Discovery of a Pederin Family Compound in a Nonsymbiotic Bloom-Forming Cyanobacterium

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.Peer reviewe

Gypsum endolithic phototrophs under moderate climate (Southern Sicily): their diversity and pigment composition

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.This project was supported by the Czech Science Foundation (Grant/Award No. 17-04270S and 21-03322S), Ministry of Education, Youth and Sports of the Czech Republic, National Programme of Sustainability I (Grant/Award No. LO1416), Charles University (Grant/Award Nos. UNCE/SCI/006 and UNCE 204069), ALGAMIC (Grant/Award No. CZ.1.05/2.1.00/19.0392). JM was supported by the Czech Science Foundation (GAČR) Project No. 22-06374S to accomplish phylogenetic and taxonomic analysis. JW was thankful for the financial support by the PGC2021-124362NB-I00 grant from MCI/AEI (Spain) and FEDER.Peer reviewe

Polyphasic analysis and secondary metabolite patterns in nostocacean cyanobacteria with different life strategies

Unbranched heterocytous cyanobacteria exhibit complex filament and colony architectures and variable life strategies from symbionts to free living planktic and non-planktic species. They are counted among microbial groups showing an extensive production of secondary metabolites, resulting in both pharmaceutically important and toxic compounds. The main focus of this thesis is to broaden our knowledge on bioactive secondary metabolite potential in this widespread group of cyanobacteria. An effective combination of methods including whole genome sequencing, bioinformatic analysis, and analytical chemistry techniques are applied to accomplish this task. The discrepancies in distribution of various classes of compounds among ecological groups defined by different life strategies are discussed. Additionally, the thesis endeavours to test multidisciplinary approaches to tackle taxonomic assignments of unresolved unbranched heterocytous cyanobacteria using morphological, phylogenetic and ecophysiological methods, including a meta-analysis of morphological traits

POLYPHASIC CHARACTERIZATION OF Anabaena spp. (CYANOBACTERIA)FROM SOKOLOC AREA, CZECH REPUBLIC

Cilj ovog rada je polifaznim pristupom, tj. kombinacijom morfološkog i molekularnog pristupa načiniti karakterizaciju 16 sojeva bentonskih iperifitonskih vrsta Anabaena spp. sakupljenih tijekom 2010. i 2011. godine na osam različitihlokaliteta iz areala Sokolov (u sjevernom djelu Češke Republike). Na temelju dobivenih rezultata morfoloških svojstava (dimenzija vegetativnih stanica, heterocita i akineta) sojevi su podijeljeni u pet morfoloških skupina (morfoloških tipova). Odabrani sojevi koji predstavljaju odvojene filogenetske klastere bazirane na 16S rRNA genu, podvrgnuti su eksperimentu s različitim vrijednostima temperature i intenziteta svjetlosti gdje nisu pokazali značajne promjene u dimenzijama vegetativnih stanica, heterocita i akineta. Za iste sojeve eksperimentalno je određen optimalan rast, koji je za tri soja (04VR10L1, 11VR10JH,01DRMII10) bio sličan, dok se za soj 12SO10CI bitno razlikovao. Za većinu sojeva,grupiranje sojeva prema 16S rRNA genu odgovaralo je grupiranju prema morfološkim svojstvima. Prema dobivenim rezultatima, možemo zaključiti da je bentonski i perifitonski rod Anabaena filogenetički heterogen. Potrebna su daljnja istraživanja s većim brojem sojeva za bolje razumijevanje bioraznolikosti Anabaena spp.Main aim of this thesis is polyphasic characterization of 16 benthic andperiphytic Anabaena strain collected from eight localities in the north part of the Czech Republic in 2010-2011 near the town Sokolov. They were studied using a polyphasic approach, a combination of morphological and molecular approaches. According to the morphological comparison, there are five morphological groups (morphological types) we can find within the group of the studied strains. Morphological characteristics of the four studied Anabaena strains were measured under various temperature and light intensity. The effect of temperature and light on the dimensions of vegetative cells, heterocytes and akinetes was weak. Four strains exposed to growth optima experiments represented distant phylogeneticclusters based on the 16S rRNA gene, three of them (04VR10L1, 11VR10JH, 01DRMII10)had really similar temperature and light optima. Only the growth optima of the fourth strain,12SO10CI , were significantly different from them. According to the results, we can conclude benthic and periphytic Anabaena genus is phylogenetically heterogeneous. Clustering of the strains based on the 16S rRNA gene corresponds to morphological differences in most cases among 16 studied strains. More extended study on a higher number of strains is necessary fora deeper understanding the biodiversity of Anabaena spp

