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Virus-like vesicles and extracellular DNA produced by hyperthermophilic archaea of the order Thermococcales
Research in Microbiology 159 (2008) 390e399 www.elsevier.com/locate/resmic
Virus-like vesicles and extracellular DNA produced by hyperthermophilic archaea of the order Thermococcales Nicolas Soler a,1, Evelyne Marguet b, Jean-Marc Verbavatz b, Patrick Forterre a,* a
Univ Paris-Sud, Institut de Ge´ne´tique et Microbiologie, CNRS, UMR 8621, F-91405 Orsay Cedex, France b IBITEC-S and CNRS URA 2096, Bat. 532, CEA-Saclay, F-91191 Gif-sur-Yvette, France Received 4 April 2008; accepted 14 April 2008 Available online 25 June 2008
Abstract Cultures of hyperthermophilic archaea (order Thermococcales) have been analyzed by electron microscopy and epifluorescence staining for the presence of virus-like particles. We found that most strains of Thermococcus and Pyrococcus produce various types of spherical membrane vesicles and unusual filamentous structures. Cellular DNA can be strongly associated with vesicles and appears as fluorescent dots by epifluorescence microscopy, suggesting that some particles assumed to be viruses in ecological studies might instead be vesicles associated with extracellular DNA. DNA in vesicle preparations is remarkably resistant to DNase treatment and thermodenaturation, indicating that association with vesicles could be an important factor determining DNA stability in natural environments. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Membrane vesicles; Thermococcales; Archaea; Hyperthermophiles; Viral ecology; Extracellular DNA; DNA resistance; Nanobacteria
1. Introduction Many new families of DNA viruses have been described that infect aerobic hyperthermophilic crenarchaea of the genera Sulfolobus and Acidianus from terrestrial hot springs [27]. These viruses exhibit unique morphologies and are strikingly different from both viruses infecting bacteria (bacterioviruses) and viruses infecting eukarya (eukaryoviruses). Virus-like particles with similar unique morphologies have also been observed in enrichment cultures of anaerobic hyperthermophilic archaea from deep-sea vents (probably euryarchaea of the order Thermococcales) [10]. However, only one archaeovirus infecting a hyperthermophilic euryarchaeon has been successfully isolated and characterized up to now, the virus PAV1 of Pyrococcus abyssi, strain GE23 [9,11]. To search
* Corresponding author. E-mail addresses: [email protected] (N. Soler), evelyne. [email protected] (E. Marguet), [email protected] (J.-M. Verbavatz), [email protected] (P. Forterre). 1 Present address: UMR2027 CNRS/Institut Curie, Universite´ Paris-Sud, Baˆtiment 110, 91405 Orsay Cedex, France. 0923-2508/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2008.04.015
for additional archaeoviruses from euryarchaeotes of the order Thermococcales (genera Pyrococcus, Thermococcus and Paleococcus), we have recently set up a collection of 70 Thermococcales strains obtained from several deep-sea hydrothermal vents located in the East-Pacific ridge [18]. About half of these strains contain putative extrachromosomal elements (either plasmids or viruses) [18]. We previously described one of these elements, the rolling-circle plasmid pTN1 from the candidate species Thermococcus nautilus [31]. In the present work, we report the results of our screening for viruses in strains in which extrachromosomal elements were previously detected. Since it is not possible to obtain lawns of Thermococcus cells, viruses cannot be screened by standard plaque formation assay. We thus directly looked for virus-like particles in culture supernatants by electron microscopy. This strategy has been successfully used in the past to isolate viruses from Sulfolobus species [39]. Previous works have indeed shown that most viruses from hyperthermophilic archaea live in a carrier state inside their host, and are regularly produced and excreted outside the cell [27]. Although we were unable to isolate viruses in this work, we were surprised to observe that most of the tested strains produce virus-like vesicles of various
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structures, including long flexible filaments. Some of these virus-like vesicles strikingly resemble membrane vesicles produced by bacterial species [2,17,20], suggesting that production of membrane vesicles can be a widespread feature of the microbial world. We observed that cellular DNA can be strongly associated with vesicles from Thermococcales and appear as fluorescent dots (virus-like) by epifluorescence microscopy. Interestingly, DNA present in vesicle preparation is highly resistant to DNase treatment and heat denaturation, suggesting that association with vesicles could be an important factor determining the stability of DNA in the environment. 2. Materials and methods 2.1. Strain cultivation Thermococcales strains were grown anaerobically in penicillin bottles for 50-ml cultures, and in Wheaton bottles for 200-ml cultures in Zillig’s broth [18]. Cultures were inoculated at 1e4% (v/v) from pure cultures isolated during the AMISTAD cruise for strains of our collection [18], or from pure cultures of type strains. Incubations were performed at 85 C overnight (15e17 h). 2.2. Vesicle preparations A 50-ml culture (stationary phase) of each strain was centrifuged at 5000 g for 20 min to remove cells. Supernatants were collected and ultracentrifuged using Nalgene tubes at 27,500 g for 3 h at 20 C in a Beckman JA17 rotor. The pellet was then resuspended with a few droplets of culture medium and analyzed by TEM. In some experiments (indicated in figure legends), a 2 h centrifugation run was performed at 110,000 g instead of 27,500 g using a SW41 Beckman rotor. For preparation of Thermococcus kodakaraensis and Thermococcus gammatolerans vVirus-Like-Particles (VLPs) 200- to 600-ml cultures in stationary phase were centrifuged at 5000 g to eliminate cells. The supernatants were then ultracentrifuged at 80,000 g for 2 h with a SW28 Beckman rotor. White pellets were resuspended in 2e6 ml of Mu buffer (200 mM NaCl, 20 mM TriseHCl pH 7.5, 1 mM CaCl2, 20 mM MgSO4), and 0.5 g/ml CsCl was added for equilibration on a centrifugation gradient at 213,000 g for 40 h with a TLS-55 Beckman rotor. White bands were recovered and dialyzed against Mu buffer to eliminate CsCl. For T. gammatolerans, these samples were submitted to micrococcal DNase digestion at 0.1 mg/ml final concentration for 1 h at 37 C. A second CsCl centrifugation gradient was then performed under the same conditions as described for the first one, in order to eliminate DNase and remaining cell debris. 2.3. Transmission electronic microscopy observations A 20-ml droplet sample was spotted onto a carbon-coated nickel grid (400 mesh, Euromedex). After 1 min, the excess liquid was removed with filter paper (Whatman) and the grid
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was rinsed with a droplet of ultrapure H2O. Negative staining was obtained by addition of a 2% (w/v) uranyl acetate droplet on the grid for 1 min, and the excess was removed again. The grid was then observed using a Philips EM208 electron microscope operated at 80 keV, coupled with an AMT CCD camera (Hamamatsu). 2.4. Analysis of DNA associated with vesicle preparations To extract DNA from vesicles, lysis buffer (50 mM Trise HCl pH 7.5, 2 mM EDTA pH 7.5, 1% SDS, 13.7% saccharose, 0.01% bromophenol blue) was added to vesicle preparations (v/v) and incubated for 10 min at 65 C, before loading on agarose gel. 2.5. DNA extraction and endonuclease restriction Total genomic DNA was purified from 50 ml culture (stationary phase) of T. gammatolerans. Cells were lysed in TENST buffer (10 mM TriseHCl pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.12% Triton X-100, 1,6% SDS) and DNA was extracted by standard procedure (phenol/chloroform extraction, isopropanol precipitation). The dried pellet was dissolved with 25 ml H2O. DNA (2e3 mg) was digested for 1 h at 37 C by the indicated restriction enzyme (Promega, 5 units). The mixture was then electrophoresed on a 0.7% agarose gel in TriseBorateeEDTA (TBE) buffer for 16 h at 1.75 V/cm, and the gel was stained with an ethidium bromide solution. 