Construction of a plasmid vector for thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

摘要

A host-vector system for Sulfolobus acidocaldarius based on the pyrEF complementation was developed. This consisted of pyrimidine auxotrophic strains and a plasmid vector, designated pSAV1. The pSAV1 vector consists of the complete pRN1 plasmid of S. islandicus, the ColE1 origin and beta-lactamase gene derived from E. coli cloning vectors, and the pyrEF operon of S. solfataricus. Purified plasmid in which the GGCC sites were methylated was used for transformation of pyrimidine auxotrophic strains of S. acidocaldarius. The transformants were selected directly on a xylose-tryptone (XT) solid medium without any prior liquid culturing, and grew as well as the wild type in uracil-free medium. After replication in S. acidocaldarius, pSAV1 was successfully recovered from cultures of transformants by the standard alkaline lysis method and could be used for transformation without further amplification or methylation. The yield of plasmid from the culture increased after exponential phase and reached about 20 ng/mL culture in the stationary phase. This suggests that the copy number of pSAV1 in a host Sulfolobus cell was at least 14, probably about 20, in the stationary phase.