High structural diversity of aeruginosins in bloom-forming cyanobacteria of the genus Planktothrix as a consequence of multiple recombination events.

Many compounds produced by cyanobacteria act as serine protease inhibitors, such as the tetrapeptides aeruginosins (Aer), which are found widely distributed. The structural diversity of Aer is intriguingly high. However, the genetic basis of this remains elusive. In this study, we explored the genetic basis of Aer synthesis among the filamentous cyanobacteria Planktothrix spp. In total, 124 strains, isolated from diverse freshwater waterbodies, have been compared regarding variability within Aer biosynthesis genes and the consequences for structural diversity. The high structural variability could be explained by various recombination processes affecting Aer synthesis, above all, the acquisition of accessory enzymes involved in post synthesis modification of the Aer peptide (e.g., halogenases, glycosyltransferases, sulfotransferases) as well as a large-range recombination of Aer biosynthesis genes, probably transferred from the bloom-forming cyanobacterium Microcystis. The Aer structural composition differed between evolutionary Planktothrix lineages, adapted to either shallow or deep waterbodies of the temperate climatic zone. Thus, for the first time among bloom-forming cyanobacteria, chemical diversification of a peptide family related to eco-evolutionary diversification has been described. It is concluded that various Aer peptides resulting from the recombination event act in chemical defense, possibly as a replacement for microcystins

Spatial divergence in the proportions of genes encoding toxic peptide synthesis among populations of the cyanobacterium Planktothrix in European lakes

It has been frequently reported that seasonal changes in toxin production by cyanobacteria are due to changes in the proportion of toxic/nontoxic genotypes in parallel to increases or decreases in population density during the seasonal cycle of bloom formation. In order to find out whether there is a relationship between the proportion of genes encoding toxic peptide synthesis and population density of Planktothrix spp. we compared the proportion of three gene regions that are indicative of the synthesis of the toxic heptapeptide microcystin (mcyB), and the bioactive peptides aeruginoside (aerB) and anabaenopeptin (apnC) in samples from 23 lakes of five European countries (n=153). The mcyB, aerB, and apnC genes occurred in 99%, 99%, and 97% of the samples, respectively, and on average comprised 60 ± 3%, 22 ± 2%, and 54 ± 4% of the total population, respectively. Although the populations differed widely in abundance (10−3–103 mm3 L−1) no dependence of the proportion of the mcyB, aerB, and apnC genes on the density of the total population was found. In contrast populations differed significantly in their average mcyB, aerB, and apnC gene proportions, with no change between prebloom and bloom conditions. These results emphasize stable population-specific differences in mcyB, aerB, and apnC proportions that are independent from seasonal influences

Distribution of toxigenic cyanobacteria in Alpine lakes and rivers as revealed by molecular screening

The increasing frequency of cyanobacteria blooms in waterbodies caused by ecosystem eutrophication could endanger human health. This risk can be mitigated by effective monitoring incorporating molecular methods. To date, most molecular studies on toxigenic cyanobacteria have been limited to microcystins (MCs), disregarding other cyanotoxins, to freshwater planktic habitats while ignoring benthic habitats, and to limited geographic areas (usually one or a few specific waterbodies). In this study, we used PCR-based methods including PCR product sequencing and chemical-analytical methods (LC-MS/MS) to screen many plankton (n = 123) and biofilm samples (n = 113) originating from 29 Alpine lakes and 18 rivers for their cyanotoxin production potential. Both mcyE (indicating MC synthesis) and anaC (indicating anatoxin (ATX) synthesis) gene fragments were able to qualitatively predict MC or ATX occurrence. The abundance of mcyE gene fragments was significantly related to MC concentrations in plankton samples (R2 = 0.61). mcyE gene fragments indicative of MC synthesis were most abundant in planktic samples (65 %) and were assigned to the genera Planktothrix and Microcystis. However, mcyE rarely occurred in biofilms of lakes and rivers, i.e., 4 % and 5 %, respectively, and were assigned to Microcystis, Planktothrix, and Nostoc. In contrast, anaC gene fragments occurred frequently in planktic samples (14 % assigned to Tychonema, Phormidium (Microcoleus), and Oscillatoria), but also in biofilms of lakes (49 %) and rivers (18 %) and were assigned to the genera Phormidium, Oscillatoria, and Nostocales. The cyrJ gene fragment indicating cylindrospermopsin synthesis occurred only once in plankton (assigned to Dolichospermum), while saxitoxin synthesis potential was not detected. For plankton samples, monomictic and less eutrophic conditions were positively related to mcyE/MC occurrence frequency, while oligomictic conditions were related to anaC/ATX frequency. The anaC/ATX frequency in biofilm was related to the lake habitats generally showing higher biodiversity as revealed from metabarcoding in a parallel stud

