Priming of microbial microcystin degradation in biomass-fed gravity driven membrane filtration biofilms.

Gravity-driven membrane (GDM) filtration is a promising tool for low-cost decentralized drinking water production. The biofilms in GDM systems are able of removing harmful chemical components, particularly toxic cyanobacterial metabolites such as microcystins (MCs). This is relevant for the application of GDM filtration because anthropogenic nutrient input and climate change have led to an increase of toxic cyanobacterial blooms. However, removal of MCs in newly developing GDM biofilms is only established after a prolonged period of time. Since cyanobacterial blooms are transient phenomena, it is important to understand MC removal in mature biofilms with or without prior toxin exposure. In this study, the microbial community composition of GDM biofilms was investigated in systems fed with water from a lake with periodic blooms of MC-producing cyanobacteria. Two out of three experimental treatments were supplemented with dead biomass of a MC-containing cyanobacterial strain, or of a non-toxic mutant, respectively. Analysis of bacterial rRNA genes revealed that both biomass-amended treatments were significantly more similar to each other than to a non-supplemented control. Therefore, it was hypothesized that biofilms could potentially be 'primed' for rapid MC removal by prior addition of non-toxic biomass. A subsequent experiment showed that MC removal developed significantly faster in mature biofilms that were pre-fed with biomass from the mutant strain than in unamended controls, indicating that MC degradation was a facultative trait of bacterial populations in GDM biofilms. The significant enrichment of bacteria related to both aerobic and anaerobic MC degraders suggested that this process might have occurred in parallel in different microniches.

Integrating phylogeny, geographic niche partitioning and secondary metabolite synthesis in bloom-forming Planktothrix.

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.

Biodegradation of microcystins during gravity-driven membrane (GDM) ultrafiltration.

Gravity-driven membrane (GDM) ultrafiltration systems require little maintenance: they operate without electricity at ultra-low pressure in dead-end mode and without control of the biofilm formation. These systems are already in use for water purification in some regions of the world where adequate treatment and distribution of drinking water is not readily available. However, many water bodies worldwide exhibit harmful blooms of cyanobacteria that severely lower the water quality due to the production of toxic microcystins (MCs). We studied the performance of a GDM system during an artificial Microcystis aeruginosa bloom in lake water and its simulated collapse (i.e., the massive release of microcystins) over a period of 21 days. Presence of live or destroyed cyanobacterial cells in the feed water decreased the permeate flux in the Microcystis treatments considerably. At the same time, the microbial biofilms on the filter membranes could successfully reduce the amount of microcystins in the filtrate below the critical threshold concentration of 1 µg L(-1) MC for human consumption in three out of four replicates after 15 days. We found pronounced differences in the composition of bacterial communities of the biofilms on the filter membranes. Bacterial genera that could be related to microcystin degradation substantially enriched in the biofilms amended with microcystin-containing cyanobacteria. In addition to bacteria previously characterized as microcystin degraders, members of other bacterial clades potentially involved in MC degradation could be identified.

Ménage-à-trois: the amoeba Nuclearia sp. from Lake Zurich with its ecto- and endosymbiotic bacteria.

We present a fascinating triad relationship between a eukaryotic amoeba and its two bacterial symbionts. The morphological characteristics of the amoeba allowed for a confident assignment to the genus Nuclearia (Opisthokonta, Nucleariidae), but species identification resulted in an ambiguous result. Sequence analysis indicated an affiliation to the species N. thermophila, however, several morphological features contradict the original description. Amoebal isolates were cultured for several years with their preferred food source, the microcystin-producing harmful cyanobacterium Planktothrix rubescens. Symbioses of the amoeba with ecto- and endosymbiotic bacteria were maintained over this period. Several thousand cells of the ectosymbiont are regularly arranged inside a layer of extracellular polymeric substances produced by the amoeba. The ectosymbiont was identified as Paucibacter toxinivorans (Betaproteobacteria), which was originally isolated by enrichment with microcystins. We found indications that our isolated ectosymbiont indeed contributed to toxin-degradation. The endosymbiont (Gammaproteobacteria, 15-20 bacteria per amoeba) is enclosed in symbiosomes inside the host cytoplasm and represents probably an obligate symbiont. We propose the name "Candidatus Endonucleariobacter rarus" for this bacterium that was neither found free-living nor in a symbiotic association. Nucleariidae are uniquely suited model organisms to study the basic principles of symbioses between opisthokonts and prokaryotes.

Molecular effects of the cyanobacterial toxin cyanopeptolin (CP1020) occurring in algal blooms: global transcriptome analysis in zebrafish embryos.

