Marine Biotechnology Research Division, Korea Institute of Ocean Science and Technology, Ansan, South Koreaa; Marine Microbiology Laboratory, Department of MarineEcosystems Dynamics, Atmosphere and Ocean Research Institute, University of Tokyo, Kashiwa, Chiba, Japanb; Marine Ecosystem Research Division, Korea Institute ofOcean Science and Technology, Ansan, South Koreac; Department of Marine Biotechnology, Korea University of Science and Technology, Daejeon, South Koread;Department of Biological Sciences, Sungkyunkwan University, Suwon, South Koreae
Extracellular vesicles (EVs) produced by a sulfur-reducing, hyperthermophilic archaeon, “Thermococcus onnurineus”NA1T, were purified and characterized. A maximum of four EV bands, showing buoyant densities between 1.1899 and 1.2828 gcm�3, were observed after CsCl ultracentrifugation. The two major EV bands, B (buoyant density at 25°C [�25] � 1.2434 g cm�3)and C (�25 � 1.2648 g cm�3), were separately purified and counted using a qNano particle analyzer. These EVs, showing differentbuoyant densities, were identically spherical in shape, and their sizes varied from 80 to 210 nm in diameter, with 120- and190-nm sizes predominant. The average size of DNA packaged into EVs was about 14 kb. The DNA of the EVs in band C was se-quenced and assembled. Mapping of the T. onnurineus NA1T EV (ToEV) DNA sequences onto the reference genome of the par-ent archaeon revealed that most genes of T. onnurineus NA1T were packaged into EVs, except for an �9.4-kb region fromTON_0536 to TON_0544. The absence of this specific region of the genome in the EVs was confirmed from band B of the sameculture and from bands B and C purified from a different batch culture. The presence of the 3=-terminal sequence and the ab-sence of the 5=-terminal sequence of TON_0536 were repeatedly confirmed. On the basis of these results, we hypothesize that theunpackaged part of the T. onnurineus NA1T genome might be related to the process that delivers DNA into ToEVs and/or themechanism generating the ToEVs themselves.
The production of extracellular vesicles (EVs) is widespreadamong all three domains of life: Eukaryota, Bacteria, and Ar-
chaea (1). Within the Archaea, EVs are produced by hyperthermo-philes such as Sulfolobus species (2, 3), Ignicoccus spp. (4) in theCrenarchaeota, and Thermococcus kodakarensis (5) in the Euryar-chaeota. Archaeal EVs released by Sulfolobus species range from 90to 230 nm in diameter and contain membrane lipids and S-layerproteins derived from the parent archaeal cell surface. An exten-sive study on the presence of EVs in cultures of hyperthermophilicarchaea (order Thermococcales), analyzed by electron microscopy,revealed that most strains of Thermococcus and Pyrococcus pro-duced various types of spherical membrane vesicles (6). Mem-brane vesicles were usually spherical in shape; however, unusualstructures such as filaments, chains of pearls, and others were alsoobserved in various sizes from 10 to 20 nm to 200 to 300 nm.Chiura reported a high level of vesicle production (ca. 3 � 1012
liter�1) from a hyperthermophilic archaeon, T. kodakarensis,which had been isolated from a hydrothermal vent, after 480 h ofculture at 70°C (5).
Since the first reported observation of microbial outer mem-brane vesicles (OMVs) from Vibrio cholerae about 50 years ago(7), the production of vesicles from microbial cells and their fea-tures have been described under various names, such as OMV,membrane vesicle or microvesicle (MV), virus-like particle or ves-icle (VLP or VLV), vector particle (VP), and so on (5, 6, 8, 9).Numerous functions have been attributed to these vesicles by sev-eral investigators. It is now well known that EVs consist of pro-teins, lipids, lipopolysaccharides, and nucleic acids (both DNAand RNA), the same as the building materials of the parent organ-isms producing the EVs. Gram-negative bacteria produce OMVsthat contain biologically active proteins and perform diverse bio-
logical processes (9, 10). Unlike other secretion mechanisms,OMVs enable bacteria to secrete insoluble molecules in additionto, and complexed with, soluble material. OMVs allow enzymes toreach distant targets in a concentrated, protected, and targetedform. OMVs also play roles in bacterial survival. Their productionis a bacterial stress response and important for nutrient acquisi-tion, biofilm development, and pathogenesis (9).
Vesicles carrying chromosomal and/or plasmid DNA havebeen reported from Gram-negative bacteria such as Escherichia,Haemophilus, Neisseria, Pseudomonas (10, 11), Ahrensia, andPseudoalteromonas species (12), the cyanobacterium Prochloro-coccus (13), and archaea such as Thermococcales strains, includingT. kodakarensis and T. nautilus (5, 14, 15). DNA was determinedto be in the interior of the vesicles by using a DNase protectionassay; however, the mechanism of DNA packaging into vesiclesremains unknown. Although the presence of DNA in EVs was
Received 10 February 2015 Accepted 22 April 2015
Accepted manuscript posted online 1 May 2015
Citation Choi DH, Kwon YM, Chiura HX, Yang EC, Bae SS, Kang SG, Lee J-H, YoonHS, Kim S-J. 2015. Extracellular vesicles of the hyperthermophilic archaeon“Thermococcus onnurineus” NA1T. Appl Environ Microbiol 81:4591–4599.doi:10.1128/AEM.00428-15.
previously controversial, several studies proved the transfer of thegenetic material to other microorganisms via vesicles. It is likelythat not all EVs carry DNA, and it is still uncertain whether all EVscontaining DNA from the parent cells are able to transfer thegenetic materials to other cells or even different organisms.
