Phylogenomic Analysis of Secondary Metabolism in the Toxic Cyanobacterial Genera Anabaena, Dolichospermum and Aphanizomenon.

Cyanobacteria produce an array of toxins that pose serious health risks to humans and animals. The closely related diazotrophic genera, Anabaena, Dolichospermum, and Aphanizomenon, frequently form poisonous blooms in lakes and brackish waters around the world. These genera form a complex now termed the Anabaena, Dolichospermum, and Aphanizomenon (ADA) clade and produce a greater array of toxins than any other cyanobacteria group. However, taxonomic confusion masks the distribution of toxin biosynthetic pathways in cyanobacteria. Here we obtained 11 new draft genomes to improve the understanding of toxin production in these genera. Comparison of secondary metabolite pathways in all available 31 genomes for these three genera suggests that the ability to produce microcystin, anatoxin-a, and saxitoxin is associated with specific subgroups. Each toxin gene cluster was concentrated or even limited to a certain subgroup within the ADA clade. Our results indicate that members of the ADA clade encode a variety of secondary metabolites following the phylogenetic clustering of constituent species. The newly sequenced members of the ADA clade show that phylogenetic separation of planktonic Dolichospermum and benthic Anabaena is not complete. This underscores the importance of taxonomic revision of Anabaena, Dolichospermum, and Aphanizomenon genera to reflect current phylogenomic understanding.

Transcriptomic and Proteomic Profiling of Anabaena sp. Strain 90 under Inorganic Phosphorus Stress.

Inorganic phosphorus (Pi) is one of the main growth-limiting factors of diazotrophic cyanobacteria. Due to human activity, the availability of Pi has increased in water bodies, resulting in eutrophication and the formation of massive cyanobacterial blooms. In this study, we examined the molecular responses of the cyanobacterium Anabaena sp. strain 90 to phosphorus deprivation, aiming at the identification of candidate genes to monitor the Pi status in cyanobacteria. Furthermore, this study increased the basic understanding of how phosphorus affects diazotrophic and bloom-forming cyanobacteria as a major growth-limiting factor. Based on RNA sequencing data, we identified 246 differentially expressed genes after phosphorus starvation and 823 differentially expressed genes after prolonged Pi limitation, most of them related to central metabolism and cellular growth. The transcripts of the genes related to phosphorus transport and assimilation (pho regulon) were most upregulated during phosphorus depletion. One of the most increased transcripts encodes a giant protein of 1,869 amino acid residues, which contains, among others, a phytase-like domain. Our findings predict its crucial role in phosphorus starvation, but future studies are still needed. Using two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found 43 proteins that were differentially expressed after prolonged phosphorus stress. However, correlation analysis unraveled an association only to some extent between the transcriptomic and proteomic abundances. Based on the present results, we suggest that the method used for monitoring the Pi status in cyanobacterial bloom should contain wider combinations of pho regulon genes (e.g., PstABCS transport systems) in addition to the commonly used alkaline phosphatase gene alone.

New structural variants of aeruginosin produced by the toxic bloom forming cyanobacterium Nodularia spumigena.

Nodularia spumigena is a filamentous diazotrophic cyanobacterium that forms blooms in brackish water bodies. This cyanobacterium produces linear and cyclic peptide protease inhibitors which are thought to be part of a chemical defense against grazers. Here we show that N. spumigena produces structurally novel members of the aeruginosin family of serine protease inhibitors. Extensive chemical analyses including NMR demonstrated that the aeruginosins are comprised of an N-terminal short fatty acid chain, L-Tyr, L-Choi and L-argininal and in some cases pentose sugar. The genome of N. spumigena CCY9414 contains a compact 18-kb aeruginosin gene cluster encoding a peptide synthetase with a reductive release mechanism which offloads the aeruginosins as reactive peptide aldehydes. Analysis of the aeruginosin and spumigin gene clusters revealed two different strategies for the incorporation of N-terminal protecting carboxylic acids. These results demonstrate that strains of N. spumigena produce aeruginosins and spumigins, two families of structurally similar linear peptide aldehydes using separate peptide synthetases. The aeruginosins were chemically diverse and we found 11 structural variants in 16 strains from the Baltic Sea and Australia. Our findings broaden the known structural diversity of the aeruginosin peptide family to include peptides with rare N-terminal short chain (C2-C10) fatty acid moieties.

The genetic basis for O-acetylation of the microcystin toxin in cyanobacteria.

Microcystins are a family of cyclic peptide toxins produced by cyanobacteria. They are responsible for the toxicosis and death of wild and domestic animals throughout the world. They display extensive variation in amino acid composition and functional group chemistry. O-acetylated microcystins are frequently produced by free-living and symbiotic strains of the genus Nostoc. Here, we show that the production of acetylated microcystins is catalyzed by an acetyl-coenzyme A-dependent O-acetyltransferase (McyL) encoded in the 57 kb microcystin synthetase gene cluster of Nostoc sp. 152. Phylogenetic analysis demonstrates that McyL belongs to a family of enzymes that inactivate antibiotics through O-acetylation. The McyL enzyme has a relaxed substrate specificity, allowing the preparation of semisynthetic microcystins. This study sheds light on the evolutionary origins and genetic diversity of an important class of enzymes involved in antibiotic resistance.

Nostophycin biosynthesis is directed by a hybrid polyketide synthase-nonribosomal peptide synthetase in the toxic cyanobacterium Nostoc sp. strain 152.

Cyanobacteria are a rich source of natural products with interesting pharmaceutical properties. Here, we report the identification, sequencing, annotation, and biochemical analysis of the nostophycin (npn) biosynthetic gene cluster. The npn gene cluster spans 45.1 kb and consists of three open reading frames encoding a polyketide synthase, a mixed polyketide nonribosomal peptide synthetase, and a nonribosomal peptide synthetase. The genetic architecture and catalytic domain organization of the proteins are colinear in arrangement, with the putative order of the biosynthetic assembly of the cyclic heptapeptide. NpnB contains an embedded monooxygenase domain linking nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) catalytic domains and predicted here to hydroxylate the nostophycin during assembly. Expression of the adenylation domains and subsequent substrate specificity assays support the involvement of this cluster in nostophycin biosynthesis. Biochemical analyses suggest that the loading substrate of NpnA is likely to be a phenylpropanoic acid necessitating deletion of a carbon atom to explain the biosynthesis of nostophycin. Biosyntheses of nostophycin and microcystin resemble each other, but the phylogenetic analyses suggest that they are distantly related to one another.