The population genetic structure and phylogeographic dispersal of Nodularia breviconcha in the Korean Peninsula based on COI and 16S rRNA genes.

A freshwater mussel, Nodularia breviconcha (Mollusca: Bivalvia: Unionida) is endemic to Korean Peninsula. It has recently been taxonomically reexamined and elevated from a subspecies of N. douglasiae to an independent species. But population genetic studies for the species have rarely been conducted. To explore the population genetic structure of N. breviconcha, the nucleotide sequences of cytochrome oxidase subunit I(COI) and 16S rRNA genes from 135 N. breviconcha individuals, including 52 from this study and 83 from Choi et al. (2020). We found 23 COI and 11 16S rRNA genes haplotypes. Phylogeny, TCS network, Principal coordinates analysis, and spatial analysis of molecular variance performed with COI gene indicated that there are exist three different genetic lineages in the N. breviconcha populations: West lineage, Southwest lineage, and Southeast lineage. According to the time calibrated phylogeny, they are likely to be diverged during the late Miocene (8-6 Ma). Geographical distribution patterns of the three genetic lineages may be related to the formation of Taebaek and Sobaek-Noryeong mountain ranges in the Korean Peninsula occurred during the Miocene (30-10 Ma). The present results of this study will be helpful not only for the conservation, but also for the exploration of the population genetic structure of endemic freshwater mussels in the Korean Peninsula.

Potent Inhibitor of Human Trypsins from the Aeruginosin Family of Natural Products.

Serine proteases regulate many physiological processes and play a key role in a variety of cancers. Aeruginosins are a family of natural products produced by cyanobacteria that exhibit pronounced structural diversity and potent serine protease inhibition. Here, we sequenced the complete genome of Nodularia sphaerocarpa UHCC 0038 and identified the 43.7 kb suomilide biosynthetic gene cluster. Bioinformatic analysis demonstrated that suomilide belongs to the aeruginosin family of natural products. We identified 103 complete aeruginosin biosynthetic gene clusters from 12 cyanobacterial genera and showed that they encode an unexpected chemical diversity. Surprisingly, purified suomilide inhibited human trypsin-2 and -3, with IC50 values of 4.7 and 11.5 nM, respectively, while trypsin-1 was inhibited with an IC50 of 104 nM. Molecular dynamics simulations suggested that suomilide has a long residence time when bound to trypsins. This was confirmed experimentally for trypsin-1 and -3 (residence times of 1.5 and 57 min, respectively). Suomilide also inhibited the invasion of aggressive and metastatic PC-3M prostate cancer cells without affecting cell proliferation. The potent inhibition of trypsin-3, together with a long residence time and the ability to inhibit prostate cancer cell invasion, makes suomilide an attractive drug lead for targeting cancers that overexpress trypsin-3. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and suggest that aeruginosins may be a source of selective inhibitors of human serine proteases.

Molecular phylogenetic, population genetic and demographic studies of Nodularia douglasiae and Nodularia breviconcha based on CO1 and 16S rRNA.

Freshwater mussels belonging to the genus Nodularia (Family Unionidae) are known to be widely distributed in East Asia. Although phylogenetic and population genetic studies have been performed for these species, there still remain unresolved questions in their taxonomic status and biogeographic distribution pathways. Here, the nucleotide sequences of CO1 and 16S rRNA were newly determined from 86 N. douglasiae and 83 N. breviconcha individuals collected on the Korean Peninsula. Based on these data, we revealed the following results: (1) N. douglasiae can be divided into the three genetic clades of A (only found in Korean Peninsula), B (widely distributed in East Asia), and C (only found in the west of China and Russia), (2) the clade A is not an independent species but a concrete member of N. douglasiae given the lack of genetic differences between the clades A and B, and (3) N. breviconcha is not a subspecies of N. douglasiae but an independent species apart from N. douglasiae. In addition, we suggested the plausible scenarios of biogeographic distribution events and demographic history of Nodularia species.