POLYPHASIC CHARACTERIZATION OF Anabaena spp. (CYANOBACTERIA)FROM SOKOLOC AREA, CZECH REPUBLIC

Cilj ovog rada je polifaznim pristupom, tj. kombinacijom morfološkog i molekularnog pristupa načiniti karakterizaciju 16 sojeva bentonskih iperifitonskih vrsta Anabaena spp. sakupljenih tijekom 2010. i 2011. godine na osam različitihlokaliteta iz areala Sokolov (u sjevernom djelu Češke Republike). Na temelju dobivenih rezultata morfoloških svojstava (dimenzija vegetativnih stanica, heterocita i akineta) sojevi su podijeljeni u pet morfoloških skupina (morfoloških tipova). Odabrani sojevi koji predstavljaju odvojene filogenetske klastere bazirane na 16S rRNA genu, podvrgnuti su eksperimentu s različitim vrijednostima temperature i intenziteta svjetlosti gdje nisu pokazali značajne promjene u dimenzijama vegetativnih stanica, heterocita i akineta. Za iste sojeve eksperimentalno je određen optimalan rast, koji je za tri soja (04VR10L1, 11VR10JH,01DRMII10) bio sličan, dok se za soj 12SO10CI bitno razlikovao. Za većinu sojeva,grupiranje sojeva prema 16S rRNA genu odgovaralo je grupiranju prema morfološkim svojstvima. Prema dobivenim rezultatima, možemo zaključiti da je bentonski i perifitonski rod Anabaena filogenetički heterogen. Potrebna su daljnja istraživanja s većim brojem sojeva za bolje razumijevanje bioraznolikosti Anabaena spp.Main aim of this thesis is polyphasic characterization of 16 benthic andperiphytic Anabaena strain collected from eight localities in the north part of the Czech Republic in 2010-2011 near the town Sokolov. They were studied using a polyphasic approach, a combination of morphological and molecular approaches. According to the morphological comparison, there are five morphological groups (morphological types) we can find within the group of the studied strains. Morphological characteristics of the four studied Anabaena strains were measured under various temperature and light intensity. The effect of temperature and light on the dimensions of vegetative cells, heterocytes and akinetes was weak. Four strains exposed to growth optima experiments represented distant phylogeneticclusters based on the 16S rRNA gene, three of them (04VR10L1, 11VR10JH, 01DRMII10)had really similar temperature and light optima. Only the growth optima of the fourth strain,12SO10CI , were significantly different from them. According to the results, we can conclude benthic and periphytic Anabaena genus is phylogenetically heterogeneous. Clustering of the strains based on the 16S rRNA gene corresponds to morphological differences in most cases among 16 studied strains. More extended study on a higher number of strains is necessary fora deeper understanding the biodiversity of Anabaena spp

Insight into functional and toxicological diversity of cyanobacterial mat communities in the Eel River

