More than just an eagle killer: The freshwater cyanobacterium Aetokthonos hydrillicola produces highly toxic dolastatin derivatives.

Cyanobacteria are infamous producers of toxins. While the toxic potential of planktonic cyanobacterial blooms is well documented, the ecosystem level effects of toxigenic benthic and epiphytic cyanobacteria are an understudied threat. The freshwater epiphytic cyanobacterium Aetokthonos hydrillicola has recently been shown to produce the "eagle killer" neurotoxin aetokthonotoxin (AETX) causing the fatal neurological disease vacuolar myelinopathy. The disease affects a wide array of wildlife in the southeastern United States, most notably waterfowl and birds of prey, including the bald eagle. In an assay for cytotoxicity, we found the crude extract of the cyanobacterium to be much more potent than pure AETX, prompting further investigation. Here, we describe the isolation and structure elucidation of the aetokthonostatins (AESTs), linear peptides belonging to the dolastatin compound family, featuring a unique modification of the C-terminal phenylalanine-derived moiety. Using immunofluorescence microscopy and molecular modeling, we confirmed that AEST potently impacts microtubule dynamics and can bind to tubulin in a similar matter as dolastatin 10. We also show that AEST inhibits reproduction of the nematode Caenorhabditis elegans. Bioinformatic analysis revealed the AEST biosynthetic gene cluster encoding a nonribosomal peptide synthetase/polyketide synthase accompanied by a unique tailoring machinery. The biosynthetic activity of a specific N-terminal methyltransferase was confirmed by in vitro biochemical studies, establishing a mechanistic link between the gene cluster and its product.

Occurrence of aetokthonotoxin producer in natural samples - A PCR protocol for easy detection.

Cyanobacteria are well known producers of bioactive metabolites, including harmful substances. The recently discovered "eagle killer" neurotoxin aetokthonotoxin (AETX) is produced by the epiphytic cyanobacterium Aetokthonos hydrillicola growing on invasive water thyme (Hydrilla verticillata). The biosynthetic gene cluster of AETX was previously identified from an Aetokthonos strain isolated from the J. Strom Thurmond Reservoir, Georgia, USA. Here, a PCR protocol for easy detection of AETX-producers in environmental samples of plant-cyanobacterium consortia was designed and tested. Three different loci of the AETX gene cluster were amplified to confirm the genetic potential for AETX production, along with two variable types of rRNA ITS regions to confirm the homogeneity of the producer´s taxonomic identity. In samples of Hydrilla from three Aetokthonos-positive reservoirs and one Aetokthonos-negative lake, the PCR of all four loci provided results congruent with the Aetokthonos presence/absence detected by light and fluorescence microscopy. The production of AETX in the Aetokthonos-positive samples was confirmed using LC-MS. Intriguingly, in J. Strom Thurmond Reservoir, recently Hydrilla free, an Aetokthonos-like cyanobacterium was found growing on American water-willow (Justicia americana). Those specimens were positive for all three aet markers but contained only minute amounts of AETX. The obtained genetic information (ITS rRNA sequence) and morphology of the novel Aetokthonos distinguished it from all the Hydrilla-hosted A. hydrillicola, likely at the species level. Our results suggest that the toxigenic Aetokthonos spp. can colonize a broader array of aquatic plants, however the level of accumulation of the toxin may be driven by host-specific interactions such as the locally hyper-accumulated bromide in Hydrilla.

Alcobiosis, an algal-fungal association on the threshold of lichenisation.

