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Applied and Environmental Microbiology, September 2005, p. 5177-5181, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5177-5181.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Microcystin Composition of the Cyanobacterium Planktothrix agardhii Changes toward a More Toxic Variant with Increasing Light Intensity

Linda Tonk,^1* Petra M. Visser,¹ Guntram Christiansen,^2, Elke Dittmann,² Eveline O. F. M. Snelder,¹ Claudia Wiedner,^1, Luuc R. Mur,¹ and Jef Huisman¹

Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands,¹ Institute for Biology, Molecular Ecology, Humboldt University of Berlin, D-10115 Berlin, Germany²

Received 19 February 2005/ Accepted 29 March 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The cyanobacterium Planktothrix agardhii, which is dominant in many shallow eutrophic lakes, can produce hepatotoxic microcystins. Currently, more than 70 different microcystin variants have been described, which differ in toxicity. In this study, the effect of photon irradiance on the production of different microcystin variants by P. agardhii was investigated using light-limited turbidostats. Both the amount of the mRNA transcript of the mcyA gene and the total microcystin production rate increased with photon irradiance up to 60 µmol m^–2 s^–1, but they started to decrease with irradiance greater than 100 µmol m^–2 s^–1. The cellular content of total microcystin remained constant, independent of the irradiance. However, of the two main microcystin variants detected in P. agardhii, the microcystin-DeRR content decreased twofold with increased photon irradiance, whereas the microcystin-DeLR content increased threefold. Since microcystin-DeLR is considerably more toxic than microcystin-DeRR, this implies that P. agardhii becomes more toxic at high light intensities.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cyanobacteria can produce a wide variety of toxic compounds, including microcystins (MC) (2, 4, 5). Microcystins are cyclic heptapeptides that can cause liver damage through inhibition of protein phosphatases (20, 41) and may promote the development of liver tumors (24). So far, more than 70 different microcystin variants have been described (7). These microcystin variants may differ in their toxicological effects. For instance, 50% lethal dose experiments with mice have indicated that MC-LR is about four times more toxic than MC-RR (12, 17).

Microcystin-producing cyanobacteria usually generate multiple microcystin variants (34). Relatively few studies (28, 30) have investigated which factors determine the specific composition of microcystin variants. Yet such studies are highly relevant given the different toxicities of the various microcystin variants. Basically, one mcy gene cluster is responsible for the synthesis of the molecular core of all microcystins in a given strain (8, 25). The different microcystin variants are synthesized nonribosomally, as variations on the same molecular core, and the synthesis is catalyzed by a large multifunctional enzyme complex consisting of peptide synthetase and polyketide synthase modules (1). The specificity of modules and various functional domains within this large enzyme complex determines the complement of microcystin variants that are produced (23). How the synthesis and activity of the entire enzyme complex is regulated is not well understood.

Several studies have revealed that the total microcystin production by cyanobacteria may vary considerably with environmental factors (27, 32, 38). Previous work has shown that the total microcystin production by Microcystis aeruginosa responds to iron (36), phosphorus (28), nitrogen (19), and photon irradiance (35, 40). Orr and Jones (29) and Long et al. (19) hypothesized that the growth rate determines the microcystin production by cyanobacteria, which provides an explanation for the impact of so many environmental factors on microcystin production. Wiedner et al. (40), however, showed that for Microcystis this relationship occurs under light-limited conditions but not under light-saturated conditions. Genetic regulation studies have revealed that increased microcystin peptide synthetase and polyketide synthase gene transcription occurs in Microcystis as a result of increasing light intensity (15). Hence, we hypothesized that light is a major factor in microcystin production.

