Characterization of HphA: the First Enzyme in the Homologation Pathway of l-Phenylalanine and l-Tyrosine.

Homologation of amino acids is the insertion or deletion of a methylene group to their side chain, which is a relatively uncommon chemical transformation observed in peptide natural product (NP) structure. Homologated amino acids can potentially make the NP more stable in a biological system, but its biosynthesis is yet to be understood. This study biochemically characterized the first of three unexplored enzymes in the homologation pathway of l-phenylalanine and l-tyrosine. Previously proposed reactions catalyzed by HphA were confirmed by reversed-phase high-performance liquid chromatography and tandem mass spectrometry analysis. The substrate profile and kinetic parameters showed high selectivity for the natural substrates and their close analogs. The comparability of HphA to homologous enzymes in primary metabolic pathways, 2-isopropylmate synthase and homocitrate synthase which are involved in l-leucine and l-lysine biosynthesis, respectively, was validated by bioinformatical and site-directed mutagenesis studies. The knowledge obtained from this study has deepened the understanding of the homologation of amino acids, which can lead to future combinatorial biosynthesis and metabolic engineering studies.

Ecotoxicological assessment of cyclic peptides produced by a Planktothrix rubescens bloom: Impact on aquatic model organisms.

Cyanobacterial blooms, a natural phenomenon in freshwater ecosystems, have increased in frequency and severity due to climate change and eutrophication. Some cyanobacteria are able to produce harmful substances called cyanotoxins. These metabolites possess different chemical structures and action mechanisms representing a serious concern for human health and the environment. The most studied cyanotoxins belong to the group of microcystins which are potent hepatotoxins. Anabaenopeptins are another class of cyclic peptides produced by certain species of cyanobacteria, including Planktothrix spp. Despite limited knowledge regarding individual effects of anabaenopeptins on freshwater organisms, reports have identified in vivo toxicity in representatives of freshwater zooplankton by cyanobacterial extracts or mixtures containing anabaenopeptins. This study focused on the isolation and toxicity evaluation of the cyanotoxins produced in the 2022 Planktothrix rubescens bloom in Averno lake, Italy. The three main cyclic peptides have been isolated and identified by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS) and optical analyses as anabaenopeptins A and B, and oscillamide Y. Ecotoxicological tests on the aquatic model organisms Daphnia magna (crustacean), Raphidocelis subcapitata (algae), and Aliivibrio fischeri (bacterium) revealed that anabaenopeptins A and B do not generate significant toxicity at environmentally relevant concentrations, being also found a stimulatory effect on R. subcapitata in the case of anabaenopeptin A. By contrast, oscillamide Y displayed toxicity. Ecological implications based on ECOSAR predictions align with experimental data. Moreover, long-term exposure bioassays on different green unicellular algae species showed that R. subcapitata was not significantly affected, while Scenedesmus obliquus and Chlorella vulgaris exhibited altered growth patterns. These results, together with the already-known background in literature, highlight the complexity of interactions between organisms and the tested compounds, which may be influenced by species-specific sensitivities, physiological differences, and modes of action, possibly affected by parameters like lipophilicity.

Shifts in phytoplankton and zooplankton communities in three cyanobacteria-dominated lakes after treatment with hydrogen peroxide.