Higher water temperatures due to climate change combined with eutrophication of inland waters promote cyanobacterial blooms. Some of the cyanobacteria produce toxins leading to drinking water contamination and fish poisoning on a global scale. Here, we focused on the molecular effects of the cyanobacterial oligopeptide cyanopeptolin CP1020, produced by Microcystis and Planktothrix strains, by means of whole-genome transcriptomics. Exposure of 72 hpf old zebrafish embryos for 96 h to 100 and 1,000 µg/L CP1020 resulted in differential transcriptional alteration of 396 and 490 transcripts (fold change ≥ 2), respectively, of which 68 gene transcripts were common. These belong to genes related to various important biological and physiological pathways. Most clearly affected were pathways related to DNA damage recognition and repair, circadian rhythm and response to light. Validation by RT-qPCR showed dose-dependent transcriptional alterations of genes belonging to DNA damage and repair and regulation of circadian rhythm. This leads to the hypothesis that CP1020 acts on DNA and has neurotoxic activity. This transcriptome analysis leads to the identification of novel and unknown molecular effects of this cyanobacterial toxin, including neurotoxicity, which may have important consequences for humans consuming contaminated drinking water.

Apoptogenic metabolites in fractions of the Benthic diatom Cocconeis scutellum parva.

Benthic diatoms of the genus Cocconeis contain a specific apoptogenic activity. It triggers a fast destruction of the androgenic gland in the early post-larval life of the marine shrimp Hippolyte inermis, leading to the generation of small females. Previous in vitro investigations demonstrated that crude extracts of these diatoms specifically activate a dose-dependent apoptotic process in human cancer cells (BT20 breast carcinoma) but not in human normal lymphocytes. Here, a bioassay-guided fractionation has been performed to detect the apoptogenic compound(s). Various HPLC separation systems were needed to isolate the active fractions, since the apoptogenic metabolite is highly active, present in low amounts and is masked by abundant but non-active cellular compounds. The activity is due to at least two compounds characterized by different polarities, a hydrophilic and a lipophilic fraction. We purified the lipophilic fraction, which led to the characterization of an active sub-fraction containing a highly lipophilic compound, whose molecular structure has not yet been identified, but is under investigation. The results point to the possible medical uses of the active compound. Once the molecular structure has been identified, the study and modulation of apoptotic processes in various types of cells will be possible.

Balgacyclamides, antiplasmodial heterocyclic peptides from Microcystis aeruguinosa EAWAG 251.

The isolation and structural characterization of three new heterocyclic and macrocyclic peptides, balgacyclamides A-C, from Microcystis aeruginosa EAWAG 251 are reported. The constitutions were determined by 2D-NMR methods and mass spectrometry, and the configurations were assigned after ozonolysis and hydrolysis by HPLC-MS methods using Marfey's method as well as GC-MS using authentic standards. Balgacyclamides A and B were active against Plasmodium falciparum K1 in the low micromolar range, while displaying low toxicity to rat myoblasts.

The toxicity and enzyme activity of a chlorine and sulfate containing aeruginosin isolated from a non-microcystin-producing Planktothrix strain.

The toxicity of six different Planktothrix strains was examined in acute toxicity assays with the crustacean Thamnocephalus platyurus. The presence of toxicity in two strains could be explained by the occurrence of microcystins. The other four Planktothrix strains were not able to produce microcystins due to different mutations in the microcystin synthetase (mcy) gene cluster. In these strains, toxicity was attributed to the presence of chlorine and sulfate containing compounds. The main representative, called aeruginosin 828A, of such a compound in the Planktothrix strain 91/1 was isolated, and structure elucidation by 2D-NMR and MS methods revealed the presence of phenyllactic acid (Pla), chloroleucine (Cleu), 2-carboxy-6-(4'-sulfo-xylosyl)-octahydroindole (Choi), and 3-aminoethyl-1-N-amidino-Δ-3-pyrroline (Aeap) residues. Aeruginosin 828A was found to be toxic for Thamnocephalus platyurus with a LC50 value of 22.4 µM, which is only slightly higher than the toxicity found for microcystins. Additionally, very potent inhibition values for thrombin (IC50 = 21.8 nM) and for trypsin (IC50 = 112 nM) have been determined for aeruginosin 828A. These data support the hypothesis that aeruginosins containing chlorine and sulfate groups, which were found in microcystin-deficient Planktothrix strains, can be considered as another class of toxins.

Suboptimal light conditions negatively affect the heterotrophy of Planktothrix rubescens but are beneficial for accompanying Limnohabitans spp.