In the present study we aimed to investigate the effect of photon irradiance on the mcyA transcript, the total microcystin production, and the microcystin composition of the filamentous cyanobacterium Planktothrix agardhii Anagn. et Kom. We selected the mcyA gene as a representative of the microcystin biosynthesis gene cluster in order to link expression of the mcy gene to total microcystin production as measured by high-performance liquid chromatography (HPLC). The experiments were carried out with continuous cultures specifically tailored to study light limitation under highly controlled conditions (14). Planktothrix was chosen because it is a widespread nuisance species (10, 31, 33), particularly in shallow turbid lakes where light is a major limiting factor (22, 27, 39). Lakes dominated by Planktothrix have significantly higher concentrations of microcystin per unit of cyanobacterial biomass than lakes dominated by other cyanobacterial species (11), yet little is known about the impact of light on Planktothrix microcystin production and composition.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Organism and culture setup.
P. agardhii strain 126/3 was kindly provided by K. Sivonen of the Department of Applied Chemistry and Microbiology, University of Helsinki (32). Cultures were checked frequently for bacterial contamination by microscopy. All experiments were performed with continuous cultures by using flat culture vessels specifically designed to study light-limited growth (14, 21); the working volume was 1.85 liters. The temperature was kept constant at 23 ± 1°C by means of a cooling element placed between the culture vessel and the light source. A continuous flow of nutrient-saturated O2 medium (37) was added to the culture to prevent nutrient limitation. Furthermore, the culture was aerated with a continuous airflow to ensure homogeneous mixing and to provide sufficient amounts of carbon dioxide for photosynthesis. The turbidostat technique was used. The pump was adjusted to keep the optical density at 750 nm constant at 0.10 to 0.15 cm^–1.

Light conditions.
The light source consisted of four white fluorescent tubes (Philips PL-L/24W/840/4P) placed in front of the culture vessel. A day-night cycle consisting of 12 h of light and 12 h of darkness was used, while all other factors were kept constant. Photon irradiance was measured using an LI-COR SA 198 quantum sensor. The specific design of the flat culture vessels allowed accurate estimation of the average photon irradiance (14). The incident irradiance (I_in) and the irradiance penetrating through the vessel (I_out) were measured at 10 evenly spaced points on the front and back surfaces of the culture vessel, respectively. The depth-averaged photon irradiance (I_avg) in the culture vessel was calculated as follows: I_avg = (I_in – I_out)/(ln I_in – ln I_out).

Experiments and sampling.
We studied the microcystin production by P. agardhii by using three time scales. First, we investigated short-term changes in microcystin composition in two diurnal experiments with a cycle consisting of 12 h of light and 12 h of darkness. In one experiment we used low light (depth-averaged irradiance, 25 µmol m^–2 s^–1), and in the other experiment we used high light (110 µmol m^–2 s^–1) during the light period. Second, on a time scale of days, we studied the transient dynamics of microcystin composition in cultures that were transferred from 25 to 65 µmol m^–2 s^–1 and from 110 to 40 µmol m^–2 s^–1 during the light period. Third, on an even longer time scale, we performed a series of steady-state experiments with depth-averaged photon irradiances of 6, 9, 17, 25, 36, 48, 74, 79, and 112 µmol m^–2 s^–1 during the light period. The optical density at 750 nm was monitored on a daily basis. When the optical density remained constant (±15%) for more than 1 week, the culture was considered to be in steady state. During a steady state, samples were taken daily for 5 days. Samples were always taken 1 h after the light was switched on. Each sample was divided into two subsamples for estimation of the biovolume and microcystin analysis.

Microcystin analysis.
Intracellular microcystin contents were analyzed by filtering 10 ml of the culture suspension in triplicate using Whatman GF/C filters (pore size, 1.2 µm). Filters were freeze-dried and stored at –20°C. Microcystin was extracted in 75% methanol (three extraction rounds) as described by Fastner et al. (9) with an extra step for grinding of the filters in a Mini Beadbeater (Biospec Products, Bartlesville, Okla.) with 0.5-mm silica beads. Dried extracts were stored at –20°C and dissolved in 50% methanol for analysis of microcystin using HPLC with photodiode array detection (Kontron Instruments). The extracts were separated using a 30 to 70% acetonitrile gradient with 0.05% trifluoroacetic acid at a flow rate of 1 ml min^–1 and a LiChrospher 100 ODS 5 µm LiChorCART 250-4 cartridge system (Merck). The different microcystin variants were identified on the basis of their characteristic UV spectra and were quantified by means of an MC-LR gravimetric standard provided by the University of Dundee.