Cyanobacteria can reach high densities in eutrophic lakes, which may cause problems due to their potential toxin production. Several methods are in use to prevent, control or mitigate harmful cyanobacterial blooms. Treatment of blooms with low concentrations of hydrogen peroxide (H2O2) is a promising emergency method. However, effects of H2O2 on cyanobacteria, eukaryotic phytoplankton and zooplankton have mainly been studied in controlled cultures and mesocosm experiments, while much less is known about the effectiveness and potential side effects of H2O2 treatments on entire lake ecosystems. In this study, we report on three different lakes in the Netherlands that were treated with average H2O2 concentrations ranging from 2 to 5 mg L-1 to suppress cyanobacterial blooms. Effects on phytoplankton and zooplankton communities, on cyanotoxin concentrations, and on nutrient availability in the lakes were assessed. After every H2O2 treatment, cyanobacteria drastically declined, sometimes by more than 99%, although blooms of Dolichospermum sp., Aphanizomenon sp., and Planktothrix rubescens were more strongly suppressed than a Planktothrix agardhii bloom. Eukaryotic phytoplankton were not significantly affected by the H2O2 additions and had an initial advantage over cyanobacteria after the treatment, when ample nutrients and light were available. In all three lakes, a new cyanobacterial bloom developed within several weeks after the first H2O2 treatment, and in two lakes a second H2O2 treatment was therefore applied to again suppress the cyanobacterial population. Rotifers strongly declined after most H2O2 treatments except when the H2O2 concentration was ≤ 2 mg L-1, whereas cladocerans were only mildly affected and copepods were least impacted by the added H2O2. In response to the treatments, the cyanotoxins microcystins and anabaenopeptins were released from the cells into the water column, but disappeared after a few days. We conclude that lake treatments with low concentrations of H2O2 can be a successful tool to suppress harmful cyanobacterial blooms, but may negatively affect some of the zooplankton taxa in lakes. We advise pre-tests prior to the treatment of lakes to define optimal treatment concentrations that kill the majority of the cyanobacteria and to minimize potential side effects on non-target organisms. In some cases, the pre-tests may discourage treatment of the lake.

Anabaenopeptins from Nostoc edaphicum CCNP1411.

Cyanobacteria of the Nostoc genus belong to the most prolific sources of bioactive metabolites. In our previous study on Nostoc edaphicum strain CCNP1411, the occurrence of cyanopeptolins and nostocyclopeptides was documented. In the current work, the production of anabaenopeptins (APs) by the strain was studied using genetic and chemical methods. Compatibility between the analysis of the apt gene cluster and the structure of the identified APs was found. Three of the APs, including two new variants, were isolated as pure compounds and tested against four serine proteases and carboxypeptidase A (CPA). The in vitro enzymatic assays showed a typical activity of this class of cyanopeptides, i.e., the most pronounced effects were observed in the case of CPA. The activity of the detected compounds against important metabolic enzymes confirms the pharmaceutical potential of anabaenopeptins.

Interspecific Interactions Drive Nonribosomal Peptide Production in Nodularia spumigena.

Nodularia spumigena is a bloom-forming cyanobacterium that produces several classes of nonribosomal peptides (NRPs) that are biologically active; however, the ecological roles of specific NRPs remain largely unknown. Here, we explored the involvement of NRPs produced by N. spumigena in interspecific interactions by coculturing the cyanobacterium and its algal competitors, the diatom Phaeodactylum tricornutum and the cryptomonad Rhodomonas salina, and measuring NRP levels and growth responses in all three species. Contrary to the expected growth suppression in the algae, it was N. spumigena that was adversely affected by the diatom, while the cryptomonad had no effect. Reciprocal effects of N. spumigena on the algae were manifested as the prolonged lag phase in R. salina and growth stimulation in P. tricornutum; however, these responses were largely attributed to elevated pH and not to specific NRPs. Nevertheless, the NRP levels in the cocultures were significantly higher than in the monocultures, with an up to 5-fold upregulation of cell-bound nodularins and exudation of nodularin and anabaenopeptin. Thus, chemically mediated interspecific interactions can promote NRP production and release by cyanobacteria, resulting in increased input of these compounds into the water. IMPORTANCE NRPs were involved in growth responses of both cyanobacteria and algae; however, the primary driver of the growth trajectories was high pH induced by N. spumigena. Thus, the pH-mediated inhibition of eukaryotic phytoplankton may be involved in the bloom formation of N. spumigena. We also report, for the first time, the reciprocal growth inhibition of N. spumigena by diatoms resistant to alkaline conditions. As all species in this study can co-occur in the Baltic Sea during summer, these findings are highly relevant for understanding ecological interactions in planktonic communities in this and other systems experiencing regular cyanobacteria blooms.

Cyanobacterial Toxins and Peptides in Lake Vegoritis, Greece.