Toxic benthic cyanobacteria have started to attract attention after long neglect due to increased reports of their potential for secondary metabolites production. Particularly, this includes potent neurotoxins in the anatoxin class. Cyanobacteria commonly grow on the surfaces on riverbeds and these biofilms consist of assemblages of cyanobacteria and other microorganisms. So far, the vast majority of studies have focused solely on the cyanobacterial component, neglecting the diversity of the whole microbial assemblage and their interactions. We sampled cyanobacterial mats from the Eel River and resolved their community composition using genome-resolved metagenomics. The analyses revealed the regular presence of a range of heterotopic and viral components alongside dominant cyanobacterial taxa. Cyanobacterial genomes often encoded biosynthetic gene clusters of known toxins, as well as other metabolites belonging to different secondary metabolite classes. Several biosynthetic gene clusters were found across examined cyanobacterial genomes, and the presence of specific heterotrophic components. Our findings raise the importance of examining and characterizing the full microbial community that exists within cyanobacterial mats to understand when cyanobacteria possess toxic gene clusters and how that is correlated with the presence of surrounding microbial assemblage

Morphological traits in nitrogen fixing heterocytous cyanobacteria: possible links between morphology and eco-physiology

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.Fil: de Tezanos Pinto, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; ArgentinaFil: Kust, Andreja. University of South Bohemia; República ChecaFil: Devercelli, Melina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto Nacional de Limnología. Universidad Nacional del Litoral. Instituto Nacional de Limnología; ArgentinaFil: Kozlíková Zapomělová, Eliška. University of South Bohemia; República Chec

Unveiling the ecological significance of algal mats and meadows: Insights into phosphorus cycling and primary production of benthic algae in post-mining lakes

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,itemporal 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 33P-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

Insight into Unprecedented Diversity of Cyanopeptides in Eutrophic Ponds Using an MS/MS Networking Approach

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

Cyanochelins, an Overlooked Class of Widely Distributed Cyanobacterial Siderophores, Discovered by Silent Gene Cluster Awakening

Cyanobacteria require iron for growth and often inhabit iron-limited habitats, yet only a few siderophores are known to be produced by them. We report that cyanobacterial genomes frequently encode polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) biosynthetic pathways for synthesis of lipopeptides featuring b-hydroxyaspartate (b-OH-Asp), a residue known to be involved in iron chelation. Iron starvation triggered the synthesis of b-OH-Asp lipopeptides in the cyanobacteria Rivularia sp. strain PCC 7116, Leptolyngbya sp. strain NIES-3755, and Rubidibacter lacunae strain KORDI 51-2. The induced compounds were confirmed to bind iron by mass spectrometry (MS) and were capable of Fe31 to Fe21 photoreduction, accompanied by their cleavage, when exposed to sunlight. The siderophore from Rivularia, named cyanochelin A, was structurally characterized by MS and nuclear magnetic resonance (NMR) and found to contain a hydrophobic tail bound to phenolate and oxazole moieties followed by five amino acids, including two modified aspartate residues for iron chelation. Phylogenomic analysis revealed 26 additional cyanochelin-like gene clusters across a broad range of cyanobacterial lineages. Our data suggest that cyanochelins and related compounds are widespread b-OH-Asp-featuring cyanobacterial siderophores produced by phylogenetically distant species upon iron starvation. Production of photolabile siderophores by phototrophic cyanobacteria raises questions about whether the compounds facilitate iron monopolization by the producer or, rather, provide Fe21 for the whole microbial community via photoreduction. IMPORTANCE All living organisms depend on iron as an essential cofactor for indispensable enzymes. However, the sources of bioavailable iron are often limited. To face this problem, microorganisms synthesize low-molecular-weight metabolites capable of iron scavenging, i.e., the siderophores. Although cyanobacteria inhabit the majority of the Earth’s ecosystems, their repertoire of known siderophores is remarkably poor. Their genomes are known to harbor a rich variety of gene clusters with unknown function. Here, we report the awakening of a widely distributed class of silent gene clusters by iron starvation to yield cyanochelins, b-hydroxy aspartate lipopeptides involved in iron acquisition. Our results expand the limited arsenal of known cyanobacterial siderophores and propose products with ecological function for a number of previously orphan gene clusters