Matti Wahlsten

Discovery of Rare and Highly Toxic Microcystins from Lichen-Associated Cyanobacterium Nostoc sp. Strain IO-102-I

ABSTRACT The production of hepatotoxic cyclic heptapeptides, microcystins, is almost exclusively reported from planktonic cyanobacteria. Here we show that a terrestrial cyanobacterium Nostoc sp. strain IO-102-I isolated from a lichen association produces six different microcystins. Microcystins were identified with liquid chromatography-UV mass spectrometry by their retention times, UV spectra, mass fragmentation, and comparison to microcystins from the aquatic Nostoc sp. strain 152. The dominant microcystin produced by Nostoc sp. strain IO-102-I was the highly toxic [ADMAdda5]microcystin-LR, which accounted for ca. 80% of the total microcystins. We assigned a structure of [DMAdda5]microcystin-LR and [d-Asp3,ADMAdda5]microcystin-LR and a partial structure of three new [ADMAdda5]-XR type of microcystin variants. Interestingly, Nostoc spp. strains IO-102-I and 152 synthesized only the rare ADMAdda and DMAdda subfamilies of microcystin variants. Phylogenetic analyses demonstrated congruence between genes involved directly in microcystin biosynthesis and the 16S rRNA and rpoC1 genes of Nostoc sp. strain IO-102-I. Nostoc sp. strain 152 and the Nostoc sp. strain IO-102-I are distantly related, revealing a sporadic distribution of toxin production in the genus Nostoc. Nostoc sp. strain IO-102-I is closely related to Nostoc punctiforme PCC 73102 and other symbiotic Nostoc strains and most likely belongs to this species. Together, this suggests that other terrestrial and aquatic strains of the genus Nostoc may have retained the genes necessary for microcystin biosynthesis.

Cyanobacteria produce a high variety of hepatotoxic peptides in lichen symbiosis

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.

Anatoxin-a Synthetase Gene Cluster of the Cyanobacterium Anabaena sp. Strain 37 and Molecular Methods To Detect Potential Producers

ABSTRACT 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.

Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

BackgroundMicrocystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1) and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis.ResultsHere we undertook a phylogenetic study to investigate the order and timing of recombination between the mcyB1 and mcyC genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the mcyB1 and mcyC adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between mcyB1 and mcyC in strains of the genera Anabaena, Microcystis, and Hapalosiphon. We also find clear evidence for independent adenylation domain conversion of mcyB1 by unrelated peptide synthetase modules in strains of the genera Nostoc and Microcystis. The recombination events replace only the adenylation domain in each case and the condensation domains of mcyB1 and mcyC are not transferred together with the adenylation domain. Our findings demonstrate that the mcyB1 and mcyC adenylation domains are recombination hotspots in the microcystin synthetase gene cluster.ConclusionRecombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains.

Direct Evidence for Production of Microcystins by Anabaena Strains from the Baltic Sea

ABSTRACT Anabaena is a filamentous, N2-fixing, and morphologically diverse genus of cyanobacteria found in freshwater and brackish water environments worldwide. It contributes to the formation of toxic blooms in freshwater bodies through the production of a range of hepatotoxins or neurotoxins. In the Baltic Sea, Anabaena spp. form late summer blooms, together with Nodularia spumigena and Aphanizomenon flos-aquae. It has been long suspected that Baltic Sea Anabaena may produce microcystins. The presence of microcystins has been reported for the coastal regions of the Baltic proper, and a recent report also indicated the presence of the toxin in the open Gulf of Finland. However, at present there is no direct evidence linking Baltic Sea Anabaena spp. to microcystin production. Here we report on the isolation of microcystin-producing strains of the genus Anabaena in the open Gulf of Finland. The dominant microcystin variants produced by these strains included the highly toxic MCYST-LR as well as [d-Asp3]MCYST-LR, [d-Asp3]MCYST-HtyR, MCYST-HtyR, [d-Asp3,Dha7]MCYST-HtyR, and [Dha7]MCYST-HtyR variants. Toxic strains were isolated from the coastal Gulf of Finland as well as from the easternmost open-sea sampling station, where there were lower salinities than at other stations. This result suggests that lower salinity may favor microcystin-producing Anabaena strains. Furthermore, we sequenced 16S rRNA genes and found evidence for pronounced genetic heterogeneity of the microcystin-producing Anabaena strains. Future studies should take into account the potential presence of microcystin-producing Anabaena sp. in the Gulf of Finland.

Highly Diverse Cyanobactins in Strains of the Genus Anabaena

ABSTRACT Cyanobactins are small, cyclic peptides recently found in cyanobacteria. They are formed through proteolytic cleavage and posttranslational modification of short precursor proteins and exhibit antitumor, cytotoxic, or multi-drug-reversing activities. Using genome project data, bioinformatics, stable isotope labeling, and mass spectrometry, we discovered novel cyclic peptides, anacyclamides, in 27 Anabaena strains. The lengths of the anacylamides varied greatly, from 7 to 20 amino acids. Pronounced sequence variation was also detected, and only one amino acid, proline, was present in all anacyclamides. The anacyclamides identified included unmodified proteinogenic or prenylated amino acids. We identified an 11-kb gene cluster in the genome of Anabaena sp. 90, and heterologous expression in Escherichia coli confirmed that this cluster was responsible for anacyclamide production. The discovery of anacyclamides greatly increases the structural diversity of cyanobactins.