Cyanotoxins (CTs) produced by cyanobacteria in surface freshwater are a major threat for public health and aquatic ecosystems. Cyanobacteria can also produce a wide variety of other understudied bioactive metabolites such as oligopeptides microginins (MGs), aeruginosins (AERs), aeruginosamides (AEGs) and anabaenopeptins (APs). This study reports on the co-occurrence of CTs and cyanopeptides (CPs) in Lake Vegoritis, Greece and presents their variant-specific profiles obtained during 3-years of monitoring (2018-2020). Fifteen CTs (cylindrospermopsin (CYN), anatoxin (ATX), nodularin (NOD), and 12 microcystins (MCs)) and ten CPs (3 APs, 4 MGs, 2 AERs and aeruginosamide (AEG A)) were targeted using an extended and validated LC-MS/MS protocol for the simultaneous determination of multi-class CTs and CPs. Results showed the presence of MCs (MC-LR, MC-RR, MC-YR, dmMC-LR, dmMC-RR, MC-HtyR, and MC-HilR) and CYN at concentrations of <1 µg/L, with MC-LR (79%) and CYN (71%) being the most frequently occurring. Anabaenopeptins B (AP B) and F (AP F) were detected in almost all samples and microginin T1 (MG T1) was the most abundant CP, reaching 47.0 µg/L. This is the first report of the co-occurrence of CTs and CPs in Lake Vegoritis, which is used for irrigation, fishing and recreational activities. The findings support the need for further investigations of the occurrence of CTs and the less studied cyanobacterial metabolites in lakes, to promote risk assessment with relevance to human exposure.

Cyanobacterial anabaenopeptin-B, microcystins and their mixture cause toxic effects on the behavior of the freshwater crustacean Daphnia magna (Cladocera).

Comparison of the toxic effects caused by the pure cyanobacterial cyclic hexapeptide anabaenopeptin-B (AN-B), the heptapeptides: microcystin-LR (MC-LR) and MC-LF as well as a binary mixture of AN-B with MC-LR on the swimming speed and hopping frequency - essential activities of Daphnia, was experimentally determined. Till now, no information on behavioral effects of AN-B and its mixture with microcystins, commonly produced by cyanobacteria, was available. Also MC-LF effect on aquatic crustaceans was determined for the first time. The results showed that AN-B exerted considerable inhibition of D. magna swimming speed and hopping frequency similar to MC-LR and MC-LF. The mixture of AN-B and MC-LR caused stronger toxic effects, than the individual oligopeptides used at the same concentration. The much lower 48 h- EC50 value of the AN-B and MC-LR mixture (0.95 ± 0.12 µg/mL) than those of individual oligopeptides AN-B (6.3 ± 0.63 µg/mL), MC-LR (4.0 ± 0.27 µg/mL), MC-LF (3.9 ± 0.20 µg/mL) that caused swimming speed inhibition explains the commonly observed stronger toxicity of complex crude cyanobacterial extracts to daphnids than individual microcystins. The obtained results indicated that AN-B, microcystins and their mixture exerted time- and concentration-dependent motility disturbances of crustaceans and they can be good candidates for evaluation of toxicity in early warning systems. Other cyanobacterial oligopeptides beyond microcystins should be considered as a real threat for aquatic organisms.

Occurrence of microcystins, anabaenopeptins and other cyanotoxins in fish from a freshwater wildlife reserve impacted by harmful cyanobacterial blooms.

Harmful algal blooms of cyanobacteria (CyanoHABs) can lead to the release of potent toxins that can seriously affect ecosystem integrity. Some freshwater watersheds are particularly at risk considering the threats to already imperiled wildlife. The consumption of tainted drinking water and contaminated food also raises concerns for human health. In the present study, a pilot survey was conducted in the riverine ecosystem of the Pike River Ecological Reserve (QC, Canada) near Missisquoi Bay, Lake Champlain. We examined the occurrence of multiclass cyanotoxins including 12 microcystins, anatoxins, cylindrospermopsin (CYN), anabaenopeptins (AP-A, AP-B), and cyanopeptolin-A in surface waters and wild-caught fish during the summer 2018. Out of the 18 targeted cyanotoxins, 14 were detected in bloom-impacted surface water samples; toxins peaked during early-mid September with the highest concentrations for MC-LR (3.8 µg L-1) and MC-RR (2.9 µg L-1). Among the 71 field-collected fish from 10 species, 30% had positive detections to at least one cyanotoxin. In positive samples, concentration ranges in fish muscle were as follows for summed microcystins (∑MCs: 0.16-9.2 µg kg-1), CYN (46-75 µg kg-1), AP-A (1.1-5.4 µg kg-1), and AP-B (0.12-5.0 µg kg-1). To the best of our knowledge, this is one the first reports of anabaenopeptins occurrence in wildlife. The maximum ∑MCs in fish was 1.15-fold higher than the World Health Organization (WHO) daily intake recommendation for adults and nearly equated the derived value for young children. The concentration of CYN was also about 3-fold higher than the limit derived from the human health guideline values.

