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In non-ribosomal peptide synthesis of cyanobacteria, promiscuous adenylation domains allow the incorporation of clickable non-natural amino acids into peptide products—namely into microcystins (MCs) or into anabaenopeptins (APs): 4-azidophenylalanine (Phe-Az), N-propargyloxy-carbonyl-L-lysine (Prop-Lys), or O-propargyl-L-tyrosine (Prop-Tyr). Subsequently, chemo-selective labeling is used to visualize the clickable cyanopeptides using Alexa Fluor 488 (A488). In this study, the time-lapse build up or decline of azide- or alkyne-modified MCs or APs was visualized during maximum growth, specifically MC biosynthesis in Microcystis aeruginosa and AP biosynthesis in Planktothrix agardhii. Throughout the time-lapse build up or decline, the A488 signal occurred with heterogeneous intracellular distribution. There was a fast increase or decrease in the A488 signal for either Prop-Tyr or Prop-Lys, while a delayed or unobservable A488 signal for Phe-Az was related to increased cell size as well as a reduction in growth and autofluorescence. The proportion of clickable MC/AP in peptide extracts as recorded by a chemical–analytical technique correlated positively with A488 labeling intensity quantified via laser-scanning confocal microscopy for individual cells or via flow cytometry at the population level. It is concluded that chemical modification of MC/AP can be used to track intracellular dynamics in biosynthesis using both analytical chemistry and high-resolution imaging.

Determining exact viral titers in a given sample is essential for many environmental and clinical applications, e.g., for studying viral ecology or application of bacteriophages for food safety. However, virus quantification is not a simple task, especially for complex environmental samples. While clonal viral isolates can be quantified with relative high accuracy using virus-specific methods, i.e., plaque assay or quantitative real-time PCR, these methods are not valid for complex and diverse environmental samples. Moreover, it is not yet known how precisely laser-based methods, i.e., epifluorescence microscopy, flow cytometry, and nanoparticle tracking analysis, quantify environmental viruses. In the present study, we compared five state-of-the-art viral quantification methods by enumerating four model viral isolates of different genome and size characteristics as well as four different environmental water samples. Although Nanoparticle tracking analysis combined with gentle staining at 30 °C could be confirmed by this study to be a reliable quantification technique for tested environmental samples, environmental samples still lack an universally applicable and accurate quantification method. Special attention has to be put on optimal sample concentrations as well as optimized sample preparations, which are specific for each method. As our results show the inefficiency when enumerating small, or single-stranded DNA or RNA viruses, the global population of viruses is presumably higher than expected.

Recently, the use of click chemistry for localization of chemically modified cyanopeptides has been introduced, i.e., taking advantage of promiscuous adenylation (A) domains in non-ribosomal peptide synthesis (NRPS), allowing for the incorporation of clickable non-natural amino acids (non-AAs) into their peptide products. In this study, time-lapse experiments have been performed using pulsed feeding of three different non-AAs in order to observe the synthesis or decline of azide- or alkyne-modified microcystins (MCs) or anabaenopeptins (APs). The cyanobacteria Microcystis aeruginosa and Planktothrix agardhii were grown under maximum growth rate conditions (r = 0.35–0.6 and 0.2–0.4 (day−1), respectively) in the presence of non-AAs for 12–168 h. The decline of the azide- or alkyne-modified MC or AP was observed via pulse-feeding. In general, the increase in clickable MC/AP in peptide content reached a plateau after 24–48 h and was related to growth rate, i.e., faster-growing cells also produced more clickable MC/AP. Overall, the proportion of clickable MC/AP in the intracellular fraction correlated with the proportion observed in the dissolved fraction. Conversely, the overall linear decrease in clickable MC/AP points to a rather constant decline via dilution by growth instead of a regulated or induced release in the course of the synthesis process.

Harmful algal blooms formed by colony-forming cyanobacteria deteriorate water resources by producing cyanotoxins, which frequently occur at high intracellular concentrations. We aimed to localize toxic microcystins (MCs) and bioactive anabaenopeptins (APs) at the subcellular level under noninvasive conditions. Since both metabolites are synthesized nonribosomally, the relaxed specificity of key enzymes catalyzing substrate activation allowed chemical labeling through a standard copper-catalyzed click chemistry reaction. The genera Planktothrix and Microcystis specifically incorporated unnatural amino acids such as N -propargyloxy-carbonyl-L-lysine or O -propargyl-L-tyrosine, resulting in modified AP or MC peptides carrying the incorporated alkyne moiety. The labeled cells were quantitatively differentiated from the unlabeled control cells. MCs and APs occurred intracellularly as distinct entities showing a cell-wide distribution but a lowered spatial overlap with natural autofluorescence. Using the immunofluorescence technique, colocalization with markers of individual organelles was utilized to relate the distribution of labeled MCs to cellular compartments, e.g., using RbcL and FtsZ (cytosol) and PsbA (thylakoids). The colocalization correlation coefficients calculated pairwise between organelles and autofluorescence were highly positive as opposed to the relatively low positive indices derived from labeled MCs. The lower correlation coefficients imply that only a portion of the labeled MC molecules were related spatially to the organelles in the cell.

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.