Blooms of Toxic Cyanobacterium Nodularia spumigena in Norwegian Fjords During Holocene Warm Periods.

In paleoecological studies, molecular markers are being used increasingly often to reconstruct community structures, environmental conditions and ecosystem changes. In this work, nodularin, anabaenopeptins and selected DNA sequences were applied as Nodularia spumigena markers to reconstruct the history of the cyanobacterium in the Norwegian fjords. For the purpose of this study, three sediment cores collected in Oslofjorden, Trondheimsfjorden and Balsfjorden were analyzed. The lack of nodularin in most recent sediments is consistent with the fact that only one report on the sporadic occurrence and low amounts of the cyanobacterium in Norwegian Fjords in 1976 has been published. However, analyses of species-specific chemical markers in deep sediments showed that thousands of years ago, N. spumigena constituted an important component of the phytoplankton community. The content of the markers in the cores indicated that the biomass of the cyanobacterium increased during the warmer Holocene periods. The analyses of genetic markers were less conclusive; they showed the occurrence of microcystin/nodularin producing cyanobacteria of Nostocales order, but they did not allow for the identification of the organisms at a species level.

Genomic and Metabolomic Analyses of Natural Products in Nodularia spumigena Isolated from a Shrimp Culture Pond.

The bloom-forming cyanobacterium Nodularia spumigena CENA596 encodes the biosynthetic gene clusters (BGCs) of the known natural products nodularins, spumigins, anabaenopeptins/namalides, aeruginosins, mycosporin-like amino acids, and scytonemin, along with the terpenoid geosmin. Targeted metabolomics confirmed the production of these metabolic compounds, except for the alkaloid scytonemin. Genome mining of N. spumigena CENA596 and its three closely related Nodularia strains-two planktonic strains from the Baltic Sea and one benthic strain from Japanese marine sediment-revealed that the number of BGCs in planktonic strains was higher than in benthic one. Geosmin-a volatile compound with unpleasant taste and odor-was unique to the Brazilian strain CENA596. Automatic annotation of the genomes using subsystems technology revealed a related number of coding sequences and functional roles. Orthologs from the Nodularia genomes are involved in the primary and secondary metabolisms. Phylogenomic analysis of N. spumigena CENA596 based on 120 conserved protein sequences positioned this strain close to the Baltic Nodularia. Phylogeny of the 16S rRNA genes separated the Brazilian CENA596 strain from those of the Baltic Sea, despite their high sequence identities (99% identity, 100% coverage). The comparative analysis among planktic Nodularia strains showed that their genomes were considerably similar despite their geographically distant origin.

Specific Chemical and Genetic Markers Revealed a Thousands-Year Presence of Toxic Nodularia spumigena in the Baltic Sea.

In the Baltic Sea, diazotrophic cyanobacteria have been present for thousands of years, over the whole brackish water phase of the ecosystem. However, our knowledge about the species composition of the cyanobacterial community is limited to the last several decades. In the current study, the presence of species-specific chemical and genetic markers in deep sediments were analyzed to increase the existing knowledge on the history of toxic Nodularia spumigena blooms in the Baltic Sea. As chemical markers, three cyclic nonribosomal peptides were applied: the hepatotoxic nodularin, which in the sea was detected solely in N. spumigena, and two anabaenopeptins (AP827 and AP883a) characteristic of two different chemotypes of this species. From the same sediment samples, DNA was isolated and the gene involved in biosynthesis of nodularin, as well as the phycocyanin intergenic spacer region (PC-IGS), were amplified. The results of chemical and genetic analyses proved for the first time the thousands-year presence of toxic N. spumigena in the Baltic Sea. They also indicated that through all this time, the same two sub-populations of the species co-existed.

Chemical and Genetic Diversity of Nodularia spumigena from the Baltic Sea.

