Allelopathic growth inhibition by the toxic, bloom-forming cyanobacterium Planktothrix rubescens

Blooms of freshwater cyanobacteria are typically accompanied by an important decrease in phytoplankton biodiversity in the water bodies where they occur. This study examines the potential production of growth-inhibiting substances by the toxic, bloom-forming cyanobacterium Planktothrix rubescens, following the observation of physical segregation between this and another cyanobacterium during previously performed mixed-culture competition experiments. Inhibition assays examining the growth of target strains exposed to donor culture filtrates showed that the growth of Planktothrix agardhii TCC 83-2, P. agardhii PMC 75.02 and Mougeotia gracillima TCC 50-2 was significantly inhibited in the presence of culture filtrate from P. rubescens TCC 29-1, isolated from Lake Bourget, France. Filtrates from P. rubescens TCC 69-6 and P. rubescens TCC 69-7, isolated from Lakes Nantua and Paladru (France), respectively, did not, however, inhibit the growth of P. agardhii TCC 83-2. This brief exploration of the allelopathic activity of P. rubescens suggests that it may potentially inhibit coexisting competitors as well as phytoplankton isolated from other freshwater ecosystems, and that this capacity may vary among different strains of Planktothrix. The potential importance of this phenomenon in pelagic competition dynamics is discussed.     

Abundance of active and inactive microcystin genotypes in populations of the toxic cyanobacterium Planktothrix spp

To investigate the abundance of active and inactive microcystin genotypes in populations of the filamentous cyanobacterium Planktothrix spp., individual filaments were grown as clonal strains in the laboratory and analysed for microcystin synthetase (mcy) genes and microcystin. Twenty-three green-pigmented strains of P. agardhii originating mostly from shallow water bodies fell into two groups, those possessing mcyA and those lacking mcyA. In contrast, all of the 49 strains that were assigned to the red-pigmented P. rubescens contained mcyA. One strain of P. agardhii and eight strains of P. rubescens contained the total microcystin synthetase gene cluster but were found inactive in microcystin synthesis. To investigate the natural abundance of inactive mcy genotypes in P. rubescens individual filaments sampled from Lake Irrsee and Lake Mondsee (Austria) were analysed directly for the presence of mcyA and microcystin by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. All filaments assigned to P. rubescens contained mcyA. The proportion of inactive microcystin genotypes in populations with a low (Irrsee) or high density (Mondsee) of P. rubescens was 5% and 21%, each. The results of this study demonstrate that P. rubescens typically contain mcy genes whereas P. agardhii have a patchy distribution of mcy genes. In both species microcystin producers co-occur with non-microcystin producers due to the absence/inactivation of mcy genes.     

Temporal variations in the dynamics of potentially microcystin-producing strains in a bloom-forming Planktothrix agardhii (Cyanobacterium) population

The concentration of microcystins (MCs) produced during blooms depends on variations in both the proportion of strains containing the genes involved in MC production and the MC cell quota (the ratio between the MC concentration and the density of cells with the mcyA genotype) for toxic strains. In order to assess the dynamics of MC-producing and non-MC-producing strains and to identify the impact of environmental factors on the relative proportions of these two subpopulations, we performed a 2-year survey of a perennial bloom of Planktothrix agardhii (cyanobacteria). Applying quantitative real-time PCR to the mcyA and phycocyanin genes, we found that the proportion of cells with the mcyA genotype varied considerably over time (ranging from 30 to 80% of the population). The changes in the proportion of cells with the mcyA genotype appeared to be inversely correlated to changes in the density of P. agardhii cells and also, to a lesser extent, to the availability of certain nutrients and the abundance of cladocerans. Among toxic cells, the MC cell quota varied throughout the survey. However, a negative correlation between the MC cell quota and the mcyA cell number during two short periods characterized by marked changes in the cyanobacterial biomass was found. Finally, only 54% of the variation in the MC concentrations measured in the lake can be explained by the dynamics of the density of cells with the MC producer genotype, suggesting that this measurement is not a satisfactory method for use in monitoring programs intended to predict the toxic risk associated with cyanobacterial proliferation.     

