Natural radioactivity of residues from groundwater treatment facilities in Finland.

The accumulation of naturally occurring radionuclides in solid residues was investigated from groundwater treatment facilities (GTFs) in Finland. Natural radionuclides U-238, Ra-226, Pb-210 and Ra-228 were found in various precipitates, sludges and filters at concentrations exceeding the general clearance level of 1 kBq kg-1used for solid materials in the European directive 2013/59/Euratom. The accumulation of natural radionuclides in different solid residues was observed even when the activity concentrations in the untreated groundwater were relatively low, and when there was no measurable change in the concentrations between raw and treated groundwater within analytical uncertainties. Based on mass and activity balance considerations this is thought to be due to the large volumes of treated water per year. The exposure of workers to natural radiation from solid residues in the regular use of a groundwater facility was found not to be likely to exceed 0.3 mSv a-1if the activity concentrations are <10 kBq kg-1for U-238, Ra-226, Pb-210 and Ra-228. The worker exposure from solid residues is therefore likely to remain below the reference level of 1 mSv a-1, and indoor radon is more of a concern for the radiation protection of workers at GTFs. However, the natural radionuclide content in the different solid residues from groundwater treatment needs to be characterised properly to be able to ensure safety in the final use of the residues with respect to the potential exposure of the public.

Radioactivity of residues from waste incineration facilities in Finland.

Waste incineration in Europe has been increasing in the past few decades as there is a need to reduce the burden on landfills and their associated environmental concerns. While incineration reduces the volume of the waste, the volume of slag and ash is still substantial. To find out potential radiation risks that incineration residues could set to workers or the public, the levels of radioactive elements in these residues were investigated from nine waste incineration plants in Finland. Natural and artificial radionuclides were detected in the residues, but in general the activity concentrations were low. This study shows that the level of Cs-137 in the fly ash from municipal waste incineration follows the pattern of 1986 fallout zones in Finland, although the levels are significantly lower than in ash from bioenergy production from the same areas. Am-241 was also detected in many samples, although the activity concentrations were very low. Based on the findings in this study, the typical ash and slag residues from municipal waste incineration do not need radiation protection measures for workers or the public even in regions that received up to 80 kBq m-2of Cs-137 fallout in 1986. The further use of these residues need not be restricted due to radioactivity. Hazardous waste incineration residues and other special cases need to be considered separately, depending on the original waste composition.

N-Prenylation of Tryptophan by an Aromatic Prenyltransferase from the Cyanobactin Biosynthetic Pathway.

Aromatic prenylation is an important step in the biosynthesis of many natural products and leads to an astonishing diversity of chemical structures. Cyanobactin pathways frequently encode aromatic prenyltransferases that catalyze the prenylation of these macrocyclic and linear peptides. Here we characterized the anacyclamide ( acy) biosynthetic gene cluster from Anabaena sp. UHCC-0232. Partial reconstitution of the anacyclamide pathway, heterologous expression, and in vitro biochemical characterization demonstrate that the AcyF enzyme, encoded in the acy biosynthetic gene cluster, is a Trp N-prenyltransferase. Bioinformatic analysis suggests the monophyletic origin and rapid diversification of cyanobactin prenyltransferase enzymes and the multiple origins of N-1 Trp prenylation in prenylated natural products. The AcyF enzyme displayed high flexibility toward a range of Trp-containing substrates and represents an interesting new tool for biocatalytic applications.

Sphaerocyclamide, a prenylated cyanobactin from the cyanobacterium Sphaerospermopsis sp. LEGE 00249.

Cyanobactins are a family of linear and cyclic peptides produced through the post-translational modification of short precursor peptides. A mass spectrometry-based screening of potential cyanobactin producers led to the discovery of a new prenylated member of this family of compounds, sphaerocyclamide (1), from Sphaerospermopsis sp. LEGE 00249. The sphaerocyclamide biosynthetic gene cluster (sph) encoding the novel macrocyclic prenylated cyanobactin, was sequenced. Heterologous expression of the sph gene cluster in Escherichia coli confirmed the connection between genomic and mass spectrometric data. Unambiguous establishment of the orientation and site of prenylation required the full structural elucidation of 1 using Nuclear Magnetic Resonance (NMR), which demonstrated that a forward prenylation occurred on the tyrosine residue. Compound 1 was tested in pharmacologically or ecologically relevant biological assays and revealed moderate antimicrobial activity towards the fouling bacterium Halomonas aquamarina CECT 5000.

