especially bromide availability-that promote toxin production.

All living organisms depend on iron as an essential cofactor for indispensable enzymes. However, the sources of bioavailable iron are often limited.

Man-made shallow fishponds in the Czech Republic have been facing high eutrophication since the 1950s. Anthropogenic eutrophication and feeding of fish have strongly affected the physicochemical properties of water and its aquatic community composition, leading to harmful algal bloom formation. In our current study, we characterized the phytoplankton community across three eutrophic ponds to assess the phytoplankton dynamics during the vegetation season. We microscopically identified and quantified 29 cyanobacterial taxa comprising non-toxigenic and toxigenic species. Further, a detailed cyanopeptides (CNPs) profiling was performed using molecular networking analysis of liquid chromatography-tandem mass spectrometry (LC-MS/MS) data coupled with a dereplication strategy. This MS networking approach, coupled with dereplication, on the online global natural product social networking (GNPS) web platform led us to putatively identify forty CNPs: fourteen anabaenopeptins, ten microcystins, five cyanopeptolins, six microginins, two cyanobactins, a dipeptide radiosumin, a cyclooctapeptide planktocyclin, and epidolastatin 12. We applied the binary logistic regression to estimate the CNPs producers by correlating the GNPS data with the species abundance. The usage of the GNPS web platform proved a valuable approach for the rapid and simultaneous detection of a large number of peptides and rapid risk assessments for harmful blooms.

Anatoxin-a, homoanatoxin-a, and dihydroanatoxin-a are potent cyanobacterial neurotoxins. They are biosynthesized in cyanobacteria from proline and acetate by a pathway involving three polyketide synthases. We report the identification of carboxy-anatoxin-a, carboxy-homoanatoxin-a, and carboxy-dihydroanatoxin-a in acidic extracts of Cuspidothrix issatschenkoi CHARLIE-1, Oscillatoria sp. PCC 6506, and Cylindrospermum stagnale PCC 7417, respectively, using liquid chromatography coupled to mass spectrometry. The structure of these carboxy derivatives was confirmed by mass spectrometry and by isotopic incorporation experiments using labeled proline and acetate. Each of these three cyanobacteria only produce one carboxy-anatoxin, suggesting that these metabolites are the product of the hydrolysis by AnaA, the type II thioesterase, of the thioesters bound to AnaG, the last polyketide synthase of the pathway. By measuring the rate of isotopic incorporation of labeled proline into carboxy-homoanatoxin-a and homoanatoxin-a produced by Oscillatoria sp. PCC 6506, we show that carboxy-homoanatoxin-a is the intracellular precursor of homoanatoxin-a, and that homoanatoxin-a is then excreted into the extracellular medium. The transformation of carboxy-homoanatoxin-a into homoanatoxin-a is a very slow two-step process, with accumulation of carboxy-homoanatoxin-a, suggesting that the decarboxylation is spontaneous and not enzymatically catalyzed. However, an unidentified and extracellular catalyst accelerates the decarboxylation when the cell extracts are prepared at neutral pH.

Heterocytous cyanobacteria are among the most prolific sources of bioactive secondary metabolites, including anabaenopeptins (APTs). A terrestrial filamentous Brasilonema sp. CT11 collected in Costa Rica bamboo forest as a black mat, was studied using a multidisciplinary approach: genome mining and HPLC-HRMS/MS coupled with bioinformatic analyses. Herein, we report the nearly complete genome consisting of 8.79 Mbp with a GC content of 42.4%. Moreover, we report on three novel tryptophan-containing APTs; anabaenopeptin 788 (1), anabaenopeptin 802 (2), and anabaenopeptin 816 (3). Furthermore, the structure of two homologues, i.e., anabaenopeptin 802 (2a) and anabaenopeptin 802 (2b), was determined by spectroscopic analysis (NMR and MS). Both compounds were shown to exert weak to moderate antiproliferative activity against HeLa cell lines. This study also provides the unique and diverse potential of biosynthetic gene clusters and an assessment of the predicted chemical space yet to be discovered from this genus.