Nodularia spumigena is a toxic, filamentous cyanobacterium occurring in brackish waters worldwide, yet forms extensive recurrent blooms in the Baltic Sea. N. spumigena produces several classes of non-ribosomal peptides (NRPs) that are active against several key metabolic enzymes. Previously, strains from geographically distant regions showed distinct NRP metabolic profiles. In this work, conspecific diversity in N. spumigena was studied using chemical and genetic approaches. NRP profiles were determined in 25 N. spumigena strains isolated in different years and from different locations in the Baltic Sea using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Genetic diversity was assessed by targeting the phycocyanin intergenic spacer and flanking regions (cpcBA-IGS). Overall, 14 spumigins, 5 aeruginosins, 2 pseudaeruginosins, 2 nodularins, 36 anabaenopeptins, and one new cyanopeptolin-like peptide were identified among the strains. Seven anabaenopeptins were new structures; one cyanopeptolin-like peptide was discovered in N. spumigena for the first time. Based on NRP profiles and cpcBA-IGS sequences, the strains were grouped into two main clusters without apparent influence of year and location, indicating persistent presence of these two subpopulations in the Baltic Sea. This study is a major step in using chemical profiling to explore conspecific diversity with a higher resolution than with a sole genetic approach.

Hiperplasia nodular focal: diagnóstico a considerar ante una masa hepática / Focal nodular hyperplasia: A diagnosis to consider in a hepatic mass

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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.

Insights into the physiology and ecology of the brackish-water-adapted Cyanobacterium Nodularia spumigena CCY9414 based on a genome-transcriptome analysis.

Nodularia spumigena is a filamentous diazotrophic cyanobacterium that dominates the annual late summer cyanobacterial blooms in the Baltic Sea. But N. spumigena also is common in brackish water bodies worldwide, suggesting special adaptation allowing it to thrive at moderate salinities. A draft genome analysis of N. spumigena sp. CCY9414 yielded a single scaffold of 5,462,271 nucleotides in length on which genes for 5,294 proteins were annotated. A subsequent strand-specific transcriptome analysis identified more than 6,000 putative transcriptional start sites (TSS). Orphan TSSs located in intergenic regions led us to predict 764 non-coding RNAs, among them 70 copies of a possible retrotransposon and several potential RNA regulators, some of which are also present in other N2-fixing cyanobacteria. Approximately 4% of the total coding capacity is devoted to the production of secondary metabolites, among them the potent hepatotoxin nodularin, the linear spumigin and the cyclic nodulapeptin. The transcriptional complexity associated with genes involved in nitrogen fixation and heterocyst differentiation is considerably smaller compared to other Nostocales. In contrast, sophisticated systems exist for the uptake and assimilation of iron and phosphorus compounds, for the synthesis of compatible solutes, and for the formation of gas vesicles, required for the active control of buoyancy. Hence, the annotation and interpretation of this sequence provides a vast array of clues into the genomic underpinnings of the physiology of this cyanobacterium and indicates in particular a competitive edge of N. spumigena in nutrient-limited brackish water ecosystems.

Diversity of peptides produced by Nodularia spumigena from various geographical regions.

Cyanobacteria produce a great variety of non-ribosomal peptides. Among these compounds, both acute toxins and potential drug candidates have been reported. The profile of the peptides, as a stable and specific feature of an individual strain, can be used to discriminate cyanobacteria at sub-population levels. In our work, liquid chromatography-tandem mass spectrometry was used to elucidate the structures of non-ribosomal peptides produced by Nodularia spumigena from the Baltic Sea, the coastal waters of southern Australia and Lake Iznik in Turkey. In addition to known structures, 9 new congeners of spumigins, 4 aeruginosins and 12 anabaenopeptins (nodulapeptins) were identified. The production of aeruginosins by N. spumigena was revealed in this work for the first time. The isolates from the Baltic Sea appeared to be the richest source of the peptides; they also showed a higher diversity in peptide profiles. The Australian strains were characterized by similar peptide patterns, but distinct from those represented by the Baltic and Lake Iznik isolates. The results obtained with the application of the peptidomic approach were consistent with the published data on the genetic diversity of the Baltic and Australian populations.

