Reassessment of the toxin profile of Cylindrospermopsis raciborskii T3 and function of putative sulfotransferases in synthesis of sulfated and sulfonated PSP toxins

Abstract

a b s t r a c tThe toxigenic freshwater cyanobacterium Cylindrospermopsis raciborskii T3 has been used as a model to study and elucidate the biosynthetic pathway of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP). There are nevertheless several inconsistencies and contradictions in the toxin profile of this strain as published by different research groups, and claimed to include carbamoyl (STX, NEO, GTX2/3), decarbamoyl (dcSTX), and N-sulfocarbamoyl (C1/2, B1) derivatives. Our analysis of the complete genome of another PSP toxin-producing cyanobacterium, Raphidiopsis brookii D9, which is closely related to C. raciborskii T3, resolved many issues regarding the correlation between biosynthetic pathways, corresponding genes and the T3 toxin profile. The putative sxt gene cluster in R. brookii D9 has a high synteny with the T3 sxt cluster, with 100% nucleotide identity among the shared genes. We also compared the PSP toxin profile of the strains by liquid chromatography coupled to mass spectrometry (LC-MS/MS). In contrast to published reports, our reassessment of the PSP toxin profile of T3 confirmed production of only STX, NEO and dcNEO. We gained significant insights via correlation between specific sxt genes and their role in PSP toxin synthesis in both D9 and T3 strains. In particular, analysis of sulfotransferase functions for SxtN (N-sulfotransferase) and SxtSUL (O-sulfotransferase) enzymes allowed us to propose an extension of the PSP toxin biosynthetic pathway from STX to the production of the derivatives GTX2/3, C1/2 and B1. This is a significantly revised view of the genetic mechanisms underlying synthesis of sulfated and sulfonated STX analogues in toxigenic cyanobacteria.

The inhibitory effect of calcium on Cylindrospermopsis raciborskii (cyanobacteria) metabolism

“…This strain was isolated from a toxic cyanobacterial bloom in Brazil (19) and has been maintained in the culture collection of the Laboratory of Cyanobacterial Ecophysiology and Toxicology (UFRJ, Brazil). The T3 strain produces STX, neosaxitoxin (NSTX) and decarbamoyl-NSTX and has been used as a type strain to elucidate the biosynthetic pathway of paralytic shellfish toxins (PSTs) (18,36). Non-axenic batch cultures of this strain were maintained in ASM-1 medium (15) at 24 ± 2 ºC, with a 12 hour L:D cycle and photon flux of 50 µmol photons.m -2 .s -1 (provided by common day light fluorescent lamps).…”

“…The PST profile of C. raciborskii T3 is STX, NSTX and dcNSTX (36). Here, we measured PST production as the sum of STX and NSTX only, since we had no dcNSTX standard available.…”

“…The presence of C. raciborskii in water supplies used by humans or livestock is potentially harmful since several strains can produce the hepatotoxic alkaloid cylindrospermopsin and other yet unidentified toxins (6,25). Differently from other geographic regions, several toxic strains isolated from Brazilian water supplies produce paralitic shellfish toxins (PSTs) (19,25,26,36).…”

Impact of Environmental Factors on the Regulation of Cyanotoxin Production

“…Bioinformatic analyses of Kellmann et al [149] indicated that the synthesis of STX begun with the multifunctional polyketide biosynthesis enzyme ( SxtA ). Only a few studies are evident about STX genes; to date STX biosynthetic gene cluster has been reported from five genera of cyanobacteria [107,108,149,150,151] and 33 genes involved in STX biosynthesis, transport and regulation were also reported in their study. The size of these gene clusters was 25.7 kb ( Raphidiopsis brookii D9) to 36 kb ( Lyngbya wollei ).…”

Diversity of cyanobacteria and cyanotoxins in Hartbeespoort Dam, South Africa

“…These findings raise the question of whether C. curvispora, R. curvata and R. mediterranea can be regarded as separate species or are most likely just rare morphotypes of C. raciborskii. Strains of Cylindrospermopsis and Raphidiopsis from Australia, Brazil, China, Japan and Thailand produce CYN, STXs or ATX (Hawkins et al 1997;Saker and Neilan 2001;Li et al 2001;Namikoshi et al 2003;Soto-Liebe et al 2010). However, all the Cylindrospermopsis and Raphidiopsis strains isolated from Hartbeespoort Dam tested negative for production of cyanotoxins and their encoding genes.…”