Ribosomal peptides are produced through the posttranslational modification of short precursor peptides. Cyanobactins are a growing family of cyclic ribosomal peptides produced by cyanobacteria. However, a broad systematic survey of the genetic capacity to produce cyanobactins is lacking. Here we report the identification of 31 cyanobactin gene clusters from 126 genomes of cyanobacteria. Genome mining suggested a complex evolutionary history defined by horizontal gene transfer and rapid diversification of precursor genes. Extensive chemical analyses demonstrated that some cyanobacteria produce short linear cyanobactins with a chain length ranging from three to five amino acids. The linear peptides were N-prenylated and O-methylated on the N and C termini, respectively, and named aeruginosamide and viridisamide. These findings broaden the structural diversity of the cyanobactin family to include highly modified linear peptides with rare posttranslational modifications.

SummaryNodularia spumigena is a filamentous nitrogen-fixing cyanobacterium that forms toxic blooms in brackish water bodies worldwide. Spumigins are serine protease inhibitors reported from a single strain of N. spumigena isolated from the Baltic Sea. These linear tetrapeptides contain non-proteinogenic amino acids including a C-terminal alcohol derivative of arginine. However, very little is known about these compounds despite the ecological importance of N. spumigena. We show that spumigins are assembled by two non-ribosomal peptide synthetases encoded in a 21 kb biosynthetic gene cluster. The compact non-ribosomal peptide synthetase features a reductive loading and release mechanism. Our analyses demonstrate that the bulk of spumigins produced by N. spumigena are released as peptide aldehydes in contrast to earlier findings. The main spumigin E variant contains an argininal residue and is a potent trypsin inhibitor. Spumigins were present in all of the N. spumigena strains isolated from the Baltic Sea and comprised up to 1% of the dry weight of the cyanobacterium. Our results demonstrate that bloom-forming N. spumigena strains produce a cocktail of enzyme inhibitors, which may explain in part the ecological success of this cyanobacterium in brackish water bodies worldwide.

Lichens are symbiotic associations between fungi and photosynthetic algae or cyanobacteria. Microcystins are potent toxins that are responsible for the poisoning of both humans and animals. These toxins are mainly associated with aquatic cyanobacterial blooms, but here we show that the cyanobacterial symbionts of terrestrial lichens from all over the world commonly produce microcystins. We screened 803 lichen specimens from five different continents for cyanobacterial toxins by amplifying a part of the gene cluster encoding the enzyme complex responsible for microcystin production and detecting toxins directly from lichen thalli. We found either the biosynthetic genes for making microcystins or the toxin itself in 12% of all analyzed lichen specimens. A plethora of different microcystins was found with over 50 chemical variants, and many of the variants detected have only rarely been reported from free-living cyanobacteria. In addition, high amounts of nodularin, up to 60 μg g −1 , were detected from some lichen thalli. This microcystin analog and potent hepatotoxin has previously been known only from the aquatic bloom-forming genus Nodularia. Our results demonstrate that the production of cyanobacterial hepatotoxins in lichen symbiosis is a global phenomenon and occurs in many different lichen lineages. The very high genetic diversity of the mcyE gene and the chemical diversity of microcystins suggest that lichen symbioses may have been an important environment for diversification of these cyanobacteria.Nostoc | Peltigera | cyanolichen | secondary metabolites | chemical defence

Significance New antifungal compounds are needed due to an increasing incidence of invasive fungal infections and resistance to many currently used drugs. Here we show that cyanobacteria are a rich source of antifungal compounds such as glycosylated lipopeptides, called hassallidins, which are commonly produced by filamentous nitrogen-fixing cyanobacteria. A diverse group of hassallidins and their complex nonribosomal biosynthesis were characterized in detail. Hassallidins and their previously unidentified biosynthetic enzymes offer new material for drug development. In addition, these compounds may have an ecological role in protecting cyanobacteria from parasitic fungi.

Cyanobacterial mass occurrences are common in fresh and brackish waters. They pose a threat to water users due to toxins frequently produced by the cyanobacterial species present. Anatoxin-a and homoanatoxin-a are neurotoxins synthesized by various cyanobacteria, e.g., Anabaena, Oscillatoria, and Aphanizomenon. The biosynthesis of these toxins and the genes involved in anatoxin production were recently described for Oscillatoria sp. strain PCC 6506 (A. Méjean et al., J. Am. Chem. Soc. 131:7512-7513, 2009). In this study, we identified the anatoxin synthetase gene cluster (anaA to anaG and orf1; 29 kb) in Anabaena sp. strain 37. The gene (81.6% to 89.2%) and amino acid (78.8% to 86.9%) sequences were highly similar to those of Oscillatoria sp. PCC 6506, while the organization of the genes differed. Molecular detection methods for potential anatoxin-a and homoanatoxin-a producers of the genera Anabaena, Aphanizomenon, and Oscillatoria were developed by designing primers to recognize the anaC gene. Anabaena and Oscillatoria anaC genes were specifically identified in several cyanobacterial strains by PCR. Restriction fragment length polymorphism (RFLP) analysis of the anaC amplicons enabled simultaneous identification of three producer genera: Anabaena, Oscillatoria, and Aphanizomenon. The molecular methods developed in this study revealed the presence of both Anabaena and Oscillatoria as potential anatoxin producers in Finnish fresh waters and the Baltic Sea; they could be applied for surveys of these neurotoxin producers in other aquatic environments.