The non-ribosomal assembly and frequent occurrence of the protease inhibitors spumigins in the bloom-forming cyanobacterium Nodularia spumigena.

Nodularia 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.

Hassallidins, antifungal glycolipopeptides, are widespread among cyanobacteria and are the end-product of a nonribosomal pathway

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. Cyanobacteria produce a wide variety of cyclic peptides, including the widespread hepatotoxins microcystins and nodularins. Another class of peptides, cyclic glycosylated lipopeptides called hassallidins, show antifungal activity. Previously, two hassallidins (A and B) were reported from an epilithic cyanobacterium Hassallia sp. and found to be active against opportunistic human pathogenic fungi. Bioinformatic analysis of the Anabaena sp. 90 genome identified a 59-kb cryptic inactive nonribosomal peptide synthetase gene cluster proposed to be responsible for hassallidin biosynthesis. Here we describe the hassallidin biosynthetic pathway from Anabaena sp. SYKE748A, as well as the large chemical variation and common occurrence of hassallidins in filamentous cyanobacteria. Analysis demonstrated that 20 strains of the genus Anabaena carry hassallidin synthetase genes and produce a multitude of hassallidin variants that exhibit activity against Candida albicans. The compounds discovered here were distinct from previously reported hassallidins A and B. The IC50 of hassallidin D was 0.29–1.0 µM against Candida strains. A large variation in amino acids, sugars, their degree of acetylation, and fatty acid side chain length was detected. In addition, hassallidins were detected in other cyanobacteria including Aphanizomenon, Cylindrospermopsis raciborskii, Nostoc, and Tolypothrix. These compounds may protect some of the most important bloom-forming and globally distributed cyanobacteria against attacks by parasitic fungi.

Analysis of an Inactive Cyanobactin Biosynthetic Gene Cluster Leads to Discovery of New Natural Products from Strains of the Genus Microcystis

Cyanobactins are cyclic peptides assembled through the cleavage and modification of short precursor proteins. An inactive cyanobactin gene cluster has been described from the genome Microcystis aeruginosa NIES843. Here we report the discovery of active counterparts in strains of the genus Microcystis guided by this silent cyanobactin gene cluster. The end products of the gene clusters were structurally diverse cyclic peptides, which we named piricyclamides. Some of the piricyclamides consisted solely of proteinogenic amino acids while others contained disulfide bridges and some were prenylated or geranylated. The piricyclamide gene clusters encoded between 1 and 4 precursor genes. They encoded highly diverse core peptides ranging in length from 7–17 amino acids with just a single conserved amino acid. Heterologous expression of the pir gene cluster from Microcystis aeruginosa PCC7005 in Escherichia coli confirmed that this gene cluster is responsible for the biosynthesis of piricyclamides. Chemical analysis demonstrated that Microcystis strains could produce an array of piricyclamides some of which are geranylated or prenylated. The genetic diversity of piricyclamides in a bloom sample was explored and 19 different piricyclamide precursor genes were found. This study provides evidence for a stunning array of piricyclamides in Microcystis, a worldwide occurring bloom forming cyanobacteria.

Natural occurrence of microcystin synthetase deletion mutants capable of producing microcystins in strains of the genus Anabaena (Cyanobacteria).

Microcystins form a large family of small cyclic heptapeptides harbouring extensive modifications in amino acid residue composition and functional group chemistry. These peptide hepatotoxins contain a range of non-proteinogenic amino acids and unusual peptide bonds, and are typically N-methylated. They are synthesized on large enzyme complexes consisting of non-ribosomal peptide synthetases and polyketide synthases in a variety of distantly related cyanobacterial genera. Here we report a 1236 bp in-frame deletion mutation in the mcyA gene of the microcystin biosynthetic pathway in nine strains of the genus Anabaena. The deletion removed almost the entire N-methyltransferase (NMT) domain. Strains of Anabaena carrying the in-frame deletion mutation incorporated mainly dehydroalanine (Dha) into the microcystins they produce while strains with full-length mcyA genes incorporated mainly N-methyldehydroalanine (Mdha). Interestingly, the strains of Anabaena lacking the NMT domain also incorporated elevated amounts of L-Ser, the precursor of Mdha and Dha, into the microcystin they produced relative to strains carrying functional NMT domains. We provide evidence for the in-frame deletion of the NMT domain without the co-conversion of the flanking adenylation domain. Our results demonstrate a further example of the strategies employed by cyanobacteria in the biosynthesis of microcystin variants.