2.6. Epifluorescence analysis using Hoechst dye Vesicle samples (40 ml) were loaded on SuperFrostÒPlus slides for 5 min. After excess removal, 40 ml Hoechst dye solution (1 mg/ml) was applied to the surface. Anti-fading solution was then added and coverglasses were mounted for observation under an Olympus Vanox AH2 microscope under UV illumination. 2.7. Epifluorescence analysis using SYBR Green I All solutions used for this protocol were filtered with 0.45, 0.2, and finally 0.02 mm filters. Thermococcales culture (1 ml) was centrifuged at 10,000 g for 20 min to eliminate cells and 500 ml supernatant were mixed with 4.5 ml ASW (Artificial Sea Water). This mixture was double filtered at 0.8/0.2 mm (AcrodiscÒ PF Syringe Filter, Pall Corporation), and then particles smaller than 0.2 mm were recovered on a 0.02 mm filter (Anodisc 25, AnoporeÒ, Whatman). In the dark, 100 ml of 10 SYBR Green I solution (diluted in H2O from 10,000, Molecular Probes) were applied to the filter for 1e1.5 h. The filter was then rinsed with H2O in a small glass Petri dish for 30 min and dried for 30 min at 37 C. Still in the dark, the filter was placed on a slide, and mounted with anti-fading solution (100 ml of 0.1% N,N-dimethhy-1,4-phenylenediamine sulfate) and a coverglass. After 30 min incubation in the
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dark, the samples were observed under the microscope with UV illumination (475 nm excitation). 2.8. Thermoprotection of DNA in the presence of vesicle preparations Samples (15 ml) were covered with mineral oil (15 ml) and incubated for the indicated time at 90 C. 12.5 ml was then recovered for electrophoretic analysis at 16 V/cm for 30 min on a 0.7% agarose gel in TBE 0.5. The gel was stained with a 1 mg/ml ethidium bromide solution. 3. Results and discussion 3.1. Production of membrane vesicles and related filamentous structures by Thermococcales We screened 34 strains from our collection of Thermococcales that harbor putative extrachromosomal elements [18] for the presence of viruses by transmission electron microscopy (TEM). Fifty milliliters of culture in stationary phase were centrifuged at 5000 g for 20 min to remove cells, and the supernatant was centrifuged for 3 h at 27,500 g to pellet putative virions. The pellets were suspended in 500 ml buffer solution and droplets were mounted on a grid and stained with uranyl acetate for examination. Surprisingly, we found that most strains (26 out of 34) produce various types of spherical vesicles (see Fig. 1 for examples). The same types of vesicles were also observed in pellets obtained after centrifugation of PEG precipitates from culture supernatants. These vesicles were usually very abundant and sometimes clustered around membranous cell debris (Fig. 2). The diameters of these vesicles were in the range of 50e100 nm. Spherical vesicles observed in this work did not resemble most known archaeoviruses [27] or virus-like particles previously described in enrichment cultures of Thermococcales [10], but some of them were strikingly similar to a round particle described as a virus from terrestrial hot springs by Rohwer and co-workers (see figure 4, bottom left in Breitbart et al. [3]) or resembled globulovirus PSV infecting the hyperthermophilic crenarchaeotes Pyrobaculum and Thermoproteus [13]. However, the sizes of these vesicles were usually heterogeneous (Fig. 1aef), which is unexpected for viral populations. Vesicles observed in this work were also clearly different from empty viral capsids previously detected in cultures of Pyrococcus furiosus (approximately 30 nm in diameter and of homogeneous sizes) [1,22]. In fact, most of them were composed of a spherical core surrounded by a membrane resembling archaeal S-layers (Fig. 1a,d,e). In several strains, we also observed filaments or short rods that looked like strings of pearls surrounded by a membrane (Fig. 3). Vesicles appear to bud from one of the two extremities of these structures as if the pearls were the progenies of the vesicle spherical core. We also detected a few cells that contained internal filamentous structures (Fig. 2b). In other strains, we observed continuous tubular structures, also surrounded by a membrane and apparently
producing vesicles at one of their extremities. In two strains, a majority of these tubular structures was mixed with rounded vesicles. These observations strongly suggest that the vesicles observed are not artifacts produced during cell lysis by collapsing membranes, but are instead biological structures that are actively produced by cells. To determine whether the production of vesicles was a specificity of strains from our collection, we also looked for their presence in four type strains of Thermococcales currently used in our laboratory, Thermococcus kodakaraensis, Thermococcus gammatolerans, Pyrococcus abyssi and Pyrococcus horikoshii. We found that all these strains produce vesicles similar to those produced by strains in our collection (see Fig. 1d,gej). It is remarkable that vesicles produced by T. kodakaraensis and T. gammatolerans clearly differed in their appearance, with vesicles from T. gammatolerans being homogeneous in size, whereas those from T. kodakaraensis were heterogeneous (Fig. 1gej). Preliminary analyses also suggested that vesicles from these two species have different lipid and protein composition (data not shown). This illustrates the variability of vesicle shapes and indicates that this feature might be used in strain (species) description. The presence of vesicles in cultures of various thermophilic archaea has been previously described, albeit only incidentally. Prangishvili et al. [26] first reported that sulfolobucin, a colicin-like compound produced by some Sulfolobus species, is associated during purification with vesicles very similar to some of those described in this report. Vesicles were also recently observed in cultures of the thermoacidophilic archaeon Aciduliprofundum boonei (see figure 2d in Reysenbach et al. [29]) and in the periplasmic space of the hyperthermophile Ignicoccus profundus (the only known archaeon with an outer membrane) [24]. All these vesicles resemble those produced by Thermococcales. In particular, an unusual structure very similar to the ‘‘string of pearls’’ observed in this work (several rounded structures surrounded by a membrane) was observed in A. boonei (see figure 2d in Reysenbach et al. [29]). These data suggest that the production of vesicles is a widely distributed feature in thermophilic archaea and could involve a conserved mechanism for their production. However, as previously mentioned, the size and shape of the vesicles also differ between closely related strains of Thermococcales, indicating that some aspects of their production might be strainspecific. Interestingly, it has been shown for a long time that many species of bacteria produce large numbers of vesicles surrounded by an outer membrane layer [2,17,20,30]. These membrane vesicles can carry various compounds such as hydrolases, toxins, antibiotics and quorum sensing factors. Recent data have shown that vesicle production is indeed a major aspect of the global response to environmental stress [21]. Stressed bacteria overproduce vesicles that are apparently used to selectively eliminate unwanted material (e.g. unfolded proteins). Based on these previous observations, we reasoned that most (possibly all) virus-like vesicles observed in this work were not viruses but vesicles of cellular origin.
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Fig. 1. Electron micrographs of vesicles purified from different Thermococcales strains (type strains and strains from our collection). (a) Thermococcus strain 9/3. (b) Thermococcus strain 29/2. (c) Thermococcus strain 28/4. (d) P. horikoschii. (e) Thermococcus strain 29/3. (f) T. nautilus 30/1. (g, h) T. kodakaraensis. (i, j) T. gammatolerans. (a, b, c, e, f) Vesicles purified by ultracentrifugation at 27,500 g. (d, gej) Vesicles purified by ultracentrifugation at 80,000 g followed by CsCl gradient centrifugation. Scale bars: 100 nm.
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Fig. 2. Electron micrographs of vesicles associated with cells or cell debris. (a) T. gammatolerans. (b) T. nautilus strain 30/1. (c) Pyrococcus strain 12/1. (d) P. horikoschii. Scale bars: 100 nm.