Several proteomic studies have been performed using Gram-negative bacterial OMVs to elucidate the biogenesis and functionsof OMVs, as well as to develop diagnostic tools, vaccines, andantibiotics effective against pathogenic bacteria (16). In contrastto the proteomic studies on vesicles, little has been done to char-acterize the DNA content of EVs. It is not yet known what genesare packaged or whether there is random or preferential genepackaging from parent organisms into EVs to enable them totransfer novel functions to other organisms. Available informa-tion on the DNA content in EVs is still too limited to understandtheir biogenesis and functions in bacterial communities.
In the present study, we report here the production of EVsfrom a hyperthermophilic archaeon, “T. onnurineus” NA1T
(ToEVs), and characterize their physical features and DNA con-tent. Specifically, we conducted extensive DNA sequencing to an-alyze the genome sequences present in ToEVs, which led to theunexpected discovery that a specific small region of the genome isnot represented in the ToEV DNA. We speculate that this findingmight be relevant to the mechanism of DNA packaging from par-ent cell to vesicle and/or to vesicle production.
MATERIALS AND METHODSIsolation of archaeal strains and cultivation of T. onnurineus strainNA1T. Sediment samples were collected from the PACMANUS hyper-thermal field (3°44=S, 151°40=E) at a depth of 1,650 m in the Manus Basinand from the caldera region (31°39.749=N, 130°46.290=E) at a depth ofabout 200 m in Kagoshima Bay, Japan. After inoculation of sedimentsamples, the enrichment cultures were grown in 25-ml serum vials con-taining 20 ml of yeast extract-peptone-sulfur (YPS) medium at 80 to 90°Cfor 3 days under anaerobic conditions (17). Colonies were randomlypicked and purified by streaking onto YPS-Phytagel four times succes-sively. Purified isolates were checked microscopically after a serial dilutionand designated NA1 and NA2 for samples from Manus Basin and KBA1for samples from Kagoshima Bay. NA1 was classified as T. onnurineusNA1T (17), and NA2 and KBA1 were tentatively classified as novel speciesof the genera Pyrococcus and Thermococcus, respectively (see Fig. S1 in thesupplemental material).
T. onnurineus NA1T was routinely cultured in a yeast extract-peptone-formate medium under anaerobic conditions at 80°C. The detailed de-scription of this medium was provided in a previous study (18). To con-centrate and purify the ToEVs, the strain was cultivated in a modifiedformate medium 1 (30), including 1.0 g liter�1 yeast extract, 13.6 g liter�1
sodium formate, and 35 g liter�1 NaCl. Trace elements and vitamins wereadded, and the pH was adjusted to 6.0 to 7.0 (18). All incubations werecarried out at 80°C under anaerobic conditions for 8 h unless otherwisestated.
Purification of extracellular vesicles. After incubation, the culturebroth (15 liters) was spun down at 10,000 � g for 30 min at 25°C using ahigh-speed centrifuge (2236HR; Gyrogen) equipped with a GRF-500-6rotor to sediment the cells. The supernatant was concentrated about 100-fold using an ultrafiltration system with a 100-kDa cutoff membrane(Millipore) after prefiltration through 0.45- and 0.2-�m-pore-size mem-brane filters (Advantec). The concentrated samples were once again fil-tered using a 0.2-�m-pore-size syringe filter, and then the samples werespun down by ultracentrifugation (Himac CS120GXL; Hitachi) at 88,000� g for 30 min at 4°C. The vesicle pellet at the bottom of the centrifugetube was resuspended overnight at 4°C in 500 �l of 1� TBT buffer (100mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2 [pH 7.4]) using a slow
rotator. The vesicle samples were treated with10 �g ml�1 (each) DNaseand RNase A (Sigma) at 25°C overnight, and heat inactivation of thenucleases was performed at 70°C for 10 min. The vesicle samples in 1�TBT buffer supplemented with 35% CsCl (Sigma) were ultracentrifugedat 174,000 � g for 20 h at 4°C using a swing bucket rotor (S52ST-0352;Hitachi). After CsCl-density gradient equilibrium ultracentrifugation, wemeasured the distance from the bottom of the tube to the surface layer orthe EV band layers, and the CsCl gradient solution in the tube was sam-pled in 0.5-cm layers from the surface layer to the bottom by the sidepuncture technique, using sterilized 1-ml syringes. At the same time, theEV bands were separately recovered from the lower boundary of eachband by the same side-puncture method. The refractive index (RI) valueof each sample was determined by using an Abbe refractometer (Atago).To calculate the buoyant density (BD) at 25°C (�25), the measured RIvalues were inserted into the following equation:
BD (g cm�3) � 10.8601 � RI � 13.4974 (1)
A second equation for correlation of the buoyant densities calculatedfrom equation 1 and the distances of each layer measured from the bottomwas generated as follows:
where d represents the distance of the middle of an EV band layer from thebottom in cm and R2 represents the coefficient of determination meantfor the relative significance of regression.