We examined the effect of light on the heterotrophic activity of the filamentous cyanobacterium Planktothrix rubescens and on its relationship with the accompanying bacteria. In situ leucine uptake by bacteria and cyanobacteria was determined in a subalpine mesotrophic lake, and natural assemblages from the zone of maximal P. rubescens abundances were incubated for 2 days at contrasting light regimes (ambient, 100× increased, dark). Planktothrix rubescens from the photic zone of the lake incorporated substantially more leucine, but some heterotrophic activity was maintained in filaments from the hypolimnion. Exposure of cyanobacteria to increased irradiance or darkness resulted in significantly lower leucine incorporation than at ambient light conditions. Highest abundances and leucine uptake of Betaproteobacteria from the genus Limnohabitans were found in the accompanying microflora at suboptimal irradiance levels for P. rubescens or in dark incubations. Therefore, two Limnohabitans strains (representing different species) were co-cultured with axenic P. rubescens at different light conditions. The abundances and leucine incorporation rates of both strains most strongly increased at elevated irradiance levels, in parallel to a decrease of photosynthetic pigment fluorescence and the fragmentation of cyanobacterial filaments. Our results suggest that Limnohabitans spp. in lakes might profit from the presence of physiologically stressed P. rubescens.

Scent of danger: floc formation by a freshwater bacterium is induced by supernatants from a predator-prey coculture.

We investigated predator-prey interactions in a model system consisting of the bacterivorous flagellate Poterioochromonas sp. strain DS and the freshwater bacterium Sphingobium sp. strain Z007. This bacterial strain tends to form a subpopulation of grazing-resistant microscopic flocs, presumably by aggregation. Enhanced formation of such flocs could be demonstrated in static batch culture experiments in the presence of the predator. The ratio of aggregates to single cells reached >0.1 after 120 h of incubation in an oligotrophic growth medium. The inoculation of bacteria into supernatants from cocultures of bacteria and flagellates (grown in oligotrophic or in rich media) also resulted in a substantially higher level of floc formation than that in supernatants from bacterial monocultures only. After separation of supernatants on a C(18) cartridge, the aggregate-inducing activity could be assigned to the 50% aqueous methanolic fraction, and further separation of this bioactive fraction could be achieved by high-pressure liquid chromatography. These results strongly suggest the involvement of one or several chemical factors in the induction of floc formation by Sphingobium sp. strain Z007 that are possibly released into the surrounding medium by flagellate grazing.

Multiple toxin production in the cyanobacterium microcystis: isolation of the toxic protease inhibitor cyanopeptolin 1020.

The isolation and structure of cyanopeptolin 1020 (hexanoic acid-Glu-N[-O-Thr-Arg-Ahp-Phe-N-Me-Tyr-Val-]) from a Microcystis strain is reported. Very potent picomolar trypsin inhibition (IC(50) = 670 pM) and low nanomolar values against human kallikrein (4.5 nM) and factor XIa (3.9 nM) have been determined for cyanopeptolin 1020. For plasmin and chymotrypsin, low micromolar concentrations were necessary for 50% inhibition. Cyanopeptolin 1020 was found to be toxic against the freshwater crustacean Thamnocephalus platyurus (LC(50) = 8.8 microM), which is in the same range as some of the well-known microcystins. These data support the hypothesis that cyanopeptolins can be considered as a second class of toxins in addition to the well-established microcystins in Microcystis.

Aggregate formation in a freshwater bacterial strain induced by growth state and conspecific chemical cues.

We investigated the induction of aggregate formation in the freshwater bacterium Sphingobium sp. strain Z007 by growth state and protistan grazing. Dialysis bag batch culture experiments were conducted in which these bacteria were grown spatially separated from bacteria or from co-cultures of bacteria and predators. In pure cultures of Sphingobium sp. strain Z007, the concentrations of single cells and aggregates inside and outside the dialysis membranes developed in a similar manner over 3 days of incubation, and the proportions of aggregates were highest during the exponential growth phase. Cell production of Sphingobium sp. strain Z007 was enhanced in the presence of another isolate, Limnohabitans planktonicus, from an abundant freshwater lineage (R-BT065) outside the bags, and even more so if that strain was additionally grazed upon by the bacterivorous flagellate Poterioochromonas sp. However, the ratios of single cells to aggregates of Sphingobium sp. strain Z007 were not affected in either case. By contrast, the feeding of flagellates on Sphingobium sp. strain Z007 outside the dialysis bags led to significantly higher proportions of aggregates inside the bags. This was not paralleled by an increase in growth rates, and all cultures were in a comparable growth state at the end of the experiment. We conclude that two mechanisms, growth state and the possible release of infochemicals by the predator, may induce aggregate formation of Sphingobium sp. strain Z007. Moreover, these infochemicals only appeared to be generated by predation on cells from the same species.