3.2. Extracellular DNA can be strongly associated with virus-like vesicles from Thermococcales Since some spherical viruses resemble vesicles, it was possible that a subset of vesicles produced by Thermococcales were indeed yet unidentified viruses. We tested this possibility for Thermococcus gammatolerans [14], which was the only strain in our hands that reproducibly produced virus-like vesicles of homogeneous size (Fig. 1i,j). The genome of T. gammatolerans has been recently sequenced (F. Confalonieri and Y. Zivanovic, personal communication) and contains an integrated element that is homologous to the putative viruses TKV2 and TKV3 of T. kodakaraensis [8]. We reasoned that the virus-like vesicles of T. gammatolerans could be bona fide virions produced from this integrated element. To test this hypothesis, vesicles of T. gammatolerans were prepared as previously described from 200 to 500 ml cultures. Cells and cell debris were removed by centrifugation at 5000 g and vesicles were pelleted by ultracentrifugation at 80,000 g. Vesicles were then centrifuged onto equilibrium cesium chloride (CsCl) gradients. We observed two close bands of visible material with a density of about 1.35 g/cm3 (Fig. 4a). The bands were collected and TEM analysis confirmed the presence of vesicles. However, the thinner upper vesicle band preparation also contained flagella and some cell debris. The material from each band was then treated with micrococcal nuclease (0.1 mg/ml 30 min at 37 C). The nuclease was inactivated by treatment with 25 mM EGTA and heating for 10 min at 70 C, and vesicles from each band were separately subjected to a second round of centrifugation in CsCl gradient. Agarose gel electrophoresis of
purified vesicles followed by staining with ethidium-bromide allowed detection of nucleic acids that remained in the well, suggesting the presence of DNA associated with material that could not enter into the gel (Fig. 4b). Interestingly, this DNA migrated as a single sharp band in the gel after treatment of vesicles by SDS, heating and proteinase K (believed to break down putative viral capsids or to open membrane vesicles) (Fig. 4b). This result a priori suggested that virus-like vesicles from T. gammatolerans were the virions of bona fide DNA viruses. DNA was then phenol-extracted from the purified vesicle preparation and digested with KpnI or ScaI (Fig. 4c). As shown in Fig. 4, and in contrast to our expectations, the restriction pattern of vesicle-associated DNA was complex and similar to that of total genomic DNA. With such an analysis, we were not able to detect any difference in nucleic acid composition between the two bands recovered from CsCl gradients. The size of the DNA associated with the vesicles was also analyzed by pulse-field gel electrophoresis and turned out to be around 20 kb, which is similar to the size of genomic DNA fragments that we prepared directly from lysed cells by standard purification procedures (data not shown). We concluded from these experiments that extracellular genomic DNA from T. gammatolerans was present in our purified vesicle preparation. However, we could not determine if the DNA was located inside the vesicles, strongly bound to their surface, or else trapped in cluster of vesicles that were only disrupted by SDS and proteinase K treatment. To test these possibilities, we first added one centrifugation step at 20,000 g after the first one at 5,000 g to remove as much cell debris as possible. We also increased the concentration of micrococcal nuclease between the two CsCl
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Fig. 3. Electron micrographs of filamentous structures. (aed) String of pearls filamentous structures. (eel) Other filamentous structures. (a) T. nautilus 30/1. (b) P. horikoschii. (c) Thermococcus strain 24/3A. (d) Thermococcus strain 31/3. (e, i) Thermococcus strain 23/200 . (f) Thermococcus strain 23/4. (g) Thermococcus strain 5/4. (h) Thermococcus strain 9/3. (j) Pyrococcus strain 32/4. (k, l) Thermococcus strain 31/3. (a, c, l) Vesicles were purified by ultracentrifugation at 27,500 g. (b, i) Vesicles were purified by ultracentrifugation at 80,000 g followed by CsCl gradient centrifugation. (d, j, k) Vesicles were purified by ultracentrifugation at 110,000 g. (e, f, g) Vesicles were purified by ultracentrifugation at 80,000 g. Scale bars: 100 nm.
gradients. We found that DNA was still present in similar amounts in vesicle preparations after treatment with 5 mg/ml of nuclease, but only in very low amounts after treatment with 100 mg/ml. Examination by TEM of vesicle preparations treated by high nuclease concentrations revealed high amounts of purified and apparently intact vesicles. We conclude that most DNA associated with purified vesicles is probably located at their surface or trapped into vesicle clusters. However, it cannot be excluded that a small subset of vesicles contained DNA, or that micrococcal nuclease at such a high concentration (100 mg/ml) can penetrate vesicles. In comparison, chromosomal DNA prepared with the standard procedure was completely degraded with 0.1 mg/ml of the enzyme. We then looked for the presence of DNA in vesicles from T. kodakaraensis, whose genome contains four integrated
putative viruses (TKV1eV4) [8]. Vesicles were purified by centrifugation on CsCl gradient as described for T. gammatolerans. We obtained only one band of purified vesicles from T. kodakaraensis after the first CsCl gradient and those purified vesicles did not contain any DNA. Besides their different shape, the presence of DNA in vesicle preparations from T. gammatolerans and its absence in vesicle preparations from T. kodakaraensis confirm that vesicles from closely related species can exhibit striking variability. We then tested for the presence of DNA in vesicles prepared from Thermococcus nautilus that harbors two extrachromosomal elements, pTN1 [31] and pTN2 (unpublished data). We again obtained only one band of material containing vesicles after CsCl gradient centrifugation. Interestingly, this material contained chromosomal DNA, as well as pTN1 and pTN2 DNA that can be
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Fig. 4. Analysis of DNA associated with purified vesicles produced by T. gammatolerans. (a) Micrograph of the bands obtained by CsCl gradient centrifugation of vesicles from T. gammatolerans supernatants. (b) Electrophoretic analysis on agarose gel of constituents of the two bands indicated by arrows in (a). (1, 2) Upper band. (3, 4) Lower band. (1, 3) 20 ml of the band material. (2, 4) 20 ml of the band material after lysis with SDS at 65 C as described in Section 2. (5) Smart ladder (Eurogentec). (6) Supercoiled DNA ladder 2e10 kb (Promega). (c) Electrophoretic analysis of restricted DNA. (1) Smart ladder (Eurogentec). (2, 4) DNA from purified vesicle preparations (after two CsCl runs). (3, 5) Total genomic DNA digestion. (2, 3) KpnI digestion. (4, 5) ScaI digestion.
visualized on agarose gels. However, this DNA was completely eliminated when vesicles were treated with 0.1 mg/ml of micrococcal nuclease. We finally screened eight other strains for the presence of DNA in vesicle preparations. We found that extracellular DNA was always present in culture supernatants and often recovered in the pellet after the ultracentrifugation steps. In two cases, DNA was also recovered in vesicle preparations after CsCl centrifugation. However, this DNA did not produce a single band, but rather, either a smear or no material visible in agarose gel after SDS and proteinase K treatment. These observations suggest that most actively growing cells of Thermococcales produce both vesicles and extracellular DNA that can be more or less strongly associated with the vesicles, depending on the strain. Finally, we conclude that most vesicles (possibly all) did not contain bona fide intravesicular DNA. Several authors have described membrane vesicles containing chromosomal and/or plasmidic DNA in various bacterial species [5,15,16,38,28]. Some reports even suggested that these vesicles can be used as carriers for genetic transformation and as possible agents of horizontal gene transfer [4,5,15,38]. However, a more recent publication reported negative results for transformation [28]. In those papers, most authors purified their vesicles without a CsCl centrifugation step and used classical DNase I treatment. In these conditions, it is clear from our experience that it is impossible to conclusively determine whether the DNA reported to be associated with vesicles was indeed located inside the vesicles, strongly bond to vesicles or trapped in vesicle clusters. 3.3. Observation of extracellular DNA by epifluorescence microscopy The rapid detection and quantification of viruses in the environment is usually performed by epifluorescence microscopy using a specific DNA dye (i.e. SYBR green or Hoechst)
to stain small particles containing DNA (assumed to be viruses) [25]. We decided to use this technique to determine whether the DNA associated with vesicles of T. gammatolerans and T. nautilus appeared as isolated virus-like brightly stained dots. Indeed, we could detect a huge amount of fluorescent dots by SYBR green staining in culture supernatants of T. nautilus that produced vesicles associated with DNA (see Fig. 5a,b). This indicates that DNA was not diluted in culture supernatants but was associated with isolated structures, most likely single vesicles or clusters of such vesicles. We also analyzed purified vesicles preparations of T. kodakaraensis and T. gammatolerans by epifluorescence microscopy using Hoechst dye. Vesicles of T. gammatolerans that are associated with DNA indeed appeared as many bright spots, whereas no spots could be detected in preparations of vesicles from T. kodakaraensis that are not associated with DNA (Fig. 5c,d). Whereas these observations are in agreement with our previous conclusion, they are also troublesome, since they indicates that vesicles can be easily confused with viruses in screenings procedures that use epifluorescence microscopy. Estimation of virus abundance is now part of all global ecological studies [32,36]. It has been repeatedly claimed over the last 15 years that viruses constitute the largest part of the biomass in both aquatic and terrestrial environments (see [7,32]). These estimations nearly always rely on visualization of viruses by epifluorescence microscopy [23,25,32]. It is usually assumed that all fluorescent particles of small size correspond to a DNA virus. However, our observations indicate that such fluorescent particles could also correspond to DNA associated with membrane vesicles. Indeed, membrane vesicles seem to be produced by most bacteria and have been detected in natural environments, including biofilms [30]. Furthermore, high amounts of extracellular DNA have been also detected in various environments (for review, see [35]) and could be involved in biofilm formation [37]. This suggests that the abundance of viruses in the environment might have been overestimated in
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Fig. 5. Epifluorescence analysis of vesicle-containing fractions. (a, b) Microscopic analysis of supernatants of T. nautilus 30/1 culture by epifluorescence staining using SYBR green I dye. Small bar represents 1 mm and large bar represents 10 mm. (b) is a magnification of part of the (a) micrograph. (c, d) Microscopic analysis of vesicle preparations from cultures of T. kodakaraensis (c) or T. gammatolerans (d) by epifluorescence staining using Hoechst dye. Bar represents 1 mm.