To remove the CsCl from the recovered EVs, the samples were placedin a sterilized Spectra/Por 4 dialysis membrane (molecular mass cutoff,12,000 to 14,000 Da; Spectrum Labs) and dialyzed five times against 100volumes of sterilized 1� TBT buffer at 4°C. The sample of purified EVswas preserved at 4°C for further experiments.
Electron microscopic observation. To examine the surface of cells forthe presence of EVs, cells of the three isolated Thermococcales strains wereobserved by scanning electron microscopy (SEM) (17). The cells wereharvested at the end of the exponential growth phase and fixed overnightat 4°C in 2% (vol/vol) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4).The fixed cells were filtered through a 0.2-�m-pore-size polycarbonatemembrane filter and then rinsed in 1� TBT buffer. Samples were sputtercoated with gold and then examined by SEM (JSM-840A; JEOL) at amagnification of �15,000 at 5 kV.
To observe the T. onnurineus NA1T cells by transmission electronmicroscopy (TEM), the cells were centrifuged at 4,700 � g for 5 min at20°C after cultivation. The cell pellets were fixed in 0.1 M cacodylatebuffer (pH 7.4) containing 2.5% (vol/vol) glutaraldehyde for 2 h and thenplaced on a carbon-coated Formvar grid (EMS). Negative staining wasperformed by adding a droplet of 2% (wt/vol) uranyl acetate onto the gridfor 10 s and then removing the excess. The grids were rinsed three timeswith droplets of ultrapure H2O and then observed using TEM (JEM1010;JEOL) operated at 80 kV (19).
Counting and measuring EVs. (i) TEM. Purified EVs were fixed over-night at 4°C with 10 mM EDTA containing 2.5% (vol/vol) glutaraldehyde,and then the sample was placed on a carbon-coated Formvar grid (EMS),placed in an ultracentrifugation tube, and spun at 46,000 � g for 90 min at20°C using an ultracentrifuge (Himac CS120GXL; Hitachi) with anS52ST-0352 rotor (19). Negative staining was performed as describedabove.
(ii) qNano. The qNano system (Izon) is a relatively new technologythat allows the detection of EVs passing through a nanopore by way ofsingle-molecule electrophoresis (20). The technology is based on theCoulter principle at the nano scale and operates by detecting transientchanges in the ionic current generated by the transport of the target par-ticles through a size-tunable nanopore in a polyurethane membrane. EVswere diluted to �10�3 with 1�-sterilized TBT buffer in a small steriletube, and the diluted sample was vigorously shaken for 1 h using a vortexmixer and measured using an NP150 nanopore aperture. When the sam-ple plugged the nanopore, the shaking time was extended after the addi-
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tion of 7.6 mM EDTA solution to disperse the vesicles. Calibration wasperformed using CP100 carboxylated polystyrene calibration particles(Izon) as a standard according to the manufacturer’s instructions.
(iii) Epifluorescence microscopy. To count the DNA-containingToEVs, the sample was stained with SYBR gold nucleic acid gel stain(Invitrogen) and examined at 1,600-fold magnification under a Zeiss epi-fluorescence microscope (21). The SYBR gold solution was added to 10 �lof ToEV sample (final SYBR gold concentration, 100�). After incubationat room temperature in the dark for 15 min, the sample was filteredthrough a 0.015-�m-pore-size polycarbonate membrane filter (What-man International, United Kingdom) with a 0.8-�m-pore-size backingmembrane filter. After drying, the filter was mounted on a glass slide, andthe ToEVs were counted under green epifluorescent excitation.
Electrophoretic analysis of nucleic acid in ToEVs. To cleanly removethe vesicle-associated DNA, EVs mixed with or without pUC19 (the ratioof EV DNA to pUC19 DNA was approximately 1:4) were treated with 10�g of nucleases for 16 h at 37°C. After heat inactivation of the nucleases at70°C for 10 min, EV DNA was extracted with a vRD kit (Geneall). Theextracted DNA was separated in a 1% agarose gel. To estimate the DNAsize and also for use in marker PCR and sequencing analysis, EVs werealways treated by the same method as described above prior to DNAextraction. To test whether the EV DNA was linear or circular in form,purified DNA was digested with endonucleases such as BamHI, EcoRI,HindIII, NotI, and SacI at 37°C for 2 h, and then the treated and non-treated DNA samples were resolved in parallel by 1% agarose gel electro-phoresis.
