RESUMEN
The nitrogen-fixing bacterium, Nostoc, is a commonly occurring cyanobacterium often found in symbiotic associations. We investigated the potential of cycad cyanobacterial endosymbionts to synthesize microcystin/nodularin. Endosymbiont DNA was screened for the aminotransferase domain of the toxin biosynthesis gene clusters. Five endosymbionts carrying the gene were screened for bioactivity. Extracts of two isolates inhibited protein phosphatase 2A and were further analyzed using electrospray ionization mass spectrometry (ESI-MS)/MS. Nostoc sp. 'Macrozamia riedlei 65.1' and Nostoc sp. 'Macrozamia serpentina 73.1' both contained nodularin. High performance liquid chromatography (HPLC) HESI-MS/MS analysis confirmed the presence of nodularin at 9.55±2.4 ng µg-1 chlorophyll a in Nostoc sp. 'Macrozamia riedlei 65.1' and 12.5±8.4 ng µg-1 Chl a in Nostoc sp. 'Macrozamia serpentina 73.1' extracts. Further scans indicated the presence of the rare isoform [L-Har(2)] nodularin, which contains L-homoarginine instead of L-arginine. Nodularin was also present at 1.34±0.74 ng ml(-1) (approximately 3 pmol per g plant ww) in the methanol root extracts of M. riedlei MZ65, while the presence of [L-Har(2)] nodularin in the roots of M. serpentina MZ73 was suggested by HPLC HESI-MS/MS analysis. The ndaA-B and ndaF genomic regions were sequenced to confirm the presence of the hybrid polyketide/non-ribosomal gene cluster. A seven amino-acid insertion into the NdaA-C1 domain of N. spumigena NSOR10 protein was observed in all endosymbiont-derived sequences, suggesting the transfer of the nda cluster from N. spumigena to terrestrial Nostoc species. This study demonstrates the synthesis of nodularin and [L-Har(2)] nodularin in a non-Nodularia species and the production of cyanobacterial hepatotoxin by a symbiont in planta.
Asunto(s)
Toxinas Bacterianas/biosíntesis , Nostoc/metabolismo , Péptidos Cíclicos/biosíntesis , Simbiosis , Zamiaceae/microbiología , Secuencia de Aminoácidos , Arginina/genética , Arginina/metabolismo , Clorofila/análisis , Clorofila A , Cromatografía Líquida de Alta Presión , ADN Bacteriano/genética , Datos de Secuencia Molecular , Familia de Multigenes , Fijación del Nitrógeno/genética , Nostoc/genética , Filogenia , Raíces de Plantas/microbiología , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Saxitoxin and its analogs cause the paralytic shellfish-poisoning syndrome, adversely affecting human health and coastal shellfish industries worldwide. Here we report the isolation, sequencing, annotation, and predicted pathway of the saxitoxin biosynthetic gene cluster in the cyanobacterium Lyngbya wollei. The gene cluster spans 36 kb and encodes enzymes for the biosynthesis and export of the toxins. The Lyngbya wollei saxitoxin gene cluster differs from previously identified saxitoxin clusters as it contains genes that are unique to this cluster, whereby the carbamoyltransferase is truncated and replaced by an acyltransferase, explaining the unique toxin profile presented by Lyngbya wollei. These findings will enable the creation of toxin probes, for water monitoring purposes, as well as proof-of-concept for the combinatorial biosynthesis of these natural occurring alkaloids for the production of novel, biologically active compounds.