Alcobiosis, the symbiosis of algae and corticioid fungi, frequently occurs on bark and wood. Algae form a layer in or below fungal basidiomata reminiscent of the photobiont layer in lichens. Identities of algal and fungal partners were confirmed by DNA barcoding. Algal activity was examined using gas exchange and chlorophyll fluorescence techniques. Carbon transfer from algae to fungi was detected as 13C, assimilated by algae, transferred to the fungal polyol. Nine fungal partners scattered across Agaricomycetes are associated with three algae from Trebouxiophycae: Coccomyxa sp. with seven fungal species on damp wood, Desmococcus olivaceus and Tritostichococcus coniocybes, both with a single species on bark and rain-sheltered wood, respectively. The fungal partner does not cause any obvious harm to the algae. Algae enclosed in fungal tissue exhibited a substantial CO2 uptake, but carbon transfer to fungal tissues was only detected in the Lyomyces-Desmococcus alcobiosis where some algal cells are tightly enclosed by hyphae in goniocyst-like structures. Unlike lichen mycobionts, fungi in alcobioses are not nutritionally dependent on the algal partner as all of them can live without algae. We consider alcobioses to be symbioses in various stages of co-evolution, but still quite different from true lichens.

A new microcystin producing Nostoc strain discovered in broad toxicological screening of non-planktic Nostocaceae (cyanobacteria).

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.

Highly divergent 16S rRNA sequences in ribosomal operons of Scytonema hyalinum (Cyanobacteria).

A highly divergent 16S rRNA gene was found in one of the five ribosomal operons present in a species complex currently circumscribed as Scytonema hyalinum (Nostocales, Cyanobacteria) using clone libraries. If 16S rRNA sequence macroheterogeneity among ribosomal operons due to insertions, deletions or truncation is excluded, the sequence heterogeneity observed in S. hyalinum was the highest observed in any prokaryotic species thus far (7.3-9.0%). The secondary structure of the 16S rRNA molecules encoded by the two divergent operons was nearly identical, indicating possible functionality. The 23S rRNA gene was examined for a few strains in this complex, and it was also found to be highly divergent from the gene in Type 2 operons (8.7%), and likewise had nearly identical secondary structure between the Type 1 and Type 2 operons. Furthermore, the 16S-23S ITS showed marked differences consistent between operons among numerous strains. Both operons have promoter sequences that satisfy consensus requirements for functional prokaryotic transcription initiation. Horizontal gene transfer from another unknown heterocytous cyanobacterium is considered the most likely explanation for the origin of this molecule, but does not explain the ultimate origin of this sequence, which is very divergent from all 16S rRNA sequences found thus far in cyanobacteria. The divergent sequence is highly conserved among numerous strains of S. hyalinum, suggesting adaptive advantage and selective constraint of the divergent sequence.

Molecular and morphological delimitation and generic classification of the family Oocystaceae (Trebouxiophyceae, Chlorophyta).

The family Oocystaceae (Chlorophyta) is a group of morphologically and ultrastructurally distinct green algae that constitute a well-supported clade in the class Trebouxiophyceae. Despite the family's clear delimitation, which is based on specific cell wall features, only a few members of the Oocystaceae have been examined using data other than morphological. In previous studies of Trebouxiophyceae, after the establishment of molecular phylogeny, the taxonomic status of the family was called into question. The genus Oocystis proved to be paraphyletic and some species were excluded from Oocystaceae, while a few other species were newly redefined as members of this family. We investigated 54 strains assigned to the Oocystaceae using morphological, ultrastructural and molecular data (SSU rRNA and rbcL genes) to clarify the monophyly of and diversity within Oocystaceae. Oonephris obesa and Nephrocytium agardhianum clustered within the Chlorophyceae and thus are no longer members of the Oocystaceae. On the other hand, we transferred the coenobial Willea vilhelmii to the Oocystaceae. Our findings combined with those of previous studies resulted in the most robust definition of the family to date. The division of the family into three subfamilies and five morphological clades was suggested. Taxonomical adjustments in the genera Neglectella, Oocystidium, Oocystis, and Ooplanctella were established based on congruent molecular and morphological data. We expect further taxonomical changes in the genera Crucigeniella, Eremosphaera, Franceia, Lagerheimia, Oocystis, and Willea in the future.