Project description:BACKGROUND:Cylindrospermopsis raciborskii is an invasive filamentous freshwater cyanobacterium, some strains of which produce toxins. Sporadic toxicity may be the result of gene deletion events, the horizontal transfer of toxin biosynthesis gene clusters, or other genomic variables, yet the evolutionary drivers for cyanotoxin production remain a mystery. Through examining the genomes of toxic and non-toxic strains of C. raciborskii, we hoped to gain a better understanding of the degree of similarity between these strains of common geographical origin, and what the primary differences between these strains might be. Additionally, we hoped to ascertain why some cyanobacteria possess the cylindrospermopsin biosynthesis (cyr) gene cluster and produce toxin, while others do not. It has been hypothesised that toxicity or lack thereof might confer a selective advantage to cyanobacteria under certain environmental conditions. RESULTS:In order to examine the fundamental differences between toxic and non-toxic C. raciborskii strains, we sequenced the genomes of two closely related isolates, CS-506 (CYN+) and CS-509 (CYN-) sourced from different lakes in tropical Queensland, Australia. These genomes were then compared to a third (reference) genome from C. raciborskii CS-505 (CYN+). Genome sizes were similar across all three strains and their G + C contents were almost identical. At least 2,767 genes were shared among all three strains, including the taxonomically important rpoc1, ssuRNA, lsuRNA, cpcA, cpcB, nifB and nifH, which exhibited 99.8-100% nucleotide identity. Strains CS-506 and CS-509 contained at least 176 and 101 strain-specific (or non-homologous) genes, respectively, most of which were associated with DNA repair and modification, nutrient uptake and transport, or adaptive measures such as osmoregulation. However, the only significant genetic difference observed between the two strains was the presence or absence of the cylindrospermopsin biosynthesis gene cluster. Interestingly, we also identified a cryptic secondary metabolite gene cluster in strain CS-509 (CYN-) and a second cryptic cluster common to CS-509 and the reference strain, CS-505 (CYN+). CONCLUSIONS:Our results confirm that the most important factor contributing to toxicity in C. raciborskii is the presence or absence of the cyr gene cluster. We did not identify any other distally encoded genes or gene clusters that correlate with CYN production. The fact that the additional genomic differences between toxic and non-toxic strains were primarily associated with stress and adaptation genes suggests that CYN production may be linked to these physiological processes.
Project description:The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX), while cylindrospermopsin (CYN), which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins.
Project description:The bloom-forming cyanobacteria, Cylindrospermopsis raciborskii, is a producer of the cytotoxic cylindrospermopsin (CYN). In this study, the growth, toxin yield, and expression of CYN biosynthesis genes of C. raciborskii were examined under varying phosphorus (P) concentrations. The results show the cell number at 0.00 and 0.01 mg·L-1 P was significantly lower than that at higher P concentrations (?0.5 mg·L-1). The chlorophyll a content, filament length, heterocyst, and akinete numbers at P ? 0.05 mg·L-1 were also significantly reduced. The intracellular and extracellular CYN concentrations and the extracellular proportions increased during the culture period, and larger values were observed at higher P concentrations. Total CYN content reached 45.34-63.83 fg·cell-1 and extracellular CYN proportion reached 11.49%-20.44% at the stationary growth phase. A significantly positive correlation was observed between CYN production and cell growth rate. Three cyr genes were expressed constantly even at P-deficient conditions. The transcription of cyr genes at P-replete conditions or after P supplementation increased from 1.18-fold to 8.33-fold. In conclusion, C. raciborskii may rapidly reorganize metabolic processes as an adaptive response to environmental P fluctuations. CYN production and cyr gene expression were constitutive metabolic processes in toxic C. raciborskii.