Asunto(s)
Genes Bacterianos/genética , Floraciones de Algas Nocivas , Toxinas de Lyngbya/biosíntesis , Familia de Multigenes/genética , Oscillatoria/genética , Oscillatoria/metabolismo , Saxitoxina/biosíntesis , Secuencia de Bases , Biología Computacional , Evolución Molecular , Toxinas de Lyngbya/química , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Saxitoxina/química , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
The recent determination of the genetic basis for the biosynthesis of the neurotoxin, saxitoxin, produced by cyanobacteria, has revealed a highly complex sequence of reactions, involving over 30 biosynthetic steps encoded by up to 26 genes clustered at one genomic locus, sxt. Insights into evolutionary-ecological processes have been found through the study of such secondary metabolites because they consist of a measurable phenotype with clear ecological consequences, synthesized by known genes in a small number of species. However, the processes involved in and timing of the divergence of prokaryotic secondary metabolites have been difficult to determine due to their antiquity and the possible frequency of horizontal gene transfer and homologous recombination. Through analyses of gene synteny, phylogenies of individual genes, and analyses of recombination and selection, we identified the evolutionary processes of this cluster in five species of cyanobacteria. Here, we provide evidence that the sxt cluster appears to have been largely vertically inherited and was therefore likely present early in the divergence of the Nostocales, at least 2,100 Ma, the earliest reliably dated appearance of a secondary metabolite. The sxt cluster has been extraordinarily conserved through stabilizing selection. Genes have been lost and rearranged, have undergone intra- and interspecific recombination, and have been subject to duplication followed by positive selection along the duplicated lineage, with likely consequences for the toxin analogues produced. Several hypotheses exist as to the ecophysiological role of saxitoxin: as a method of chemical defense, cellular nitrogen storage, DNA metabolism, or chemical signaling. The antiquity of this gene cluster indicates that potassium channels, not sodium channels, may have been the original targets of this compound. The extraordinary conservation of the machinery for saxitoxin synthesis, under radically changing environmental conditions, shows that it has continued to play an important adaptive role in some cyanobacteria.
Asunto(s)
Secuencia Conservada/genética , Neurotoxinas/genética , Saxitoxina/genética , Animales , Cianobacterias/clasificación , Cianobacterias/genética , Evolución Molecular , Eliminación de Gen , Duplicación de Gen , Genes Bacterianos/fisiología , Humanos , Familia de Multigenes , Neurotoxinas/biosíntesis , Neurotoxinas/clasificación , Neurotoxinas/envenenamiento , Filogenia , Bloqueadores de los Canales de Potasio/metabolismo , Bloqueadores de los Canales de Potasio/envenenamiento , Canales de Potasio/metabolismo , Recombinación Genética , Saxitoxina/biosíntesis , Saxitoxina/clasificación , Saxitoxina/envenenamiento , Selección Genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Sintenía/genéticaRESUMEN
Saxitoxin (STX) and its 57 analogs are a broad group of natural neurotoxic alkaloids, commonly known as the paralytic shellfish toxins (PSTs). PSTs are the causative agents of paralytic shellfish poisoning (PSP) and are mostly associated with marine dinoflagellates (eukaryotes) and freshwater cyanobacteria (prokaryotes), which form extensive blooms around the world. PST producing dinoflagellates belong to the genera Alexandrium, Gymnodinium and Pyrodinium whilst production has been identified in several cyanobacterial genera including Anabaena, Cylindrospermopsis, Aphanizomenon Planktothrix and Lyngbya. STX and its analogs can be structurally classified into several classes such as non-sulfated, mono-sulfated, di-sulfated, decarbamoylated and the recently discovered hydrophobic analogs--each with varying levels of toxicity. Biotransformation of the PSTs into other PST analogs has been identified within marine invertebrates, humans and bacteria. An improved understanding of PST transformation into less toxic analogs and degradation, both chemically or enzymatically, will be important for the development of methods for the detoxification of contaminated water supplies and of shellfish destined for consumption. Some PSTs also have demonstrated pharmaceutical potential as a long-term anesthetic in the treatment of anal fissures and for chronic tension-type headache. The recent elucidation of the saxitoxin biosynthetic gene cluster in cyanobacteria and the identification of new PST analogs will present opportunities to further explore the pharmaceutical potential of these intriguing alkaloids.