Species composition and cyanotoxin production in periphyton mats from three lakes of varying trophic status.

In lakes, benthic micro-algae and cyanobacteria (periphyton) can contribute significantly to total primary productivity and provide important food sources for benthic invertebrates. Despite recognition of their importance, few studies have explored the diversity of the algal and cyanobacterial composition of periphyton mats in temperate lakes. In this study, we sampled periphyton from three New Zealand lakes: Tikitapu (oligotrophic), Okareka (mesotrophic) and Rotoiti (eutrophic). Statistical analysis of morphological data showed a clear delineation in community structure among lakes and highlighted the importance of cyanobacteria. Automated rRNA intergenic spacer analysis (ARISA) and 16S rRNA gene clone libraries were used to investigate cyanobacterial diversity. Despite the close geographic proximity of the lakes, cyanobacterial species differed markedly. The 16S rRNA gene sequence analysis identified eight cyanobacterial OTUs. A comparison with other known cyanobacterial sequences in GenBank showed relatively low similarities (91-97%). Cyanotoxin analysis identified nodularin in all mats from Lake Tikitapu. ndaF gene sequences from these samples had very low (≤ 89%) homology to sequences in other known nodularin producers. To our knowledge, this is the first detection of nodularin in a freshwater environment in the absence of Nodularia. Six cyanobacteria species were isolated from Lake Tikitapu mats. None were found to produce nodularin. Five of the species shared low (< 97%) 16S rRNA gene sequence similarities with other cultured cyanobacteria.

Characterization of nifH gene expression, modification and rearrangement in Nodularia spumigena strain AV1.

The annually reoccurring blooms that characterize the surface waters of the Baltic Sea are dominated by filamentous, heterocystous cyanobacteria such as Nodularia spumigena. In a previous study, we have demonstrated that N. spumigena strain AV1 differentiates heterocysts in the absence of detectable nitrogen fixation activity, an unusual physiological trait that is clearly distinct from other well-studied cyanobacteria. To further analyze the uncoupling between these two processes, we analyzed the gene expression and modification of the nitrogenase enzyme (the enzyme responsible for nitrogen fixation) in N. spumigena AV1, as well as in several other N. spumigena strains. Here, we demonstrate the occurrence of two nifH gene copies in N. spumigena strain AV1, only one of which is located in a complete nifHDK cluster and several NifH protein forms. Furthermore, we demonstrate the occurrence of a DNA rearrangement mechanism acting within the nifH gene copy located in the nifHDK cluster and present only in the strains exhibiting the previously reported uncoupling between heterocyst differentiation and nitrogen fixation processes. These data stress the existence of a distinct and complex regulatory circuit related to nitrogen fixation in this ecologically significant bloom-forming cyanobacterium.

A new quantitative PCR assay for the detection of hepatotoxigenic cyanobacteria.

Toxin-producing cyanobacteria are a worldwide threat to both human and animal health. The hepatotoxins microcystin and nodularin are the most commonly occurring toxins produced by bloom-forming cyanobacteria. They are cyclic peptides that are synthesized nonribosomally by a multienzyme complexes encoded within the microcystin (mcyS) and nodularin (ndaS) synthetase gene clusters. Early detection of potentially toxic blooms would allow for pre-emptive action to reduce consumer exposure to cyanotoxins. We have developed a quantitative PCR (qPCR) assay based on SYBR-green chemistry for the detection of potentially hepatotoxic cyanobacteria spanning all known microcystin and nodularin producing taxa using primers specifically targeting mcyE and ndaF. The qPCR assay was validated against previously analyzed cyanobacterial bloom samples. Whole cell qPCR using cultured M. aeruginosa PCC7806 and non-toxic M. aeruginosa UTEX2386 had a sensitivity of 1000 cells ml⁻¹. In summary, we have developed a robust and sensitive molecular method for the detection and quantification of hepatotoxigenic cyanobacteria in bloom samples. This technology offers several advantages over traditional and contemporary testing protocols currently used to assess water quality.