Antibiotic effects of three strains of chrysophytes (Ochromonas, Poterioochromonas) on freshwater bacterial isolates.

We investigated the antibiotic effects of extracts of freeze-dried biomass and culture supernatants from the mixotrophic chrysophyte species Ochromonas danica, Poterioochromonas sp. strain DS, and Poterioochromonas malhamensis on bacterial strains isolated from lake water. Methanolic biomass extracts inhibited the growth of all tested strains, albeit to a different extent, whereas aqueous biomass extracts only affected bacteria of the genus Flectobacillus. The antibiotic action of supernatants from flagellate cultures could be mostly attributed to lipophilic substances, but the growth of bacteria affiliated with Flectobacillus and Sphingobium was also affected by hydrophilic compounds. A comparison of biomass extracts from light- and dark-adapted cultures of Poterioochromonas sp. strain DS showed that the growth-inhibiting factor was unrelated to chlorophyll derivatives. Supernatants from a dark-adapted, phagotrophically grown flagellate culture had stronger antibiotic effects and affected more bacterial strains than the supernatant from a light-adapted culture. Significant growth reduction of a Flectobacillus isolate was already induced by extremely low concentrations of lipophilic extracts from these supernatants. Our results show that metabolites of the studied flagellates - either released actively or during cell lysis - may selectively affect the growth of some aquatic bacteria even in very small doses and thus potentially affect microbial community composition. Moreover, the antibiotic potential of mixotrophic chrysophytes may change with their nutritional mode.

Production of the antifungal compound pyrrolnitrin is quorum sensing-regulated in members of the Burkholderia cepacia complex.

Members of the genus Burkholderia are known for their ability to suppress soil-borne fungal pathogens by the production of various antibiotic compounds. In this study we investigated the role of N-acylhomoserine lactone (AHL)-dependent quorum sensing (QS) in the expression of antifungal traits. Using a quorum quenching approach, that is, by heterologous expression of the Bacillus sp. AiiA lactonase, we show that expression of antifungal activities is AHL-dependent in the large majority of the investigated strains belonging to various Burkholderia species. We demonstrate that in certain strains of Burkholderia ambifaria, Burkholderia pyrrocinia and Burkholderia lata, one of the QS-regulated antifungal agents is pyrrolnitrin (prn), a common broad-spectrum antibiotic that is also produced by some Pseudomonas and Serratia species. To investigate the underlying molecular mechanisms of AHL-dependent prn production in better detail, we inactivated the AHL synthase cepI as well as cepR, which encodes the cognate AHL receptor protein, in B. lata 383. Both QS mutants no longer produced prn as assessed by gas chromatography-mass spectrometry analysis and as a consequence were unable to inhibit growth of Rhizoctonia solani. Using fusions of the lacZ gene to the promoter of the prnABCD operon, which directs the synthesis of prn, we demonstrate that expression of prn is positively regulated by CepR at the level of transcription.

Isolation of aerucyclamides C and D and structure revision of microcyclamide 7806A: heterocyclic ribosomal peptides from Microcystis aeruginosa PCC 7806 and their antiparasite evaluation.

Aerucyclamides C and D were isolated from the cyanobacterium Microcystis aeruginosa PCC 7806, and their structures established by NMR spectroscopy and chemical transformation and degradation. Acidic hydrolysis of aerucyclamide C (CF(3)CO(2)H, H(2)O) resulted in microcyclamide 7806A. This chemical evidence combined with spectroscopic and physical data suggest a structure revision for microcyclamide 7806A, which incorporates an O-acylated Thr ammonium residue instead of the originally proposed methyl oxazoline ring. We have prepared microcyclamide 7806B upon basic and acidic treatment of microcyclamide 7806A, which suggests that both these compounds are hydrolysis products of aerucyclamide C and that the aerucyclamides A-D are the actual metabolites produced via ribosomal peptide synthesis in M. aeruginosa PCC 7806. Antiplasmodial evaluation established submicromolar IC(50) values for aerucyclamide B against Plasmodium falciparum; low micromolar values for aerucyclamide C were found against Trypanosoma brucei rhodesiense. The compounds were selective for the parasites over a cell line of L6 rat myoblasts and are thus considered for further study as antimalarial agents.

Isolation and structure determination of two microcystins and sequence comparison of the McyABC adenylation domains in Planktothrix species.

