SHUTTLE VECTORS FOR HYPERTHERMOPHILIC ARCHAEA

Progress in understanding the basic molecular, biochemical, and physio logical characteristics of archaeal hyperthermophiles has been limited by the lack of suitable expression vectors. Here, we report the const ruction of versatile shuttle vectors that can be maintained, and selec ted for, in both archaea and bacteria. The primary construct, pAG1, wa s produced by ligating portions of the archaeal cryptic plasmid pGT5 a nd the bacterial plasmid pUC19, both of which exhibit high copy number s. A second vector construct, pAG2, was generated, with a reduced copy number in Escherichia coli, by introducing the Rom/Rop gene from pBR3 22 into pAG1. After transformation, both pAG1 and pAG2 were stably mai ntained and propagated in the euryarchaeote Pyrococcus furiosus, the c renarchaeote Sulfolobus acidocaldarius, and in Escherichia coli. An ar chaeal selective marker, the alcohol dehydrogenase gene from Sulfolobu s solfataricus, was isolated by polymerase chain reaction (PCR) amplif ication and cloned into the two constructs. They were stably maintaine d and expressed in the two archaea and conferred resistance to butanol and benzyl alcohol. However, the vector pAG21, deriving from pAG2, pr oved the more stable in E. coli probably due to its lower copy number in the bacterium. Conditions are presented for the use of the vectors which, potentially, can be used for other hyperthermophilic archaea.