The rapid emergence of resistance in pathogenic bacteria together with a steep decline in economic incentives has rendered a new wave in the drug development by the pharmaceutical industry and researchers. Since cyanobacteria are recognized as wide producers of pharmaceutically important compounds, we investigated thirty-four cyanobacterial extracts prepared by solvents of different polarities for their antimicrobial potential. Almost all tested cyanobacterial strains exhibited some degree of antimicrobial bioactivity, with more general effect on fungal strains compared with bacteria. Surprisingly ~50% of cyanobacterial extracts exhibited specific activity against one or few bacterial indicator strains with Gram-positive bacteria being more affected. Extracts of two most promising strains were subjected to activity-guided fractionation and determination of the minimum inhibitory concentration (MIC) against selected bacterial and fungal isolates. Multiple fractions were responsible for their antimicrobial effect with MIC reaching low-micromolar concentrations and in some of them high level of specificity was recorded. Twenty-six bioactive fractions analyzed on LC-HRMS/MS and Global Natural Product Social Molecular Networking (GNPS) online workflow using dereplication resulted in identification of only forty-nine peptide spectrum matches (PSMs) with eleven unique metabolites spectrum matches (MSMs). Interestingly, only three fractions from Nostoc calcicola Lukešová 3/97 and four fractions from Desmonostoc sp. Cc2 showed the presence of unique MSMs suggesting the presence of unknown antimicrobial metabolites among majority of bioactive fractions from both the strains. Our results highlight potential for isolation and discovery of potential antimicrobial bioactive lead molecules from cyanobacterial extracts.

Puwainaphycins (PUWs) and minutissamides (MINs) are structurally analogous cyclic lipopeptides possessing cytotoxic activity. Both types of compound exhibit high structural variability, particularly in the fatty acid (FA) moiety. Although a biosynthetic gene cluster responsible for synthesis of several PUW variants has been proposed in a cyanobacterial strain, the genetic background for MINs remains unexplored. Herein, we report PUW/MIN biosynthetic gene clusters and structural variants from six cyanobacterial strains. Comparison of biosynthetic gene clusters indicates a common origin of the PUW/MIN hybrid nonribosomal peptide synthetase and polyketide synthase. Surprisingly, the biosynthetic gene clusters encode two alternative biosynthetic starter modules, and analysis of structural variants suggests that initiation by each of the starter modules results in lipopeptides of differing lengths and FA substitutions. Among additional modifications of the FA chain, chlorination of minutissamide D was explained by the presence of a putative halogenase gene in the PUW/MIN gene cluster of Anabaena minutissima strain UTEX B 1613. We detected PUW variants bearing an acetyl substitution in Symplocastrum muelleri strain NIVA-CYA 644, consistent with an O-acetyltransferase gene in its biosynthetic gene cluster. The major lipopeptide variants did not exhibit any significant antibacterial activity, and only the PUW F variant was moderately active against yeast, consistent with previously published data suggesting that PUWs/MINs interact preferentially with eukaryotic plasma membranes. IMPORTANCE Herein, we deciphered the most important biosynthetic traits of a prominent group of bioactive lipopeptides. We reveal evidence for initiation of biosynthesis by two alternative starter units hardwired directly in the same gene cluster, eventually resulting in the production of a remarkable range of lipopeptide variants. We identified several unusual tailoring genes potentially involved in modifying the fatty acid chain. Careful characterization of these biosynthetic gene clusters and their diverse products could provide important insight into lipopeptide biosynthesis in prokaryotes. Some of the variants identified exhibit cytotoxic and antifungal properties, and some are associated with a toxigenic biofilm-forming strain. The findings may prove valuable to researchers in the fields of natural product discovery and toxicology.

Benthic cyanobacteria recognized as producers of natural products, including cyanotoxins, have been neglected for systematic toxicological studies. Thus, we have performed a broad study investigating cyanotoxin potential of 311 non-planktic nostocacean representatives combining molecular and chemical analyses. Out of these, a single strain Nostoc sp. Treb K1/5, was identified as a new microcystin producer. Microcystins [Asp3]MC-YR, [Asp3]MC-FR, [Asp3]MC-HtyR and Ala-Leu/Ile-Asp-Arg-Adda-Glu-Mdha are reported for the first time from the genus Nostoc. All the studied strains were also analyzed for the occurrence of nodularins, cylindrospermopsin and (homo)anatoxin-a, yet no novel producer has been discovered. Our findings indicate rare occurrence of the common cyanotoxins in non-planktic nostocaceae which is in contrast with frequent reports of cyanotoxin producers among phylogenetically closely related planktic cyanobacteria.

