GENERAL VECTORS FOR ARCHAEAL HYPERTHERMOPHILES - STRATEGIES BASED ON A MOBILE INTRON AND A PLASMID

Although there are currently no cloning and expression vectors availab le for archaeal hyperthermophiles, small cryptic plasmids have been ch aracterized for these organisms as well as viruses and introns capable of spreading between cells. Below, we review the recent progress in a dapting these genetic elements as vectors for Pyrococcus furiosus and Sulfolobus acidocaldarius. An efficient and reliable transformation pr ocedure is described for both organisms. The potential of the mobile i ntron from Desulfurococcus mobilis, inserted into the bacterial vector pUC 18 to generate a new type of vector, was investigated in S. acido caldarius. A polylinker was inserted upstream from the open reading fr ame encoding the homing enzyme I-DmoI. Both the polylinker and a 276 b p fragment of the tetracycline gene from pBR322 could be inserted into the intron-plasmid construct and spreading still occurred in the cult ure of S, acidocaldarius. Experiments are in progress to test the co-m obility of the alcohol dehydrogenase and beta-galactosidase genes from Sulfolobus species with the intron. A shuttle vector pCSV1 was also p roduced by fusing the pGT5 plasmid from Pyrococcus abyssi and the bact erial vector pUC19 which, on transformation, is stable in both organis ms without selection. Growth inhibition studies indicate that both P. furiosus and S, acidocaldarius are sensitive to the antibiotics carbom ycin, celesticetin, chloramphenicol and thiostrepton as well as butano l and butylic alcohol. Spontaneous mutants resistant to these drugs ha ve been isolated carrying single site mutations in their 23S rRNA gene , they include mutants of S, acidocaldarius resistant to chloramphenic ol, carbomycin and celesticetin with the mutation C2452U and thiostrep ton-resistant mutants of P. furiosus carrying the mutation A1067G (bot h numbers corresponding to Escherichia coli 23S rRNA). These mutated g enes are being developed as selective markers. Moreover, two beta-gala ctosidase genes from P. furiosus have been cloned as possible phenotyp ic markers, one of these exhibits maximum activity at 95 degrees C wit h O-nitrophenyl beta-D-galactopyranoside as substrate.