Unmarked gene deletion and host–vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus

Abstract

Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce an S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation. One mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the beta-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out, and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus-E. coli plasmid shuttle vectors were constructed, which genetically complemented DeltapyrEFDeltalacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.

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

“…However, considering that the copy number of the shuttle vector (Deng et al, 2009) presumably contributes to the overexpression of HMG-CoA reductase, we were not certain whether sim R would work well as a selection marker in gene knockout manipulations. This study confirms that simvastatin can be used as a resistance marker in a gene knockout system.…”

“…S. islandicus competent cells were prepared and transformed with approximately 0.5-1 mg circular or linearized DNA by electroporation, as described elsewhere (Deng et al, 2009), with the following minor modifications: (i) the host wild-type M.16.4 strain was used for transformation; (ii) after transformation with knockout plasmids, the cells were regenerated in 1 ml base salt solution without shaking at 78 uC for about 2 h and then transferred into a plastic flask (Corning) with 20 ml pre-warmed DT liquid medium which contained 12 mM simvastatin. The transformed cells were enriched in 20 ml DT in the presence of 12 mM simvastatin for three rounds (4 days per round) to select and concentrate simvastatinresistant strains.…”

“…The transformed cells were enriched in 20 ml DT in the presence of 12 mM simvastatin for three rounds (4 days per round) to select and concentrate simvastatinresistant strains. To identify lacS mutants, aliquots of the cultures were spread on DT plates by taking advantage of two-layer cultivation and then sprayed with 2 mg ml 21 X-Gal solution (Deng et al, 2009). For the isolation of pyrEF deletion mutants, the cultures were spread on the DT plates with 20 mg uracil ml 21 and 50 mg 5-FOA ml 21 .…”

“…With the limited success in the use of antibiotics in Archaea (Aravalli & Garrett, 1997;Cannio et al, 1998;Contursi et al, 2003), genetic tools including shuttlevector and gene-disruption systems have been described in Sulfolobus species, most of which are based on either uracil or lactose selection (Berkner et al, 2007;Deng et al, 2009;Sakofsky et al, 2011;Worthington et al, 2003). For example, a primary system in Sulfolobus acidocaldarius utilizes uracil selection, in which spontaneous uracil auxotrophs in the pyrEF genes are selected for by resistance to the toxic compound 5-fluoroorotic acid (5-FOA).…”

“…For example, a primary system in Sulfolobus acidocaldarius utilizes uracil selection, in which spontaneous uracil auxotrophs in the pyrEF genes are selected for by resistance to the toxic compound 5-fluoroorotic acid (5-FOA). Transformants of these auxotrophs are then selected for by complementation through addition of a functional pyrEF cassette (Grogan, 2009 allelic replacement and markerless gene deletion, have been constructed in S. islandicus Rey15A and S. acidocaldarius (Ajon et al, 2011;Deng et al, 2009;Li et al, 2011;Sakofsky et al, 2011;Zhang et al, 2010).…”