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. [PUBLICATION ABSTRACT]

Toxic freshwater cyanobacteria form harmful algal blooms that can cause acute toxicity to humans and livestock. Globally distributed, bloom-forming cyanobacteria Planktothrix either retain or lose the mcy gene cluster (encoding the synthesis of the secondary metabolite hepatotoxin microcystin or MC), resulting in a variable spatial/temporal distribution of (non)toxic genotypes. Despite their importance to human well-being, such genotype diversity is not being mapped at scales relevant to nature. We aimed to reveal the factors influencing the dispersal of those genotypes by analyzing 138 strains (from Europe, Russia, North America and East Africa) for their (i) mcy gene cluster composition, (ii) phylogeny and adaptation to their habitat and (iii) ribosomally and nonribosomally synthesized oligopeptide products. Although all the strains from different species contained at least remnants of the mcy gene cluster, various phylogenetic lineages evolved and adapted to rather specific ecological niches (for example, through pigmentation and gas vesicle protein size). No evidence for an increased abundance of specific peptides in the absence of MC was found. MC and peptide distribution rather depended on phylogeny, ecophysiological adaptation and geographic distance. Together, these findings provide evidence that MC and peptide production are primarily related to speciation processes, while within a phylogenetic lineage the probability that strains differ in peptide composition increases with geographic distance.

The human health risks posed by exposure to cyanobacterial toxins such as microcystin (MC) through water and fish consumption remain poorly described. During the last two decades, coastal regions of Lake Victoria such as Nyanza Gulf (Kisumu Bay) have shown severe signs of eutrophication with blooms formed by Microcystis producing MC. In this study, the spatial variability in MC concentration in Kisumu Bay was investigated which was mostly caused by Microcystis buoyancy and wind drifting. Small fish (<6 cm) mainly composed of Rastrineobola argentea were examined for MC content by means of biological methods such as ELISA and protein phosphatase inhibition assay (PPIA) and partly by chemical-analytical methods such as LC-MS/MS. Overall, the MC content in small fish was related to the MC content observed in the seston. When comparing the MC content in the seston in relation to dry weight with the MC content in small fish the latter was found three orders of magnitude decreased. On average, the ELISA-determined MC contents exceeded the PPIA-determined MC contents by a factor of 8.2 ± 0.5 (SE) while the MC contents as determined by LC-MS/MS were close to the detection limit. Using PPIA, the MC content varied from 25–109 (mean 62 ± 7) ng/g fish dry weight in Kisumu Bay vs. 14 ± 0.8 ng MC/g in the more open water of L. Victoria at Rusinga channel. Drying the fish under the sun showed little effect on MC content, although increased humidity might indirectly favor photocatalyzed MC degradation.

The disturbing effect of a short-term cooling period during summer on planktonic bacterial community structure of an alpine lake was investigated using 16S rDNA pyrosequencing. Proteobacteria, Actinobacteria, and Bacteroidetes constituted the most abundant phyla. During the sampling period (from July to August 2010), a sudden cooling period with high precipitation occurred, as indicated by a decrease in conductivity, calcium, and dissolved organic carbon concentration resulting from increased runoff. The relative abundance of Actinobacteria, Betaproteobacteria, and Cyanobacteria decreased during this short-term cooling period. Instead, a rapid shift from Betaproteobacteria to Gammaproteobacteria occurred, which was mainly caused by an increase of Acinetobacter rhizosphaerae. Soon after the short-term cooling period, warmer weather conditions got re-established and Betaproteobacteria recovered and became again dominant. Non-metric multi-dimensional scaling analysis and Venn diagrams revealed a planktonic bacterial community composition with high similarity at the beginning and the end of the growing season. Air temperature and precipitation were significantly correlated with the observed variation in operational taxonomic unit (OTU) relative abundance. It is concluded that, in response to the short-term cooling period, a distinct planktonic bacterial OTU community developed. It rapidly diminished, however, as summer conditions became re-established, implying the recovery of the original bacterial community structure.

A guide to state-of-the-art molecular tools for monitoring and managing the toxigenicity of cyanobacteria Runaway eutrophication and climate change has made the monitoring and management of toxigenic organisms in the world's bodies of water more urgent than ever. In order to influence public policy regarding the detection and quantification of those organisms, it is incumbent upon scientists to raise the awareness of policy makers concerning the increased occurrence of toxigenic cyanobacteria and the threats they pose. As molecular methods can handle many samples in short time and help identify toxigenic organisms, they are reliable, cost-effective tools available for tracking toxigenic cyanobacteria worldwide. This volume arms scientists with the tools they need to track toxigenicity in surface waters and food supplies and, hopefully, to develop new techniques for managing the spread of toxic cyanobacteria. This handbook offers the first comprehensive treatment of molecular tools for monitoring toxigenic cyanobacteria. Growing out of the findings of the landmark European Cooperation in Science and Technology Cyanobacteria project (CYANOCOST), it provides detailed, practical coverage of the full array of available molecular tools and protocols, from water sampling, nucleic acid extraction, and downstream analysis-including PCR and qPCR based methods-to genotyping (DGGE), diagnostic microarrays, and community characterization using next-gen sequencing techniques.-Offers an overview of the latest trends in the field, while providing a foundation for understanding and applying the tools and techniques described -Provides detailed coverage of the full range of molecular tools currently available, with expert guidance on the analysis and interpretation of results -Includes step-by-step guidance on standard operational procedures, including molecular tests used in environmental monitoring, with individual chapters devoted to each procedure -Complements the published Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis from the CyanoCOST project This handbook is an indispensable working resource for scientists, lab technicians, and water management professionals and an excellent text/reference for graduate students and supervisors who use molecular tools. It will also be of great value to environmental health and protection officials and policy makers.