EV DNA sequencing and reference assembly. DNA was extractedfrom collected ToEVs with minimal contaminants (T. onnurineus NA1T
itself and any other organisms, as determined by epifluorescence micros-copy and TEM) using a vRD kit. EV DNA (ca. 100 ng) was used for libraryconstruction according to the manufacturer’s instructions (Ion Torrent,San Francisco, CA), which was followed by sequencing using the NGStechnique on an Ion Torrent Personal Genome Machine (Life Technolo-gies, San Francisco, CA). We obtained 420.76 Mb (2,578,006 reads; meanread length, 202 bp) of high quality reads (Q20) from an overall total of�521.82 Mb of sequence data. Only high-quality reads were used forreference genome assembly on the complete T. onnurineus NA1T genome(NC_011529) using CLC Genomics Workbench v.5.5.1 (CLC Bio, Aar-hus, Denmark).
Genome-spanning gene primers and amplification. To determinethe presence of T. onnurineus NA1T genes in ToEVs, 36 paired primerswere designed at intervals of �50 kb on the basis of the known genomicsequences of T. onnurineus NA1T (see Table S1 in the supplemental ma-terial). Purified DNAs of ToEVs, and of T. onnurineus NA1T as a refer-ence, were amplified for 35 cycles of 94°C for 1 min, 55°C for 1 min, and72°C for 2 min, and the products were separated on a 1% agarose gel.
GenBank accession numbers. The GenBank/EMBL/DDBJ accessionnumbers for the 16S rRNA gene sequences of Pyrococcus sp. strain NA2 andThermococcus sp. strain KBA1 are CP002670 and KP299296, respectively.
RESULTSMorphological characteristics of ToEVs. Three Thermococcalesstrains were cultivated, and the cell surfaces were observed bySEM. Although no bud-like structures, presumptive EVs, werefound on Pyrococcus sp. strain NA2, both T. onnurineus NA1T andThermococcus sp. strain KBA1 had EV-like structures buddingfrom the surfaces of the cells (see Fig. S2 in the supplementalmaterial). Presumptive EVs were more abundant on the cell sur-faces of the KBA1 strain than on the NA1 strain.
Of the two vesicle-producing Thermococcus strains, T. onnu-rineus NA1T was chosen for further examination as an EV pro-ducer in the present study because the parent microorganism hadalready been taxonomically identified and studied by genomic andproteomic approaches (17, 22, 23). TEM images of negativelystained cells appeared to show that the vesicles budded from thesurfaces of parent cells (Fig. 1A and B). A lumen-like structurewithin the developing bud protruded from the parent cell (Fig.1A), and then these buds were pinched from the cell surface (Fig.1B) and liberated into the surrounding milieu as EVs. During thebudding of a vesicle, it is likely that the electron-dense body (EDB)moves from the parent cell to the bud’s lumen, as the vesicle con-tinues to grow and finally is released from the parent cell. A uniquechain of vesicles connected to a host cell (Fig. 1C) was also ob-served.
To purify the EVs, the culture broth (15 liters) of T. onnurineusNA1T cells was centrifuged to eliminate the parent cells, and thenthe supernatant was concentrated by a tangential-flow membranefilter system as described in Materials and Methods. The concen-trated ToEVs were further purified by CsCl gradient ultracentrif-ugation. After the separation of vesicles in the CsCl gradient, wefound four distinct bands having different buoyant densities, withtwo major bands having buoyant densities of 1.2434 g cm�3 (bandB) and 1.2648 g cm�3 (band C). The two additional bands (A andD) were occasionally seen in large-volume batch cultures (100 to150 liters) but not both bands at the same time, even when theculture conditions were the same. For example, one lighter vesicleband, band A (�25 � 1.1899 g cm�3), was located above band Band a heavier band, D (�25 � 1.2828 g cm�3), was located belowband C. However, these two bands were not consistently present,
FIG 1 TEM images of negatively stained T. onnurineus NA1T cells. (A) Vesicle protruding from the parent cell surface, early stage. (B) Vesicle bud almost pinchedoff from the surface of parent cell, about to be released as an EV. (C) Vesicles connected to the parent cell as a chain. The highly electron-dense cytoplasmicmaterials appear as a dark color inside the cell and EV. Arrows indicate ToEVs. Scale bar, 1 �m.
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and we did not obtain enough of these vesicles for further exper-iments. Thus, we report here only on the ToEVs of the two majorbands, B and C, for which sufficient vesicles were purified.
The ToEVs of bands B and C were negatively stained and ex-amined by TEM. There was no morphological difference in theToEVs from bands B and C. Both ToEVs were spherical in shapewithout any tail (Fig. 2), and we could not find any typical virus-like structure associated with the ToEVs. According to the num-ber of ToEVs counted from purified bands B and C by qNano, thevesicle production levels were estimated to be 2.7 � 1010 and 3.7 �1010 liter�1 of culture medium, respectively, and the band B/bandC production ratio was about 2:3. The size and number of ToEVsin the subsamples were counted and measured by two methods,TEM and qNano. The sizes were determined to be in the range of
80 to 210 nm and 120 to 550 nm by TEM and qNano, respectively(Fig. 3). Although the size range of EVs measured by qNano seemsto be slightly greater than that determined by TEM, no significantdifference between the two measurements was detected byStudent t test (P � 0.05). The size distribution of ToEVs inband B was identical to that of ToEVs in band C, as confirmedby qNano. The mode value for vesicles from both bands was140 nm.