Anabaenopeptins from Cyanobacteria in Freshwater Bodies of Greece.

Cyanobacteria are photosynthetic microorganisms that are able to produce a large number of secondary metabolites. In freshwaters, under favorable conditions, they can rapidly multiply, forming blooms, and can release their toxic/bioactive metabolites in water. Among them, anabaenopeptins (APs) are a less studied class of cyclic bioactive cyanopeptides. The occurrence and structural variety of APs in cyanobacterial blooms and cultured strains from Greek freshwaters were investigated. Cyanobacterial extracts were analyzed with LC-qTRAP MS/MS using information-dependent acquisition in enhanced ion product mode in order to obtain the fragmentation mass spectra of APs. Thirteen APs were detected, and their possible structures were annotated based on the elucidation of fragmentation spectra, including three novel ones. APs were present in the majority of bloom samples (91%) collected from nine Greek lakes during different time periods. A large variety of APs was observed, with up to eight congeners co-occurring in the same sample. AP F (87%), Oscillamide Y (87%) and AP B (65%) were the most frequently detected congeners. Thirty cyanobacterial strain cultures were also analyzed. APs were only detected in one strain (Microcystis ichtyoblabe). The results contribute to a better understanding of APs produced by freshwater cyanobacteria and expand the range of structurally characterized APs.

High Levels of Anabaenopeptins Detected in a Cyanobacteria Bloom from N.E. Spanish Sau-Susqueda-El Pasteral Reservoirs System by LC-HRMS.

The appearance of a bloom of cyanobacteria in the Sau-Susqueda-El Pasteral system (River Ter, NE Spain) in the autumn of 2015 has been the most recent episode of extensive bloom detected in Catalonia. This system is devoted mainly to urban supply, regulation of the river, irrigation and production of hydroelectric energy. In fact, it is one of the main supply systems for the metropolitan area of cities such as Barcelona and Girona. An assessment and management plan was implemented in order to minimize the risk associated to cyanobacteria. The reservoir was confined and periodic sampling was carried out. Low and high toxicity was detected by cell bioassays with human cell lines. Additionally, analysis studies were performed by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-high resolution mass spectrometry (LC-HRMS). A microcystin target analysis and suspect screening of microcystins, nodularins, cylindrosperpmopsin and related cyanobacterial peptides by LC-HRMS were applied. The results for the analysis of microcystins were negative (<0.3 µg/L) in all the surface samples. Only traces of microcystin-LR, -RR and -dmRR were detected by LC-HRMS in a few ng/L from both fractions, aqueous and sestonic. In contrast, different anabaenopeptins and oscillamide Y at unusually high concentrations (µg-mg/L) were observed. To our knowledge, no previous studies have detected these bioactive peptides at such high levels. The reliable identification of these cyanobacterial peptides was achieved by HRMS. Although recently these peptides are detected frequently worldwide, these bioactive compounds have received little attention. Therefore, more studies on these substances are recommended, especially on their toxicity, health risk and presence in water resources.

Discovery of Unusual Cyanobacterial Tryptophan-Containing Anabaenopeptins by MS/MS-Based Molecular Networking.

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.

Blooms of Toxic Cyanobacterium Nodularia spumigena in Norwegian Fjords During Holocene Warm Periods.

