SULFOLOBUS SOLFATARICUS AS SOURCE OF GLYCOSYL HYDROLASES WITH BIOTECHNOLOGICAL POTENTIAL

The pSSVx genetic element from Sulfolobus islandicus REY15/4 is a hybrid between a plasmid and a fusellovirus, able to be maintained in nonintegrative form and to spread when the helper SSV2 virus is present in the cells. In this work, the satellite virus was engineered to obtain an Escherichia coli–Sulfolobus solfataricus shuttle vector for gene transfer and expression in S.solfataricus by fusing site-specifically the pSSVx chromosome with an E.coli plasmid replicon and the ampicillin resistance gene. The pSSVx-based vector was proven functional like the parental virus, namely it was able to spread efficiently through infected S.solfataricus cells. Moreover, the hybrid plasmid stably transformed S.solfataricus and propagated with no rearrangement, recombination or integration into the host chromosome. The high copy number of the artificial genetic element was found comparable with that calculated for the wild-type pSSVx in the new host cells, with no need of genetic markers for vector maintenance in the cells and for transfomant enrichment. The newly constructed vector was also shown to be an efficient cloning vehicle for the expression of passenger genes in S.solfataricus. In fact, a derivative plasmid carrying an expression cassette of the lacS gene encoding the b-glycosidase from S.solfataricus under the control of the Sulfolobus chaperonine (thermosome tf55) heat shock promoter was also able to drive the expression of a functional enzyme. Complementation of the b-galactosidase deficiency in a deletion mutant strain of S.solfataricus demonstrated that lacS gene was an efficient marker for selection of single transformants on solid minimal lactose medium. INTRODUCTION Host/virus interaction modes have provided windows to study microbial diversity (1) as well as genetic processes at the molecular level, in particular for prokaryotes, and hence have helped in clarifying the physiological mechanisms, the dependence on the specific biochemical environment and evolution of their host cells (2,3). Very few viruses have been identified from Archaea (4) as compared with Bacteria and Eukarya and detailed description has been reported for those from hyperthermophilic archaea (5,6) with representatives that replicate in the genus Sulfolobus being the majority within the kingdom Crenarchaeota (6–8). To date, the Fuselloviridae are the most widespread on earth in the Sulfolobus genus with viruses sharing similar morphology as well as DNA genome size and organization (9–12). Sulfolobus spindle-shaped virus 1 (SSV1) is the best stud- ied member of this family and demonstrated to be temperate both in Sulfolobus shibatae and in non-natural but related Sulfolobus hosts, such as Sulfolobus solfataricus (13,14); infection, integration of DNA into the host chromosome and production of virions cause apparently no phenotype change but a significant growth retardation of the host cells which can be visualized as turbid plaques around propagation foci on plated lawns of indicator host cells (14–18). More recently, another fusellovirus, SSV2 from Sulfolobus islandicus strain 15/4 was isolated, characterized and its complete genomic sequence determined. SSV2 shares with SSV1 similar morphology, replication and DNA size (19). The overall genome architecture is conserved but the low similarity in the sequences should be responsible for the higher copy number and the lack of a strong ultraviolet induction of episomal SSV2 DNA and particle production, as well as for the different integration of the SSV2 genome which occurs into the host chromosome at the site of a glycyl tRNA instead of arginyl-tRNA (12). S.islandicus REY15/4 harbors also a small plasmid, pSSVx, assigned to the pRN family (20,21) of Sulfolobales plasmids; pSSVx is also capable of spreading in the cell cultures of *To whom correspondence should be addressed at Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via Pietro Castellino 111, 80131, Naples, Italy. Tel: +39 081 613 2285; Fax: +39 081 613 2248; Email: r.cannio@ibp.cnr. it