Advantages of qNano are that it is time saving and less labori-ous to count the number of vesicles and to measure the size forcomparison with TEM images. However, when vesicles aggre-gated into clumps, qNano likely recognized these as single vesicles,giving an incorrect size and number. qNano showed the size ofseveral vesicles to be bigger than 200 nm, which was not found
FIG 2 TEM images of negatively stained ToEVs in band B (A) and ToEVs in band C (B). ToEVs were suspended in TBT buffer, adsorbed to a carbon-coated grid,and negatively stained for 30 s with 2% uranyl acetate. Scale bars, 100 nm.
FIG 3 Size distribution of ToEVs estimated by qNano and TEM.�, qNano/ToEVs of band B;Œ, qNano/ToEVs of band C;Œ, TEM/ToEVs of band C. The figuredepicts the diameter of vesicles (in nm) versus normalized counts of vesicles (%). The size distribution of ToEVs bigger than 300 nm is not presented.
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from the TEM observations on samples purified using a 0.2-�m-pore-size membrane filter (Fig. 3). In the size distribution profilefrom the qNano analysis, we found two or three peaks in thedistribution curve that were larger than 200 nm, which likely wereaggregates of two, three, or even more vesicles. Therefore, thenumber of vesicles in the present study was counted using onlythose with a diameter smaller than 210 nm. The qNano counts ofToEVs would obviously be underestimates if the vesicle aggrega-tion in the sample was serious. This drawback could be minimizedby disaggregating vesicle samples by vigorous shaking. Further-more, treatment of the vesicle sample with a nonionic detergentcould also enhance disaggregation.
According to the manufacturer’s information, the NP150nanopore used in this experiment should be able to count theparticles efficiently in the range between 75 and 300 nm. However,vesicles smaller than 120 nm could not be confirmed by qNano,whereas TEM analysis showed such vesicles to be ca. 5% of thetotal, when the vesicles were not shrunk by the preparation pro-cedure for the TEM sample. It is possible that qNano might un-derestimate the total count of ToEVs ca. 5% due to a detectionlimit for particles smaller than 100 nm. This drawback of theqNano method was also pointed out by Momen-Heravi et al. (20).
The numbers of nucleic acid-containing ToEVs of bands B andC were counted by epifluorescence microscopy with the SYBRgold staining method. The ratios of the number of SYBR gold-stained vesicles to the total number of vesicles counted by qNanowere ca. 58% 6% and 81% 4% (n � 3), respectively, for bandsB and C.
Biochemical characteristics of ToEVs. To test whether ToEVscontain DNA or not, nucleic acids were extracted from ToEVs of
band C. The electrophoretic analysis of nucleic acids from ToEVswas performed after treatment with a combination of DNase andRNase (Fig. 4). The sample of nucleic acids purified from ToEVswithout any DNase and RNase showed DNA and RNA bands(lane 2); however, no band was found when it was treated withboth enzymes (lane 5). When this sample was pretreated withDNase (lane 3) and RNase (lane 4), the DNA and RNA bands,respectively, disappeared. These results strongly support the viewthat ToEVs carry DNA and RNA.
The DNA isolated from the vesicles could either be originatingfrom inside bona fide vesicles or be from contaminated surfacesand/or free DNA from the surroundings. To confirm the origin ofDNA associated with the EVs, purified ToEVs with or withoutadded pUC19 DNA were treated with DNase I. Total DNA ex-tracted from ToEVs plus pUC19 DNA without DNase I showedthree distinctive bands (see Fig. S3, lane 3, in the supplementalmaterial); however, only one band (near 10 kb) remained afterDNase I treatment (see Fig. S3, lanes 2 and 4, in the supplementalmaterial).
To estimate the size of the DNA in the vesicles, the ToEV DNA,which was apparently packaged from the T. onnurineus NA1T ge-nome into the EVs, was extracted, purified, and then separated ina 1% agarose gel (Fig. 5). The mobility of the ToEV DNA bandtreated by endonucleases prior to the gel electrophoresis did notshow any difference compared to the untreated DNA. This indi-cates that the ToEV DNA is linear in form. Based on the findingthat ToEV has linear double-stranded DNA, the size of the DNAwas estimated to be �14 kb from the relationship between molec-ular weight and the distance of DNA band migration. The DNApackaged into ToEVs was 1/100 the size of the genome of theparent microorganism (1,848 kb).