Strains of the toxic and bloom-forming Nodularia spumigena (cyanobacteria) can degrade methylphosphonate and release methane.

Nodularia spumigena is a nitrogen-fixing cyanobacterium that forms toxic blooms in the Baltic Sea each summer and the availability of phosphorous is an important factor limiting the formation of these blooms. Bioinformatic analysis identified a phosphonate degrading (phn) gene cluster in the genome of N. spumigena suggesting that this bacterium may use phosphonates as a phosphorus source. Our results show that strains of N. spumigena could grow in medium containing methylphosphonic acid (MPn) as the sole source of phosphorous and released methane when growing in medium containing MPn. We analyzed the total transcriptomes of N. spumigena UHCC 0039 grown using MPn and compared them with cultures growing in Pi-replete medium. The phnJ, phosphonate lyase gene, was upregulated when MPn was the sole source of phosphorus, suggesting that the expression of this gene could be used to indicate the presence of bioavailable phosphonates. Otherwise, growth on MPn resulted in only a minor reconstruction of the transcriptome and enabled good growth. However, N. spumigena strains were not able to utilize any of the anthropogenic phosphonates tested. The phosphonate utilizing pathway may offer N. spumigena a competitive advantage in the Pi-limited cyanobacterial blooms of the Baltic Sea.

Cyclic peptide production using a macrocyclase with enhanced substrate promiscuity and relaxed recognition determinants.

Macrocyclic peptides have promising therapeutic potential but the scaling up of their chemical synthesis is challenging. The cyanobactin macrocyclase PatGmac is an efficient tool for production but is limited to substrates containing 6-11 amino acids and at least one thiazoline or proline. Here we report a new cyanobactin macrocyclase that can cyclize longer peptide substrates and those not containing proline/thiazoline and thus allows exploring a wider chemical diversity.

A Unique Tryptophan C-Prenyltransferase from the Kawaguchipeptin Biosynthetic Pathway.

Cyanobactins are a rapidly growing family of linear and cyclic peptides produced by cyanobacteria. Kawaguchipeptins A and B, two macrocyclic undecapeptides reported earlier from Microcystis aeruginosa NIES-88, are shown to be products of the cyanobactin biosynthetic pathway. The 9 kb kawaguchipeptin (kgp) gene cluster was identified in a 5.26 Mb draft genome of Microcystis aeruginosa NIES-88. We verified that this gene cluster is responsible for the production of the kawaguchipeptins through heterologous expression of the kgp gene cluster in Escherichia coli. The KgpF prenyltransferase was overexpressed and was shown to prenylate C-3 of Trp residues in both linear and cyclic peptides in vitro. Our findings serve to further enhance the structural diversity of cyanobactins to include tryptophan-prenylated cyclic peptides.

Genome mining expands the chemical diversity of the cyanobactin family to include highly modified linear peptides.

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.

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.

Cyanobactins-ribosomal cyclic peptides produced by cyanobacteria.

Cyanobactins are small cyclic peptides that are produced by a diverse selection of cyanobacteria living in symbioses as well as terrestrial, marine, or freshwater environments. They include compounds with antimalarial, antitumor, and multidrug reversing activities and potential as pharmaceutical leads. Cyanobactins are produced through the proteolytic cleavage and cyclization of precursor peptides coupled with further posttranslational modifications such as heterocyclization, oxidation, or prenylation of amino acids. Cyanobactin gene clusters encode two proteases which cleave and cyclisize the precursor peptide as well as proteins participating in posttranslational modifications. The bioinformatic mining of cyanobacterial genomes has led to the discovery of novel cyanobactins. Heterologous expression of these gene clusters provided insights into the role of the genes participating in the biosynthesis of cyanobactins and facilitated the rational design of novel peptides. Enzymes participating in the biosynthesis of cyanobactins may prove useful as catalysts for producing novel cyclic peptides in the future. The recent discovery of the cyanobactin biosynthetic pathway in cyanobacteria extends our knowledge of their potential as producers of interesting metabolites.

Highly diverse cyanobactins in strains of the genus Anabaena.

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.

Widespread occurrence and lateral transfer of the cyanobactin biosynthesis gene cluster in cyanobacteria.

Cyanobactins are small cyclic peptides produced by cyanobacteria. Here we demonstrate the widespread but sporadic occurrence of the cyanobactin biosynthetic pathway. We detected a cyanobactin biosynthetic gene in 48 of the 132 strains included in this study. Our results suggest that cyanobactin biosynthetic genes have a complex evolutionary history in cyanobacteria punctuated by a series of ancient horizontal gene transfer events.