Characterization Of Endosymbiont From Deep Sea Hydrothermal Vent Tubeworm Ridgeia Picesae

Ridgeia piscesae is a vestimentiferan tubeworm that inhabits deep sea hydrothermal vents along Explorer, Juan de Fuca and Gorda Ridges in the northeastern Pacific Ocean. The known endosymbionts of R. piscesae at hydrothermal vents were within the subdivisionγ-proteobacteria. However, many studies were conducted against the hypothesis of"single taxon endosymbiont within the trophsome".The primary goal of this study was to phylogenetically characterize the endosymbionts in the trophosome of R. piscesae collected from Endeavor ventfield on Juan de Fuca Ridge. The endosymbionts were isolated from the tubeworm by differential centrifugation and their 16S rRNA genes were amplified with universal bacterial 16S rRNA primers. The genes were subsequently sequenced and the 16S rDNA clone library was screened by restriction fragment length polymorphism (RFLP) analysis to identify distinct clone types. The RFLP analysis identified only one clone family that fell within the subdivision ofε-proteobacteria. To confirm the autotrophy of the endosymbiont, the trophosome section of tubeworm were stained by haematoxylin/eosin dye mixture and only one type of coccoid cells was observed under microscope. Besides, the genes of RuBisCO formⅠ(cbbL) andⅡ(cbbM) were both detected from the endosymbiont DNA, indicating that this endosymbiont utilized a Calvin-Benson Cycle for carbon fixation. Detection of ATP sulfurylase gene and other sulfur metabolism related genes also suggested that this autotrophic symbiosis was mediated by sulfide oxidation process.On the other hand, the cosmid library of genomic DNA extracted from R. piscesae endosymbionts was constructed. Clones resistant to heavy metal of arsenic, lead and copper were screened. The single clone that was resistant to arsenic revealed high enzyme activity and was subjected to cosmid vector sequencing. The gene cluster of arsenic resistance enzyme was then identified.