General vectors for archaeal hyperthermophiles: Strategies based on a mobile intron and a plasmid

Although there are currently no cloning and expression vectors available for archaeal hyperthermophiles, small cryptic plasmids have been characterized for these organisms as well as viruses and introns capable of spreading between cells. Below, we review the recent progress in adapting these genetic elements as vectors for Pyrococcus furiosus and Sulfolobus acidocaldarius. An efficient and reliable transformation procedure is described for both organisms. The potential of the mobile intron from Desulfurococcus mobilis, inserted into the bacterial vector pUC18 to generate a new type of vector, was investigated in S. acidocaldarius. A polylinker was inserted upstream from the open reading frame encoding the homing enzyme I-DmoI. Both the polylinker and a 276 bp fragment of the tetracycline gene from pBR322 could be inserted into the intron-plasmid construct and spreading still occurred in the culture of S. acidocaldarius. Experiments are in progress to test the co-mobility of the alcohol dehydrogenase and β-galactosidase genes from Sulfolobus species with the intron. A shuttle vector pCSV1 was also produced by fusing the pGT5 plasmid from Pyrococcus abyssi and the bacterial vector pUC19 which, on transformation, is stable in both organisms without selection. Growth inhibition studies indicate that both P. furiosus and S. acidocaldarius are sensitive to the antibiotics carbomycin, celesticetin, chloramphenicol and thiostrepton as well as butanol and butylic alcohol. Spontaneous mutants resistant to these drugs have been isolated carrying single site mutations in their 23S rRNA gene; they include mutants of S. acidocaldarius resistant to chloramphenicol, carbomycin and celesticetin with the mutation C2452U and thiostrepton-resistant mutants of P. furiosus carrying the mutation A1067G (both numbers corresponding to Escherichia coli 23S rRNA). These mutated genes are being developed as selective markers. Moreover, two β-galactosidase genes from P. furiosus have been cloned as possible phenotypic markers; one of these exhibits maximum activity at 95°C with O-nitrophenyl β-d-galactopyranoside as substrate.