Phylogenetic analysis of intracellular bacteria of a harmful marine microalga, Heterocapsa circularisquama (Dinophyceae)

Heterocapsa circularisquama, a noxious marine dinoflagellate, has frequently caused red tides and killed cultured bivalves in western Japanese embayments. Observations by electron and epifluorescence microscopy revealed that many bacterial particles were detected inside the H. circularisquama cells. To elucidate the identity and origin of the intracellular bacteria associated with H, circularisquama, bacterial 16S ribosomal RNA genes (16S rDNA) were directly amplified by polymerase chain reaction from 5 clonal cultures of the algal strains that had been isolated from different localities. After cloning, randomly selected clones including the bacterial 16S rDNA fragments were sequenced. The results showed that intracellular bacterial populations consisted of only 2 ribotypes of bacteria, even though the algal strains were established from different localities. One ribotype (bac-G), which was dominant in the intracellular bacterial population, belonged to the gamma-proteobacteria group, and the other (bac-F) clustered with the Flexibacter-Cytophaga-Bacteroides group, Both of these are novel species of endosymbiotic bacteria because of their unique 16S rDNA sequences. Furthermore, the populations of extracellularly associated bacteria were also composed of bac-G and bac-F, indicating that they originated from the intracellular bacteria. Fluorescence in situ hybridization targeting 16S rRNA indicated that bac-G appeared to localize on the algal nuclear surface, while bac-F was distributed in the cytoplasmic space of algal cells. These results strongly suggest that only a few species of specific bacteria reside and share their habitat in the H. circularisquama cells as endosymbionts.