title: The application of molecular techniques to investigate the production of sodium channel blocking toxins by heterotrophic marine bacteria
Creators: name: Hosie, Arthur Hugh Frank
Publisher: name: University of Glasgow
Year: 1998

description: 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 the fluorescence detected by the chemical fluorescence assay was not caused by any of these toxins. Although the compound detected by the chemical fluorescence assay has not been fully characterised, this assay was used in conjunction with a mouse neuroblastoma cell sodium channel blocking assay to screen almost 1,000 A862::miniTn5CmlacZ exconjugants for decreased SCB toxin production. Seven exconjugants were identified which had a significantly lower fluorescence than A862 wt. Inverse polymerase chain reaction was used successfully to amplify the DNA sequence adjacent to the transposon insertion in five of these exconjugants. Subsequent sequence analysis assigned putative functions to two genes located near the insertion site of different exconjugants. The significance of these two genes and their products to SCB toxin production is unclear. It is possible that the serine protease (Ssp), encoded by one of the genes, could be involved in the post-translational modification of another protein involved in either the post-translational modification of a further protein required for toxin biosynthesis, or alternatively, it could increase the pool of biosynthetic precursors by degrading peptides which are precursors of toxin biosynthesis. The product of the second gene, a H-NS homologue, is possibly involved in the regulation of expression of other genes involved in SCB toxin biosynthesis as H-NS is known to affect transcription of a number of genes. This is the first description of a H-NS in marine bacteria of the family Vibrionaceae, and therefore further extends the range of organisms known to produce H-NS homologues, which until recently were only described in enterobacteria. Single specific primer PCR using degenerate primers designed to consensus sequences of known sulfotransferase enzymes, was used in an attempt to amplify from strain A862, genes encoding potential sulfotransferase enzymes involved in PSP biosynthesis. Although SSP-PCR products were obtained, none contained significant homology to known sulfotransferase genes. However, this negative result does not indicate that these enzymes are not present in this organism. If partial amino acid sequences become available for sulfotransferases or other enzymes involved in SCB biosynthesis, the SSP-PCR described here may be used to identify the genes which encode these enzymes.