Evelyne Marguet

Virus-like vesicles and extracellular DNA produced by hyperthermophilic archaea of the order Thermococcales

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.

Molecular diversity of new Thermococcales isolates from a single area of hydrothermal deep-sea vents as revealed by randomly amplified polymorphic DNA fingerprinting and 16S rRNA gene sequence analysis

ABSTRACT Members of the Thermococcales are anaerobic Archaea belonging to the kingdom Euryarchaea that are studied in many laboratories as model organisms for hyperthermophiles. We describe here a molecular analysis of 86 new Thermococcales isolates collected from six different chimneys of a single hydrothermal field located in the 13°N 104°W segment of the East Pacific ridge at a depth of 2,330 m. These isolates were sorted by randomly amplified polymorphic DNA (RAPD) fingerprinting into nine groups, and nine unique RAPD profiles were obtained. One RAPD group corresponds to new isolates of Thermococcus hydrothermalis, whereas all other groups and isolates with unique profiles are different from the 22 reference strains included in this study. Analysis of 16S rRNA gene sequences of representatives of each RAPD group and unique profiles showed that one group corresponds to Pyrococcus strains, whereas all the other isolates are Thermococcus strains. We estimated that our collection may contain at least 11 new species. These putative species, isolated from a single area of hydrothermal deep-sea vents, are dispersed in the 16S rRNA tree among the reference strains previously isolated from diverse hot environments (terrestrial, shallow water, hydrothermal vents) located around the world, suggesting that there is a high degree of dispersal of Thermococcales. About one-half of our isolates contain extrachromosomal elements that could be used to search for novel replication proteins and to develop genetic tools for hyperthermophiles.

Protection of DNA by salts against thermodegradation at temperatures typical for hyperthermophiles

Abstract The effect of physiological concentrations of KCl and MgCl2 on the chemical stability of double-stranded and single-stranded DNA has been studied at temperatures typical for hyperthermophiles. These two salts protect both double and single-stranded DNA against heat-induced cleavage by inhibiting depurination. High KCl concentrations also protect DNA cleavage at apurinic sites, while high MgCl2 concentrations stimulate this cleavage. It has been previously proposed that salt protects double-stranded DNA against depurination by stabilizing the double helix. However, the inhibition of the depurination of single-stranded DNA by KCl and MgCl2 indicates that this effect is more probably due to a direct interaction of salts with purine nucleotides. These results suggest that the number and nature of heat-induced DNA lesions which have to be repaired might be quite different from one hyperthermophile to another, depending on their intracellular salt concentration. High salt concentrations might be also useful to protect DNA in long polymerase chain reaction (PCR) experiments and for long-term preservation.

Two novel families of plasmids from hyperthermophilic archaea encoding new families of replication proteins

Thermococcales (phylum Euryarchaeota) are model organisms for physiological and molecular studies of hyperthermophiles. Here we describe three new plasmids from Thermococcales that could provide new tools and model systems for genetic and molecular studies in Archaea. The plasmids pTN2 from Thermococcus nautilus sp. 30-1 and pP12-1 from Pyrococcus sp. 12-1 belong to the same family. They have similar size (approximately 12 kb) and share six genes, including homologues of genes encoded by the virus PAV1 from Pyrococcus abyssi. The plasmid pT26-2 from Thermococcus sp. 26-2 (21.5 kb), that corresponds to another plasmid family, encodes many proteins having homologues in virus-like elements integrated in several genomes of Thermococcales and Methanococcales. Our analyses confirm that viruses and plasmids are evolutionary related and co-evolve with their hosts. Whereas all plasmids previously isolated from Thermococcales replicate by the rolling circle mechanism, the three plasmids described here probably replicate by the theta mechanism. The plasmids pTN2 and pP12-1 encode a putative helicase of the SFI superfamily and a new family of DNA polymerase, whose activity was demonstrated in vitro, whereas pT26-2 encodes a putative new type of helicase. This strengthens the idea that plasmids and viruses are a reservoir of novel protein families involved in DNA replication.

Plasmids, viruses and virus-like membrane vesicles from Thermococcales

Several families of plasmids and viruses (PVs) have now been described in hyperthermophilic archaea of the order Thermococcales. One family of plasmids replicates by the rolling circle mechanism, whereas most other PVs probably replicate by the θ mode. PVs from Thermococcales encode novel families of DNA replication proteins that have only detectable homologues in other archaeal PVs. PVs from different families share a common gene pool and co-evolve with their hosts. Most Thermococcales also produce virus-like membrane vesicles similar to eukaryotic microparticles (ectosomes). Some membrane vesicles of Thermococcus nautilus harbour the plasmid pTN1, suggesting that vesicles can be involved in plasmid transfer between species.

