Editor: Patricia Sobecky
Pulsed nitrogen supply induces dynamic changes in the amino acid composition and microcystin production of the harmful cyanobacterium Planktothrix agardhii
Article first published online: 23 JUL 2010
© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiology Ecology
Volume 74, Issue 2, pages 430–438, November 2010
How to Cite
Van de Waal, D. B., Ferreruela, G., Tonk, L., Van Donk, E., Huisman, J., Visser, P. M. and Matthijs, H. C.P. (2010), Pulsed nitrogen supply induces dynamic changes in the amino acid composition and microcystin production of the harmful cyanobacterium Planktothrix agardhii. FEMS Microbiology Ecology, 74: 430–438. doi: 10.1111/j.1574-6941.2010.00958.x
Present addresses: Dedmer B. Van de Waal, Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany. Linda Tonk, Centre for Marine Studies, University of Queensland, St Lucia, Qld 4072, Australia.
- Issue published online: 23 JUL 2010
- Article first published online: 23 JUL 2010
- Received 24 September 2009; revised 8 June 2010; accepted 5 July 2010.Final version published online 23 August 2010.
- amino acids;
- carbon to nitrogen ratio;
- harmful cyanobacteria;
Planktothrix agardhii is a widespread harmful cyanobacterium of eutrophic waters, and can produce the hepatotoxins [Asp3]microcystin-LR and [Asp3]microcystin-RR. These two microcystin variants differ in their first variable amino acid position, which is occupied by either leucine (L) or arginine (R). Although microcystins are extensively investigated, little is known about the mechanisms that determine the production of different microcystin variants. We hypothesize that enhanced nitrogen availability will increase the intracellular content of the nitrogen-rich amino acid arginine, and thereby promote the production of the variant [Asp3]microcystin-RR. To test this hypothesis, we transferred P. agardhii strain 126/3 from nitrogen-replete to nitrogen-deficient conditions, and after 2 weeks of growth under nitrogen deficiency, we added a nitrogen pulse. We found a rapid increase in the cellular nitrogen to carbon ratio and the amino acids aspartic acid and arginine, indicative of cyanophycin synthesis. This was followed by a more gradual increase of the total amino acid content connected to balanced growth. As expected, the [Asp3]microcystin-RR variant increased strongly after the nitrogen pulse, while the [Asp3]microcystin-LR increased to a much lesser extent. We conclude that sudden nitrogen enrichment affects the amino acid composition of harmful cyanobacteria, which, in turn, affects the production and composition of their microcystins.
Aquatic ecosystems throughout the world have been enriched with nutrients derived from urban, industrial and agricultural activities (Vitousek et al., 1997; Galloway et al., 2004; Glibert et al., 2005). This anthropogenic eutrophication, in combination with global warming, facilitates the proliferation of harmful cyanobacteria (Dokulil & Teubner, 2000; Glibert et al., 2005; Jöhnk et al., 2008; Paerl & Huisman, 2008), which have become an increasing nuisance in many freshwater lakes and brackish waters (Reynolds, 1987; Carmichael, 2001; Huisman et al., 2005). Dense cyanobacterial blooms can contain very high toxin concentrations, posing a major threat to birds, mammals and human health (Chorus & Bartram, 1999; Carmichael, 2001; Codd et al., 2005).
Several harmful cyanobacteria produce microcystins, a family of oligopeptides that can cause serious damage to the liver (Sivonen & Jones, 1999; Carmichael, 2001; Codd et al., 2005). Microcystins consist of seven amino acids, of which two amino acid positions are variable, whereas the other five positions are more conserved (Welker & Von Döhren, 2006). Thus far, 89 microcystin variants have been described (Welker & Von Döhren, 2006). These variants may differ in their toxicity (Sivonen & Jones, 1999; Chen et al., 2006; Hoeger et al., 2007). Yet, little is known about the mechanisms that determine the production of the different microcystin variants.