The extracellular microcystin concentrations were below the detection limit of the HPLC (2.5 ng of microcystin). Therefore, extracellular microcystin concentrations were determined using an enzyme-linked immunosorbent assay according to the protocol of a Microcystin Plate kit (catalog no. EP 022; EnviroLogix Inc.). Since the enzyme-linked immunosorbent assay approach does not distinguish between different microcystin variants, we were unable to quantify the different extracellular microcystins.

Biovolume and growth rate.
The lengths of the filaments were measured by using Image Analysis (Qwin, Leica Microsystems) and samples stored in Lugol's iodine. The average diameter of P. agardhii filaments was estimated to be 3.3 µm (standard deviation, 0.3 µm; n = 147). The biovolume (BV) of the filaments was calculated as follows: BV = 0.5Lr², where L is the filament length and r is the filament diameter. The specific growth rate (µ) was calculated by using the following equation:

where x₁ and x₂ are the estimated biomasses (measured as biovolume) at times t₁ and t₂, respectively, and D is the dilution rate (in h^–1). The microcystin production rate was calculated by multiplying the microcystin content by the specific growth rate.

RNA extraction and quantification.
Samples (25 ml) were taken from four continuous cultures in steady state grown at different levels of photon irradiance. These samples (three samples per continuous culture) were added to tubes filled to 25 ml with ice. The tubes were centrifuged, and the pellets were frozen with liquid nitrogen and stored at –20°C until RNA extraction. Total RNA was extracted by using a method similar to that described by Kaebernick et al. (15), using Trizol reagent (Gibco BRL, Life Technologies, Rockville, Md.) after the pellet was crushed in a precooled mortar. Phenol extraction and precipitation were performed according to the manufacturer's protocol. RNA was quantified using Northern analysis.

RPA.
Primers McyA-Cd 1R (5'-AAAAGTGTTTTATTAGCGGCTCAT-3') and McyA-Cd 1F (5'-AAAATTAAAAGCCGTATCAAA-3') were used to amplify a 300-bp fragment of the mcyA gene of P. agardhii (6) as described by Hisbergues et al. (13). This fragment was ligated into the pGEM-T cloning vector (Promega). The probe for the mRNA protection assay (RPA) was prepared by in vitro transcription (Maxiscript; Ambion, Austin, Tex.) and labeled with [-³²P]UTP. The RPA was performed by using a method similar to that described by Kaebernick et al. (15) according to the manufacturer's instructions (Boehringer). The probe was mixed with different amounts of mRNA standardized by 16S RNA gel electrophoresis. The transcripts were analyzed by polyacrylamide gel electrophoresis, using 12.5% Gel 40 and 16.8 g of urea in 40 ml (total volume) combined with a Tris-borate-EDTA buffer. The gel was run for 2.5 h at 30 mA and subsequently exposed to X-ray film overnight at –80°C. The pixel densities of the bands were estimated with image analysis software (Qwin, Leica Microsystems). The background density was subtracted from the density of each band, and the highest pixel density was defined as 100%.

Data analysis.
The data were analyzed by means of stepwise multiple regression, using polynomials to estimate higher-order terms. Higher-order terms were added only if they improved the fit significantly at the 0.05 level.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Diurnal and transient dynamics.
The microcystins of P. agardhii consisted mainly of MC-DeRR and lower concentrations of MC-DeLR. Traces of other microcystin variants were observed, but their concentrations barely exceeded the detection level. The MC-DeRR and MC-DeLR contents showed no distinct diurnal fluctuations, despite the imposed day-night cycles (Fig. 1A). In the transient-state experiments, in which cultures were transferred from low light to high light and vice versa, the MC-DeRR and MC-DeLR contents adjusted to the new light conditions within 6 to 8 days (Fig. 1B and C). This shows that the relevant time scale for changes in microcystin composition is on the order of several days.

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FIG. 1. Time scales of microcystin dynamics. (A) Diurnal changes in microcystin contents at two levels of photon irradiance. Symbols: , MC-DeRR at 25 µmol m–2 s–1; , MC-DeLR at 25 µmol m–2 s–1; •, MC-DeRR at 110 µmol m–2 s–1; , MC-DeLR at 110 µmol m–2 s–1. (B and C) Changes in MC-DeRR content (solid symbols) and MC-DeLR content (open symbols) during transient states from 25 to 65 µmol m–2 s–1 (B) and from 110 to 40 µmol m–2 s–1 (C). The error bars indicate standard deviations (n = 3).

