The application of molecular techniques to investigate the production of sodium channel blocking toxins by heterotrophic marine bacteria

Historically the production of tetrodotoxin has been attributed to Puffer fish and the production of paralytic shellfish poisons to dinoflagellates and fresh water cyanobacterial species, especially at times of monospecific blooms. In the last decade, however, a broad spectrum of bacterial species have been reported to produce tetrodotoxin. Similarly, heterotrophic bacteria have been reported to produce paralytic shellfish poisons in culture, although little is known about the biosynthetic pathways of these toxins. In this study the production of sodium channel blocking toxins by heterotrophic marine bacteria was investigated by transposon mutagenesis and other molecular techniques. The suitability of several bacterial isolates for mutagenesis with a variety of transposons was evaluated by phenotype characterisation with regard to galactosidase activity and spontaneous antibiotic resistance, resulting in the choice of three strains (Alteromonas tetraodonis GFC, and sea water isolates A912 and A862, the latter being a putative Shewanella sp.) for mutagenesis with three different transposon constructs. Mutagenesis of A. tetraodonis GFC with miniMu(tetr) was hindered by a high frequency of spontaneous resistance of the Escherichia coli donor strain to particular antibiotics; the use of this transposon construct was not pursued. The present methods employed for the detection of sodium channel blocking toxins were not suitable for processing the large number of bacterial samples to be screened for loss of toxin production. A chemical fluorescence assay (Bates and Rapoport, 1975) was adapted for assaying bacterial cultures for paralytic shellfish poisons. However, only strain A862 contained a compound with fluorescent properties indicative of PSP toxins. Although post-column oxidation HPLC analysis indicated that strain A862 may produce neoSTX, GTX1/4, B2, C2, C3 and C4 toxins, pre-column oxidation high performance liquid chromatography analysis of this strain showed that