some studies. Our finding that cellular DNA can be strongly bound to vesicles with sizes in the range of 50e200 nm also suggests that DNA assumed to be of viral origin in some metagenomic studies could be contaminated by cellular DNA. It should therefore be very useful for designing a general strategy to discriminate between viruses and vesicles in ecological surveys. However, this might prove to be difficult considering the heterogeneity of viruses and virus-like vesicles. Furthermore, the situation might be different from one environment to another. For instance, it might be easier to discriminate between head and tailed viruses and bacterial membrane vesicles in aquatic environments, considering the typical morphology of caudavirales and the absence of lipids in their virions. In contrast, it might be more difficult to discriminate between spherical viruses and vesicles in other environments, such as terrestrial hot springs. Proof of principle analysis has shown a good correlation between direct epifluorescent counts and transmission electron microscopy counts of head and tailed particles in an aquatic environment [12], suggesting that vesicles might be scarce in such biotopes, but this might not be the case in other environments. In any case, it will be important in the future to determine whether virus-like vesicles are indeed present in various natural environments (including aquatic ones), to determine their nature and to quantify their abundance. 3.4. Protection of vesicle-associated DNA against thermodenaturation Extracellular DNA present in the environment is more resistant to enzymatic degradation than free DNA, but the reason for this increase in stability is unclear (for review, see [35]). It
has been suggested that DNA is stabilized by absorption onto minerals or to unspecified ‘‘organic matter’’. In this work, we observed that DNA associated with the vesicles produced by Thermococcales is more resistant to micrococcal nuclease than free DNA. This suggests that the association of extracellular DNA to vesicles could be an important factor protecting it from degradation. Since the vesicles observed in this work are produced by hyperthermophilic archaea, it was interesting to determine whether they could also protect DNA against thermodenaturation. Indeed, it is unclear how stable extracellular DNA can exist in hydrothermal environments. Topologically closed DNA (e.g. plasmid) is stable at temperatures typical for hyperthermophiles (at least up to 107 C) but linear DNA fragments are denatured at 70e80 C [19]. We incubated vesicle preparations of T. gammatolerans (collected after the first ultracentrifugation step) at 90 C from 5 to 20 min and loaded then on an agarose gel. As shown in Fig. 6a, most DNA associated with vesicles remained in the wells and was still present in similar amounts after 20 min of incubation at 90 C. In contrast, free chromosomal DNA was rapidly degraded into smaller pieces (producing smears) and had nearly completely disappeared after 5 min at 90 C. We performed a similar experiment with a preparation of T. nautilus vesicles (Fig. 6b). In that case, most DNA associated with the vesicles again remained in the well with the vesicles, but a small portion was released and migrated as a single diffuse band slightly above the position of purified T. nautilus chromosomal DNA. As shown in Fig. 6b, the amount of both DNA associated with vesicles and released DNA running in the gel remained stable after incubation for 15 min at 90 C, (lane 4), whereas genomic DNA was completely denatured after only 5 min (lane 5). This result clearly indicates that extracellular
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Fig. 6. Thermoprotection of DNA in the presence of vesicle preparations. (a) Total DNA (2e5) and vesicle preparation (6e9) of T. gammatolerans. Samples were respectively incubated 0 min (2 and 6), 1 min (3 and 7), 3 min (4 and 8) or 5 min (5 and 9) at 90 C. (1) Smart ladder (Eurogentec). (b) Vesicle preparation of T. nautilus 30/1. (1, 2, 3, 4) Samples were respectively incubated 0, 5, 10 and 15 min at 90 C.
DNA can be protected at high temperature environments via its association with vesicles produced by hyperthermophilic archaea. It is tempting to speculate that such stabilized extracellular DNA could be involved in lateral gene transfer between hyperthermophiles. Indeed, the size of DNA fragments associated with vesicles (around 20 kb) is compatible with the idea that such extracellular DNA can be involved in natural transformation. In conclusion, we have shown here that most Thermococcales strains produce virus-like vesicles of various shapes, including unusual filamentous structures. This is reminiscent of membrane vesicles production by many bacterial species. Eukaryotic cells also produce vesicles that have been called exosomes [33]. It will be interesting to determine if archaeal, eukaryal and bacterial vesicles are analogous or homologous structures. In the latter case, vesicle production might be an ancient feature of cells and possibly already occurred in LUCA (the last universal common ancestor) [6]. It would also be interesting to investigate the possibility that such ubiquitous vesicles have been labeled ‘‘nanobacteria’’ in some studies [34]. From a survey of vesicle structures in Archaea, it seems that vesicle structure and production exhibit both features that are conserved in widely divergent species (e.g. string of pearl structures in some Thermococcales and in A. boonei) and features that could differ between closely related strains or species. Further work is now required to determine the biochemical content of vesicles and the nature of their association with DNA, and to obtain a better appreciation of their abundance and role in natural environments. Acknowledgments We thank Claire Geslin and Me´lusine Gaillard for help in the epifluorescent experiments, and Je´ril De´grouard and Danielle Jaillard from the ‘‘Centre Commun de Microscopie Electronique’’ (CCME) of the Universite´ Paris-Sud for their support in electron microscopy analyses. We are grateful to Florence Lorieux for technical help in some experiments. We thank David Prangishvili and Simonetta Gribaldo for English corrections and critical reading of the manuscript. This work would not
have been possible without the participation of one of us (E.M.) in the AMISTAD expedition organized by the CNRS (Centre National de la Recherche Scientifique) and the IFREMER (Institut Franc¸ais de Recherche et d’Exploitation de la Mer). This work was supported by the ACI-BCNS150 grant, the Agence Nationale de la Recherche with grant GIS Ge´nomique Marine (ANR-05-BLAN-0408-01), and by the OPV CNRS program (Origine des plane`tes et de la vie). NS was supported by a PhD grant from the Ministe`re de l’Enseignement supe´rieur et de la Recherche. References [1] Akita, F., Chong, K.T., Tanaka, H., Yamashita, E., Miyazaki, N., Nakaishi, Y., Suzuki, M., Namba, K., Ono, Y., Tsukihara, T., Nakagawa, A. (2007) The crystal structure of a virus-like particle from the hyperthermophilic archaeon Pyrococcus furiosus provides insight into the evolution of viruses. J. Mol. Biol. 368, 1469e1483. [2] Beveridge, T.J. (1999) Structures of gram-negative cell walls and their derived membrane vesicles. J. Bacteriol. 181, 4725e4733. [3] Breitbart, M., Wegley, L., Leeds, S., Schoenfeld, T., Rohwer, F. (2004) Phage community dynamics in hot springs. Appl. Environ. Microbiol. 70, 1633e1640. [4] Chiura, H.X. (2002) Broad host range xenotrophic gene transfer by viruslike particles from a hot spring. Microb. Environ. 17, 53e58. [5] Dorward, D.W., Garon, C.F., Judd, R.C. (1989) Export and intercellular transfer of DNA via membrane blebs of Neisseria gonorrhoeae. J. Bacteriol. 171, 2499e2505. [6] Forterre, P., Gribaldo, S. (2007) The origin and evolution of terrestrial life. HFSP J 1, 156e168. [7] Fuhrman, J.A. (1999) Marine viruses and their biogeochemical and ecological effects. Nature 399, 541e548. [8] Fukui, T., Atomi, H., Kanai, T., Matsumi, R., Fujiwara, S., Imanaka, T. (2005) Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes. Genome Res. 15, 352e363. [9] Geslin, C., Le Romancer, M., Erauso, G., Gaillard, M., Perrot, G., Prieur, D. (2003) PAV1, the first virus-like particle isolated from a hyperthermophilic euryarchaeote, ‘‘Pyrococcus abyssi. J. Bacteriol. 185, 3888e3894. [10] Geslin, C., Le Romancer, M., Gaillard, M., Erauso, G., Prieur, D. (2003) Observation of virus-like particles in high temperature enrichment cultures from deep-sea hydrothermal vents. Res. Microbiol. 154, 303e307. [11] Geslin, C., Gaillard, M., Flament, D., Rouault, K., Le Romancer, M., Prieur, D., Erauso, G. (2007) Analysis of the first genome of
