Kaarina Sivonen

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing

The cyanobacterial phylum encompasses oxygenic photosynthetic prokaryotes of a great breadth of morphologies and ecologies; they play key roles in global carbon and nitrogen cycles. The chloroplasts of all photosynthetic eukaryotes can trace their ancestry to cyanobacteria. Cyanobacteria also attract considerable interest as platforms for “green” biotechnology and biofuels. To explore the molecular basis of their different phenotypes and biochemical capabilities, we sequenced the genomes of 54 phylogenetically and phenotypically diverse cyanobacterial strains. Comparison of cyanobacterial genomes reveals the molecular basis for many aspects of cyanobacterial ecophysiological diversity, as well as the convergence of complex morphologies without the acquisition of novel proteins. This phylum-wide study highlights the benefits of diversity-driven genome sequencing, identifying more than 21,000 cyanobacterial proteins with no detectable similarity to known proteins, and foregrounds the diversity of light-harvesting proteins and gene clusters for secondary metabolite biosynthesis. Additionally, our results provide insight into the distribution of genes of cyanobacterial origin in eukaryotic nuclear genomes. Moreover, this study doubles both the amount and the phylogenetic diversity of cyanobacterial genome sequence data. Given the exponentially growing number of sequenced genomes, this diversity-driven study demonstrates the perspective gained by comparing disparate yet related genomes in a phylum-wide context and the insights that are gained from it.

Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes

ABSTRACT Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanjärvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanjärvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and Anabaena. The main microcystin producer in Lake Tuusulanjärvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of Anabaena. Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.

Effect of nitrogen and phosphorus on growth of toxic and nontoxic Microcystis strains and on intracellular microcystin concentrations.

The growth and intracellular microcystin concentration of two hepatotoxic and two nontoxic axenic Microcystis strains were measured in batch cultures with variable nitrogen (0.84-84 mg L(-1)) and phosphorus (0.05-5.5 mg L(-1)) concentrations. Growth was estimated by measuring dry weight, optical density, chlorophyll a, and cellular protein concentration. Microcystin concentrations in cells and in culture medium were measured by HPLC analysis. Both nontoxic strains needed less nutrients for their growth at low nutrient concentrations. With high nutrient concentrations the toxic strains grew better than the nontoxic strains. Growth and intracellular microcystin concentration did not correlate in the hepatotoxic strains. Multivariate regression analysis together with mathematical modeling revealed a significant interactive effect of nitrogen and phosphorus, which partly explains the controversial results obtained in previous studies. In this study we have shown that variation of nitrogen and phosphorus concentrations influence the growth and the microcystin production of Microcystis strains and that the strains differ in their response to nutrients. High levels of nitrogen and phosphorus in freshwaters may favor the growth of toxic Microcystis strains over nontoxic ones.

Toxic cyanobacteria (blue-green algae) in Finnish fresh and coastal waters

A survey of the occurrence of toxic blooms of cyanobacteria in Finnish fresh and coastal waters was made during 1985 and 1986. Toxicity of the freeze-dried water bloom samples was tested by mouse-bioassay (i.p.). Forty-four per cent (83/188) of the bloom samples were found to be lethally toxic. Hepatotoxic blooms (54) were almost twice as common as neurotoxic ones (29). Anabaena was the most frequently found genus in toxic and non-toxic blooms and it was present in all neurotoxic samples. Statistical associations were found between hepatotoxicity and incidence of Microcystis aeruginosa, M. viridis, M. wesenbergii, Anabaena flos-aquae and Anabaena spiroides. Neurotoxicity was statistically associated with Anabaena lemmermannii, Anabaena flos-aquae and Gomphosphaeria naegeliana. Isolation of strains of cyanobacteria confirmed the occurrence of hepatotoxic and neurotoxic strains of Anabaena, as well as hepatotoxic strains of Microcystis and Oscillatoria species.Toxic blooms caused cattle poisonings at three different lakes during the study period. Toxic blooms also occurred in drinking water sources. Our study shows that toxic cyanobacteria are more common in Finnish lakes than would be expected on the basis of animal poisonings. The results of this study show the existence of toxic cyanobacteria in Finnish water supplies and the need for their continued study as agents of water based disease.

Molecular characterization of planktic cyanobacteria of Anabaena, Aphanizomenon, Microcystis and Planktothrix genera

Toxic and non-toxic cyanobacterial strains from Anabaena, Aphanizomenon, Calothrix, Cylindrospermum, Nostoc, Microcystis, Planktothrix (Oscillatoria agardhii), Oscillatoria and Synechococcus genera were examined by RFLP of PCR-amplified 16S rRNA genes and 16S rRNA gene sequencing. With both methods, high 16S rRNA gene similarity was found among planktic, anatoxin-a-producing Anabaena and non-toxic Aphanizomenon, microcystin-producing and non-toxic Microcystis, and microcystin-producing and non-toxic Planktothrix strains of different geographical origins. The respective sequence similarities were 99.9-100%, 94.2-99.9% and 99.3-100%. Thus the morphological characteristics (e.g. Anabaena and Aphanizomenon), the physiological (toxicity) characteristics or the geographical origins did not reflect the level of 16S rRNA gene relatedness of the closely related strains studied. In addition, cyanobacterial strains were fingerprinted with repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR. All the strains except two identical pairs of Microcystis strains had different band profiles. The overall grouping of the trees from the 16S rRNA gene and the REP- and ERIC-PCR analyses was similar. Based on the 16S rRNA gene sequence analysis, four major clades were formed. (i) The clade containing filamentous heterocystous cyanobacteria was divided into three discrete groups of Anabaena/Aphanizomenon, Anabaena/Cylindrospermum/ Nodularia/Nostoc and Calothrix strains. The three other clades contained (ii) filamentous non-heterocystous Planktothrix, (iii) unicellular non-heterocystous Microcystis and (iv) Synechococcus strains.