Transposons inactivate biosynthesis of the nonribosomal peptide microcystin in naturally occurring Planktothrix spp

The filamentous cyanobacteria Planktothrix spp. occur in the temperate region of the Northern hemisphere. The red-pigmented Planktothrix rubescens bacteria occur in deep, physically stratified, and less eutrophic lakes. Planktothrix is a known producer of the toxic heptapeptide microcystin (MC), which is produced nonribosomally by a large enzyme complex consisting of peptide synthetases and polyketide synthases encoded by a total of nine genes (mcy genes). Planktothrix spp. differ in their cellular MC contents as well as the production of MC variants; however, the mechanisms favoring this diversity are not understood. Recently, the occurrence of Planktothrix strains containing all mcy genes but lacking MC has been reported. In this study, 29 such strains were analyzed to find out if mutations of the mcy genes lead to the inability to synthesize MC. Two deletions, spanning 400 bp (in mcyB; one strain) and 1,869 bp (in mcyHA; three strains), and three insertions (IS), spanning 1,429 bp (in mcyD; eight strains), 1,433 bp (in mcyEG; one strain), and 1,433 bp (in mcyA; one strain), were identified. Though found in different genes and different isolates and transcribed in opposite directions, IS were found to be identical and contained conserved domains assigned to transposable elements. Using mutation-specific primers, two insertions (in mcyD and mcyA) and one deletion (in mcyHA) were found regularly in populations of P. rubescens in different lakes. The results demonstrate for the first time that different mutations resulting in inactivation of MC synthesis do occur frequently and make up a stable proportion of the mcy gene pool in Planktothrix populations over several years.     

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.     

Grazing of two toxic Planktothrix species by Daphnia pulicaria: potential for bloom control and transfer of microcystins

The role of zooplankton in the control of cyanobacterial bloomsand the transfer of cyanotoxins to higher trophic levels areof great importance to the management of water resources. Manystudies have focused on the cyanobacterium Microcystis, butfew have examined the interactions between zooplankton and filamentouscyanobacteria. In this study, we provide experimental evidencefor the potential grazing of two toxic strains of filamentouscyanobacteria, Planktothrix rubescens and P. agardhii, by Daphniapulicaria, and for transfer of toxins in the planktonic foodchain. We determined clearance rates (CRs) by adult and juvenileD. pulicaria of the two Planktothrix strains, Scenedesmus acutusand a mixture of S. acutus cells with P. rubescens culture filtrate.Filament lengths were analyzed, and microcystin (MCY) presencein Daphnia was assessed using the Protein Phosphatase-2A (PP-2A)Inhibition Assay. The two Planktothrix strains were equallygrazed by D. pulicaria, but at lower CRs than S. acutus. Potentialanti-grazer toxins in P. rubescens filtrate did not inhibitDaphnia grazing. Small P. rubescens (<100 µm) filamentswere preferentially grazed by adult D. pulicaria, suggestingtheir limited ability to control a Planktothrix population duringa bloom. Large quantities of MCYs were found in unstarved Daphniapreviously exposed to Planktothrix, whereas quantities weresignificantly smaller in individuals starved for 24 h beforepreservation. This indicated a potential for transfer of toxinsin the food chain by Daphnia, especially immediately after ingestionof toxic cyanobacteria.     

Amino acid availability determines the ratio of microcystin variants in the cyanobacterium Planktothrix agardhii

Cyanobacteria are capable of producing multiple microcystin variants simultaneously. The mechanisms that determine the composition of microcystin variants in cyanobacteria are still debated. [Asp³]microcystin-RR contains arginine at the position where the more toxic [Asp³]microcystin-LR incorporates leucine. We cultured the filamentous cyanobacterium Planktothrix agardhii strain 126/3 with and without external addition of leucine and arginine. Addition of leucine to the growth medium resulted in a strong increase of the [Asp³]microcystin LR/RR ratio, while addition of arginine resulted in a decrease. This demonstrates that amino acid availability plays a role in the synthesis of different microcystin variants. Environmental changes affecting cell metabolism may cause differences in the intracellular availability of leucine and arginine, which can thus affect the production of microcystin variants. Because leucine contains one nitrogen atom while arginine contains four nitrogen atoms, we hypothesized that low nitrogen availability might shift the amino acid composition in favor of leucine, which might explain seasonal increases in the [Asp³]microcystin LR/RR ratio in natural populations. However, when a continuous culture of P. agardhii was shifted from nitrogen-saturated to a nitrogen-limited mineral medium, leucine and arginine concentrations decreased, but the leucine/arginine ratio did not change. Accordingly, while the total microcystin concentration of the cells decreased, we did not observe changes in the [Asp³]microcystin LR/RR ratio in response to nitrogen limitation.     