Growth rates.
The specific growth rate (µ) of P. agardhii strain 126/3, as measured during steady-state conditions in continuous cultures, is a unimodal function of photon irradiance (Fig. 2) (multiple regression for µ: 0.002 + 0.0006I – 4 x 10^–6: ²; R² = 0.97; n = 9; P < 0.01). The specific growth rate was limited by photon irradiance at least up to 60 µmol m^–2 s^–1, whereas at levels greater than 100 µmol m^–2 s^–1 growth seemed to be inhibited by photon irradiance.

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FIG. 2. Specific growth rate of P. agardhii grown under light-limited conditions as a function of photon irradiance. The solid line is based on multiple regression. The error bars indicate standard deviations (n = 5).

mRNA transcripts and microcystin production.
Samples were taken from four of the steady-state cultures to perform mRNA protection assays in order to estimate the effects of different light intensities on the amount of the mcyA transcript. This experiment revealed a pattern similar to the specific growth rate pattern. The transcriptional response of the mcyA gene increased with photon irradiance up to 60 µmol m^–2 s^–1 (Fig. 3). At levels greater than 100 µmol photons m^–2 s^–1 the mcyA gene produced less mRNA transcript. This pattern was further matched by the total microcystin production, which also increased with photon irradiance up to 60 µmol m^–2 s^–1 and decreased at 100 µmol m^–2 s^–1 (Fig. 3).

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FIG. 3. Total microcystin production rate () and amount of mcyA transcript () of P. agardhii plotted against photon irradiance. The error bars indicate standard deviations (n = 5).

Microcystin composition.
In all experiments, the extracellular microcystin concentration was less than 1% of the total microcystin concentration in the cultures. Thus, almost all microcystin was intracellular. The total microcystin content of P. agardhii in the steady-state experiments did not change significantly with photon irradiance (Fig. 4) (linear regression for total microcystin content: R² = 0.39; n = 9; P > 0.05). However, the composition of the microcystin variants did change with photon irradiance. The MC-DeRR content decreased with increased photon irradiance (Fig. 4) (linear regression for MC-DeRR: 2.37 – 0.008I; R² = 0.81; n = 9; P < 0.01), whereas the MC-DeLR content increased with increased photon irradiance (Fig. 4) (linear regression for MC-DeLR: 0.162 + 0.004I; R² = 0.93; n = 9; P < 0.01). As a result, the ratio of MC-DeLR to MC-DeRR increased more than sixfold with increased photon irradiance, as accurately described by a linear relationship (linear regression for LR/RR ratio: 0.044 + 0.0034I; R² = 0.97; n = 9; P < 0.001). The MC-DeRR production rate (P_MC-DeRR) was a unimodal function of photon irradiance, with the maximum production rate at a photon irradiance in the range from 50 to 80 µmol m^–2 s^–1 (Fig. 5, upper panel) (multiple regression for P_MC-DeRR: –0.0032 + 0.0013I – 1 x 10^–5I²; R² = 0.98; n = 9; P < 0.01). The MC-DeLR production rate (P_MC-DeLR) increased but was saturated with increased photon irradiance (Fig. 5, lower panel) (multiple regression for P_MC-DeLR: –0.0024 + 0.0003I – 2 x 10^–6I²; R² = 0.96; n = 9; P < 0.05).

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FIG. 4. Total microcystin content (), MC-DeRR content (), and MC-DeLR content (•) of P. agardhii as a function of photon irradiance. The error bars indicate standard deviations (n = 5). The solid lines for MC-DeRR and MC-DeLR are based on linear regression. The line for MC-Total indicates the mean.

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FIG. 5. Rates of production of MC-DeRR (upper panel) and MC-DeLR (lower panel) in P. agardhii as a function of photon irradiance. The error bars indicate standard deviations (n = 5). The solid lines are based on multiple regression.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our results reveal that there is a direct relationship between the transcription rate of the mcyA gene and the total microcystin production rate of P. agardhii (Fig. 3). Both gene transcription and total microcystin production increased with increased photon irradiance up to 60 µmol m^–2 s^–1. At levels greater than 100 µmol m^–2 s^–1 both rates decreased with increased photon irradiance. This result confirms previous findings with Microcystis which showed that light exerts major control over the transcription of the mcy gene complex (15). The close correspondence between mcyA transcription and microcystin production is consistent with previous findings (8, 25, 26) which showed that one mcy gene cluster is responsible for the synthesis of the core of all microcystin variants.

