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

Karina Stucken,Uwe John,Allan Cembella,Katia Soto-Liebe,Mónica Vásquez

doi:10.3390/toxins6061896

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.

Highlights

Cyanobacterial blooms often dominate the photosynthetic plankton in eutrophic freshwater and coastal brackish waters
We explored the relationship between toxin production and transcriptional regulation of ntcA and of the main putative genes for toxin biosynthesis and transport in C. raciborskii CS-505 and R. brookii D9
Experiments were performed from cultures adapted to grow on nitrate in a five-day period to observe the early response to N-deprivation of CS-505 and D9

Summary

Introduction

Cyanobacterial blooms often dominate the photosynthetic plankton in eutrophic freshwater and coastal brackish waters. Many cyanobacteria fix dinitrogen (N2), and can survive and even thrive in N-depleted environments. Cyanobacteria produce a wide array of toxic secondary metabolites (cyanotoxins) [2,3], among them, the hepatotoxic microcystins and nodularins, the cytotoxic cylindrospermopsins and apratoxins, the dermatotoxins lyngbyatoxins, and the neurotoxic anatoxins. Putative gene clusters have been identified for the biosynthesis of these toxins; all encode for large multienzymatic complexes of non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS) or by hybrid NRPS/PKS complexes [4,5,6,7,8,9]. The paralytic shellfish poisoning toxins (PSTs) are neurotoxins produced in freshwater by cyanobacteria and in marine environments by dinoflagellates [10]. The putative biosynthesis pathway of PSTs is directed by a novel PKS and additional non-NRPS/PKS enzymes [11]

Results

Discussion

Conclusion