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Virus-like vesicles and extracellular DNA produced by hyperthermophilic archaea of the order Thermococcales Nicolas Soler a,1, Evelyne Marguet b, Jean-Marc Verbavatz b, Patrick Forterre a,* a
Univ Paris-Sud, Institut de Ge´ne´tique et Microbiologie, CNRS, UMR 8621, F-91405 Orsay Cedex, France b IBITEC-S and CNRS URA 2096, Bat. 532, CEA-Saclay, F-91191 Gif-sur-Yvette, France Received 4 April 2008; accepted 14 April 2008 Available online 25 June 2008
Abstract Cultures of hyperthermophilic archaea (order Thermococcales) have been analyzed by electron microscopy and epifluorescence staining for the presence of virus-like particles. We found that most strains of Thermococcus and Pyrococcus produce various types of spherical membrane vesicles and unusual filamentous structures. Cellular DNA can be strongly associated with vesicles and appears as fluorescent dots by epifluorescence microscopy, suggesting that some particles assumed to be viruses in ecological studies might instead be vesicles associated with extracellular DNA. DNA in vesicle preparations is remarkably resistant to DNase treatment and thermodenaturation, indicating that association with vesicles could be an important factor determining DNA stability in natural environments. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Membrane vesicles; Thermococcales; Archaea; Hyperthermophiles; Viral ecology; Extracellular DNA; DNA resistance; Nanobacteria
1. Introduction Many new families of DNA viruses have been described that infect aerobic hyperthermophilic crenarchaea of the genera Sulfolobus and Acidianus from terrestrial hot springs [27]. These viruses exhibit unique morphologies and are strikingly different from both viruses infecting bacteria (bacterioviruses) and viruses infecting eukarya (eukaryoviruses). Virus-like particles with similar unique morphologies have also been observed in enrichment cultures of anaerobic hyperthermophilic archaea from deep-sea vents (probably euryarchaea of the order Thermococcales) [10]. However, only one archaeovirus infecting a hyperthermophilic euryarchaeon has been successfully isolated and characterized up to now, the virus PAV1 of Pyrococcus abyssi, strain GE23 [9,11]. To search
* Corresponding author. E-mail addresses: [email protected] (N. Soler), evelyne. [email protected] (E. Marguet), [email protected] (J.-M. Verbavatz), [email protected] (P. Forterre). 1 Present address: UMR2027 CNRS/Institut Curie, Universite´ Paris-Sud, Baˆtiment 110, 91405 Orsay Cedex, France. 0923-2508/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2008.04.015
for additional archaeoviruses from euryarchaeotes of the order Thermococcales (genera Pyrococcus, Thermococcus and Paleococcus), we have recently set up a collection of 70 Thermococcales strains obtained from several deep-sea hydrothermal vents located in the East-Pacific ridge [18]. About half of these strains contain putative extrachromosomal elements (either plasmids or viruses) [18]. We previously described one of these elements, the rolling-circle plasmid pTN1 from the candidate species Thermococcus nautilus [31]. In the present work, we report the results of our screening for viruses in strains in which extrachromosomal elements were previously detected. Since it is not possible to obtain lawns of Thermococcus cells, viruses cannot be screened by standard plaque formation assay. We thus directly looked for virus-like particles in culture supernatants by electron microscopy. This strategy has been successfully used in the past to isolate viruses from Sulfolobus species [39]. Previous works have indeed shown that most viruses from hyperthermophilic archaea live in a carrier state inside their host, and are regularly produced and excreted outside the cell [27]. Although we were unable to isolate viruses in this work, we were surprised to observe that most of the tested strains produce virus-like vesicles of various
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structures, including long flexible filaments. Some of these virus-like vesicles strikingly resemble membrane vesicles produced by bacterial species [2,17,20], suggesting that production of membrane vesicles can be a widespread feature of the microbial world. We observed that cellular DNA can be strongly associated with vesicles from Thermococcales and appear as fluorescent dots (virus-like) by epifluorescence microscopy. Interestingly, DNA present in vesicle preparation is highly resistant to DNase treatment and heat denaturation, suggesting that association with vesicles could be an important factor determining the stability of DNA in the environment. 2. Materials and methods 2.1. Strain cultivation Thermococcales strains were grown anaerobically in penicillin bottles for 50-ml cultures, and in Wheaton bottles for 200-ml cultures in Zillig’s broth [18]. Cultures were inoculated at 1e4% (v/v) from pure cultures isolated during the AMISTAD cruise for strains of our collection [18], or from pure cultures of type strains. Incubations were performed at 85 C overnight (15e17 h). 2.2. Vesicle preparations A 50-ml culture (stationary phase) of each strain was centrifuged at 5000 g for 20 min to remove cells. Supernatants were collected and ultracentrifuged using Nalgene tubes at 27,500 g for 3 h at 20 C in a Beckman JA17 rotor. The pellet was then resuspended with a few droplets of culture medium and analyzed by TEM. In some experiments (indicated in figure legends), a 2 h centrifugation run was performed at 110,000 g instead of 27,500 g using a SW41 Beckman rotor. For preparation of Thermococcus kodakaraensis and Thermococcus gammatolerans vVirus-Like-Particles (VLPs) 200- to 600-ml cultures in stationary phase were centrifuged at 5000 g to eliminate cells. The supernatants were then ultracentrifuged at 80,000 g for 2 h with a SW28 Beckman rotor. White pellets were resuspended in 2e6 ml of Mu buffer (200 mM NaCl, 20 mM TriseHCl pH 7.5, 1 mM CaCl2, 20 mM MgSO4), and 0.5 g/ml CsCl was added for equilibration on a centrifugation gradient at 213,000 g for 40 h with a TLS-55 Beckman rotor. White bands were recovered and dialyzed against Mu buffer to eliminate CsCl. For T. gammatolerans, these samples were submitted to micrococcal DNase digestion at 0.1 mg/ml final concentration for 1 h at 37 C. A second CsCl centrifugation gradient was then performed under the same conditions as described for the first one, in order to eliminate DNase and remaining cell debris. 2.3. Transmission electronic microscopy observations A 20-ml droplet sample was spotted onto a carbon-coated nickel grid (400 mesh, Euromedex). After 1 min, the excess liquid was removed with filter paper (Whatman) and the grid
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was rinsed with a droplet of ultrapure H2O. Negative staining was obtained by addition of a 2% (w/v) uranyl acetate droplet on the grid for 1 min, and the excess was removed again. The grid was then observed using a Philips EM208 electron microscope operated at 80 keV, coupled with an AMT CCD camera (Hamamatsu). 2.4. Analysis of DNA associated with vesicle preparations To extract DNA from vesicles, lysis buffer (50 mM Trise HCl pH 7.5, 2 mM EDTA pH 7.5, 1% SDS, 13.7% saccharose, 0.01% bromophenol blue) was added to vesicle preparations (v/v) and incubated for 10 min at 65 C, before loading on agarose gel. 2.5. DNA extraction and endonuclease restriction Total genomic DNA was purified from 50 ml culture (stationary phase) of T. gammatolerans. Cells were lysed in TENST buffer (10 mM TriseHCl pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.12% Triton X-100, 1,6% SDS) and DNA was extracted by standard procedure (phenol/chloroform extraction, isopropanol precipitation). The dried pellet was dissolved with 25 ml H2O. DNA (2e3 mg) was digested for 1 h at 37 C by the indicated restriction enzyme (Promega, 5 units). The mixture was then electrophoresed on a 0.7% agarose gel in TriseBorateeEDTA (TBE) buffer for 16 h at 1.75 V/cm, and the gel was stained with an ethidium bromide solution. 