Description: 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.

Description: Cylindrospermopsis raciborskii is a filamentous, potentially toxic bloom-forming cyanobacterium in tropical and temperate freshwaters. We compared the cylindrospermopsin (CYN) producing C. raciborskii strain CS-505 (Australia) against a PSP toxin producing strain D9 (Brazil), originally identified as conspecific. Cylindrospermopsis raciborskii forms a monophyletic group comprising identities 〉99% for the 16S rRNA gene, whereas strains D9 and CS-505 are 99.7% similar. We sequenced the complete genome of these strains, the first for any PSP- or CYN-toxin producing cyanobacterium. The genome sizes are the smallest known for filamentous cyanobacteria (3.3 and 3.9 Mbp for D9 and CS-505, respectively), but exhibit an apparently large size difference. Genome comparison yielded 471 and 801 strain-specific genes for D9 and CS-505, respectively, and revealed remarkable differences between strains. These differences are mainly related to signatures for high genomic plasticity in CS-505, gene loss in D9 (such as the lack of nitrogen fixation genes), and genes for secondary metabolite production, specifically in the STX and CYN clusters. Functional analysis via gene expression studies using microarrays and qPCR have supported the function of the identified toxin-related gene clusters as well as indicating fundamental differences in phenotypic expression between the strains.

Description: Paralytic shellfish poisoning (PSP) is a syndrome related to consumption of shellfish contaminated by toxigenic microalgae that biosynthesize the tetrahydropurine neurotoxin saxitoxin (STX) and/or various derivatives. Dinoflagellates are the main producers of PSP toxins in marine environments but PSP toxin production is also well described from freshwater cyanobacteria. Although the pathways involved in STX biosynthesis remain poorly known, studies with labeled precursors have suggested that dinoflagellates and cyanobacteria share this biosynthetic pathway. In contrast to the complexity of the dinoflagellate genome (3000215000 Mbp), the simplicity of cyanobacterial genomes (from 1.8 to 13.6 Mb) offers an alternative model for understanding the pathways involved in toxin production and gene regulation. The cyanobacterium Cylindrospermopsis raciborskii (genome size ca. 2.2 Mb) comprises strains able to produce either PSP toxins or the hepatotoxin cylindrospermopsin (CYN), as well as non-toxigenic strains. We have fully sequenced the complete genome of two strains of C. raciborskii that share 99.7% of their 16S rRNA sequences but differ in toxin profile (a CYN- versus a PSP toxin-producer). This project includes a study of: i) comparative genomics to identify the main differences between these strains that could be related to toxin production or regulation; and ii) physiological conditions that modify the toxin profile and its genetic regulation in the CYN- versus PSP toxin-producing strains using whole genome microarrays. The data for the cyanobacterium should be useful to evaluate homology of PSP toxin production among Cyanobacteria and Dinoflagellates and may assist in identifying the key genes responsible for PSP toxin synthesis in Dinoflagellates.

Description: Cylindrospermopsis raciborskii and Raphidiopsis sp. are filamentous freshwater bloom forming cyanobacteria, which can co-exist in the same bloom, morphologically distinguishable because of the terminal heterocyst (N fixation cells) in C. raciborskii. C. raciborskii comprise strains which produce either Cylindrospermopsin (CYN) an hepatotoxin, Paralytic Shellfish Poisoning toxins (PSP toxins) or do not produce toxins. On the other hand, the few strains described from Raphidiopsis genus produce anatoxin-a (neurotoxin), CYN, or do not produce toxins. Regardless of these phenotypic differences, C. raciborskii and Raphidiopsis sp. are not differentiable at 16S rRNA sequence level (similarity higher than 99%). However, analysis of the 16S23S rRNA Internal Transcribed Spacer (ITS), from C. raciborskii strains (CYN-producing or non toxic) and one PSP toxin-producing strain morphologically classified as C. raciborskii strain D9 showed a 28bp deletion signature of Raphidiopsis strains in D9. Further analysis demonstrated that D9 was not able to fix nitrogen; supporting D9 classification within the Raphidiopsis genus. We sequenced the complete genome from C. raciborskii CS-505, a CYN-producing, and Raphidiopsis sp. D9, finding high similarity between both genomes. Although the genome size is different (3.9 and 3.3 Mbp), these strains share around 2500 genes with a percentage of similarity higher than 97% between them. The differences are restricted to mobility elements and genome repair. Is the nitrogen fixation a phenotypic character strong enough to classify these organisms in two different genuses? We propose that both species must be re-classified within one genus, Cylindrospermopsis.

Description: Cylindrospermopsis raciborskii is a filamentous, potentially toxic, bloom-forming cyanobacterium in tropical and temperate freshwaters. Its high competition and ability of produce toxins have made of this cyanobacterium one of the most studied in the ecological and public health fields. A clonal culture described as C. raciborskii strain D9 (Brazil), produces Paralytic Shellfish Poisoning (PSP) toxins; however, differed from C. raciborskii due to the presence of anomalous heterocysts (N-fixing specialized cells characteristic of the species). C. raciborskii strain CS-505 (Australia) produces cylindrospermopsin (CYN). In spite of these phenotypic differences, strains D9 and CS-505 present 99.7% of identity at the 16S rRNA gene. We have sequenced the complete genome of these toxic strains; the first for cyanobacteria producing either PSP- or CYN-toxins. The genome sizes, the smallest found for filamentous cyanobacteria, are 3.3 and 3.9 Mbp for D9 and CS-505, respectively, fairly different for so closely related organisms. The genome comparison revealed a large amount of transposition elements and repeated sequences, as well as secondary metabolite pathways in CS-505 genome, demonstrating a high plasticity and signatures of large genome species. In contrast, strain D9 presents a high number of genes involved in DNA modification (protection) and repair and lacks all the nitrogen fixation genes. In summary, it seems the strain D9 have undergone genome reduction and implies a resistance of strain D9 to the acquisition of new functions and also raise the possibility of a misclassification of this strain.