Microcystins (MCs) are toxic heptapeptides found in cyanobacteria and share the common structure cyclo(-d-Ala(1)-l-X(2)-d-isoMeAsp(3)-l-Z(4)-Adda(5)-d-isoGlu(6)-Mdha(7)). The letters X and Z in the general formula above represent a wide range of l-amino acids that occupy positions 2 and 4, respectively. In general the variation in structural variants is due to the exchange of amino acids in position 7, 2, and 4. In the present work we report two homotyrosine (Hty)-containing microcystin variants, [d-Asp(3),(E)-Dhb(7)]-MC-HtyY (1) and [d-Asp(3),(E)-Dhb(7)]-MC-HtyHty (2), which were isolated from strain No80 of Planktothrix rubescens. Their structures were elucidated using amino acid analysis as well as 1D and 2D NMR techniques. The adenylation domains of McyABC involved in amino acid activation in positions 7, 2, and 4 of the microcystin molecule, respectively, were compared with corresponding genes of Planktothrix strain CYA126/8 producing [d-Asp(3),Mdha(7)]-MC-RR and [d-Asp(3),Mdha(7)]-MC-LR. While the adenylation domain comparison of McyAB between the two Planktothrix strains revealed considerable DNA recombination, the adenylation domain of McyC showed only a single amino acid substitution, which was correlated with the replacement of Arg by Hty in position 4 of the microcystin molecule.

Aerucyclamides A and B: isolation and synthesis of toxic ribosomal heterocyclic peptides from the cyanobacterium Microcystis aeruginosa PCC 7806.

Two new modified hexacyclopeptides, aerucyclamides A and B, were isolated from the toxic freshwater cyanobacterium Microcystis aeruginosa PCC 7806. The constitution was assigned by spectroscopic methods, and the configuration determined by chemical degradation and analysis by Marfey's method combined with chemical synthesis. Synthetic aerucyclamide B was obtained through oxidation of aerucyclamide A (MnO2, benzene). The aerucyclamides were found to be toxic to the freshwater crustacean Thamnocephalus platyurus, exhibiting LC50 values for congeners A and B of 30.5 and 33.8 microM, respectively.

Potent algicides based on the cyanobacterial alkaloid nostocarboline.

[structure: see text] Nostocarboline and seven derivatives were prepared and displayed minimal inhibitory concentration (MIC) values >or=100 nM against the growth of Microcystis aeruginosa PCC 7806, Synechococcus PCC 6911, and Kirchneriella contorta SAG 11.81, probably via the inhibition of photosynthesis. The natural product hybrid nostocarboline/ciprofloxacin displayed additional antibacterial activity against several Gram-negative bacteria (MICs >or=0.7 microM). Nostocarboline can thus be considered a potent, selective, readily available, natural algicide.

Cyanopeptolin 954, a chlorine-containing chymotrypsin inhibitor of Microcystis aeruginosa NIVA Cya 43.

A new depsipeptide, cyanopeptolin 954 (1), was isolated from the freshwater cyanobacterium Microcystis aeruginosa NIVA Cya 43. The structure of the compound was elucidated by chemical and spectroscopic analyses, including 2D NMR and GC-MS of the hydrolysate. The major structural differences compared to previously characterized heptadepsipeptides of Microcystis are the replacement of the basic amino acid in position 4 by L-leucine, the presence of L-phenylalanine in position 6, and the uncommon residue 3'-chloro-N-Me-L-tyrosine in position 7. Cyanopeptolin 954 inhibited chymotrypsin with an IC50 value of 45 nM. Nostopeptin BN920, formerly isolated from the cyanobacterium Nostoc,(1) was isolated from the same strain of Microcystis, and a cis amide bond between Phe (6) and N-Me-Tyr (7) was shown. Nostopeptin BN920 inhibited chymotrypsin with an IC50 value of 31 nM.

High crustacean toxicity of microcystin congeners does not correlate with high protein phosphatase inhibitory activity.

Microcystins are strong toxins and efficient inhibitors of eukaryotic protein phosphatases. To determine structure related properties of six different microcystin congeners, we applied standardized inhibition assays for the protein phosphatases 1 and 2A, and an acute toxicity assay with Thamnocephalus platyurus. Protein phosphatase inhibition and acute toxicity did not correlate with each other. While the inhibition of the protein phosphatases 1 and 2A was much weaker for [D-Asp3,(E)-Dhb7]microcystin-RR than for the other congeners, the toxicity was one of the highest. [D-Asp3]microcystin-LR exhibited only small differences to microcystin-LR. The data show that mechanisms other than the inhibition of protein phosphatases, such as uptake, transport, detoxification or other target sites may have a strong modulating effect on the toxicity of a microcystin congener for a particular animal. Structural changes can offset or even reverse the specific toxicity of microcystin congeners.