2.6. Epifluorescence analysis using Hoechst dye Vesicle samples (40 ml) were loaded on SuperFrostÒPlus slides for 5 min. After excess removal, 40 ml Hoechst dye solution (1 mg/ml) was applied to the surface. Anti-fading solution was then added and coverglasses were mounted for observation under an Olympus Vanox AH2 microscope under UV illumination. 2.7. Epifluorescence analysis using SYBR Green I All solutions used for this protocol were filtered with 0.45, 0.2, and finally 0.02 mm filters. Thermococcales culture (1 ml) was centrifuged at 10,000 g for 20 min to eliminate cells and 500 ml supernatant were mixed with 4.5 ml ASW (Artificial Sea Water). This mixture was double filtered at 0.8/0.2 mm (AcrodiscÒ PF Syringe Filter, Pall Corporation), and then particles smaller than 0.2 mm were recovered on a 0.02 mm filter (Anodisc 25, AnoporeÒ, Whatman). In the dark, 100 ml of 10 SYBR Green I solution (diluted in H2O from 10,000, Molecular Probes) were applied to the filter for 1e1.5 h. The filter was then rinsed with H2O in a small glass Petri dish for 30 min and dried for 30 min at 37 C. Still in the dark, the filter was placed on a slide, and mounted with anti-fading solution (100 ml of 0.1% N,N-dimethhy-1,4-phenylenediamine sulfate) and a coverglass. After 30 min incubation in the
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dark, the samples were observed under the microscope with UV illumination (475 nm excitation). 2.8. Thermoprotection of DNA in the presence of vesicle preparations Samples (15 ml) were covered with mineral oil (15 ml) and incubated for the indicated time at 90 C. 12.5 ml was then recovered for electrophoretic analysis at 16 V/cm for 30 min on a 0.7% agarose gel in TBE 0.5. The gel was stained with a 1 mg/ml ethidium bromide solution. 3. Results and discussion 3.1. Production of membrane vesicles and related filamentous structures by Thermococcales We screened 34 strains from our collection of Thermococcales that harbor putative extrachromosomal elements [18] for the presence of viruses by transmission electron microscopy (TEM). Fifty milliliters of culture in stationary phase were centrifuged at 5000 g for 20 min to remove cells, and the supernatant was centrifuged for 3 h at 27,500 g to pellet putative virions. The pellets were suspended in 500 ml buffer solution and droplets were mounted on a grid and stained with uranyl acetate for examination. Surprisingly, we found that most strains (26 out of 34) produce various types of spherical vesicles (see Fig. 1 for examples). The same types of vesicles were also observed in pellets obtained after centrifugation of PEG precipitates from culture supernatants. These vesicles were usually very abundant and sometimes clustered around membranous cell debris (Fig. 2). The diameters of these vesicles were in the range of 50e100 nm. Spherical vesicles observed in this work did not resemble most known archaeoviruses [27] or virus-like particles previously described in enrichment cultures of Thermococcales [10], but some of them were strikingly similar to a round particle described as a virus from terrestrial hot springs by Rohwer and co-workers (see figure 4, bottom left in Breitbart et al. [3]) or resembled globulovirus PSV infecting the hyperthermophilic crenarchaeotes Pyrobaculum and Thermoproteus [13]. However, the sizes of these vesicles were usually heterogeneous (Fig. 1aef), which is unexpected for viral populations. Vesicles observed in this work were also clearly different from empty viral capsids previously detected in cultures of Pyrococcus furiosus (approximately 30 nm in diameter and of homogeneous sizes) [1,22]. In fact, most of them were composed of a spherical core surrounded by a membrane resembling archaeal S-layers (Fig. 1a,d,e). In several strains, we also observed filaments or short rods that looked like strings of pearls surrounded by a membrane (Fig. 3). Vesicles appear to bud from one of the two extremities of these structures as if the pearls were the progenies of the vesicle spherical core. We also detected a few cells that contained internal filamentous structures (Fig. 2b). In other strains, we observed continuous tubular structures, also surrounded by a membrane and apparently
producing vesicles at one of their extremities. In two strains, a majority of these tubular structures was mixed with rounded vesicles. These observations strongly suggest that the vesicles observed are not artifacts produced during cell lysis by collapsing membranes, but are instead biological structures that are actively produced by cells. To determine whether the production of vesicles was a specificity of strains from our collection, we also looked for their presence in four type strains of Thermococcales currently used in our laboratory, Thermococcus kodakaraensis, Thermococcus gammatolerans, Pyrococcus abyssi and Pyrococcus horikoshii. We found that all these strains produce vesicles similar to those produced by strains in our collection (see Fig. 1d,gej). It is remarkable that vesicles produced by T. kodakaraensis and T. gammatolerans clearly differed in their appearance, with vesicles from T. gammatolerans being homogeneous in size, whereas those from T. kodakaraensis were heterogeneous (Fig. 1gej). Preliminary analyses also suggested that vesicles from these two species have different lipid and protein composition (data not shown). This illustrates the variability of vesicle shapes and indicates that this feature might be used in strain (species) description. The presence of vesicles in cultures of various thermophilic archaea has been previously described, albeit only incidentally. Prangishvili et al. [26] first reported that sulfolobucin, a colicin-like compound produced by some Sulfolobus species, is associated during purification with vesicles very similar to some of those described in this report. Vesicles were also recently observed in cultures of the thermoacidophilic archaeon Aciduliprofundum boonei (see figure 2d in Reysenbach et al. [29]) and in the periplasmic space of the hyperthermophile Ignicoccus profundus (the only known archaeon with an outer membrane) [24]. All these vesicles resemble those produced by Thermococcales. In particular, an unusual structure very similar to the ‘‘string of pearls’’ observed in this work (several rounded structures surrounded by a membrane) was observed in A. boonei (see figure 2d in Reysenbach et al. [29]). These data suggest that the production of vesicles is a widely distributed feature in thermophilic archaea and could involve a conserved mechanism for their production. However, as previously mentioned, the size and shape of the vesicles also differ between closely related strains of Thermococcales, indicating that some aspects of their production might be strainspecific. Interestingly, it has been shown for a long time that many species of bacteria produce large numbers of vesicles surrounded by an outer membrane layer [2,17,20,30]. These membrane vesicles can carry various compounds such as hydrolases, toxins, antibiotics and quorum sensing factors. Recent data have shown that vesicle production is indeed a major aspect of the global response to environmental stress [21]. Stressed bacteria overproduce vesicles that are apparently used to selectively eliminate unwanted material (e.g. unfolded proteins). Based on these previous observations, we reasoned that most (possibly all) virus-like vesicles observed in this work were not viruses but vesicles of cellular origin.
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Fig. 1. Electron micrographs of vesicles purified from different Thermococcales strains (type strains and strains from our collection). (a) Thermococcus strain 9/3. (b) Thermococcus strain 29/2. (c) Thermococcus strain 28/4. (d) P. horikoschii. (e) Thermococcus strain 29/3. (f) T. nautilus 30/1. (g, h) T. kodakaraensis. (i, j) T. gammatolerans. (a, b, c, e, f) Vesicles purified by ultracentrifugation at 27,500 g. (d, gej) Vesicles purified by ultracentrifugation at 80,000 g followed by CsCl gradient centrifugation. Scale bars: 100 nm.
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Fig. 2. Electron micrographs of vesicles associated with cells or cell debris. (a) T. gammatolerans. (b) T. nautilus strain 30/1. (c) Pyrococcus strain 12/1. (d) P. horikoschii. Scale bars: 100 nm.