Microcystins are produced stepwise, by nonribosomal peptide synthetases and polyketide synthases, which are large modular constructed enzymes in which each module is responsible for a cycle of polypeptide or polyketide chain elongation (Tillett et al., 2000; Börner & Dittmann, 2005). The unique flexible binding pocket in the first module of the McyB enzyme enables the incorporation of different amino acids at the two variable amino acid positions (Christiansen et al., 2003; Welker & Von Döhren, 2006). For instance, in [Asp3]microcystin-LR, the first and the second variable amino acid positions are occupied by leucine (L) and arginine (R), while in [Asp3]microcystin-RR, both positions are occupied by arginine (Sivonen & Jones, 1999; Hesse & Kohl, 2001). Recently, we showed that the addition of leucine increased the [Asp3]microcystin-LR/RR ratio of the filamentous cyanobacterium Planktothrix agardhii, while the addition of arginine reduced this ratio (Tonk et al., 2008). Hence, the availability of different amino acids plays a role in the production of different microcystin variants.
Arginine contains four bound nitrogen atoms, whereas leucine contains only one nitrogen atom. Hence, [Asp3]microcystin-RR is a relatively nitrogen-rich microcystin variant. It is well known that the microcystin production of harmful cyanobacteria is favored by nitrogen enrichment (Long et al., 2001; Downing et al., 2005; Van de Waal et al., 2009). Interestingly, many cyanobacteria can store excess nitrogen in the polypeptide cyanophycin, consisting of an aspartic acid backbone and arginine side groups (Allen, 1984; Oppermann-Sanio & Steinbüchel, 2002). Cyanophycin synthesis is especially stimulated when nitrogen-deficient cyanobacteria are suddenly exposed to conditions of nitrogen excess (Allen, 1984; Tapia et al., 1996; Oppermann-Sanio & Steinbüchel, 2002; Maheswaran et al., 2006).
Pulsed nitrogen enrichment is a common phenomenon in many aquatic ecosystems. For instance, rain showers may flush nitrogen into lakes through enhanced surface runoff and discharge of upstream waters, storms may mix nitrogen-rich water from the hypolimnion into the surface layers of stratified lakes and fertilization of nearby agricultural fields may spill excess nitrogen into surface waters. It seems likely that such nitrogen pulses will temporarily increase the intracellular availability of arginine to form the nitrogen-storage polymer cyanophycin in harmful cyanobacteria. Will these changes in the amino acid composition be reflected in their cellular microcystin composition?
Earlier, we described changes in the microcystin composition of P. agardhii in response to enhanced light availability (Tonk et al., 2005) and externally added amino acids (Tonk et al., 2008), and that of Microcystis aeruginosa in response to externally added nitrogen (Van de Waal et al., 2009). Our results were consistent with the hypothesis that the production of [Asp3]microcystin-RR is favored by an enhanced availability of nitrogen and the nitrogen-rich amino acid arginine. Contrary to expectation, however, neither the intracellular leucine/arginine ratio nor the [Asp3]microcystin-LR/RR ratio changed when nitrogen availability was gradually reduced in a continuous-culture experiment with P. agardhii (Tonk et al., 2008).
In this study, we use the same P. agardhii strain as in Tonk et al. (2008). However, instead of a gradual reduction in nitrogen availability, we now add a nitrogen pulse. We hypothesize that a nitrogen pulse will lead to a transient increase in the intracellular arginine content, which is built into cyanophycin, and will shift the microcystin variant composition toward [Asp3]microcystin-RR. To test this hypothesis, we exposed nitrogen-starved P. agardhii cells to a nitrogen pulse, and determined how this affected their intracellular composition of amino acids and microcystin variants.
Materials and methods
The filamentous cyanobacterium P. agardhii strain 126/3 was provided by the Division of Microbiology, University of Helsinki. The microcystins produced by this P. agardhii strain are the demethylated variants [Asp3]microcystin-LR and [Asp3]microcystin-RR.
