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

Abstract

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 (⌬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.

Archaeal orthologs of Cdc45 and GINS form a stable complex that stimulates the helicase activity of MCM

“…When required, Brock's medium was solidified with 0.7% Gelrite (GmbH). Transformations with of S. islandicus with expression plasmid pSSR-Cdc45 and subsequent growth were as described (29). Yeast two-hybrid assays were performed in strain AH109, as described in the Matchmaker handbook (Clontech).…”

A broadly applicable gene knockout system for the thermoacidophilic archaeon Sulfolobus islandicus based on simvastatin selection

“…Construction of the HMG-CoA reductase overexpression cassette (simvastatin-resistance cassette, sim R ) and the pyrEF and lacS knockout plasmids was carried out as described below. The simvastatinresistance cassette was amplified from the Sulfolobus-E. coli shuttle vector pSSR (Zheng et al, 2012) with primers sim R -For/sim R -Rev, containing SphI/NcoI and MluI/SalI sites, respectively (Tables 1 and S1). This sequence consisted of a strong constitutive promoter from S. acidocaldarius 7d and a copy of hmg-coA from S. tokodaii (Zheng et al, 2012).…”

“…The simvastatinresistance cassette was amplified from the Sulfolobus-E. coli shuttle vector pSSR (Zheng et al, 2012) with primers sim R -For/sim R -Rev, containing SphI/NcoI and MluI/SalI sites, respectively (Tables 1 and S1). This sequence consisted of a strong constitutive promoter from S. acidocaldarius 7d and a copy of hmg-coA from S. tokodaii (Zheng et al, 2012). The Sto hmg-coA was selected to limit homologous recombination between the plasmid and the endogenous hmg-coA sequence in S. islandicus M.16.4, which share only 69.8 % identity.…”

“…It has been reported that 16 mM simvastatin can completely inhibit the cell growth of S. islandicus Rey15A, indicating that simvastatin is stable in environments with high temperature and low pH (Zheng et al, 2012). To further verify the possibility of using simvastatin as a broadly selectable marker, strains including S. islandicus M.16.4, S. islandicus M.16.27, S. islandicus Y.G.57.14, S. islandicus L.S.2.15, S. solfataricus P2, S. acidocaldarius, S. tokodaii and M. sedula were tested.…”

“…This selection strategy has now been exploited in another hyperthermophilc euryarchaeon, Pyrococcus furious (optimal temperature 100 u C), with great success (Lipscomb et al, 2011;Waege et al, 2010). Recently, simvastatin has been used to rescue lethal deletion mutants in S. islandicus by transformation with a shuttle vector overexpressing HMG-CoA reductase from Sulfolobus tokodaii under the control of a constitutive promoter from S. acidocaldarius 7d (Zheng et al, 2012). This antibiotic marker can potentially be applied in gene knockout analysis; however, it was not known whether overexpression from a single chromosomal copy with the engineered constitutive promoter would be sufficient to confer resistance to simvastatin.…”