Contrasting microcystin production and cyanobacterial population dynamics in two Planktothrix-dominated freshwater lakes

Microcystin concentrations in two Dutch lakes with an important Planktothrix component were related to the dynamics of cyanobacterial genotypes and biovolumes. Genotype composition was analysed by using denaturing gradient gel electrophoresis (DGGE) profiling of the intergenic transcribed spacer region of the rrn operon (rRNA-ITS), and biovolumes were measured by using microscopy. In Lake Tjeukemeer, microcystins were present throughout summer (maximum concentration 30 microg l(-1)) while cyanobacterial diversity was low and very constant. The dominant phototroph was Planktothrix agardhii. In contrast, Lake Klinckenberg showed a high microcystin peak (up to 140 microg l(-1)) of short duration. In this lake, cyanobacterial diversity was higher and very dynamic with apparent genotype successions. Several genotypes derived from DGGE field profiles matched with genotypes from cultures isolated from field samples. The microcystin peak measured in Lake Klinckenberg could be confidently linked to a bloom of Planktothrix rubescens, as microscopic and genotypic analysis showed identity of bloom samples and a toxin-producing P. rubescens culture. Toxin-producing genotypes were detected in the microbial community before they reached densities at which they were detected by using microscopy. Cyanobacterial biovolumes provided additional insights in bloom dynamics. In both lakes, the microcystin content per cell was highest at the onset of the blooms. Our results suggest that while genotypic characterization of a lake can be valuable for detection of toxic organisms, for some lakes a monitoring of algal biomass has sufficient predictive value for an assessment of toxin production.     

Collapse of a Planktothrix agardhii perennial bloom and microcystin dynamics in response to reduced phosphate concentrations in a temperate lake

Planktothrix agardhii dynamics, microcystin concentration and limnological variables were monitored every 2 weeks for 2 years (2004-2006) in a shallow hypereutrophic artificial lake (BNV, Viry-Chatillon, France). Time-series analysis identified two components in the P. agardhii biomass dynamics: (1) a significant decreasing trend in P. agardhii biomass (65% of the overall variance) and (2) a residual component without significant seasonal periodicity. A path-analysis model was built to determine the main factors controlling the P. agardhii dynamics over the period studied. The model explained 66% of P. agardhii biomass changes. The decreasing trend in P. agardhii biomass was significantly related to a decrease in the PO4(3-) concentration resulting from an improved treatment of the incoming watershed surface water. The residual component was related to zooplankton dynamics (cyclopoid abundances), supporting the hypothesis of a top-down control of P. agardhii, but only when the biomass was low. Forty-nine percent of the variability in the microcystin (MC) concentration (min:<0.1 microg equivalent MC-LR L(-1); max: 7.4 microg equivalent MC-LR L(-1)) could be explained by changes in the P. agardhii biomass. The highest toxin content was observed when P. agardhii biomass was the lowest, which suggests changes in the proportion of microcystin-producing and -nonproducing subpopulations and/or the physiological status of cells.     

Seasonal patterns of microcystin-producing and non-producing Planktothrix rubescens genotypes in a deep pre-alpine lake

The filamentous cyanobacterium Planktothrix rubescens produces secondary metabolites called microcystins (MC) that are potent toxins for most eukaryotes, including zooplankton grazers, cattle and humans. P. rubescens occurs in many deep and thermally stratified lakes throughout Europe. In Lake Zurich (Switzerland), it re-appeared in the 1970s concomitant with decreasing eutrophication. Since then, P. rubescens has become the dominant species in this major drinking water reservoir, where it forms massive metalimnetic blooms during late summer. These cyanobacteria harbor subpopulations of non-MC producers, but little is known about the environmental factors affecting the success of such genotypes. The non-MC-producing subpopulation of P. rubescens was studied using a quantitative real-time PCR (qPCR) assay on the MC synthetase (mcy) gene cluster that targets a deletion on the mcyH and mcyA genes, which inactivates MC biosynthesis. Two complementary qPCR assays were used to assess the total population abundance (based on the 16S rDNA gene) and the mcy gene copy number (based on a conserved region in the adenylation domain of the mcyB gene). The objective was to evaluate the seasonal patterns of the share of non-MC-producing filaments in the total P. rubescens population. The mcyHA mutants were present in low proportions (up to 14%) throughout the year. Their highest relative abundances occurred during the winter mixis, when total concentrations of P. rubescens were minimal. The MC deficient mutants seemed to better survive in sparse populations, possibly because of lower grazing pressure and a consequently reduced need for MC-mediated protection. Alternatively, the mutants might cope better with the sub-optimal, stressful pressure and light conditions during the winter mixis. Altogether, our results suggest that subtle trade-offs might seasonally determine the proportions of non-MC producers within P. rubescens populations.     