Three batch cultures of 400 mL were grown on a rotatory shaker in 2-L Erlenmeyer flasks at 21±1 °C. The cultures were supplied with O2 medium, originally defined for Oscillatoria species (Van Liere & Mur, 1978). Nitrogen was provided as nitrate at a concentration of 6 mM for nitrogen-rich conditions and at 0.2 mM for nitrogen-deficient conditions, while all other nutrients were added at saturating concentrations. Light was supplied by white fluorescent tubes (Philips TL-M 40W/33RS) at an average incoming irradiance of 26±1 μmol photons m−2 s−1. Biomass was estimated every 2 days by determining the total biovolume of P. agardhii filaments according to Tonk et al. (2008) using a Casy 1 TTC automated cell counter (Schärfe System GmbH) with a 150-μm capillary (Rohrlack & Utkilen, 2007). The specific growth rate, μ, was calculated according to the following equation:
where x1 and x2 represent the total biovolumes of P. agardhii at times t1 and t2, respectively. The shift from the exponential growth phase to the stationary growth phase was determined by a strong decline in the specific growth rate.
When the cultures in nitrogen-replete conditions reached the exponential growth phase, samples were taken during three consecutive days. After the third day of sampling, the cultures were pooled and centrifuged at 3500 g for 20 min. The supernatant was removed and pellets were washed twice with nitrogen-deficient O2 mineral medium containing 0.2 mM of nitrate. Washed cells were redistributed in three 2-L Erlenmeyer flasks to continue growth under nitrogen-deficient conditions until a stationary phase was reached, and subsequently, samples were taken for three consecutive days. Nitrogen deficiency was detected by the decrease of the nitrogen-rich pigment phycocyanin (Allen, 1984). Estimates of phycocyanin and chlorophyll-a were based on A627 nm and A438 nm, respectively, using an Aminco DW-2000 double-beam spectrophotometer (Olis Inc.). After this period of nitrogen-limited growth, a pulse of nitrate was added to the cultures to obtain the initial concentration of 6 mM nitrate at once. Culture growth resumed and samples were taken daily until the end of the exponential growth phase was reached. All samples were analyzed as described below.
For determination of the intracellular microcystin contents, aliquots of the culture suspension were filtrated through a Whatman GF/C filter (pore size ∼1.2 μm) in triplicate. The filters were freeze-dried and intracellular microcystins were extracted in three rounds with 75% MeOH according to Fastner et al. (1998) with an additional grinding step using 0.5 mm beads and a Mini Beadbeater (BioSpec Products Inc.). Dried extracts were dissolved in 50% MeOH before analysis by HPLC with photodiode array detection (Kontron Instruments Ltd). Separation of the different microcystin structural variants was carried out using a LiChrospher 100 ODS 5 μm LiChorCART 250-4 cartridge system (Merck) and using a 30–70% acetonitrile gradient in milli-Q water with 0.05% trifluoroacetic acid at a flow rate of 1 mL min−1. The different microcystin variants were identified by their characteristic UV spectra and quantified using a microcystin-LR and microcystin-RR gravimetrical standard provided by the University of Dundee. Extracellular microcystin concentrations were below the detection limit of the HPLC (2.5 ng of microcystin) and were considered to be negligible, as they typically comprise <3% of the total microcystin concentration (Long et al., 2001; Wiedner et al., 2003; Tonk et al., 2005).
Analysis of carbon and nitrogen content
The cellular carbon and nitrogen contents were estimated in aliquots of the culture suspension, in triplicate. To collapse the gas vesicles, samples were pressurized at 10 bar and centrifuged at 2000 g for 15 min. The supernatant was removed and the pellet was resuspended in milli-Q water, transferred into Eppendorf tubes and centrifuged for 5 min at 15 000 g. Then, the supernatant was removed and pellets were freeze-dried for dry weight determination. The carbon and nitrogen content of homogenized freeze-dried cell powder was analyzed using a Vario EL Elemental Analyzer (Elementar Analysensysteme GmbH).
Amino acid analysis
An aliquot of culture material was hydrolyzed with 30% HCl at 110 °C for 12 h. Subsequently, the extract was vaporized to dryness under vacuum at 40 °C, and a borate buffer was added to maintain a constant pH of 9.8. After derivatization with o-phthaldialdehyde and N-isobutyrylcysteine as in Fitznar et al. (1999), amino acids were analyzed by reversed-phase HPLC. The Waters Alliance 2690 separation module (Waters Corporation) was equipped with a Nova-Pak C18 3.9 × 150 mm column (Waters Corporation) with an Alltech Allsphere ODS-1 guard column (Alltech Associates) and a Waters fluorescence detector 474 (Waters Corporation). Amino acid concentrations in the extract were calculated based on a series of standard amino acid solutions (Sigma-Aldrich).