Biodiversity patterns of cyanobacterial oligotypes in lakes and rivers: results of a large-scale metabarcoding survey in the Alpine region

In this work, we characterised the cyanobacterial communities in the plankton and littoral biofilm of 38 lakes and in the biofilm of 21 rivers in the Alps and surrounding subalpine regions by 16S rRNA gene metabarcoding. We found little overlap in the distribution of amplicon sequence variants (ASVs) between the three habitats and between water bodies. The differences were caused by environmental filtering acting on the selection of the most abundant ASVs and a high contribution of rare oligotypes. The differentiation of community and genotype composition from specific water bodies was explained to a significant extent by environmental variables and morphometry. The taxonomic consistency of ASVs classified under the same genus name was assessed by phylogenetic analyses performed on three representative dominant genera, namely Cyanobium, Tychonema and Planktothrix. The analyses revealed eco-evolutionary adaptations in lakes and rivers, including some evidence for a polyphyletic nature. Monitoring individual genotypes in relation to environmental conditions will be useful to define the ecological amplitude of these taxa. However, the persistence or ephemeral nature of some of the rarest and most unusual ASVs has remained unknow

Nontoxic Strains of Cyanobacteria Are the Result of Major Gene Deletion Events Induced by a Transposable Element

Blooms that are formed by cyanobacteria consist of toxic and nontoxic strains. The mechanisms that result in the occurrence of nontoxic strains are enigmatic. All the nontoxic strains of the filamentous cyanobacterium Planktothrix that were isolated from 9 European countries were found to have lost 90% of a large microcystin synthetase (mcy) gene cluster that encoded the synthesis of the toxic peptide microcystin (MC). Those strains still contain the flanking regions of the mcy gene cluster along with remnants of the transposable elements that are found in between. The majority of the strains still contain a gene coding for a distinct thioesterase type II (mcyT), which is putatively involved in MC synthesis. The insertional inactivation of mcyT in an MC-producing strain resulted in the reduction of MC synthesis by 94 ± 2% (1 standard deviation). Nontoxic strains that occur in shallow lakes throughout Europe form a monophyletic lineage. A second lineage consists of strains that contain the mcy gene cluster but differ in their photosynthetic pigment composition, which is due to the occurrence of strains that contain phycocyanin or large amounts of phycoerythrin in addition to phycocyanin. Strains containing phycoerythrin typically occur in deep-stratified lakes. The rare occurrence of gene cluster deletion, paired with the evolutionary diversification of the lineages of strains that lost or still contain the mcy gene cluster, needs to be invoked in order to explain the absence or dominance of toxic cyanobacteria in various habitats

Toxic/Bioactive Peptide Synthesis Genes Rearranged by Insertion Sequence Elements Among the Bloom-Forming Cyanobacteria Planktothrix

It has been generally hypothesized that mobile elements can induce genomic rearrangements and influence the distribution and functionality of toxic/bioactive peptide synthesis pathways in microbes. In this study, we performed in depth genomic analysis by completing the genomes of 13 phylogenetically diverse strains of the bloom-forming freshwater cyanobacteria Planktothrix spp. to investigate the role of insertion sequence (IS) elements in seven pathways. Chromosome size varied from 4.7–4.8 Mbp (phylogenetic Lineage 1 of P. agardhii/P. rubescens thriving in shallow waterbodies) to 5.4–5.6 Mbp (Lineage 2 of P. agardhii/P. rubescens thriving in deeper physically stratified lakes and reservoirs) and 6.3–6.6 Mbp (Lineage 3, P. pseudagardhii/P. tepida including planktic and benthic ecotypes). Although the variation in chromosome size was positively related to the proportion of IS elements (1.1–3.7% on chromosome), quantitatively, IS elements and other paralogs only had a minor share in chromosome size variation. Thus, the major part of genomic variation must have resulted from gene loss processes (ancestor of Lineages 1 and 2) and horizontal gene transfer (HGT). Six of seven peptide synthesis gene clusters were found located on the chromosome and occurred already in the ancestor of P. agardhii/P. rubescens, and became partly lost during evolution of Lineage 1. In general, no increased IS element frequency in the vicinity of peptide synthesis gene clusters was observed. We found a higher proportion of IS elements in ten breaking regions related to chromosomal rearrangements and a tendency for colocalization of toxic/bioactive peptide synthesis gene clusters on the chromosome

