Masters Thesis

Investigating symbiotic bacteria as a source of tetrodotoxin in the rough-skinned newt, Taricha granulosa

Tetrodotoxin (TTX) is a highly potent neurotoxin found in diverse group of genetically unrelated organisms. Given that convergent evolution of TTX biosynthesis genes in such a wide range of phylogenetically distinct organisms is unlikely, the occurrence of TTX in most TTX-bearing organisms may result from either dietary uptake of bacterially-produced TTX or symbiosis with TTX-producing bacteria. Although TTX-producing bacteria have been isolated from most TTX-containing organisms, no microbial producer of TTX has previously been identified in the rough-skinned newt, Taricha granulosa. The objective of this study is therefore to determine if different species of symbiotic bacteria that reside on the rough-skinned newt are associated with different levels of tetrodotoxin. To this end, both culture-independent and culture-dependent approaches were utilized to investigate the potential relationship between resident members of the cutaneous flora and TTX levels in T. granulosa. Symbiotic microbes that inhabit the gastrointestinal (GI) tract of T. granulosa were also identified for the first time in this study. Using culture-independent techniques, the dominant microorganism residing in the cutaneous flora of the rough-skinned was identified as Psuedomonas. spp. while the dominant species residing in the gastrointestinal flora was identified as Gemella spp. Furthermore, Staphylococcus spp., Clostridium spp., Bacillus amyloliquefaciens, and Rumnicoccus gauvreauii were also shown to inhabit the intestinal flora of T. granulosa. PCR-based methods were utilized as a culture-dependent approach to screen bacteria collected from the skin of newts for genes that may be involved in the biosynthesis of TTX. Four isolates from high TTX-containing newts were found to posses genes encoding enzymes that could potentially play key roles in the biosynthesis of TTX. In addition to evaluating the bacterial origin hypothesis of TTX in T. granulosa, which has important evolutionary implications, this study may also aid in the identification of the genes involved in the biosynthesis of TTX that may enable large scale production of the toxin for practical purposes such as the treatment of pain and epilepsy.

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