The particle SSV1 from the extremely thermophilic archaeon Sulfolobus is a virus: demonstration of infectivity and of transfection with viral DNA.

Abstract

The lemon-shaped "virus-like" particle SSV1 produced by the thermophilic archaeon Sulfolobus shibatae has not previously been observed to infect any host. Using a plaque assay suitable for the extreme growth conditions of this archaeon, we have shown infection of Sulfolobus solfataricus by SSV1. Upon infection, the viral genome was always found integrated into a tRNA gene of the host chromosome, a situation similar to that in S. shibatae, proving that site-specific integration is involved in establishing the lysogenic state. As in S. shibatae, UV-irradiation of lysogenized S. solfataricus led to virus production apparently not accompanied by cell lysis. We have also demonstrated the efficient uptake of exogenous DNA and its expression in Sulfolobus by transfecting S. solfataricus with SSV1 DNA by electroporation. Transfection efficiencies of up to 10' transfectants per lag of DNA were obtained.Much of our present knowledge of the molecular genetics of bacteria is based on work with phage host systems. In Archaea (previously named archaebacteria; see ref. 1) only a few virus host systems have been sufficiently investigated to serve this purpose (for reviews see refs. 2 and 3).In one of the two kingdoms, the Euryarchaeota (1), two phage/host systems have been studied in some molecular detail: the temperate phage DH of Halobacterium halobium (4-6) and the transducing phage TM1 of Methanobacterium thermoautotrophicum (7,8). In the other kingdom of the archaeal domain, the Crenarchaeota (1), comprising extreme thermophiles, a family of viruses of the anaerobic host Thermoproteus tenax, TTV1 through TTV4 (2, 9, 10), is of limited usefulness, because no plaque assay is available. The UV-inducible particle SSV1 (11) of the aerobic extremely thermophilic host Sulfolobus shibatae has been thoroughly investigated on the molecular genetic level. The SSV1 DNA of 15.5 kilobase pairs (kbp), which is stably carried within the host cells both as covalently closed circular DNA and integrated in the chromosome (12-14), has been totally sequenced (15). All transcripts and their respective promoters and terminators have been identified (16)(17)(18). The viral nature of this particle has, however, remained obscure, since infection of an exogenous host could not be demonstrated and attempts to cure the "lysogenic" host S. shibatae were unsuccessful. Moreover the possibility of developing a plaque assay seemed unlikely, because the UV-inducible virus production does not involve cell lysis (11), and plating of thermophilic archaea as lawns required for scoring plaque formation has not previously been described.The detailed knowledge ofits genome makes SSV1 suitable not only for studies of virus-host interaction but also for the development of a vector system for extreme thermophiles. In the absence of appropriate genetic markers, phage DNA transfection could provide a good approach for the establishment and optimization of transformation conditions, as it did in halophiles (19).In this study we show that SSV1 is indeed a virus b...

The regulatory role of DNA supercoiling in nucleoprotein complex assembly and genetic activity

“…This might also apply to integrative recombination catalyzed by the phage λ integrase (Thompson et al 1988;Crisona et al 1999), as well as other reactions catalyzed by the members of integrase superfamily of site-specific recombinases (Abremski and Hoess 1985;Dove and Dorman 1994;Corcoran and Dorman 2009). On the other hand, the recombination by C-terminally-modified SSV1 virus integrase from a thermophilic archaeon characterized by positively supercoiled DNA is more efficient with fully relaxed/linear DNA substrates, and is also directionally highly promiscuous in vitro (Muskhelishvili et al 1993), implicating additional, presently unknown, factors in imparting directionality on the SSV1 integrase-driven recombination in vivo (Schleper et al 1992). …”

Structural Organization of Essential Iron-Sulfur Clusters in the Evolutionarily Highly Conserved ATP-binding Cassette Protein ABCE1

“…To transfer the araS promoter together with the gene to be expressed into the virus-based vector, the BlnI/EagI insert from pSVA30 was ligated into pMJ02 (19), resulting in the plasmid pSVA31. Electroporation of S. solfataricus pyrEF mutant PH1-16 with pSVA31 and the isolation of single transformants were carried out as described (20,21). Integration of the viral vector into the genome was confirmed by Southern blot analysis using standard procedures.…”

Two novel conjugative plasmids from a single strain of Sulfolobus

“…Due to this early identification, S. solfataricus and its relatives have become model organisms for fundamental studies of Archaea. Studies of the genus Sulfolobus have been instrumental in understanding archaeal mechanisms of transposition (Martusewitsch et al, 2000), transfection (Schleper et al, 1992), transformation (Aravalli & Garrett, 1997;Cannio et al, 1998;Elferink et al, 1996;Stedman et al, 1999) and conjugation (Reilly & Grogan, 2001;Schleper et al, 1995). An impressive variety of mobile genetic elements has recently been discovered in Archaea in general, and in Sulfolobus in particular: viruses, autonomous insertion sequence (IS) elements, non-autonomous miniature inverted repeat transposable elements (MITEs), small nonconjugative plasmids and large conjugative plasmids (Brugger et al, 2002;Prangishvili et al, 2001;Rice et al, 2001;Zillig et al, 1998).…”