Variability in the response of the cyanobacterium Nodularia spumigena to nitrogen supplementation.

Nodularia spumigena is a filamentous cyanobacterium that is commonly found in brackish water bodies. The species is capable of fixing atmospheric nitrogen in specialized cells termed heterocysts. N. spumigena dominates the annual toxic summer blooms in the Baltic Sea causing environmental and economical problems. We have previously demonstrated that N. spumigena strain AV1 exhibits a different response to the presence of combined nitrogen as compared to model cyanobacteria such as Nostoc PCC7120 and Nostoc punctiforme by uncoupling between nitrogen fixation and heterocyst differentiation.In order to assess whether or not the behaviour of N. spumigena strain AV1 is characteristic of N. spumigena populations, especially in the Baltic Sea, we have investigated the effect of nitrate and ammonium ions on growth, nitrogen fixation activity and presence of heterocysts in eight different Nodularia strains. Our results show that most of the strains retain heterocysts in the absence of nitrogenase activity indicating that uncoupling between nitrogen fixation and heterocyst differentiation is most likely a common behaviour among N. spumigena strains, especially in the Baltic Sea. In addition, there are variations between strains in regard to nitrate uptake.

Morphological, toxicological and molecular characterization of a benthic Nodularia isolated from Atlantic estuarine environments.

A polyphasic study of a benthic Nodularia isolate (LEGE06071) from an Atlantic environment, specifically salt pans, was performed. LEGE06071 resembled both type strains of Nodularia sphaerocarpa and Nodularia harveyana, while ACOI00729 (purchased isolate) was identified as N. sphaerocarpa. The length and width of vegetative cells varied from 3.10 to 3.15 microm and from 3.71 to 4.25 microm, respectively, while heterocyts were 3.91-4.89 microm long and 4.20-4.74 microm wide. None of the isolates had aerotopes. The sequencing of the 16S rRNA gene from the two Nodularia isolates indicated that they belonged neither to Nodularia spumigena nor N. harveyana. Nodularin and other cyanotoxin synthesis-associated genes could not be detected, nor could nodularin production be detected by ELISA. However, MALDI-TOF analysis of LEGE06071 revealed the presence of other compounds, namely, glycolipids. Hence, toxicological screenings against Artemia nauplii, Escherichia coli and Salmonella typhimurium were performed. Toxic effects could only be observed against Artemia, with 48 h-LC(50) values for the aqueous and crude extract of methanol of 53.21 mg ml(-1) and 17.81 mg ml(-1), respectively. This study presents the first evidence of a non-nodularin-producing Nodularia isolate in Atlantic salt pan ecosystems and its potential ecotoxicity against Artemia sp.

Expression of the nodularin synthetase genes in the Baltic Sea bloom-former cyanobacterium Nodularia spumigena strain AV1.

Cyanobacterial blooms in the Baltic Sea are a common phenomenon and are formed by the heterocystous, filamentous species Nodularia spumigena. The toxicity of these blooms is attributed to the hepatotoxin nodularin, produced by N. spumigena. Little is known regarding the regulatory mechanisms or environmental signaling that control nodularin production. Here we report the characterization of the transcriptional expression pattern of the nodularin synthetase gene cluster (nda) during phosphate depletion, and nitrogen supplementation. Real-time PCR analysis of these genes revealed that while cells continuously expressed the nda cluster, the expression of all nda genes increased when cells were subjected to phosphate depletion, and decreased in the presence of ammonium. In contrast to the shifts in expression, the intracellular and extracellular nodularin concentrations did not vary significantly during the treatments.