Our observation that an isolated strain of Planktothrix produces different structural variants of microcystin can be explained by the multispecificity of single domains of the microcystin biosynthesis complex. In particular, the first module of McyB can incorporate a variety of different amino acids at the variable X position within the microcystin structure (6). At this variable position, microcystin-DeRR contains arginine, whereas microcystin-DeLR contains leucine. Strikingly, our results show that the composition of microcystin variants may change substantially with light conditions. The content of MC-DeRR decreased with increased photon irradiance, whereas the content of MC-DeLR increased (Fig. 4). Transient-state experiments confirmed these results (Fig. 1B and C). Why does Planktothrix produce less arginine-based microcystins but more leucine-based microcystins at higher irradiance? Perhaps a conformational change in the substrate-binding pocket of the first module of the McyB enzyme could lead to a change in the substrate specificity of the module. Biochemical assays with the McyB enzyme could elucidate this possibility. A plausible alternative explanation is that different light conditions induce changes in the composition of available amino acids. Increased photosynthesis at a high light intensity may raise the C/N ratio of the cells. This would favor leucine over arginine, since relatively less nitrogen would be available to synthesize the nitrogen-rich arginine molecule, resulting in a shift in microcystin synthesis from MC-DeRR to MC-DeLR. This hypothesis can be tested by measuring the amino acid concentrations in cells grown at different levels of photon irradiance.

Previous studies with Microcystis aeruginosa PCC 7806 showed that its total microcystin content increased with irradiance under light-limited conditions (40). M. aeruginosa contains mainly the microcystin variants microcystin-LR and microcystin-DeLR. Our findings with Planktothrix revealed patterns similar to the Microcystis patterns for MC-DeLR, as the MC-DeLR content of Planktothrix increased with increased irradiance. However, in Planktothrix the increase in MC-DeLR was accompanied by a decrease in MC-DeRR. As a result, in contrast to Microcystis, the total microcystin content of P. agardhii remained constant while the ratio of microcystin-DeLR to microcystin-DeRR increased more than sixfold with increased irradiance. According to mouse bioassays, MC-DeLR is at least four times more toxic than MC-DeRR (12, 17). Thus, while the total microcystin content of P. agardhii was not affected by light, the toxicity of P. agardhii actually increased with increased photon irradiance.

Lakes dominated by microcystin-producing cyanobacteria may show considerable seasonal variability in total microcystin concentrations (3, 10, 16). It could be suggested that the observed variability results from physiological changes in the total microcystin content of P. agardhii (for instance, changes induced by changing light conditions). However, our results do not support this hypothesis, as photon irradiance had no significant influence on the total microcystin content of P. agardhii. Likewise, the diurnal experiments showed that short-term changes in photon irradiance have little effect on the total microcystin content. It is more likely that the variability in total microcystin concentration commonly observed in Planktothrix-dominated lakes results from a succession of closely related Planktothrix genotypes that differ in microcystin production. This is consistent with recent findings of Kurmayer et al. (18) on the coexistence of toxic and nontoxic P. agardhii strains in shallow lakes. However, our results show that monitoring of the different strains of P. agardhii is not sufficient to predict the toxicity of a P. agardhii bloom. Even the toxicity of a single P. agardhii strain is quite variable, since the composition of intracellular microcystin variants may change in response to environmental conditions.

In conclusion, P. agardhii is a widespread cyanobacterium of shallow, turbid lakes. Light conditions in shallow lakes may change on a time scale of days to weeks due to changes in cloudiness or wind-induced resuspension of sediments. Our findings show that such changes in light conditions may profoundly affect the microcystin composition and thereby the toxicity of P. agardhii. The harmful cyanobacterium P. agardhii produces a more toxic variant during periods of sunny weather, when recreational activities in lakes are most attractive.