To confirm that the purified ToEVs were produced by the T.onnurineus NA1T cells, 16S rRNA genes extracted from ToEVswere amplified with primer TON_1979 coding for 16S rRNA andsequenced. The EVs contained the same 16S rRNA gene sequence
FIG 4 Electrophoretic analysis of ToEV nucleic acids without or with DNaseand RNase treatment. Lane 1, 1-kb DNA ladder; lane 2, no treatment withDNase and RNase; lanes 3 and 5, nucleic acids of ToEVs treated with DNase I;lanes 4 and 5, nucleic acids of ToEVs treated with RNase A. White arrowsindicate two rRNAs in lanes 2 and 3.
FIG 5 Size estimation of DNA from ToEVs. Lane 1, lambda/HindIII DNAladder; lane 2, total nucleic acids isolated from ToEVs; lane 3, 1-kb DNAladder. Molecular sizes are indicated in kilobases. The lower two discrete bandsin lane 2 represent RNA bands as shown in Fig. 4.
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as the parent archaeon. This confirms observations that vesicleswere produced by the parent archaeal cell. It appears that DNA ispackaged into the lumen of the protruding bud before the vesiclesare freed from the parent cell. To determine whether any region ofthe genome is preferentially packaged from the parent cells intoToEVs, the extracted ToEV DNA was amplified using pairedprimers for 36 protein-encoding genes that were selected so as tospan the T. onnurineus NA1T genome at regular intervals (seeTable S1 in the supplemental material). Of the 36 parent markers,35 markers (only TON_0544 was an exception) were representedin the DNA from the EVs of bands C (see Fig. S4 in the supple-mental material) and B, while the DNA of the parent cells thatproduced the vesicles showed the presence of all the markers, in-cluding TON_0544.
Analysis of the ToEV DNA. To determine whether only openreading frame (ORF) TON_0544 was missing from the collective“genome” of ToEVs or whether any other gene was also excludedduring the DNA packaging process from the parent cells to EVs,the entire DNA complement encapsulated in the vesicles (band C)was sequenced. These ToEV DNA sequence reads (total of 2.58
million reads) were assembled on the reference genome of T. on-nurineus NA1T, for a total of 1,847,607 bp (NC_011529 [22]).From the assembled ToEV DNA sequence, all gene sequences inthe vesicles were derived from the parent cell. There were no otherarchaeal, prokaryotic, eukaryotic, or inserted viral sequences.Mapping of reads on the reference genome revealed that 99.5% ofthe T. onnurineus NA1T genome was covered with a 246.8� meancoverage (standard deviation, 56.4�; maximum, 766�), and0.5% of the genome was not covered by any reads (Fig. 6). Fromthe distribution of ToEV DNA sequences on the genome map ofT. onnurineus NA1T, there was a reproducible overabundance ofreads from the region of the predicted chromosomal replicationorigin (oriC locates at TON_1644 [24]) (Fig. 6A). The 0.5% un-covered part of the genome existed as a single region of �9,400 bp(TON_0536 to TON_0544 of NC_011529). ORFs fromTON_0536 (875 bp) to TON_0544 (1,221 bp) were assigned ashydrogenase (gamma subunit), sulfhydrogenase (beta subunit),hypothetical formate transporter, hypothetical formate dehydro-genase (alpha subunit), oxidoreductase iron-sulfur protein,4Fe-4S cluster-binding protein, glutamate synthase (beta chain-related oxidoreductase), 4Fe-4S cluster-binding protein, and al-cohol dehydrogenase, respectively. Furthermore, the 3= region ofTON_0536 (258 bp) was found in the ToEV DNA sequences, butthe 5= region was not (617 bp).
A question was raised whether the same region (�9.4 kb) of theT. onnurineus NA1T genome was repeatedly missing in the ToEVspurified from different batch cultures. If only occasionally a cer-tain part of the genome fails to get packaged into ToEVs, it ispossible that the excluded region could be recovered from anotherbatch of EVs. DNA was extracted from ToEVs that were purifiedfrom the broth of another batch culture of T. onnurineus NA1T. Itwas amplified using primers for TON_0494, the TON_0536 3=region (forward, 5=-CGGCGTTGATACTATGTC-3=; reverse, 5=-GATGTATAAGGCGGTCTTC-3=, size 200 bp), TON_0598, andTON_1979, which existed in the previous ToEV DNA, and prim-ers for the TON_0536 5= region (forward, 5=-GAGATCCTTCATGGCTTC-3=; reverse, 5=-CATGTGTCACGACAATCC-3=, size500 bp) and TON_0544, which were not present.
The absence of a specific region of the genome in the vesicleswas confirmed by PCR using the ToEV DNA of band C purified
FIG 7 Gel electrophoresis for PCR products of ToEVs amplified by specificprimers. Lane 1, lambda/HindIII DNA ladder; lane 2, TON_0494; lane 3, 3=region of TON_0536; lane 4, 5= region of TON_0536; lane 5, TON_0544; lane6, TON_0598; lane 7, TON_1979 (16S rRNA genes); lane 8, 1-kb DNA ladder.The molecular sizes are indicated.