In paleoecological studies, molecular markers are being used increasingly often to reconstruct community structures, environmental conditions and ecosystem changes. In this work, nodularin, anabaenopeptins and selected DNA sequences were applied as Nodularia spumigena markers to reconstruct the history of the cyanobacterium in the Norwegian fjords. For the purpose of this study, three sediment cores collected in Oslofjorden, Trondheimsfjorden and Balsfjorden were analyzed. The lack of nodularin in most recent sediments is consistent with the fact that only one report on the sporadic occurrence and low amounts of the cyanobacterium in Norwegian Fjords in 1976 has been published. However, analyses of species-specific chemical markers in deep sediments showed that thousands of years ago, N. spumigena constituted an important component of the phytoplankton community. The content of the markers in the cores indicated that the biomass of the cyanobacterium increased during the warmer Holocene periods. The analyses of genetic markers were less conclusive; they showed the occurrence of microcystin/nodularin producing cyanobacteria of Nostocales order, but they did not allow for the identification of the organisms at a species level.

The Effects of Cyanobacterial Bloom Extracts on the Biomass, Chl-a, MC and Other Oligopeptides Contents in a Natural Planktothrix agardhii Population.

Blooms of the cyanobacterium Planktothrix agardhii are common in shallow, eutrophic freshwaters. P. agardhii may produce hepatotoxic microcystins (MCs) and many other bioactive secondary metabolites belonging mostly to non-ribosomal oligopeptides. The aim of this work was to study the effects of two extracts (Pa-A and Pa-B) of P. agardhii-predominated bloom samples with different oligopeptide profiles and high concentration of biogenic compounds on another natural P. agardhii population. We hypothesised that the P. agardhii biomass and content of oligopeptides in P. agardhii is shaped in a different manner by diverse mixtures of metabolites of different P. agardhii-dominated cyanobacterial assemblages. For this purpose, the biomass, chlorophyll a and oligopeptides content in the treated P. agardhii were measured. Seven-day microcosm experiments with four concentrations of the extracts Pa-A and Pa-B were carried out. Generally, aeruginosins (AERs), cyanopeptolins (CPs) and anabaenopeptins (APs) were the most numerous peptides; however, only 16% of them were common for both extracts. The addition of the extracts resulted in similar effects on P. agardhii: an increase in biomass, Chl-a and MC content in the exposed P. agardhii as well as changes in its oligopeptide profile were observed. MCs present in the extracts did not inhibit accumulation of P. agardhii biomass, and did not have any negative effect on MC and Chl-a content. No evidence for bioaccumulation of dissolved peptides in the P. agardhii exposed was found. As the two tested extracts differed considerably in oligopeptide composition, but contained similar high concentrations of nutrients, it seems that biogenic compounds, not oligopeptides themselves, positively influenced the mixed natural P. agardhii population.

A Data-Independent Methodology for the Structural Characterization of Microcystins and Anabaenopeptins Leading to the Identification of Four New Congeners.

Toxin-producing cyanobacteria are responsible for the presence of hundreds of bioactive compounds in aquatic environments undergoing increasing eutrophication. The identification of cyanotoxins is still emerging, due to the great diversity of potential congeners, yet high-resolution mass spectrometry (HRMS) has the potential to deepen this knowledge in aquatic environments. In this study, high-throughput and sensitive on-line solid-phase extraction ultra-high performance liquid chromatography (SPE-UHPLC) coupled to HRMS was applied to a data-independent acquisition (DIA) workflow for the suspect screening of cyanopeptides, including microcystin and anabaenopeptin toxin classes. The unambiguous characterization of 11 uncommon cyanopeptides was possible using a characterization workflow through extensive analysis of fragmentation patterns. This method also allowed the characterization of four unknown cyanotoxins ([Leu1, Ser7] MC-HtyR, [Asp3]MC-RHar, AP731, and AP803). The quantification of 17 common cyanotoxins along with the semi-quantification of the characterized uncommon cyanopeptides resulted with the identification of 23 different cyanotoxins in 12 lakes in Canada, United Kingdom and France. The concentrations of the compounds varied between 39 and 41,000 ng L-1. To our knowledge, this is the first DIA method applied for the suspect screening of two families of cyanopeptides simultaneously. Moreover, this study shows the great diversity of cyanotoxins in lake water cyanobacterial blooms, a growing concern in aquatic systems.