The pederin family includes a number of bioactive compounds isolated from symbiotic organisms of diverse evolutionary origin. Pederin is linked to beetle-induced dermatitis in humans, and pederin family members possess potent antitumor activity caused by selective inhibition of the eukaryotic ribosome. Their biosynthesis is accomplished by a polyketide/nonribosomal peptide synthetase machinery employing an unusual trans-acyltransferase mechanism. Here, we report a novel pederin type compound, cusperin, from the free-living cyanobacterium Cuspidothrix issatschenkoi (earlier Aphanizomenon). The chemical structure of cusperin is similar to that of nosperin recently isolated from the lichen cyanobiont Nostoc sharing the tehrahydropyran moiety and major part of the linear backbone. However, the cusperin molecule is extended by a glycine residue and lacks one hydroxyl substituent. Pederins were previously thought to be exclusive to symbiotic relationships. However, C. issatschenkoi is a nonsymbiotic planktonic organism and a frequent component of toxic water blooms. Cusperin is devoid of the cytotoxic activity reported for other pederin family members. Hence, our findings raise questions about the role of pederin analogues in cyanobacteria and broaden the knowledge of ecological distribution of this group of polyketides.

Heterocytous cyanobacteria are able to fix nitrogen (in heterocytes) and to produce dormant cells (akinetes). Heterocyte and akinete shape, size, and relative position have taxonomical relevance and possibly ecological value too. We collected-from literature and nature-and compared morphological data on vegetative cells, heterocytes, and akinetes across four genera taxonomically separated from Anabaena. In average, heterocyte size doubled that of vegetative cells-probably because of extra cell wall deposition. Heterocyte morphology was remarkably similar across genera, both in size and shape (spherical). The latter may decrease oxygen diffusion from adjoining vegetative cells. Akinetes were huge (one order of magnitude bigger) compared to vegetative cells, probably because of its massive genome replication, extra deposition of wall layers, allocation of storage and number of vegetative cells fused during akinete differentiation. Akinete shape was mostly cylindrical, or oval, but rarely spherical. In line with molecular data, we found morphological differences between Anabaena (non-aerotopated, soil or benthic) and Dolichospermum (aerotopated, planktonic), including vegetative cell size, and akinete size, shape, and relative position to the heterocyte. Differences may relate to adaptations to their contrasting environments (benthic versus planktic). Further research is needed to generalize our results to other heterocytous genera.

Bacterial lipopeptides, which contain beta-amino fatty acids, are an abundant group of bacterial secondary metabolites exhibiting antifungal and/or cytotoxic properties. Here we have developed an LC-HRMS/MS method for the selective detection of beta-amino fatty acid containing cyclic lipopeptides. The method was optimized using the lipopeptides iturin A and puwainaphycin F, which contain fatty acids of similar length but differ in the amino acid composition of the peptide cycle. Fragmentation energies of 10-55 eV were used to obtain the amino acid composition of the peptide macrocycle. However, fragmentation energies of 90-130 eV were used to obtain an intense fragment specific for the beta-amino fatty acid (CnH2n+2N+). The method allowed the number of carbons and consequently the length of the beta-amino fatty acid to be estimated. We identified 21 puwainaphycin variants differing in fatty acid chain in the crude extract of cyanobacterium Cylindrospermum alatosporum using this method. Analogously 11 iturin A variants were detected. The retention time of the lipopeptide variants showed a near perfect linear dependence (R-2=0.9995) on the length of the fatty acid chain in linear separation gradient which simplified the detection of minor variants. We used the method to screen 240 cyanobacterial strains and identified lipopeptides from 8 strains. The HPLC-HRMS/MS method developed here provides a rapid and easy way to detecting novel variants of cyclic lipopeptides. (C) 2016 Elsevier B.V. All rights reserved.

Four benthic Anabaena strains isolated from different localities in the Czech Republic were examined from the morphological, phylogenetic, and physiological points of view. The results of combined analysis showed distinct morphological dissimilarity between the studied strains, which were further found to belong to different phylogenetic groups based on the 16S rRNA gene phylogeny. To assess the temperature and light (irradiance) optima of the strains, we exposed them to various combinations of these two parameters. The experiment revealed unexpectedly high temperature and light optima for some of the strains, while others showed optima that were similar to those of previously studied planktic species of related heterocytous genera. Our study is the first of its kind to be applied to benthic Anabaena strains. Our results indicate that the benthic Anabaena genus is much more complex than previously thought and provide novel insights into the biology and ecology of benthic Anabaena species. With the collection of more data, we expect the genus Anabaena will be split into several new monophyletic taxa, each covering distinct morpho- and ecotypes.