FIG 6 (A) Coverage of T. onnurineus NA1T genome by DNA sequencing reads for ToEVs. The mean coverage was about 247�, with a 56.4� standard deviationand a maximum coverage of 766�. An arrow indicates the predicted chromosomal replication origin (oriC) of T. onnurineus NA1 at the specific genomicposition of 1,508,116 bp. (B) Expanded unrecovered region (�9.4 kb) of the T. onnurineus NA1T genome. No reads for ToEVs were obtained for ORFs of the T.onnurineus NA1T genome from TON_0536 to TON_0544.
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from the independent batch culture (Fig. 7). We confirmed thesequence of the PCR product of TON_0544 against the genomicDNA of the parent cell that had produced the vesicles in the samebatch culture and the nonspecific PCR products of TON_0544(lane 5) against ToEV DNA. These results clearly indicated thatthe 5= region of TON_0536, as well as TON_0544, of the T. onnu-rineus NA1T genome had not been packaged into the ToEVs,whereas TON_0494, the 3=-region of TON_0536, TON_0598, andTON_1979 gave the same result seen previously. ToEV DNA fromband B was also examined using these specific primer sets, and theresult was the same as for DNA from ToEV band C (see Fig. S5 inthe supplemental material).
DISCUSSION
The presence of vesicles in cultures of various thermophilic ar-chaea has been described previously, but only superficially (25,26). According to an extensive electron microscopy study of ves-icles produced from hyperthermophilic archaea of the order Ther-mococcales, most isolated strains, 26 of 34, produced various typesof spherical vesicles (6). In the present study, we confirmed thattwo of three Thermococcales isolates were able to produce vesicles(see Fig. S2 in the supplemental material). Based on this result andthe report of Soler et al. (6), the production of vesicles is a widelydistributed feature in hyperthermophilic Thermococcales andcould involve a conserved mechanism for their production.
The morphological features of such vesicles are similar regard-less of the parent microorganism (archaea, bacteria, or eu-karyotes) producing them (1). The ToEVs were spherical, withouta tail (Fig. 1 and 2), and they had the same vesicle morphology asthose previously reported from other microorganisms, except fortheir size. It is well established that Gram-negative bacterial MVsrange from 10 to 300 nm in diameter and that MVs derived fromGram-positive bacteria, such as Bacillus spp., are between 50 and150 nm in diameter (1). Archaeal MVs, such as those released bySulfolobus species, range from 90 to 230 nm in diameter. Eukary-otic microbial vesicles, derived from fungi and parasites, includeat least two vesicle populations, exosomes and shedding MVs,which range from 40 to 100 nm and 100 to 1,000 nm in diameter,respectively (1). ToEVs in the present study ranged between 80and 210 nm, as determined by TEM. These EVs were clearly dif-ferent from the morphology of a phage or virus which possesses ahead, tail, tail fibers, or a structurally unique capsid. The vesiclesare likely produced by a budding process analogous to that seen inyeast and different from the mechanism of virus production. TheToEVs did not show a double-bilayer structure such as reportedfor the OMVs of the psychrotolerant, Gram-negative bacterium,Shewanella vesiculosa M7T (27). Archaeal cells have a different cellmembrane and wall architecture than Gram-negative bacteria.The Thermococcus cell wall consists of a single plasma membraneand an outer protein matrix called the S layer, without a periplas-mic space, and thus cannot form a bilayered structure. ToEVswere surrounded by a thick, somewhat electron-transparent layer,likely the S layer, and an electron-dense single-layer plasma mem-brane, which is not easily distinguished from the darkly stainingcytoplasmic contents inside the ToEVs (Fig. 2). Similar to thechain of vesicles that was observed in the present study (Fig. 1C),there are several reports on unique forms of vesicles, such as tubeshaped, branched, chain of pearls, and so on (6). Interestingly, noEDB was observed in this chain of vesicles, while the parent cell
showed a “beads on a string” EDB that resembled a nucleosome-like structure.
It has been argued that most vesicles of Thermococcales strainsdo not contain bona fide intravesicular DNA, but rather that theDNA is strongly bound to vesicles or trapped in vesicle clusters(6). In the present study, we confirmed that the purified ToEVswere free of any DNA except for DNA in the lumens of the EVs(see Fig. S3 in the supplemental material). Consistent with this,the stability of DNA in the lumens of EVs against heat and nu-cleases indicates that DNA within EVs is protected from hostileconditions, which would increase the efficiency of a putative ves-icle-mediated DNA delivery to a recipient cell.
