Reverse transcription PCR amplification of cyanobacterial symbiont 16S rRNA sequences from single non-photosynthetic eukaryotic marine planktonic host cells

A reverse transcription-polymerase chain reaction (RT-PCR) method was developed for amplifying prokaryotic symbiont rRNA sequences from individual marine planktonic eukaryotic host cells belonging to the tintinnid genus Codonella, the spongiose radiolarian genus Dictyocoryne, and several Dinophysoid genera (Amphisolenia, Citharistes, Dinophysis, Histioneis, Ornithocercus). Of the 93 sequences obtained from 67 clone libraries, 26% were most similar to a variety of heterotrophic bacteria and 4% were most similar to plastids. Most (70%) of the sequences had the greatest similarity to cyanobacterial 16S rRNA sequences. Several (10%) of these had low sequence identity (<94%) to cyanobacteria in the database, suggesting that they represent novel cyanobacterial lineages. Fifty-three percent of the cyanobacterial sequences were most closely related (>96% identical) to Synechococcus sp., and three were >99% identical to Prochlorococcus sp. Subgroups of sequences most similar to Synechococcus sp. were derived from multiple host types, indicating that the same symbiont was capable of forming an association with a variety of host organisms. Sixteen libraries yielded two or three divergent (from 2% to 25% different) sequence types, indicating the presence of mixed assemblages in single host cells. This study provides the first insight into the phylogenetic diversity of the symbiotic cyanobacteria of open ocean protists.