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)-10(3) mm(3) 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.     

Seasonal dynamics and depth distribution of Planktothrix spp. in Lake Steinsfjorden (Norway) related to environmental factors

To investigate factors and mechanisms regulating toxin-producingpopulations of Planktothrix, we conducted a field study (2001–04)in the mesotrophic Lake Steinsfjorden, South-eastern Norway.The occurring species, Planktothrix rubescens and P. agardhii,had similar depth distributions and seasonal dynamics, bothforming metalimnetic blooms in 10–14 m depth. By comparingthe resource availability and temperature in Lake Steinsfjordenwith demands determined in laboratory studies, temperature andlight were identified as the most important factors controllinggrowth and depth distribution of Planktothrix spp. In addition,macronutrients, especially nitrogen, may have limited growthin periods. A lowering of nutrient supplies over time couldin addition to the prevailing suboptimal temperature and lightconditions prevent the population of Planktothrix spp. fromforming blooms. On two occasions, a major decrease in Planktothrixspp. abundance in the open water could be linked to a transporttowards the banks of Lake Steinsfjorden with subsequent decompositionin the littoral zone. Our results show that the depth distributionand seasonal dynamics of Planktothrix spp. in Lake Steinsfjordenis controlled by environmental factors in a similar way as inother Nordic, Central European and North American lakes.     

Competition between microcystin- and non-microcystin-producing Planktothrix agardhii (cyanobacteria) strains under different environmental conditions

The factors that control the production of microcystins (hepatotoxins) during cyanobacterial blooms, and the function of these metabolites remain largely unknown. In an attempt to provide answers to these questions, we compared the fitness of microcystin (MC)-producing and non-MC-producing Planktothrix agardhii strains under various experimental conditions. More specifically, we investigated the effects of temperature, light intensity and nitrate concentrations on several MC-producing and non-MC-producing strains in monoculture and competition experiments. In the monoculture experiments, no significant difference in cell growth rates was found for any of the environmental conditions tested. On the other hand, at the end of the competition experiments, we found that when the environmental conditions limited cell growth, MC-producing strains were clearly winning out over the non-MC-producing ones. This suggested that, under growth-limiting conditions, the benefits of producing MC outweigh the cost. Moreover, the reverse was found under non-growth-limiting conditions, suggesting that under environmental conditions that favour cyanobacterial growth, the cost of MC production must outweigh its benefits. These findings suggest that environmental factors may have an indirect effect on the MC production rate, and on the selection of MC-producing and non-MC-producing strains, via their direct impact on both the cell growth rate and the cell densities in the cultures. Several hypotheses have been advanced concerning the possible function of MCs, but none of them seems to be supported by our data.     

A Dhb-microcystin from the filamentous cyanobacterium Planktothrix rubescens

A Dhb-microcystin variant was isolated from the filamentous cyanobacterium Planktothrix rubescens. Its structure was elucidated as (E)-Dhb-microcystin-HilR ([D-Asp3, (E)-Dhb7]microcystin-HilR) on the basis of spectral data and amino acid analysis after acid hydrolysis.     

Molecular Mechanisms of the Response to 4-tert-Octylphenol-Induced Stress in a Cyanobacterium Planktothrix agardhii

Microbiology - The molecular mechanisms of stress response of Planktothrix agardhii, one of the dominant cya-nobacteria in the basins of the Northwestern Russia and other regions during...     

Species-specific real-time PCR cell number quantification of the bloom-forming cyanobacterium Planktothrix agardhii

A species-specific method to detect and quantify Planktothrix agardhii was developed by combining the SYBR Green I real-time polymerase chain reaction technique with a simplified DNA extraction procedure for standard curve preparation. Newly designed PCR primers were used to amplify a specific fragment within the rpoC1 gene. Since this gene exists in single copy in the genome, it allows the direct achievement of cell concentrations. The cell concentration determined by real-time PCR showed a linear correlation with the cell concentration determined from direct microscopic counts. The detection limit for cell quantification of the method was 8?cells?μL(-1), corresponding to 32 cells per reaction. Furthermore, the real-time qPCR method described in this study allowed a successful quantification of P. agardhii from environmental water samples, showing that this protocol is an accurate and economic tool for a rapid absolute quantification of the potentially toxic cyanobacterium P. agardhii.     