Benthic Diatom Communities in an Alpine River Impacted by Waste Water Treatment Effluents as Revealed Using DNA Metabarcoding

Freshwater ecosystems are continuously affected by anthropogenic pressure. One of the main sources of contamination comes from wastewater treatment plant (WWTP) effluents that contain wide range of micro- and macropollutants. Chemical composition, toxicity levels and impact of treated effluents (TEs) on the recipient aquatic ecosystems may strongly differ depending on the wastewater origin. Compared to urban TEs, hospital ones may contain more active pharmaceutical substances. Benthic diatoms are relevant ecological indicators because of their high species and ecological diversity and rapid response to human pressure. They are routinely used for water quality monitoring. However, there is a knowledge gap on diatom communities’ development and behavior in treated wastewater in relation to prevailing micro- and macropollutants. In this study, we aim to (1) investigate the response of diatom communities to urban and hospital TEs, and (2) evaluate TEs effect on communities in the recipient river. Environmental biofilms were colonized in TEs and the recipient river up- and downstream from the WWTP output to study benthic diatoms using DNA metabarcoding combined with high-throughput sequencing (HTS). In parallel, concentrations of nutrients, pharmaceuticals and seasonal conditions were recorded. Diatom metabarcoding showed that benthic communities differed strongly in their diversity and structure depending on the habitat. TE sites were generally dominated by few genera with polysaprobic preferences belonging to the motile guild, while river sites favored diverse communities from oligotrophic and oligosaprobic groups. Seasonal changes were visible to lower extent. To categorize parameters important for diatom changes we performed redundancy analysis which suggested that communities within TE sites were associated to higher concentrations of beta-blockers and non-steroidal anti-inflammatory drugs in urban effluents vs. antibiotics and orthophosphate in hospital effluents. Furthermore, indicator species analysis showed that 27% of OTUs detected in river downstream communities were indicator for urban or hospital TE sites and were absent in the river upstream. Finally, biological diatom index (BDI) calculated to evaluate the ecological status of the recipient river suggested water quality decrease linked to the release of TEs. Thus, in-depth assessment of diatom community composition using DNA metabarcoding is proposed as a promising technique to highlight the disturbing effect of pollutants in Alpine rivers

Global solutions to regional problems : collecting global expertise to address the problem of harmful cyanobacterial blooms. A Lake Erie case study

In early August 2014, the municipality of Toledo, OH (USA) issued a ‘do not drink’ advisory on their water supply directly affecting over 400,000 residential customers and hundreds of businesses (Wilson, 2014). This order was attributable to levels of microcystin, a potent liver toxin, which rose to 2.5 μg L⁻¹ in finished drinking water. The Toledo crisis afforded an opportunity to bring together scientists from around the world to share ideas regarding factors that contribute to bloom formation and toxigenicity, bloom and toxin detection as well as prevention and remediation of bloom events. These discussions took place at an NSF- and NOAA-sponsored workshop at Bowling Green State University on April 13 and 14, 2015. In all, more than 100 attendees from six countries and 15 US states gathered together to share their perspectives. The purpose of this review is to present the consensus summary of these issues that emerged from discussions at the Workshop. As additional reports in this special issue provide detailed reviews on many major CHAB species, this paper focuses on the general themes common to all blooms, such as bloom detection, modeling, nutrient loading, and strategies to reduce nutrients.This is the publisher’s final pdf. The published article is copyrighted by Elsevier and can be found at: http://www.journals.elsevier.com/harmful-algae/Keywords: Nitrogen, CHAB, Phosphorus, Microcystin, Cyanobacteria, Lake Eri