Development of a Simvastatin Selection Marker for a Hyperthermophilic Acidophile, Sulfolobus islandicus

摘要

We report here a novel selectable marker for the hyperthermophilic crenarchaeon Sulfolobus islandicus . The marker cassette is composed of the sac7d promoter and the hmg gene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (P_( sac7d ) -hmg ), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting the pyrEF gene of the expression vector pSeSD for P_( sac7d ) -hmg with which the Sulfolobus expression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization of Sulfolobus transformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His_(6)-tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers ( pyrEF and hmg ) was subsequently exploited for genetic analysis. A herA merodiploid strain of S. islandicus was constructed using pyrEF marker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (Δ herA ) cells generated from the herA merodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability of S. islandicus . To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.