Genetic Variability of Microcystin Biosynthesis Genes in Planktothrix as Elucidated from Samples Preserved by Heat Desiccation during Three Decades

Historic samples of phytoplankton can provide information on the abundance of the toxigenic genotypes of cyanobacteria in dependence on increased or decreased eutrophication. The analysis of a time-series from preserved phytoplankton samples by quantitative PCR (qPCR) extends observation periods considerably. The analysis of DNA from heat-desiccated samples by qPCR can be aggravated by point substitutions or the fragmentation of DNA introduced by the high temperature. In this study, we analyzed whether the heat desiccation of the cellular material of the cyanobacterium Planktothrix sp. introduced potential errors to the template DNA that is used for qPCR within (i) 16S rDNA and phycocyanin genes and (ii) the mcyA gene indicative of the incorporation of either dehydrobutyrine (Dhb) or N-methyl-dehydroalanine (Mdha) in position 7, and (ii) the mcyB gene, which is indicative of homotyrosine (Hty) in position 2 of the microcystin (MC) molecule. Due to high temperature desiccation, the deterioration of the DNA template quality was rather due to fragmentation than due to nucleotide substitutions. By using the heat-desiccated samples of Lake Zürich, Switzerland the abundance of the Dhb, Mdha and Hty genotypes was determined during three decades (1977-2008). Despite major changes in the trophic state of the lake resulting in a major increase of the total Planktothrix population density, the proportion of these genotypes encoding the synthesis of different MC congeners showed high stability. Nevertheless, a decline of the most abundant mcyA genotype indicative of the synthesis of Dhb in position 7 of the MC molecule was observed. This decline could be related to the gradual incline in the proportion of a mutant genotype carrying a 1.8kbp deletion of this gene region. The increase of this mcyA (Dhb) gene deletion mutant has been minor so far, however, and likely did not affect the overall toxicity of the population.     

Interlaboratory comparison of Taq Nuclease Assays for the quantification of the toxic cyanobacteria Microcystis sp

The application of quantitative real-time PCR has been proposed for the quantification of toxic genotypes of cyanobacteria. We have compared the Taq Nuclease Assay (TNA) in quantifying the toxic cyanobacteria Microcystis sp. via the intergenic spacer region of the phycocyanin operon (PC) and mcyB indicative of the production of the toxic heptapeptide microcystin between three research groups employing three instruments (ABI7300, GeneAmp5700, ABI7500). The estimates of mcyB genotypes were compared using (i) DNA of a mcyB containing strain and a non-mcyB containing strain supplied in different mixtures across a low range of variation (0-10% of mcyB) and across a high range of variation (20-100%), and (ii) DNA from field samples containing Microcystis sp. For all three instruments highly significant linear regression curves between the proportion of the mcyB containing strain and the percentage of mcyB genotypes both within the low range and within the high range of mcyB variation were obtained. The regression curves derived from the three instruments differed in slope and within the high range of mcyB variation mcyB proportions were either underestimated (0-50%) or overestimated (0-72%). For field samples cell numbers estimated via both TNAs as well as mcyB proportions showed significant linear relationships between the instruments. For all instruments a linear relationship between the cell numbers estimated as PC genotypes and the cell numbers estimated as mcyB genotypes was observed. The proportions of mcyB varied from 2 to 28% and did not differ between the instruments. It is concluded that the TNA is able to provide quantitative estimates on mcyB genotype numbers that are reproducible between research groups and is useful to follow variation in mcyB genotype proportion occurring within weeks to months.     

The uptake of amino acids by the cyanobacterium Planktothrix rubescens is stimulated by light at low irradiances

The rates of uptake of five amino acids--alanine, glutamate, glycine, leucine and serine--by axenic cultures of the cyanobacterium Planktothrix rubescens were measured over a range of irradiances using the (14)C-labelled amino acids at the nanomolar concentrations observed in Lake Zürich. The rates in the light exceeded the dark rates by as much as two- to ninefold. The light-affinity constants for stimulation were similar, indicating a similar process for each of the five amino acids. The E(k) (light saturation irradiance) for light stimulation was only 1 micromol m(-2) s(-1), less than the compensation point for photosynthesis and autotrophic growth, and much lower than the E(k) for either process. The E(k) for amino acid uptake was also less than the irradiance at which filaments obtain neutral buoyancy, which determines the depth at which they stratify and the irradiance they receive. This indicates that stimulation of amino acid uptake by light of low irradiances provides a mechanism for supplementing growth of filaments stratifying deep in the metalimnion, which, while able to grow at low irradiances, are often left with insufficient light to sustain them. Acetate uptake was also stimulated by light, but the kinetics differed.