Effects of secondary metabolites produced by different cyanobacterial populations on the freshwater zooplankters Brachionus calyciflorus and Daphnia pulex.

Cyanobacterial blooms in eutrophic water bodies are a worldwide problem. Combined effects of mixtures of secondary metabolites produced by different cyanobacterial species on aquatic fauna are still not well recognised. We compared the survivorship of Brachionus calyciflorus Pallas (Rotifera) and Daphnia pulex Leyding (Cladocera) exposed to pure microcystin LR (MC-LR), anatoxin-a (ANTX) and to five extracts obtained from bloom-forming cyanobacteria Microcystis, Planktothrix and Dolichospermum. The obtained results revealed different response of the organisms to high concentrations of pure MC-LR, ANTX and complex cyanobacterial extracts. The extracts' toxicity to invertebrates was higher than that exerted by pure cyanotoxins and was dependent on the composition of cyanobacterial metabolites: Microcystis spp. extract containing anabaenopeptins A and B, aeruginosamide, four variants of cyanopeptolins and five MCs was not toxic to either of the organisms, whereas Planktothrix agardhii extract (I), containing anabaenopeptins A, B, F, 915, oscillamide Y, five different aeruginosins and four variants of MC was more toxic to daphnids than to rotifers. The extracts of another P. agarhdii (II) biomass and two different biomass samples of Dolichospermum spp. also affected survivorship of the rotifer and cladoceran, however, to various extent. It strongly suggests that non-ribosomal oligopeptides, other than MCs, had essential contribution to the observed toxicity to invertebrates and their effects on particular species or populations can vary depending on the secondary metabolite profiles of cyanobacteria.

Anabaenopeptins and cyanopeptolins induce systemic toxicity effects in a model organism the nematode Caenorhabditis elegans.

Cyanobacterial blooms represent a significant risk to environmental and human health due to their production of toxic secondary metabolites, cyanopeptides. Anabaenopeptins and cyanopeptolins are cyanopeptides increasingly detected in surface waters at concentrations exceeding regulatory toxicity levels for other cyanotoxins such as microcystins. Yet their toxicity to aquatic organisms are not well understood. Here we assessed the toxicological effects of three anabaenopeptins (AP-A, AP-B, and AP-F) and three cyanopeptolins (CYP-1007, CYP-1020, and CYP-1041) to a model organism the nematode Caenorhabditis elegans. Examined toxicity endpoints included reproduction, hatching time, growth rate, lifespan, and age-related vulval integrity. Microcystin RR (MC-RR) and microginin 690 were also included in the study for comparisons. At an identical mass concentration (10 µg/L, corresponding to a molar concentration ranging 0.01-0.014 µM depending on the specific peptide), anabaenopeptins (APs) showed the greatest toxicity among all cyanopeptides tested. APs decreased worm reproduction by 23%-34% and shortened worm lifespan by 5 days (a 30% reduction) compared to the controls. APs also induced a remarkable age-related vulval integrity defect (Avid phenotype) in the worm, where over 95% of exposed worms developed the phenotype, compared to a less than 15% in control worms. CYPs showed similar toxicity as MC-RR, and Microginin 690 was the least toxic. These findings suggest that APs and CYPs may pose significant health risks to aquatic organisms. More toxicological studies of these cyanopeptides using different species across different trophic levels are needed to gain a thorough understanding of their potential impact on ecological systems and human health.

Total Synthesis of Keramamides†A and L from a Common Precursor by Late-Stage Indole Synthesis and Configurational Revision.

The marine natural products keramamide A and L, members of the class of anabaenopeptin-type peptides, were synthesized for the first time by a convergent and flexible route. The installation of the substituted tryptophan moieties was accomplished at the very end of the synthesis on the cyclic peptides, and thus enabled the synthesis of both natural products from one common precursor. The preparation of several epimers clearly indicates that the originally proposed relative configurations of both Keramamides A and L were not correct.