At the onset of the experiment, the cyanobacterial cells were characterized by a high phycocyanin to chlorophyll-a ratio (PC : Chl-a ratio) and a high cellular nitrogen to carbon ratio (N : C ratio), both indicative of nitrogen-replete conditions (Fig. 1). After cells were transferred to nitrate-depleted conditions on day 2, cultures reached a nitrogen-limited stationary phase on day 12 of the experiment (Fig. 1a). Nitrogen limitation became evident from the gradually decreasing PC : Chl-a ratios (Fig. 1b) and cellular N : C ratios (Fig. 1c). After addition of nitrogen as a nitrate pulse, both the PC : Chl-a ratio and the cellular N : C ratio reverted to values measured at the start of the experiment within 10–15 days.
Likewise, the total amino acid content decreased during the nitrogen-deplete conditions, and then increased gradually to values measured at the initial nitrogen-replete conditions within 13 days after the nitrate pulse (Fig. 2a). N : C ratios of the total amino acid pool increased from 0.26 to 0.29 after the nitrate pulse, and then returned to 0.26 toward the end of the experiment (Table 1). Both l-arginine and l-aspartic acid increased more rapidly than other amino acids and reached high values already within 1 and 2 days after the nitrate pulse (Fig. 2b and c). This resulted in a transient increase in the relative contents of l-arginine and l-aspartic acid (Fig. 3). The relative l-arginine content increased from ∼5% of the total amino acids to >10% (Fig. 3a), while l-aspartic acid increased from ∼10% of the total amino acids to nearly 18% (Fig. 3b). An increase of l-arginine and l-aspartic acid is indicative of cyanophycin production (Allen, 1984; Oppermann-Sanio & Steinbüchel, 2002; Maheswaran et al., 2006). l-Leucine and other amino acids showed a more gradual increase, comparable to the pattern observed in the total amino acid content and consistent with resuming cell growth (Figs 2d and 3c; Table 1).
|Amino acid (AA)||N : C ratio (M)||Average content (μg mm−3)|
|Start (day 0)||Before nitrogen-pulse (day 16)||After nitrogen-pulse (day 17)||End (day 31)|
|d-Alanine||1 : 3||0.07||0.13||0.16||0.29|
|l-Alanine||1 : 3||4.11||1.32||1.54||4.43|
|l-Arginine||4 : 6||3.45||0.97||3.24||3.64|
|d-Aspartic acid||1 : 4||0.32||0.09||0.19||0.26|
|l-Aspartic acid||1 : 4||4.98||1.96||3.96||5.49|
|d-Glutamic acid||1 : 5||0.53||0.25||0.28||0.54|
|l-Glutamic acid||1 : 5||5.96||2.05||2.47||6.34|
|l-Glycine||1 : 2||2.76||0.96||1.12||3.03|
|l-Histidine||3 : 6||0.85||0.16||0.20||0.48|
|l-Isoleucine||1 : 6||2.62||0.91||1.07||2.89|
|l-Leucine||1 : 6||4.68||1.53||1.80||5.07|
|l-Lysine||2 : 6||2.54||0.75||0.89||2.59|
|l-Methionine||1 : 5||0.91||0.10||0.09||0.59|
|l-Phenylalanine||1 : 9||2.28||0.84||0.97||2.54|
|d-Phenylalanine||1 : 9||0.04||0.01||0.01||0.03|
|l-Serine||1 : 3||2.65||0.81||0.94||2.72|
|l-Threonine||1 : 4||2.92||1.04||1.20||3.15|
|l-Tyrosine||1 : 9||2.62||0.76||0.77||2.79|
|l-Valine||1 : 5||3.02||1.02||1.20||3.22|
|Total N : C ratio AA (M)||0.26||0.26||0.29||0.26|
The total cellular microcystin content was strongly correlated with the cellular amino acid content (Fig. 4; Pearson's product-moment correlation: r=0.958, n=9, P<0.0001). However, the amount of amino acids allocated to microcystins was negligible; it remained below 0.5% of the total amino acid pool. During the first 15 days, when cells had been transferred to nitrogen-deplete conditions, both the [Asp3]microcystin-RR and the [Asp3]microcystin-LR content decreased. After the nitrate pulse, [Asp3]microcystin-RR gradually increased to values measured at the initial nitrogen-replete conditions (Fig. 5a). However, the [Asp3]microcystin-LR content increased only slightly and remained far below the concentration measured at the initial nitrogen-replete conditions (Fig. 5b).