 
This study was supported by grants from the European Union within the programs CYANOTOX, TOPIC, and PEPCY. The investigations performed by P.M.V. and J.H. were supported by the Earth and Life Science Foundation (ALW), which is subsidized by The Netherlands Organization for Scientific Research (NWO).

We thank Kaarina Sivonen for providing the P. agardhii strain used in this study and Geoff Codd and coworkers of the University of Dundee for providing the MC-LR gravimetric standard. Furthermore, we are grateful to Jan-Christoph Kehr and Michael Hisbergues for their help with the mRNA protection assay, to Eugenia Sampayo-Garrido and Mariska Weijerman for their contributions to the experimental work, and to Hans Matthijs, Klaus Jöhnk, Jolanda Verspagen, and Edwin Kardinaal for discussions.

 
* Corresponding author. Mailing address: Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands. Phone: 31 20 5256027. Fax: 31 20 5257064. E-mail: ltonk{at}science.uva.nl.

Present address: Department of Chemistry, University of Hawaii, Honolulu, HI 96822.

Present address: Leibniz Institute of Freshwater Ecology and Inland Fisheries, Alte Fischerhütte 2, 16775 Neuglobsow, Germany.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Börner, T., and E. Dittmann. 2005. Molecular biology of cyanobacterial toxins: genetic basis of microcystin production, p. 25-40. In J. Huisman, H. C. P. Matthijs, and P. M. Visser (ed.), Harmful cyanobacteria. Springer, Dordrecht, The Netherlands.
2
2. Botes, D. P., A. A. Tuinman, P. L. Wessels, C. C. Viljoen, H. Kruger, D. H. Williams, S. Santikarn, R. J. Smith, and S. J. Hammond. 1984. The structure of cyanoginosin-LA, a cyclic peptide from the cyanobacterium Microcystis aeruginosa. J. Chem. Soc. Perkin Trans. 1:2311-2319.
3
3. Briand, J. F., C. Robillot, C. Quiblier-Lloberas, and C. Bernard. 2002. A perennial bloom Planktothrix agardhii (Cyanobacteria) in a shallow eutrophic French lake: limnological and microcystin production studies. Arch. Hydrobiol. 153:605-622.
4
4. Carmichael, W. W. 1997. The cyanotoxins. Adv. Bot. Res. 27:211-256.
5
5. Carmichael, W. W., S. M. F. O. Azevedo, J. S. An, R. J. R. Molica, E. M. Jochimsen, S. Lau, K. L. Rinehart, G. R. Shaw, and G. K. Eaglesham. 2001. Human fatalities from cyanobacteria: chemical and biological evidence for cyanotoxins. Environ. Health Perspect. 109:663-668.[Medline]
6
6. Christiansen, G., J. Fastner, M. Erhard, T. Börner, and E. Dittmann. 2003. Microcystin biosynthesis in Planktothrix: genes, evolution, and manipulation. J. Bacteriol. 185:564-572.[Abstract/Free Full Text]
7
7. Codd, G. A., J. Lindsay, F. M. Young, L. F. Morrison, and J. S. Metcalf. 2005. Harmful cyanobacteria: from mass mortalities to management measures, p. 1-24. In J. Huisman, H. C. P. Matthijs, and P. M. Visser (ed.), Harmful cyanobacteria. Springer, Dordrecht, The Netherlands.
8
8. Dittmann, E., B. A. Neilan, M. Erhard, H. von Döhren, and T. Börner. 1997. Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis aeruginosa PCC 7806. Mol. Microbiol. 26:779-787.[CrossRef][Medline]
9
9. Fastner, J., I. Flieger, and U. Nuemann. 1998. Optimised extraction of microcystins from field samples: a comparison of different solvents and procedures. Water Res. 32:3177-3181.[CrossRef]
10
10. Fastner, J., M. Erhard, W. W. Carmichael, F. Sun, K. L. Rinehart, H. Rönicke, and I. Chorus. 1999. Characterization and diversity of microcystins in natural blooms and strains of the genera Microcystis and Planktothrix from German freshwaters. Arch. Hydrobiol. 145:147-163.
