Gene transfer and regulation in Sulfolobus solfataricus.

The mechanisms and regulation of the complex fundamental cell processes in thermophilic Archaea, such as transcription, translation, replication, secretion, cell growth and signalling have been only partially elucidated. This is mainly due to the lack of the tools needed to study their molecular biology, including appropriate vector-transformation systems, genetic and selectable markers, and mutant production and screening methods. Sulfolobus solfataricus was chosen as a model host system for the study of gene transfer and regulation since it is a good recipient for viruses and plasmids; moreover, the knowledge of its genomic organisation by genome sequencing, gene cloning and expression, is in good progress. An E.coli/Sulfolobus shuttle vector, the pEXSs, was obtained, inserting in an E.coli plasmid the genomic replication sequence of the S. shibate virus particle SSV1 and using a thermoadapted version of the hygromicin phosphotransferase as the selective acquired phenotype. In order to obtain a higher copy number plasmid, the natural S. islandicus multicopy plasmid pRN1 was used to construct an E.coli/Sulfolobus shuttle vector, the pHYG* by fusing it with the E.coli plasmid pGEM7zf(+) and providing it with the hygromycin phosphotransferase gene as selectable marker; since it has been demonstrated the pHYG* to be instable and to suffer rearrangment and/or deletions, attemps are being done to overcome recombination events. Furthermore, transformation systems based on the use of different E. coli host strains, and/or extrachromosomal genetic elements, and/or selectable phenotype using adh genes from thermophilic sources as transformation marker, are underway. In order to study the transcriptional regulation in Sulfolobus solfataricus, we examined the expression of the alcohol dehydrogenase gene (adh). Primer extension analysis allowed the identification of the transcriptional initiation site and the consensus TATA box. The effect of nutrients and toxic substrates was investigated at RNA and protein level and it was shown that specific messenger RNA levels were strictly dependent on growth phase, nutrient composition, and substrate concentration. ADH enzyme activities in extracts of the cells grown in the same different conditions were proportional to the intensity of the specific Northern hybridization bands, revealing that the transcription of the adh gene is finely regulated at transcriptional level