Specific Chemical and Genetic Markers Revealed a Thousands-Year Presence of Toxic Nodularia spumigena in the Baltic Sea.

In the Baltic Sea, diazotrophic cyanobacteria have been present for thousands of years, over the whole brackish water phase of the ecosystem. However, our knowledge about the species composition of the cyanobacterial community is limited to the last several decades. In the current study, the presence of species-specific chemical and genetic markers in deep sediments were analyzed to increase the existing knowledge on the history of toxic Nodularia spumigena blooms in the Baltic Sea. As chemical markers, three cyclic nonribosomal peptides were applied: the hepatotoxic nodularin, which in the sea was detected solely in N. spumigena, and two anabaenopeptins (AP827 and AP883a) characteristic of two different chemotypes of this species. From the same sediment samples, DNA was isolated and the gene involved in biosynthesis of nodularin, as well as the phycocyanin intergenic spacer region (PC-IGS), were amplified. The results of chemical and genetic analyses proved for the first time the thousands-year presence of toxic N. spumigena in the Baltic Sea. They also indicated that through all this time, the same two sub-populations of the species co-existed.

Spatial analysis of toxic or otherwise bioactive cyanobacterial peptides in Green Bay, Lake Michigan.

Cyanobacterial harmful algal blooms (cyanoHABs) are a growing problem in freshwater systems worldwide. CyanoHABs are well documented in Green Bay, Lake Michigan but little is known about cyanoHAB toxicity. This study characterized the diversity and spatial distribution of toxic or otherwise bioactive cyanobacterial peptides (TBPs) in Green Bay. Samples were collected in 2014 and 2015 during three cruises at sites spanning the mouth of the Fox River north to Chambers Island. Nineteen TBPs were analyzed including 11 microcystin (MC) variants, nodularin, three anabaenopeptins, three cyanopeptolins and microginin-690. Of the 19 TBPs, 12 were detected in at least one sample, and 94% of samples had detectable TBPs. The most prevalent TBPs were MCRR and MCLR, present in 94% and 65% of samples. The mean concentration of all TBPs was highest in the Fox River and lower bay, however, the maximum concentration of all TBPs occurred in the same sample north of the lower bay. MCs were positively correlated with chlorophyll and negatively correlated with distance to the Fox River in all cruises along a well-established south-to-north trophic gradient in Green Bay. The mean concentration of MC in the lower bay across all cruises was 3.0 +/- 2.3 µg/L. Cyanopeptolins and anabaenopeptins did not trend with the south-north trophic gradient or varied by cruise suggesting their occurrence is driven by different environmental factors. Results from this study provides evidence that trends in TBP concentration differ by congener type over a trophic gradient.

Morphologic, Phylogenetic and Chemical Characterization of a Brackish Colonial Picocyanobacterium (Coelosphaeriaceae) with Bioactive Properties.

Despite their cosmopolitan distribution, knowledge on cyanobacteria in the family Coelosphaeriaceae is limited. In this study, a single species culture of a coelosphaeran cyanobacterium isolated from a brackish rock pool in the Baltic Sea was established. The strain was characterized by morphological features, partial 16S rRNA sequence and nonribosomal oligopeptide profile. The bioactivity of fractionated extracts against several serine proteases, as well as protein-serine/threonine phosphatases was studied. Phylogenetic analyses of the strain suggested a close relationship with Snowella litoralis, but its morphology resembled Woronichinia compacta. The controversial morphologic and phylogenetic results demonstrated remaining uncertainties regarding species division in this cyanobacteria family. Chemical analyses of the strain indicated production of nonribosomal oligopeptides. In fractionated extracts, masses and ion fragmentation spectra of seven possible anabaenopeptins were identified. Additionally, fragmentation spectra of cyanopeptolin-like peptides were collected in several of the fractions. The nonribosomal oligopeptide profile adds another potential identification criterion in future inter- and intraspecies comparisons of coelosphaeran cyanobacteria. The fractionated extracts showed significant activity against carboxypeptidase A and trypsin. Inhibition of these important metabolic enzymes might have impacts at the ecosystem level in aquatic habitats with high cyanobacteria densities.