Quantitative real-time PCR detection of toxic Nodularia cyanobacteria in the Baltic Sea.

A specific quantitative real-time PCR (qPCR) method was developed for the quantification of hepatotoxin nodularin-producing Nodularia, one of the main bloom-forming cyanobacteria in the Baltic Sea. Specific PCR primers were designed for subunit F of the nodularin synthetase gene (ndaF), which encodes the NdaF subunit of the nodularin synthetase gene complex needed for nodularin production. The qPCR method was applied to water samples (a total of 120 samples) collected from the Baltic Sea in July 2004. As few as 30 ndaF gene copies ml(-1) of seawater could be detected, and thus, the method was very sensitive. The ndaF gene copy numbers and nodularin concentrations were shown to correlate in the Baltic seawater, indicating the constant production of nodularin by Nodularia. This qPCR method for the ndaF gene can be used for detailed studies of Nodularia blooms and their formation. ndaF gene copies and nodularin were detected mostly in the surface water but also in deeper water layers (down to 30 m). Toxic Nodularia blooms are not only horizontally but also vertically widely distributed, and thus, the Baltic fauna is extensively exposed to nodularin.

Characterization of PPTNs, a cyanobacterial phosphopantetheinyl transferase from Nodularia spumigena NSOR10.

The phosphopantetheinyl transferases (PPTs) are a superfamily of essential enzymes required for the synthesis of a wide range of compounds, including fatty acids, polyketides, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways by transfer of a phosphopantetheinyl moiety. The diverse PPT superfamily can be divided into two families based on specificity and conserved sequence motifs. The first family is typified by the Escherichia coli acyl carrier protein synthase (AcpS), which is involved in fatty acid synthesis. The prototype of the second family is the broad-substrate-range PPT Sfp, which is required for surfactin biosynthesis in Bacillus subtilis. Most cyanobacteria do not encode an AcpS-like PPT, and furthermore, some of their Sfp-like PPTs belong to a unique phylogenetic subgroup defined by the PPTs involved in heterocyst differentiation. Here, we describe the first functional characterization of a cyanobacterial PPT based on a structural analysis and subsequent functional analysis of the Nodularia spumigena NSOR10 PPT. Southern hybridizations suggested that this enzyme may be the only PPT encoded in the N. spumigena NSOR10 genome. Expression and enzyme characterization showed that this PPT was capable of modifying carrier proteins resulting from both heterocyst glycoplipid synthesis and nodularin toxin synthesis. Cyanobacteria are a unique and vast source of bioactive metabolites; therefore, an understanding of cyanobacterial PPTs is important in order to harness the biotechnological potential of cyanobacterial natural products.

Community structure of the bacteria associated with Nodularia sp. (Cyanobacteria) aggregates in the Baltic Sea.

The community structure of the bacteria associated with Nodularia spumigena (Mertens) cyanobacterial aggregates in the Baltic Sea was studied with temperature gradient gel electrophoresis (TGGE), using a 16S rRNA gene fragment as a target. Various developmental stages of the aggregates and free-floating cyanobacterial filaments were sampled to reveal possible changes in associated microbial community structure during development and senescence of the aggregates. The microbial community structures of all samples differed, and the communities of young and decaying aggregates were separated by cluster analysis of the TGGE fingerprint data. Sequencing of the TGGE fragments indicated the presence of bacteria from the alpha-, beta-, and gamma-proteobacterial groups, as well as members of Cytophaga-Flexibacter-Bacteroides lineages and gram-positive Actinobacteria spp. The majority of the Nodularia-associated sequences were not closely related to previously reported 16S rDNA sequences from the Baltic Sea or any other environment. The structure of the bacterial assemblage reflects the environmental changes associated with the succession and decay of the cyanobacterial aggregates. In addition, the sequence data suggest that the N. spumigena (Mertens) blooms in the Baltic Sea may host thus far uncharacterized bacterial species.