Browsing by Author "Cembella, Allan"

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    Impact of Nitrogen Sources on Gene Expression and Toxin Production in the Diazotroph Cylindrospermopsis raciborskii CS-505 and Non-Diazotroph Raphidiopsis brookii D9
    (2014) Stucken, Karina; John, Uwe; Cembella, Allan; Soto-Liebe, Katia; Vasquez, Monica
    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.
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    PSP toxin release from the cyanobacterium Raphidiopsis brookii D9 (Nostocales) can be induced by sodium and potassium ions
    (2012) Soto-Liebe, Katia; Mendez, Marco A.; Fuenzalida, Loreto; Krock, Bernd; Cembella, Allan; Vasquez, Monica
    Paralytic shellfish poisoning (PSP)toxins are a group of naturally occurring neurotoxic alkaloids produced among several genera of primarily freshwater cyanobacteria and marine dinoflagellates. Although saxitoxin (STX) and analogs are all potent Na+ channel blockers in vertebrate cells, the functional role of these compounds for the toxigenic microorganisms is unknown. Based upon the known importance of monovalent cations (such as sodium) in the maintenance of cellular homeostasis and ion channel function, we examined the effect of high extracellular concentrations of these ions on growth, cellular integrity, toxin production and release to the external medium in the filamentous fresh-water cyanobacterium, Raphidiopsis brookii D9; a gonyautoxins (GTX2/3) and SIX producing toxigenic strain. We observed a toxin export in response to high (17 mM) NaCl and KCl concentrations in the growth medium that was not primarily related to osmotic stress effects, compared to the osmolyte mannitol. Addition of exogenous PSP toxins with the same compositional profile as the one produced by R. brookii D9 was able to partially mitigate this effect of high Na+ (17 mM). The PSP toxin biosynthetic gene cluster (sxt) in D9 has two genes (sxtF and sxtM) that encode for a MATE (multidrug and toxic compound extrusion) transporter. This protein family, represented by NorM in the bacterium Vibrio parahaemolyticus, confers resistance to multiple cationic toxic agents through Na+/drug antiporters. Conserved domains for Na+ and drug recognition have been described in NorM. For the D9 sxt cluster, the Na+ recognition domain is conserved in both SxtF and SxtM, but the drug recognition domain differs between them. These results suggest that PSP toxins are exported directly in response to the presence of monovalent cations (Na+, K+) at least at elevated concentrations. Thus, the presence of both genes in the sxt cluster from strain D9 can be explained as a selective recognition mechanism by the SxtF/M transporters for GTX2/3 and SIX. We propose that these toxins in cyanobacteria could act extracellularly as a protective mechanism to ensure homeostasis against extreme salt variation in the environment. (C) 2012 Elsevier Ltd. All rights reserved.
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    Reassessment of the toxin profile of Cylindrospermopsis raciborskii T3 and function of putative sulfotransferases in synthesis of sulfated and sulfonated PSP toxins
    (PERGAMON-ELSEVIER SCIENCE LTD, 2010) Soto Liebe, Katia; Murillo, Alejandro A.; Krock, Bernd; Stucken, Karina; Fuentes Valdes, Juan J.; Trefault, Nicole; Cembella, Allan; Vasquez, Monica
    The 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 (SIX, NEO, GTX2/3), decarbamoyl (dcSTX), and N-sulfocarbamoyl (C1/2, 81) 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 SIX, 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 SIX 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. (C) 2010 Elsevier Ltd. All rights reserved.