The ToEV DNA averaged �14 kb in size (Fig. 5), sufficient toencode multiple proteins. For comparison, vesicles of T. nautilus30-1 harbored the endogenous plasmid pTN1, which was derivedfrom the host archaeal cells, and was �3.4 kb in size (14), whereasDNA �310 kb in size was reported from the vesicles of T. koda-karensis B41 (8). The DNA size of the ToEVs was within the sizerange of DNA found in vesicles produced by two hyperthermo-philic archaea, T. nautilus and T. kodakarensis. The sizes of DNAsin vesicles from the bacteria Prochlorococcus (13) and Aquifex sp.(5) have been reported to be about 3 and 406 kb, respectively. Thevesicle DNA fragments from the cyanobacterium ProchlorococcusMED4 were heterogeneous in size, with some measuring at least 3kb (13). It is obvious that the DNA size in vesicles showed a widerange depending on the parent organism producing the vesicles.Although smaller than the exceptionally large molecules of T.kodakarensis and Aquifex sp., ToEVs contained genomic DNA ofsufficient size for gene transfer, because DNA exceeding 1 kb inlength can be utilized as an information molecule when it is trans-formed into recipient cells (28, 29).
Even though the presence of DNA in vesicles has been reportedfrequently, information about the genome sequences found invesicles has remained surprisingly scarce. As far as we know, therehave been only two reports of sequence data for DNA retrievedfrom vesicles; a Gram-negative alphaproteobacterium, Ahrensiakielensis B9; a gammaproteobacterium, Pseudoalteromonas ma-rina mano4; and a cyanobacterium, Prochlorococcus MED4 (12,13). Since both of these studies amplified the vesicle DNA to con-struct libraries, an artifact could not be entirely ruled out. In thepresent study, we sequenced DNA from about 1.7 � 1011 ToEVsfrom archaeal vesicles without amplification. Hagemann et al.(12) obtained about 30 and 46 kb of sequence information fromvesicle genome libraries of A. kielensis and P. marina, respectively.All sequences were prokaryotic except for one sequence showingsignificant similarity with a eukaryotic gene. Biller et al. (13) foundthat vesicle DNA corresponded to broadly distributed regions ofthe MED4 genome, representing �50% of the entire chromo-somal sequence. The sequence data obtained for ToEV DNA in-dicated that all sequences were derived from the parent cell DNA.We found almost all regions of the T. onnurineus NA1T genome(the whole genome is about 1.85 Mb), representing �99.5% of theentire chromosomal sequence (Fig. 6) in EV DNA. Our resultssuggest that the frequency of packaging specific DNA sequencesinto ToEVs has a possible link to the chromosomal replicationstep, since we noted an overabundance of reads from a region of T.onnurineus NA1T encoding the chromosomal replication origin(oriC), as indicated by an arrow in Fig. 6A. No information existson whether the DNA fragment formation for packaging is a ran-dom or organized process. From our results, we can speculate
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either that DNA fragments from near the origin are more abun-dant in the cell or that the DNA fragments averaging about 14 kbstart to fragment from the replication origin, and therefore thosefragments are preferentially packaged from the parent cells to ves-icles. However, this result is the exact opposite of the finding of anoverabundance for vesicle DNA sequences at the predicted chro-mosome terminus location of Prochlorococcus MED4 (13). Thisopposite result might be an artifact due to extensive amplificationof vesicle DNA. It might also reflect differences in the parent or-ganisms, archaea and bacteria.
The absence of 0.5% of the parent genomic sequence betweenTON_0536 and TON_0544 in the ToEVs is not an artifact. Theabsence of this region was confirmed by the PCR result forTON_0544 from the vesicle DNAs of bands B and C even when thevesicles were isolated from two different batch cultures (see Fig. S4and S5 in the supplemental material; Fig. 7). Furthermore, the 5=region of TON_0536 (617 bp) was repeatedly missing from thevesicle DNA purified from a different batch culture, while the 3=region (258 bp) was present in the DNA analysis, suggesting the 5=region was very specifically not delivered into the vesicle (see Fig.S5 in the supplemental material; Fig. 7). There are a number ofpossible explanations for how this region of the genome was miss-ing from the DNA of vesicles. One possibility might be that thisregion of the genome was not present in the genome of the parentcells due to a genetic instability of the microorganism. However,we confirmed that the missing DNA fragment was present in thegenome of the parent cell (see Fig. S4 and S5 in the supplementalmaterial). A second possibility is that this region of the genomecould not be replicated and fragmented because it was directlybound to a protein that fulfills an important function such as thefragmentation of DNA or packaging of DNA fragments into ves-icles, and therefore it was not physically moved into the vesiclelumen.
On the basis of our results, we hypothesize that the unpackagedpart of the T. onnurineus NA1T genome plays an indispensablerole in the process for DNA delivery into ToEVs and/or the mech-anism of ToEV production. We are now constructing T. onnu-rineus NA1T mutants lacking the unpackaged genomic region andwill investigate the effect of such mutations on ToEV productionand ToEV DNA composition in the near future.
ACKNOWLEDGMENTS
We thank E. S. Choi for the EV counting by TEM and J. P. van der Meer forEnglish correction.
This study was supported by the KIOST in-house programs (PE99314,PE99252, and PE99263), as well as the Marine and Extreme GenomeResearch Center Program and the Development of Biohydrogen Produc-tion Technology Program of the Ministry of Ocean and Fisheries, Repub-lic of Korea.
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