Our results show dynamic changes in the cellular composition of amino acids and microcystin variants in response to changing nitrogen availability. Upon addition of a nitrate pulse to nitrogen-deficient P. agardhii cells, we observed a rapid increase in the cellular N : C ratio and in the relative contents of l-arginine and l-aspartic acid. This makes it plausible that nitrogen is stored in the polypeptide cyanophycin (Allen & Hutchison, 1980; Allen, 1984; Mackerras et al., 1990). Cyanophycin then serves as nitrogen storage until cells resume balanced growth. Indeed, after cells resumed growth, the relative contribution of l-arginine and l-aspartic acid to the total amino acid pool declined, which was accompanied by a gradual increase of the other amino acids. This indicates that part of the nitrogen temporarily stored in cyanophycin is invested in protein synthesis for growth, and is reallocated into other nitrogen-rich compounds, such as the accessory pigment phycocyanin that is functional in light harvesting for photosynthesis (Fig. 1b). It is likely that hydrolysis of cyanophycin provides the cell temporarily with a high availability of l-arginine, which can be incorporated into the l-arginine-rich variant [Asp3]microcystin-RR. This explains the increase of the [Asp3]microcystin-RR content to nearly the same values as those at the start of the experiment. But why does the [Asp3]microcystin-LR content remain low and does not recover to its initial value? We suggest that this is due to the strong transient increase in l-arginine, which competes with l-leucine for incorporation into the general microcystin structure. Thus, we conclude that changes in the microcystin composition of P. agardhii in response to nitrogen enrichment are mediated by changes in the intracellular availability of amino acids.
Our results might seem to contrast with those of Tonk et al. (2008), where transition of a continuous culture with P. agardhii from nitrogen-saturated to nitrogen-limited conditions increased neither the intracellular leucine/arginine ratio nor the [Asp3]microcystin-LR/RR ratio. Likewise, the reduction in nitrogen availability during the first 15 days of our current experiment did not yield an increased leucine/arginine ratio or [Asp3]microcystin-LR/RR ratio. However, Tonk et al. (2008) and the initial part of our current experiment concerned a reduction in nitrogen availability. Nitrogen depletion will arrest amino acid synthesis, leading to an overall reduction of all intracellular amino acids, including both leucine and arginine (Fig. 2). In contrast, the addition of a nitrogen pulse will specifically favor the synthesis of arginine and aspartic acid rather than leucine (Fig. 3), as arginine and aspartic acid are important components of the storage molecule cyanophycin.
To clarify the underlying physiological mechanisms, we propose a scheme of nitrogen and carbon assimilation in microcystin-producing cyanobacteria based on known physiological pathways and the results of our experiments (Fig. 6). Inorganic carbon is assimilated via the Calvin cycle to form low-molecular sugars such as glucose, which are subsequently converted to pyruvate during glycolysis. Inorganic nitrogen is reduced to ammonium, which is subsequently incorporated into carbon skeletons through the glutamine synthetase–glutamate synthase cycle. Conversion of glutamine to glutamate makes use of 2-oxoglutarate, which is derived from pyruvate in the citric acid cycle (Vázquez-Bermúdez et al., 2000; Flores & Herrero, 2005).
