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Purification and Characterization of extracellular vesicles from a 2 hyperthermophilic archaeon Thermococcus onnurineus NA1T

Dong Hee Choia, Yong Min Kwona, Hiroshi Xavier Chiurab, Eun Chan Yang, Seung Seob Bae, Sung Gyun Kanga, Jung-Hyun Leea, Hwan Su Yoone and Sang-Jin Kima,

Applied and Environmental Microbiology, 2015-

Extracellular vesicles (EVs) produced by a sulfur-reducing, hyperthermophilic archaeon, Thermococcus onnurineus NA1T (To), were purified and characterized. A maximum of four EV bands, showing buoyant densities between 1.1899 and 1.2828 g cm-3, were observed after CsCl ultracentrifugation. The two major EV bands, B (ρ25=1.2434 g cm-3) and C (ρ25=1.2648 g cm-3), were separately purified and counted using a qNano particle analyzer. The EVs showing different buoyant densities were identically spherical in shape, and their size varied from 80 to 210 nm in diameter, with the 120 and 190 nm sizes predominant. The average size of DNA packaged into EVs was about 14 kb. The DNA of the EVs in band C was sequenced and assembled. The mapping of the ToEV DNA sequences onto the reference genome of the parent archaeon revealed that most genes of T. onnurineus NA1T were packaged into EVs, except for an approximately 9.4 kb region encoded from TON_0536 to 0544. The absence of this specific region of the genome in the EVs was confirmed from band B of the same culture and from bands B and C purified from a different batch culture. The presence of the 5’ -terminal sequence and the absence of the 3’ -terminal sequence of TON_0536 were repeatedly confirmed. On the basis of those results, we hypothesize that the unpackaged part of the T. onnurineus NA1T genome might be related to the process that delivers DNA into ToEVs and/or the mechanism generating the ToEVs themselves.

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