Features of the extracellular vesicles (EVs) produced from a hyperthermophilic archaeon, Thermococcusonnurineus NA1

Abstract

A sulfur-reducing hyperthermophilic archaeon, Thermococcus onnurineus NA1, is able to produce extracellular vesicles (EV) under anaerobic cultivation at 80 oC. The EVs were concentrated from 15 L of culture broth to ca. 100-fold by the ultrafiltration system and major two bands were purified through the CsCl-density gradient ultracentrifugation. Their buoyant density ranged from 1.350 to 1.354 gcm -3 and 1.354 to 1.358 gcm -3, respectively. After subsequent purification of the two bands, the both bands EVs were directly counted by the Izon qNano system. The final number of the concentrated EVs was 4.0&#9747 1011 particles ml-1 and 5.5&#9747 1011 particles ml-1 , respectively. Hence EVs were produced at the rate of 6.4&#9747 107 particles ml-1 of culture medium. The EVs were spherical in shape and the size varied from 120 nm to 220 nm in diameter, and both vesicles showed identical shape and size. The vesicles between 150 nm and 170 nm in size were major population. DNA contents of EVs were examined by the 1% agarose gel electrophoresis after DNA extraction (QIAGEN). The complete genome sequence of the EVs was performed by Ion Torrent and then assembled. Comparing the sequences between the whole genome of parent archaea and EVs it was revealed that most genes of T. onnurineus NA1 genome were packaged into EVs except approximately 9.4 kb from TON_0536 to 0544 which were assigned as hydrogenase (gamma subunit), sulfhyafiltration system and major two bands were purified through the CsCl-density gradient ultracentrifugation. Their buoyant density ranged from 1.350 to 1.354 gcm -3 and 1.354 to 1.358 gcm -3, respectively. After subsequent purification of the two bands, the both bands EVs were directly counted by the Izon qNano system. The final number of the concentrated EVs was 4.0&#9747 1011 particles ml-1 and 5.5&#9747 1011 particles ml-1 , respectively. Hence EVs were produced at the rate of 6.4&#9747 107 particles ml-1 of culture medium. The EVs were spherical in shape and the size varied from 120 nm to 220 nm in diameter, and both vesicles showed identical shape and size. The vesicles between 150 nm and 170 nm in size were major population. DNA contents of EVs were examined by the 1% agarose gel electrophoresis after DNA extraction (QIAGEN). The complete genome sequence of the EVs was performed by Ion Torrent and then assembled. Comparing the sequences between the whole genome of parent archaea and EVs it was revealed that most genes of T. onnurineus NA1 genome were packaged into EVs except approximately 9.4 kb from TON_0536 to 0544 which were assigned as hydrogenase (gamma subunit), sulfhy