Accordingly, synthesis of amino acids tightly involves cellular nitrogen and carbon metabolism. Interestingly, glutamate is a direct precursor for arginine synthesis (Fig. 6). A high rate of glutamate formation under nitrogen-pulsed conditions may therefore promote the production of arginine. Moreover, a nitrogen pulse will also generate a high demand for 2-oxoglutarate to serve as carbon skeleton in the glutamine synthetase–glutamate synthase cycle. This drain on 2-oxoglutarate may deplete the pyruvate availability for leucine synthesis (Fig. 6). Hence, nitrogen excess will enhance arginine over leucine synthesis, which would particularly favor the production of [Asp3]microcystin-RR. Conversely, a high availability of light energy and inorganic carbon but low nitrogen availability will enhance the pool size of pyruvate, which is a direct precursor for leucine synthesis (Fig. 6). Higher cellular levels of leucine will promote the production of [Asp3]microcystin-LR. Hence, changes in nitrogen or carbon assimilation will affect the synthesis of amino acids in different ways, with implications for the production of different microcystin variants.
Given these underlying physiological processes, our findings relating the amino acid composition with the microcystin composition of P. agardhii are likely to offer an explanation for the microcystin composition of other harmful cyanobacterial species as well. A recent laboratory study showed that nitrate addition resulted in increased cellular N : C ratios in the harmful cyanobacterium M. aeruginosa (Van de Waal et al., 2009). This increase in the N : C ratio was accompanied by an increased microcystin content, particularly of the nitrogen-rich microcystin-RR variant. Moreover, a survey of several Microcystis-dominated lakes showed that the relative microcystin-RR content increased with the seston N : C ratio in these lakes (Van de Waal et al., 2009). It seems plausible that the enhanced production of microcystin-RR with increasing cellular N : C ratios in Microcystis is mediated by changes in the l-arginine content. Yet, study of a wider range of cyanobacterial species producing a larger variety of microcystin variants is needed to fully corroborate the role of the intracellular amino acid composition in the production of different microcystin variants.
Understanding the production of different microcystin variants is important, because microcystin variants differ in toxicity. Their acute toxicity is estimated by lethal dose 50% (LD50) assays on mice. A lower LD50 (the intraperitoneal dose lethal for 50% of the mouse population) indicates a higher toxicity. Microcystin-LR (LD50=33–73 μg kg−1) is substantially more toxic than microcystin-RR (LD50=310–630 μg kg−1), while the toxicity of [Asp3]microcystin-LR (LD50=160–300 μg kg−1) and [Asp3]microcystin-RR (LD50=250–360 μg kg−1) is more comparable (Sivonen & Jones, 1999; Chen et al., 2006; Hoeger et al., 2007). Typically, the methylated variants microcystin-LR and microcystin-RR are found in Microcystis blooms (Fastner et al., 1999; Kemp & John, 2006; Van de Waal et al., 2009), whereas the demethylated variants [Asp3]microcystin-LR and [Asp3]microcystin-RR occur in Planktothrix blooms (Fastner et al., 1999; Wiedner et al., 2002; Welker et al., 2004). This implies that the toxicity of Planktothrix blooms is relatively independent of the microcystin composition but is mainly determined by the total microcystin content. However, with microcystin-LR being substantially more toxic than microcystin-RR, the situation is different for Microcystis-dominated lakes. The toxicity of Microcystis blooms will be very sensitive to the microcystin composition. For instance, a shift from microcystin-RR toward microcystin-LR would make a Microcystis bloom much more toxic.
This study investigated the impact of a nitrogen pulse on the microcystin variants of P. agardhii. In view of recent findings with Microcystis (Van de Waal et al., 2009), discussed above, we speculate that Microcystis and other microcystin-producing cyanobacteria will respond in a similar way. Our results indicate that dynamic changes in nitrogen availability modify the amino acid composition of harmful cyanobacteria, which will affect the production and composition of their microcystin variants, with potential implications for the toxicity of harmful cyanobacterial blooms.
The research of D.B.V.d.W., L.T., J.H. and P.M.V. was supported by the Earth and Life Sciences Foundation (ALW), subsidized by the Netherlands Organization for Scientific Research (NWO). In addition, L.T. and P.M.V. were supported by a European Union grant within the program PEPCY. We thank Prof. Kaarina Sivonen of the University of Helsinki for providing P. agardhii strain 126/3 and Prof. Geoff Codd of the University of Dundee for providing microcystin gravimetrical standards.
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