11
11. Fastner, J., U. Neumann, B. Wirsing, J. Weckesser, C. Wiedner, B. Nixdorf, and I. Chorus. 1999. Microcystins (hepatotoxic heptapeptides) in German freshwater bodies. Environ. Toxicol. 14:13-22.
12
12. Harada, K. I. 1996. Chemistry and detection of microcystins, p. 103-148. In M. F. Watanabe, K. I. Harada, W. W. Carmichael, and H. Fujiki (ed.), Toxic microcystins. CRC Press, New York, N.Y.
13
13. Hisbergues, M., G. Christiansen, L. Rouhiainen, K. Sivonen, and T. Börner. 2003. PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera. Arch. Microbiol. 180:402-410.[CrossRef][Medline]
14
14. Huisman, J., H. C. P. Matthijs, P. M. Visser, H. Balke, C. A. M. Sigon, J. Passarge, F. J. Weissing, and L. R. Mur. 2002. Principles of the light-limited chemostat: theory and ecological applications. Antonie Leeuwenhoek 81:117-133.
15
15. Kaebernick, M., B. A. Neilan, T. Börner, and E. Dittmann. 2000. Light and the transcriptional response of the microcystin biosynthesis gene cluster. Appl. Environ. Microbiol. 66:3387-3392.[Abstract/Free Full Text]
16
16. Kardinaal, W. E. A., and P. M. Visser. 2005. Dynamics of cyanobacterial toxins: sources of variability in microcystin concentrations, p. 41-64. In J. Huisman, H. C. P. Matthijs, and P. M. Visser (ed.), Harmful cyanobacteria. Springer, Dordrecht, The Netherlands.
17
17. Kotak, B. G., A. K-Y. Lam, E. E. Prepas, S. L. Kenefick, and S. E. Hrudey. 1995. Variability of the hepatotoxin microcystin-LR in hypereutrophic drinking water lakes. J. Phycol. 31:248-263.[CrossRef]
18
18. Kurmayer, R., G. Christiansen, J. Fastner, and T. Börner. 2004. Abundance of active and inactive microcystin genotypes in populations of the toxic cyanobacterium Planktothrix spp. Environ. Microbiol. 6:831-841.[CrossRef][Medline]
19
19. Long, B. M., G. J. Jones, and P. T. Orr. 2001. Cellular microcystin content in N-limited Microcystis aeruginosa can be predicted from growth rate. Appl. Environ. Microbiol. 67:278-283.[Abstract/Free Full Text]
20
20. MacKintosh, C., K. A. Beattie, S. Klumpp, P. Cohen, and G. A. Codd. 1990. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. FEBS Lett. 264:187-192.[CrossRef][Medline]
21
21. Matthijs, H. C. P., H. Balke, U. M. Hes, B. M. A. Kroon, L. R. Mur, and R. A. Binot. 1996. Application of light-emitting diodes in bioreactors: flashing light effects and energy economy in algal culture (Chlorella pyrenoidosa). Biotechnol. Bioeng. 50:98-107.[CrossRef]
22
22. Mur, L. R., and H. Schreurs. 1995. Light as a selective factor in the distribution of phytoplankton species. Water Sci. Technol. 32:25-34.
23
23. Neilan, B. A., E. Dittmann, L. Rouhainen, R. A. Bass, V. Schaub, K. Sivonen, and T. Börner. 1999. Nonribosomal peptide synthesis and toxigenicity of cyanobacteria. J. Bacteriol. 181:4089-4097.[Abstract/Free Full Text]
24
24. Nishiwaki-Matsushima, R., T. Ohta, S. Nishiwaki, M. Suganuma, K. Koyhama, T. Ishika, W. W. Carmichael, and H. Fujiki. 1992. Liver-tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. J. Cancer Res. Clin. Oncol. 118:420-424.[CrossRef][Medline]
25
25. Nishizawa, T., M. Asayama, K. Fujii, K. Harada, and M. Shirai. 1999. Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp. J. Biochem. 126:520-526.[Abstract/Free Full Text]
26
26. Nishizawa, T., A. Ueda, M. Asayama, K. Fujii, K. Harada, K. Ochi, and M. Shirai. 2000. Polyketide synthase gene coupled to the peptide synthetase module involved in the biosynthesis of the cyclic heptapeptide microcystin. J. Biochem. 127:779-789.[Abstract/Free Full Text]