Genes Coding for Hepatotoxic Heptapeptides (Microcystins) in the Cyanobacterium Anabaena Strain 90

ABSTRACT The cluster of microcystin synthetase genes from Anabaena strain 90 was sequenced and characterized. The total size of the region is 55.4 kb, and the genes are organized in three putative operons. The first operon (mcyA-mcyB-mcyC) is transcribed in the opposite direction from the second operon (mcyG-mcyD-mcyJ-mcyE-mcyF-mcyI) and the third operon (mcyH). The genes mcyA, mcyB, and mcyC encode nonribosomal peptide synthetases (NRPS), while mcyD codes for a polyketide synthase (PKS), and mcyG and mcyE are mixed NRPS-PKS genes. The genes mcyJ, mcyF, and mcyI are similar to genes coding for a methyltransferase, an aspartate racemase, and a d-3-phosphoglycerate dehydrogenase, respectively. The region in the first module of mcyB coding for the adenylation domain was found to be 96% identical with the corresponding part of mcyC, suggesting a recent duplication of this fragment and a replacement in mcyB. In Anabaena strain 90, the order of the domains encoded by the genes in the two sets (from mcyG to mcyI and from mcyA to mcyC) is colinear with the hypothetical order of the enzymatic reactions for microcystin biosynthesis. The order of the microcystin synthetase genes in Anabaena strain 90 differs from the arrangement found in two other cyanobacterial species, Microcystis aeruginosa and Planktothrix agardhii. The average sequence match between the microcystin synthetase genes of Anabaena strain 90 and the corresponding genes of the other species is 74%. The identity of the individual proteins varies from 67 to 81%. The genes of microcystin biosynthesis from three major producers of this toxin are now known. This makes it possible to design probes and primers to identify the toxin producers in the environment.

PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera

Microcystins are harmful hepatotoxins produced by many, but not all strains of the cyanobacterial genera Anabaena, Microcystis, Anabaena, Planktothrix, and Nostoc. Waterbodies have to be monitored for the mass development of toxic cyanobacteria; however, because of the close genetic relationship of microcystin-producing and non-producing strains within a genus, identification of microcystin-producers by morphological criteria is not possible. The genomes of microcystin-producing cells contain mcy genes coding for the microcystin synthetase complex. Based on the sequence information of mcy genes from Microcystis and Planktothrix, a primer pair for PCR amplification of a mcyA gene fragment was designed. PCR with this primer pair is a powerful means to identify microcystin-producing strains of the genera Anabaena, Microcystis, and Planktothrix. Moreover, subsequent RFLP analysis of the PCR products generated genus-specific fragments and allowed the genus of the toxin producer to be identified. The assay can be used with DNA from field samples.

The effect of water treatment processes on the removal of hepatotoxins fromMicrocystis andOscillatoria cyanobacteria: A laboratory study

The behaviour of hepatotoxins fromMicrocystis andOscillatoria cyanobacteria in some common water treatment processes was investigated on the laboratory scale in order to obtain data on their potential transfer to drinking water. Two toxins were separated from both of the freeze-dried cyanobacterial materials used in the experiments, a natural bloom consisting ofM. wesenbergii andM. viridis and a laboratory-grown culture ofO. agardhii. The concentrations of all four toxins before the treatments were between 30 and 60 μg/1. The investigated water treatment processes, selected from among methods applied in Finland, were: (1) Al2(SO4)3 flocculation with sand filtration and chlorination; (2) FeCl3 flocculation with sand filtration and chlorination; (3) addition of activated carbon powder with Al2(SO4)3 flocculation, sand filtration and chlorination; (4) Al2(SO4) flocculation with sand filtration, activated carbon filtration and chlorination; and (5) ozonation with Al2(SO4)3 flocculation, sand filtration and chlorination. The conventional flocculation-filtration-chlorination procedures resulted in a relatively small decrease in the toxin concentrations. Activated carbon powder in low doses did not improve the results, but activated carbon filtration as well as ozonation completely removed the toxins. The toxin concentrations were determined by HPLC. The proper functioning of the treatment processes was monitored by measurement of the KMnO4 value, turbidity and flocculation chemical residues.

Persistence of cyanobacterial hepatotoxin, microcystin-LR in particulate material and dissolved in lake water

Abstract The persistence of cyanobacterial hepatotoxin, microcystin-LR, was investigated in Lake Tuusulanjarvi in southern Finland from August to October, 1993 and 1994. The amount of toxin in particulate material and dissolved in water were determined by HPLC from samples collected from mesocosm enclosures and from the surrounding lake water. In the beginning of the experiments over 80% of the phytoplankton biomass consisted of cyanobacteria. The main species were Microcystis wesenbergii (Chroococcales, Cyanobacteria), M. viridis and M. aeruginosa. The microcystin-LR concentration in particulate material varied from 2.7 to 3.2 μg l−1 and the corresponding concentration of microcystin LR dissolved in water from 0.06 to 0.21 μg l−1. The cyanobacterial biomass decreased towards the middle of September and simultaneously the microcystin concentration in freeze dried particulate material decreased below the detection limit of 10 μg g−1, corresponding 0.01 μg l−1. Dissolved microcystin-LR was detected in a concentration range of 1 to 5 ng l−1 even at the end of the experiments in October, when the cyanobacterial biomass was less than 1 mg l−1. Thus, dissolved microcystin was more persistent compared to microcystin in particulate material: the decimal reduction time for dissolved toxin was 30 d and for toxin in particulate material about 15 d.