3.2. Extracellular DNA can be strongly associated with virus-like vesicles from Thermococcales Since some spherical viruses resemble vesicles, it was possible that a subset of vesicles produced by Thermococcales were indeed yet unidentified viruses. We tested this possibility for Thermococcus gammatolerans [14], which was the only strain in our hands that reproducibly produced virus-like vesicles of homogeneous size (Fig. 1i,j). The genome of T. gammatolerans has been recently sequenced (F. Confalonieri and Y. Zivanovic, personal communication) and contains an integrated element that is homologous to the putative viruses TKV2 and TKV3 of T. kodakaraensis [8]. We reasoned that the virus-like vesicles of T. gammatolerans could be bona fide virions produced from this integrated element. To test this hypothesis, vesicles of T. gammatolerans were prepared as previously described from 200 to 500 ml cultures. Cells and cell debris were removed by centrifugation at 5000 g and vesicles were pelleted by ultracentrifugation at 80,000 g. Vesicles were then centrifuged onto equilibrium cesium chloride (CsCl) gradients. We observed two close bands of visible material with a density of about 1.35 g/cm3 (Fig. 4a). The bands were collected and TEM analysis confirmed the presence of vesicles. However, the thinner upper vesicle band preparation also contained flagella and some cell debris. The material from each band was then treated with micrococcal nuclease (0.1 mg/ml 30 min at 37 C). The nuclease was inactivated by treatment with 25 mM EGTA and heating for 10 min at 70 C, and vesicles from each band were separately subjected to a second round of centrifugation in CsCl gradient. Agarose gel electrophoresis of
purified vesicles followed by staining with ethidium-bromide allowed detection of nucleic acids that remained in the well, suggesting the presence of DNA associated with material that could not enter into the gel (Fig. 4b). Interestingly, this DNA migrated as a single sharp band in the gel after treatment of vesicles by SDS, heating and proteinase K (believed to break down putative viral capsids or to open membrane vesicles) (Fig. 4b). This result a priori suggested that virus-like vesicles from T. gammatolerans were the virions of bona fide DNA viruses. DNA was then phenol-extracted from the purified vesicle preparation and digested with KpnI or ScaI (Fig. 4c). As shown in Fig. 4, and in contrast to our expectations, the restriction pattern of vesicle-associated DNA was complex and similar to that of total genomic DNA. With such an analysis, we were not able to detect any difference in nucleic acid composition between the two bands recovered from CsCl gradients. The size of the DNA associated with the vesicles was also analyzed by pulse-field gel electrophoresis and turned out to be around 20 kb, which is similar to the size of genomic DNA fragments that we prepared directly from lysed cells by standard purification procedures (data not shown). We concluded from these experiments that extracellular genomic DNA from T. gammatolerans was present in our purified vesicle preparation. However, we could not determine if the DNA was located inside the vesicles, strongly bound to their surface, or else trapped in cluster of vesicles that were only disrupted by SDS and proteinase K treatment. To test these possibilities, we first added one centrifugation step at 20,000 g after the first one at 5,000 g to remove as much cell debris as possible. We also increased the concentration of micrococcal nuclease between the two CsCl
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Fig. 3. Electron micrographs of filamentous structures. (aed) String of pearls filamentous structures. (eel) Other filamentous structures. (a) T. nautilus 30/1. (b) P. horikoschii. (c) Thermococcus strain 24/3A. (d) Thermococcus strain 31/3. (e, i) Thermococcus strain 23/200 . (f) Thermococcus strain 23/4. (g) Thermococcus strain 5/4. (h) Thermococcus strain 9/3. (j) Pyrococcus strain 32/4. (k, l) Thermococcus strain 31/3. (a, c, l) Vesicles were purified by ultracentrifugation at 27,500 g. (b, i) Vesicles were purified by ultracentrifugation at 80,000 g followed by CsCl gradient centrifugation. (d, j, k) Vesicles were purified by ultracentrifugation at 110,000 g. (e, f, g) Vesicles were purified by ultracentrifugation at 80,000 g. Scale bars: 100 nm.
gradients. We found that DNA was still present in similar amounts in vesicle preparations after treatment with 5 mg/ml of nuclease, but only in very low amounts after treatment with 100 mg/ml. Examination by TEM of vesicle preparations treated by high nuclease concentrations revealed high amounts of purified and apparently intact vesicles. We conclude that most DNA associated with purified vesicles is probably located at their surface or trapped into vesicle clusters. However, it cannot be excluded that a small subset of vesicles contained DNA, or that micrococcal nuclease at such a high concentration (100 mg/ml) can penetrate vesicles. In comparison, chromosomal DNA prepared with the standard procedure was completely degraded with 0.1 mg/ml of the enzyme. We then looked for the presence of DNA in vesicles from T. kodakaraensis, whose genome contains four integrated
putative viruses (TKV1eV4) [8]. Vesicles were purified by centrifugation on CsCl gradient as described for T. gammatolerans. We obtained only one band of purified vesicles from T. kodakaraensis after the first CsCl gradient and those purified vesicles did not contain any DNA. Besides their different shape, the presence of DNA in vesicle preparations from T. gammatolerans and its absence in vesicle preparations from T. kodakaraensis confirm that vesicles from closely related species can exhibit striking variability. We then tested for the presence of DNA in vesicles prepared from Thermococcus nautilus that harbors two extrachromosomal elements, pTN1 [31] and pTN2 (unpublished data). We again obtained only one band of material containing vesicles after CsCl gradient centrifugation. Interestingly, this material contained chromosomal DNA, as well as pTN1 and pTN2 DNA that can be
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Fig. 4. Analysis of DNA associated with purified vesicles produced by T. gammatolerans. (a) Micrograph of the bands obtained by CsCl gradient centrifugation of vesicles from T. gammatolerans supernatants. (b) Electrophoretic analysis on agarose gel of constituents of the two bands indicated by arrows in (a). (1, 2) Upper band. (3, 4) Lower band. (1, 3) 20 ml of the band material. (2, 4) 20 ml of the band material after lysis with SDS at 65 C as described in Section 2. (5) Smart ladder (Eurogentec). (6) Supercoiled DNA ladder 2e10 kb (Promega). (c) Electrophoretic analysis of restricted DNA. (1) Smart ladder (Eurogentec). (2, 4) DNA from purified vesicle preparations (after two CsCl runs). (3, 5) Total genomic DNA digestion. (2, 3) KpnI digestion. (4, 5) ScaI digestion.
visualized on agarose gels. However, this DNA was completely eliminated when vesicles were treated with 0.1 mg/ml of micrococcal nuclease. We finally screened eight other strains for the presence of DNA in vesicle preparations. We found that extracellular DNA was always present in culture supernatants and often recovered in the pellet after the ultracentrifugation steps. In two cases, DNA was also recovered in vesicle preparations after CsCl centrifugation. However, this DNA did not produce a single band, but rather, either a smear or no material visible in agarose gel after SDS and proteinase K treatment. These observations suggest that most actively growing cells of Thermococcales produce both vesicles and extracellular DNA that can be more or less strongly associated with the vesicles, depending on the strain. Finally, we conclude that most vesicles (possibly all) did not contain bona fide intravesicular DNA. Several authors have described membrane vesicles containing chromosomal and/or plasmidic DNA in various bacterial species [5,15,16,38,28]. Some reports even suggested that these vesicles can be used as carriers for genetic transformation and as possible agents of horizontal gene transfer [4,5,15,38]. However, a more recent publication reported negative results for transformation [28]. In those papers, most authors purified their vesicles without a CsCl centrifugation step and used classical DNase I treatment. In these conditions, it is clear from our experience that it is impossible to conclusively determine whether the DNA reported to be associated with vesicles was indeed located inside the vesicles, strongly bond to vesicles or trapped in vesicle clusters. 3.3. Observation of extracellular DNA by epifluorescence microscopy The rapid detection and quantification of viruses in the environment is usually performed by epifluorescence microscopy using a specific DNA dye (i.e. SYBR green or Hoechst)
to stain small particles containing DNA (assumed to be viruses) [25]. We decided to use this technique to determine whether the DNA associated with vesicles of T. gammatolerans and T. nautilus appeared as isolated virus-like brightly stained dots. Indeed, we could detect a huge amount of fluorescent dots by SYBR green staining in culture supernatants of T. nautilus that produced vesicles associated with DNA (see Fig. 5a,b). This indicates that DNA was not diluted in culture supernatants but was associated with isolated structures, most likely single vesicles or clusters of such vesicles. We also analyzed purified vesicles preparations of T. kodakaraensis and T. gammatolerans by epifluorescence microscopy using Hoechst dye. Vesicles of T. gammatolerans that are associated with DNA indeed appeared as many bright spots, whereas no spots could be detected in preparations of vesicles from T. kodakaraensis that are not associated with DNA (Fig. 5c,d). Whereas these observations are in agreement with our previous conclusion, they are also troublesome, since they indicates that vesicles can be easily confused with viruses in screenings procedures that use epifluorescence microscopy. Estimation of virus abundance is now part of all global ecological studies [32,36]. It has been repeatedly claimed over the last 15 years that viruses constitute the largest part of the biomass in both aquatic and terrestrial environments (see [7,32]). These estimations nearly always rely on visualization of viruses by epifluorescence microscopy [23,25,32]. It is usually assumed that all fluorescent particles of small size correspond to a DNA virus. However, our observations indicate that such fluorescent particles could also correspond to DNA associated with membrane vesicles. Indeed, membrane vesicles seem to be produced by most bacteria and have been detected in natural environments, including biofilms [30]. Furthermore, high amounts of extracellular DNA have been also detected in various environments (for review, see [35]) and could be involved in biofilm formation [37]. This suggests that the abundance of viruses in the environment might have been overestimated in
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Fig. 5. Epifluorescence analysis of vesicle-containing fractions. (a, b) Microscopic analysis of supernatants of T. nautilus 30/1 culture by epifluorescence staining using SYBR green I dye. Small bar represents 1 mm and large bar represents 10 mm. (b) is a magnification of part of the (a) micrograph. (c, d) Microscopic analysis of vesicle preparations from cultures of T. kodakaraensis (c) or T. gammatolerans (d) by epifluorescence staining using Hoechst dye. Bar represents 1 mm.