Asunto(s)
Toxinas Marinas/farmacología , Neurotoxinas/toxicidad , Saxitoxina/toxicidad , Alcaloides/metabolismo , Alcaloides/farmacología , Alcaloides/toxicidad , Animales , Humanos , Toxinas Marinas/metabolismo , Toxinas Marinas/toxicidad , Neurotoxinas/metabolismo , Neurotoxinas/farmacología , Saxitoxina/análogos & derivados , Saxitoxina/farmacología , Intoxicación por Mariscos/etiologíaRESUMEN
BACKGROUND: Saxitoxin and its analogues collectively known as the paralytic shellfish toxins (PSTs) are neurotoxic alkaloids and are the cause of the syndrome named paralytic shellfish poisoning. PSTs are produced by a unique biosynthetic pathway, which involves reactions that are rare in microbial metabolic pathways. Nevertheless, distantly related organisms such as dinoflagellates and cyanobacteria appear to produce these toxins using the same pathway. Hypothesised explanations for such an unusual phylogenetic distribution of this shared uncommon metabolic pathway, include a polyphyletic origin, an involvement of symbiotic bacteria, and horizontal gene transfer. RESULTS: We describe the identification, annotation and bioinformatic characterisation of the putative paralytic shellfish toxin biosynthesis clusters in an Australian isolate of Anabaena circinalis and an American isolate of Aphanizomenon sp., both members of the Nostocales. These putative PST gene clusters span approximately 28 kb and contain genes coding for the biosynthesis and export of the toxin. A putative insertion/excision site in the Australian Anabaena circinalis AWQC131C was identified, and the organization and evolution of the gene clusters are discussed. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed. CONCLUSION: The PST biosynthesis gene cluster presents a mosaic structure, whereby genes have apparently transposed in segments of varying size, resulting in different gene arrangements in all three sxt clusters sequenced so far. The gene cluster organizational structure and sequence similarity seems to reflect the phylogeny of the producer organisms, indicating that the gene clusters have an ancient origin, or that their lateral transfer was also an ancient event. The knowledge we gain from the characterisation of the PST biosynthesis gene clusters, including the identity and sequence of the genes involved in the biosynthesis, may also afford the identification of these gene clusters in dinoflagellates, the cause of human mortalities and significant financial loss to the tourism and shellfish industries.
Asunto(s)
Anabaena/genética , Aphanizomenon/genética , Familia de Multigenes , Neurotoxinas/biosíntesis , Saxitoxina/análogos & derivados , Saxitoxina/biosíntesis , Anabaena/clasificación , Anabaena/metabolismo , Aphanizomenon/clasificación , Aphanizomenon/metabolismo , Australia , Secuencia de Bases , Vías Biosintéticas , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Genes Bacterianos/genética , Secuencias Invertidas Repetidas , Datos de Secuencia Molecular , Estructura Molecular , Neurotoxinas/química , Filogenia , ARN Ribosómico 16S/genética , Saxitoxina/química , Análisis de Secuencia de ADNRESUMEN
To investigate the potential for differential accumulation of paralytic shellfish toxins (PSTs) in various tissues of the akoya pearl oyster, Pinctada imbricata, two feeding trials were carried out using the PST-producing dinoflagellate, Alexandrium minutum. When fed with A. minutum at concentrations between 100 and 1300 cells ml(-1), the maximum clearance by P. imbricata was shown to occur at a density of 300 cells ml(-1). When fed twice daily at this rate for up 12 days, P. imbricata accumulated analogues of gonyautoxins (GTXs): GTXs 1,4 and 2,3. The levels of GTXs in the viscera increased progressively on days 4, 8 and 12 to peak at 17.9+/-4.47 microg STX-equivalent 100 g(-1) biomass. Following 12 days of depuration, in the absence of A. minutum, GTX levels fell by approximately 65% to 6.0+/-2.20 microg STX-equivalent 100 g(-1) biomass. No GTX was found in the oysters at the start of the trial or in untreated controls. The accumulation of GTX was found to be tissue specific. No GTX was detected in the muscle tissue of P. imbricata during the feeding trial.