Hyperthermophilic archaea produce membrane vesicles that can transfer DNA

Thermococcales are hyperthermophilic archaea found in deep-sea hydrothermal vents. They have been recently reported to produce membrane vesicles (MVs) into their culture medium. Here, we have characterized the mode of production and determined the biochemical composition of MVs from two species of Thermococcales, Thermococcus gammatolerans and Thermococcus kodakaraensis. We observed that MVs are produced by a budding process from the cell membrane reminiscent of ectosome (microparticle) formation in eukaryotes. MVs and cell membranes from the same species have a similar protein and lipid composition, confirming that MVs are produced from cell membranes. The major protein present in cell membranes and MVs of both species is the oligopeptide binding protein OppA. This protein is also abundant in MVs from cells grown in minimal medium, suggesting that OppA could be involved in processes other than peptides scavenging. We have previously shown that MVs from Thermococcales harbour DNA and protect DNA against thermodegradation. Here, we show that T. kodakaraensis cells transformed with the shuttle plasmid pLC70 release MVs harbouring this plasmid. Notably, these MVs can be used to transfer pLC70 into plasmid-free cells, suggesting that MVs could be involved in DNA transfer between cells at high temperature.

Fake virus particles generated by fluorescence microscopy

Many laboratories are actively studying the abundance and roles of viruses in natural ecosystems. In these studies, the presence and number of viral particles is usually determined using fluorescent dyes. However, DNA associated with membrane-derived vesicles (MVs), gene transfer agents (GTAs), or cell debris can produce fluorescent dots that can be confused with viral particles. We suspect that fluorescence counting can lead to overestimation of virus numbers and even suggest the presence of viruses when there are none. Future studies in environmental virology should acknowledge this point and consider how to bypass this problem. Besides trying to improve discrimination between virions and MVs, we suggest adopting less holistic approaches, focusing on the detection of known virus groups and the isolation of new viruses from a broader range of hosts.

The rolling‐circle plasmid pTN1 from the hyperthermophilic archaeon Thermococcus nautilus

The hyperthermophilic archaeon Thermococcus nautilus carries a plasmid, pTN1, which encodes a rolling‐circle (RC) replication initiator protein of 74 kDa (Rep74) and an orphan protein of 24 kDa (p24). The Rep74 protein is homologous to the Rep75 protein encoded by the RC plasmid pGT5 from Pyrococcus abyssi. Comparative analysis of Rep74 and Rep75 sequences shows that these proteins correspond to a new family of RC initiators formed by the fusion of a Rep domain with an N‐terminal domain of unknown function. Surprisingly, the Rep domain of Rep74/75 is more closely related to transposases encoded by IS elements than to Rep proteins of other RC plasmids. The p24 protein contains a hydrophobic segment, a highly charged region and a zinc finger motif. A recombinant p24 protein lacking the hydrophobic segment binds and condenses both single‐ and double‐stranded DNA, and forms DNA aggregates with extreme compaction at high protein to DNA ratio. In addition to encoding proteins of significant interest, pTN1 is remarkable by being the only characterized plasmid isolated from a Thermococcus strain, thus being useful to develop genetic tools in Thermococcus kodakaraensis for which gene disruption methods became recently available.

Membrane vesicles in natural environments: a major challenge in viral ecology

The production of extracellular membrane vesicles (hereafter called MVs) is a universal cellular feature, common to the three domains of life (Deatherage and Cookson, 2012). In particular, it has been known for decades that bacteria, including marine species, produce MVs in the laboratory but also in biofilms or during infections (Schooling and Beveridge, 2006; Deatherage and Cookson, 2012). However, until recently, the presence of MVs in natural environments has been largely overlooked by molecular ecologists. This attitude is probably going to change with a recent report in Science in which Biller et al. (2014) demonstrate the abundance of bacterial vesicles in marine ecosystems. They first show that several strains from the numerically dominant marine phytoplankter Prochlorococcus produce large amounts of MVs in the laboratory, suggesting that marine phototrophic bacteria also produce MVs in their natural environment. This hypothesis has been directly tested by examining two distinct ocean water samples for the presence of MVs. As expected, the authors succeeded to isolate abundant MVs from these two samples, with concentrations ranging from 105 to 106 vesicles ml−1 of sea water. This is similar to the lower range of concentrations reported for viral particles in the oceans (105 to 109 virus-like particles/ml; Suttle, 2007). It is therefore surprising that Biller et al. (2014) observed only negligible number of apparent tailed phages (or gene transfer agents (GTA)) in their vesicle-rich ocean samples even though the methods they used to isolate MVs were similar to those traditionally employed for the isolation of viral particles. Indeed, the dimensions, morphology and molecular composition of MVs are very similar to those of some virions (Forterre et al., 2013). This observation suggests that MVs could outnumber true viral particles in some marine environments.

Extracellular membrane vesicles harbouring viral genomes.

Cells from the three domains of life produce extracellular membrane vesicles (MVs), suggesting that MV production is a fundamental aspect of cellular physiology. We have recently shown that MVs produced by the hyperthermophilic archaeon Thermococcus kodakaraensis can be used as vehicles to transfer exogenous recombinant plasmid DNA from cell to cell. Here, we show that Thermococcus nautilus, which harbours three plasmids, pTN1, pTN2 and pTN3, produces MVs, and that some of them selectively incorporate pTN1 and pTN3. Interestingly, pTN3 represents the genome of a defective virus, which encodes signature proteins common to a large group of viruses infecting hosts from all three cellular domains. However, preparations of MVs produced by T. nautilus have a protein composition similar to that of classical MVs from Thermococcales and do not contain the viral major capsid protein encoded by pTN3. Our results suggest that MVs can serve as vehicles for the intercellular transport of viral genomes and facilitate recombination between viral, plasmid and/or cellular chromosomes in the absence of viral infection.