27
27. Nixdorf, B., U. Mischke, and J. Rücker. 2003. Phytoplankton assemblages and steady state in deep and shallow eutrophic lakes: an approach to differentiate the habitat properties of Oscillatoriales. Hydrobiologia 502:111-121.[CrossRef]
28
28. Oh, H.-M., S. J. Lee, M.-H. Jang, and B.-D. Yoon. 2000. Microcystin production by Microcystis aeruginosa in a phosphorus-limited chemostat. Appl. Environ. Microbiol. 66:176-179.[Abstract/Free Full Text]
29
29. Orr, P. T., and G. J. Jones. 1998. Relationship between microcystin production and cell division rates in nitrogen-limited Microcystis aeruginosa cultures. Limnol. Oceanogr. 43:1604-1614.
30
30. Rapala, J., K. Sivonen, C. Lyra, and S. I. Niemelä. 1997. Variation of microcystins, cyanobacterial hepatotoxins, in Anabaena spp. as a function of growth stimuli. Appl. Environ. Microbiol. 63:2206-2212.[Abstract]
31
31. Scheffer, M., S. Rinaldi, A. Gragnani, L. R. Mur, and E. H. van Nes. 1997. On the dominance of filamentous cyanobacteria in shallow, turbid lakes. Ecology 78:272-282.[CrossRef]
32
32. Sivonen, K. 1990. Effect of light, temperature, nitrate, orthophosphate and bacteria on growth of and hepatotoxin production by Oscillatoria agardhii strains. Appl. Environ. Microbiol. 56:2658-2666.[Abstract/Free Full Text]
33
33. Sivonen, K., S. I. Niemelä, R. M. Niemi, L. Lepistö, T. H. Luoma, and L. A. Räsäsnen. 1990. Toxic cyanobacteria (blue-green algae) in Finnish fresh and coastal waters. Hydrobiologia 190:267-275.[CrossRef]
34
34. Sivonen, K., and G. Jones. 1999. Cyanobacterial toxins, p. 41-111. In I. Chorus and J. Bartram (ed.), Toxic cyanobacteria in water: a guide to their public health consequences, monitoring and management. E. & F. N. Spon, London, England.
35
35. Utkilen, H., and N. Gjølme. 1992. Toxin production by Microcystis aeruginosa as a function of light in continuous cultures and its ecological significance. Appl. Environ. Microbiol. 58:1321-1325.[Abstract/Free Full Text]
36
36. Utkilen, H., and N. Gjølme. 1995. Iron-stimulated toxin production in Microcystis aeruginosa. Appl. Environ. Microbiol. 61:797-800.[Abstract]
37
37. Van Liere, L., and L. R. Mur. 1978. Light-limited cultures of the blue-green alga Oscillatoria agardhii. Mitt. Int. Ver. Limnol. 21:158-167.
38
38. Watanabe, M. F., and S. Oishi. 1985. Effects of environmental factors on toxicity of a cyanobacterium (Microcystis aeruginosa) under culture conditions. Appl. Environ. Microbiol. 49:1342-1344.[Abstract/Free Full Text]
39
39. Wiedner, C., B. Nixdorf, R. Heinze, B. Wirsing, U. Neumann, and J. Weckesser. 2002. Regulation of cyanobacteria and microcystin dynamics in polymictic shallow lakes. Arch. Hydrobiol. 155:383-400.
40
40. Wiedner, C., P. M. Visser, J. Fastner, J. S. Metcalf, G. A. Cod, and L. R. Mur. 2003. Effects of light on the microcystin content of Microcystis strain PCC 7806. Appl. Environ. Microbiol. 69:1475-1481.[Abstract/Free Full Text]
41
41. Yoshizawa, S., R. Matsushima, M. F. Watanabe, K. Harada, A. Ichihara, W. W. Carmichael, and H. Fujiki. 1990. Inhibition of protein phosphatases by microcystin and nodularin associated with hepatotoxicity. J. Cancer Res. Clin. Oncol. 116:609-614.[CrossRef][Medline]

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Applied and Environmental Microbiology, September 2005, p. 5177-5181, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5177-5181.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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