Choi Dong Hee, Kwon Yong Min, Chiura Hiroshi Xavier, Yang Eun Chan, Bae Seung Seob, Kang Sung Gyun, Lee Jung-Hyun, Yoon Hwan Su, Kim Sang-Jin
Marine Biotechnology Research Division, Korea Institute of Ocean Science and Technology, Ansan, South Korea.
Marine Microbiology Laboratory, Department of Marine Ecosystems Dynamics, Atmosphere and Ocean Research Institute, University of Tokyo, Kashiwa, Chiba, Japan.
Appl Environ Microbiol. 2015 Jul;81(14):4591-9. doi: 10.1128/AEM.00428-15. Epub 2015 May 1.
Extracellular vesicles (EVs) produced by a sulfur-reducing, hyperthermophilic archaeon, "Thermococcus onnurineus" NA1(T), 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 (buoyant density at 25°C [ρ(25)] = 1.2434 g cm(-3)) and C (ρ(25) = 1.2648 g cm(-3)), were separately purified and counted using a qNano particle analyzer. These EVs, showing different buoyant densities, were identically spherical in shape, and their sizes varied from 80 to 210 nm in diameter, with 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. Mapping of the T. onnurineus NA1(T) EV (ToEV) DNA sequences onto the reference genome of the parent archaeon revealed that most genes of T. onnurineus NA1(T) were packaged into EVs, except for an ∼9.4-kb region from TON_0536 to TON_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 3'-terminal sequence and the absence of the 5'-terminal sequence of TON_0536 were repeatedly confirmed. On the basis of these results, we hypothesize that the unpackaged part of the T. onnurineus NA1(T) genome might be related to the process that delivers DNA into ToEVs and/or the mechanism generating the ToEVs themselves.
对嗜热超嗜热硫还原古菌“温泉嗜热栖热菌”NA1(T)产生的细胞外囊泡（EVs）进行了纯化和表征。在氯化铯超速离心后，观察到最多四条EV条带，其浮力密度在1.1899至1.2828 g cm(-3)之间。两条主要的EV条带，B（25°C时的浮力密度[ρ(25)] = 1.2434 g cm(-3)）和C（ρ(25) = 1.2648 g cm(-3)），分别进行了纯化，并使用qNano颗粒分析仪进行计数。这些具有不同浮力密度的EVs形状均为球形，直径在80至210 nm之间变化，其中以120 nm和190 nm大小为主。包装到EVs中的DNA平均大小约为14 kb。对条带C中的EVs DNA进行了测序和组装。将温泉嗜热栖热菌NA1(T) EV（ToEV）DNA序列映射到亲本古菌的参考基因组上，结果显示，除了从TON_0536到TON_0544的约9.4 kb区域外，温泉嗜热栖热菌NA1(T)的大多数基因都被包装到了EVs中。从同一培养物的条带B以及从不同批次培养物中纯化得到的条带B和C中，均证实了EVs中该基因组特定区域的缺失。反复证实了TON_0536的3'-末端序列存在而5'-末端序列缺失。基于这些结果，我们推测温泉嗜热栖热菌NA1(T)基因组未包装的部分可能与将DNA递送到ToEVs的过程和/或产生ToEVs本身的机制有关。
