Specimens of the toxic ribbon worm Cephalothrix simula from the Sea of Japan were screened for tetrodotoxin-producing bacteria. A single TTX-producing bacterial strain (No 1839) was isolated from tissues of C. simula and studied by immunohistochemical methods (including immunoelectron and immunofluorescent microscopies) with anti-TTX antibodies. Sequencing of 16S rRNA gene of the strain 1839 showed that it is most likely Bacillus sp. CU040510-015 and Bacillus asahii. Based on its morphological and biochemical properties, however we suppose that the isolated Bacillus sp. 1839 should be classified as representing a new species. Microdistribution of TTX in bacterial cell was investigated under electron microscope by immunoenzymatic methods. TTX was concentrated in the forespore and free spores, but it was not detected in the vegetative cells of Bacillus sp. 1839. We suggest that release of free mature spores from sporangium of Bacillus sp. 1839 leads to appearance of toxin in tissues of C. simula. Confocal laser-scanning microscopy (CLSM) method with anti-TTX antibodies can be recommended for preliminary detection of apparent TTX accumulation.Graphical abstract
Microdistribution of tetrodotoxin in Bacillus sp 1839. Transmission electron micrographs showed that some bacteria with mature forespore were TTX-gold labeled. Cytoplasm of mother cell was slightly labeled. Coat and cortex of the forespore were heavy labeled, whereas forespore core was moderately labeled.