Diversity of CRISPR systems in the euryarchaeal Pyrococcales.

Pyrococcales are members of the order Thermococcales, a group of hyperthermophilic euryarchaea that are frequently found in deep sea hydrothermal vents. Infectious genetic elements, such as plasmids and viruses, remain a threat even in this remote environment and these microorganisms have developed several ways to fight their genetic invaders. Among these are the recently discovered CRISPR systems. In this review, we have combined and condensed available information on genetic elements infecting the Thermococcales and on the multiple CRISPR systems found in the Pyrococcales to fight them. Their organization and mode of action will be presented with emphasis on the Type III-B system that is the only CRISPR system known to target RNA molecules in a process reminiscent of RNA interference. The intriguing case of Pyrococcus abyssi, which is among the rare strains to present a CRISPR system devoid of the universal cas1 and cas2 genes, is also discussed.

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.

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.

PAV1, the first virus-like particle isolated from a hyperthermophilic euryarchaeote, "Pyrococcus abyssi".

We describe the first virus-like particle of a hyperthermophilic euryarchaeote which was discovered in a strain of "Pyrococcus abyssi" previously characterized in our laboratory. This particle, named PAV1, is lemon-shaped (120 nm x 80 nm), with a short tail terminated by fibers, and resembles the virus SSV1, the type member of the Fuselloviridae, isolated from Sulfolobus shibatae. Sensitivity of the virus-like particle to organic solvents and detergents suggested that the envelope of PAV1 may contain lipids in addition to proteins. It contains a double-stranded circular DNA of 18 kb which is also present in high copy number in a free form in the host cytoplasm. No integrated form of the PAV1 genome could be detected in the host chromosome. Under standard growth conditions, the host cells continuously release PAV1 particles into the culture supernatant without spontaneous lysis, with a maximum reached in the late stationary phase. UV, gamma irradiation, treatment with mitomycin C, and various physiological stresses had no effect on PAV1 production. Screening of a large number of Thermococcales isolates did not permit to find a sensitive host. These results suggest that PAV1 persists in the host strain in a stable carrier state rather than a prophage.