some studies. Our finding that cellular DNA can be strongly bound to vesicles with sizes in the range of 50e200 nm also suggests that DNA assumed to be of viral origin in some metagenomic studies could be contaminated by cellular DNA. It should therefore be very useful for designing a general strategy to discriminate between viruses and vesicles in ecological surveys. However, this might prove to be difficult considering the heterogeneity of viruses and virus-like vesicles. Furthermore, the situation might be different from one environment to another. For instance, it might be easier to discriminate between head and tailed viruses and bacterial membrane vesicles in aquatic environments, considering the typical morphology of caudavirales and the absence of lipids in their virions. In contrast, it might be more difficult to discriminate between spherical viruses and vesicles in other environments, such as terrestrial hot springs. Proof of principle analysis has shown a good correlation between direct epifluorescent counts and transmission electron microscopy counts of head and tailed particles in an aquatic environment [12], suggesting that vesicles might be scarce in such biotopes, but this might not be the case in other environments. In any case, it will be important in the future to determine whether virus-like vesicles are indeed present in various natural environments (including aquatic ones), to determine their nature and to quantify their abundance. 3.4. Protection of vesicle-associated DNA against thermodenaturation Extracellular DNA present in the environment is more resistant to enzymatic degradation than free DNA, but the reason for this increase in stability is unclear (for review, see [35]). It
has been suggested that DNA is stabilized by absorption onto minerals or to unspecified ‘‘organic matter’’. In this work, we observed that DNA associated with the vesicles produced by Thermococcales is more resistant to micrococcal nuclease than free DNA. This suggests that the association of extracellular DNA to vesicles could be an important factor protecting it from degradation. Since the vesicles observed in this work are produced by hyperthermophilic archaea, it was interesting to determine whether they could also protect DNA against thermodenaturation. Indeed, it is unclear how stable extracellular DNA can exist in hydrothermal environments. Topologically closed DNA (e.g. plasmid) is stable at temperatures typical for hyperthermophiles (at least up to 107 C) but linear DNA fragments are denatured at 70e80 C [19]. We incubated vesicle preparations of T. gammatolerans (collected after the first ultracentrifugation step) at 90 C from 5 to 20 min and loaded then on an agarose gel. As shown in Fig. 6a, most DNA associated with vesicles remained in the wells and was still present in similar amounts after 20 min of incubation at 90 C. In contrast, free chromosomal DNA was rapidly degraded into smaller pieces (producing smears) and had nearly completely disappeared after 5 min at 90 C. We performed a similar experiment with a preparation of T. nautilus vesicles (Fig. 6b). In that case, most DNA associated with the vesicles again remained in the well with the vesicles, but a small portion was released and migrated as a single diffuse band slightly above the position of purified T. nautilus chromosomal DNA. As shown in Fig. 6b, the amount of both DNA associated with vesicles and released DNA running in the gel remained stable after incubation for 15 min at 90 C, (lane 4), whereas genomic DNA was completely denatured after only 5 min (lane 5). This result clearly indicates that extracellular
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Fig. 6. Thermoprotection of DNA in the presence of vesicle preparations. (a) Total DNA (2e5) and vesicle preparation (6e9) of T. gammatolerans. Samples were respectively incubated 0 min (2 and 6), 1 min (3 and 7), 3 min (4 and 8) or 5 min (5 and 9) at 90 C. (1) Smart ladder (Eurogentec). (b) Vesicle preparation of T. nautilus 30/1. (1, 2, 3, 4) Samples were respectively incubated 0, 5, 10 and 15 min at 90 C.
DNA can be protected at high temperature environments via its association with vesicles produced by hyperthermophilic archaea. It is tempting to speculate that such stabilized extracellular DNA could be involved in lateral gene transfer between hyperthermophiles. Indeed, the size of DNA fragments associated with vesicles (around 20 kb) is compatible with the idea that such extracellular DNA can be involved in natural transformation. In conclusion, we have shown here that most Thermococcales strains produce virus-like vesicles of various shapes, including unusual filamentous structures. This is reminiscent of membrane vesicles production by many bacterial species. Eukaryotic cells also produce vesicles that have been called exosomes [33]. It will be interesting to determine if archaeal, eukaryal and bacterial vesicles are analogous or homologous structures. In the latter case, vesicle production might be an ancient feature of cells and possibly already occurred in LUCA (the last universal common ancestor) [6]. It would also be interesting to investigate the possibility that such ubiquitous vesicles have been labeled ‘‘nanobacteria’’ in some studies [34]. From a survey of vesicle structures in Archaea, it seems that vesicle structure and production exhibit both features that are conserved in widely divergent species (e.g. string of pearl structures in some Thermococcales and in A. boonei) and features that could differ between closely related strains or species. Further work is now required to determine the biochemical content of vesicles and the nature of their association with DNA, and to obtain a better appreciation of their abundance and role in natural environments. Acknowledgments We thank Claire Geslin and Me´lusine Gaillard for help in the epifluorescent experiments, and Je´ril De´grouard and Danielle Jaillard from the ‘‘Centre Commun de Microscopie Electronique’’ (CCME) of the Universite´ Paris-Sud for their support in electron microscopy analyses. We are grateful to Florence Lorieux for technical help in some experiments. We thank David Prangishvili and Simonetta Gribaldo for English corrections and critical reading of the manuscript. This work would not
have been possible without the participation of one of us (E.M.) in the AMISTAD expedition organized by the CNRS (Centre National de la Recherche Scientifique) and the IFREMER (Institut Franc¸ais de Recherche et d’Exploitation de la Mer). This work was supported by the ACI-BCNS150 grant, the Agence Nationale de la Recherche with grant GIS Ge´nomique Marine (ANR-05-BLAN-0408-01), and by the OPV CNRS program (Origine des plane`tes et de la vie). NS was supported by a PhD grant from the Ministe`re de l’Enseignement supe´rieur et de la Recherche. References [1] Akita, F., Chong, K.T., Tanaka, H., Yamashita, E., Miyazaki, N., Nakaishi, Y., Suzuki, M., Namba, K., Ono, Y., Tsukihara, T., Nakagawa, A. (2007) The crystal structure of a virus-like particle from the hyperthermophilic archaeon Pyrococcus furiosus provides insight into the evolution of viruses. J. Mol. Biol. 368, 1469e1483. [2] Beveridge, T.J. (1999) Structures of gram-negative cell walls and their derived membrane vesicles. J. Bacteriol. 181, 4725e4733. [3] Breitbart, M., Wegley, L., Leeds, S., Schoenfeld, T., Rohwer, F. (2004) Phage community dynamics in hot springs. Appl. Environ. Microbiol. 70, 1633e1640. [4] Chiura, H.X. (2002) Broad host range xenotrophic gene transfer by viruslike particles from a hot spring. Microb. Environ. 17, 53e58. [5] Dorward, D.W., Garon, C.F., Judd, R.C. (1989) Export and intercellular transfer of DNA via membrane blebs of Neisseria gonorrhoeae. J. Bacteriol. 171, 2499e2505. [6] Forterre, P., Gribaldo, S. (2007) The origin and evolution of terrestrial life. HFSP J 1, 156e168. [7] Fuhrman, J.A. (1999) Marine viruses and their biogeochemical and ecological effects. Nature 399, 541e548. [8] Fukui, T., Atomi, H., Kanai, T., Matsumi, R., Fujiwara, S., Imanaka, T. (2005) Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes. Genome Res. 15, 352e363. [9] Geslin, C., Le Romancer, M., Erauso, G., Gaillard, M., Perrot, G., Prieur, D. (2003) PAV1, the first virus-like particle isolated from a hyperthermophilic euryarchaeote, ‘‘Pyrococcus abyssi. J. Bacteriol. 185, 3888e3894. [10] Geslin, C., Le Romancer, M., Gaillard, M., Erauso, G., Prieur, D. (2003) Observation of virus-like particles in high temperature enrichment cultures from deep-sea hydrothermal vents. Res. Microbiol. 154, 303e307. [11] Geslin, C., Gaillard, M., Flament, D., Rouault, K., Le Romancer, M., Prieur, D., Erauso, G. (2007) Analysis of the first genome of
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