Impact of nitrogen sources on gene expression and toxin production in the diazotroph Cylindrospermopsis raciborskii CS-505 and non-diazotroph Raphidiopsis brookii D9

Abstract

Different environmental nitrogen sources play selective roles in the development of cyanobacterial blooms and noxious effects are often exacerbated when toxic cyanobacteria are dominant. Cylindrospermopsis raciborskii CS-505 (heterocystous, nitrogen fixing) and Raphidiopsis brookii D9 (non-N₂ fixing) produce the nitrogenous toxins cylindrospermopsin (CYN) and paralytic shellfish toxins (PSTs), respectively. These toxin groups are biosynthesized constitutively by two independent putative gene clusters, whose flanking genes are target for nitrogen (N) regulation. It is not yet known how or if toxin biosynthetic genes are regulated, particularly by N-source dependency. Here we show that binding boxes for NtcA, the master regulator of N metabolism, are located within both gene clusters as potential regulators of toxin biosynthesis. Quantification of intra- and extracellular toxin content in cultures at early stages of growth under nitrate, ammonium, urea and N-free media showed that N-sources influence neither CYN nor PST production. However, CYN and PST profiles were altered under N-free medium resulting in a decrease in the predicted precursor toxins (doCYN and STX, respectively). Reduced STX amounts were also observed under growth in ammonium. Quantification of toxin biosynthesis and transport gene transcripts revealed a constitutive transcription under all tested N-sources. Our data support the hypothesis that PSTs and CYN are constitutive metabolites whose biosynthesis is correlated to cyanobacterial growth rather than directly to specific environmental conditions. Overall, the constant biosynthesis of toxins and expression of the putative toxin-biosynthesis genes supports the usage of qPCR probes in water quality monitoring of toxic cyanobacteria.

Figure 1

Growth curves of CS-505 (A,B) and D9 (C,D). Curves were plotted by chlorophyll a and dry weight per unit culture volume. Values are shown as the average of three biological replicates; error bars indicate ±SD from the mean (n = 3).

Figure 2

C:N ratios of CS-505 (A) and D9 (B) grown under alternative N-regimes. Values are shown as a percentage of variation with respect to t = 0; error bars indicate ±SD from the mean (n = 3). Values significantly different from t = 0 (one way ANOVA, Tukey’s HSD post hoc test p < 0.01) are marked with a * symbol.

Figure 3

Toxin production by CS-505 grown under four alternative N-regimes. (A) Total CYN + doCYN content over the time in the intra- and extracellular component per unit culture volume; (B) total toxin content normalized to biomass (dry weight); (C) and (D) intra- and extracellular ratios of CYN:doCYN, respectively.

Figure 4

Specific toxin production rate as function of specific growth rate in the four N regimes for CS-505 (A) and D9 (B).

Figure 5

Toxin production by D9 grown under four alternative N-regimes. (A) Total PST content over the time in the intra- and extracellular component per unit culture volume; (B) total toxin content normalized to biomass (dry weight); (C) and (D) intra- and extracellular STX content; (E) and (F) intra- and extracellular STX:GTX2/3 ratios.