Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea

Abstract

The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolobus–Escherichia coli shuttle vectors based on the small multicopy plasmid pRN1 from Sulfolobus islandicus. Selection in suitable uracil auxotrophs is provided through inclusion of pyrEF genes in the plasmid. The shuttle vectors do not integrate into the genome and do not rearrange. The plasmids allow functional overexpression of genes, as could be demonstrated for the β-glycosidase (lacS) gene of S. solfataricus. In addition, we demonstrate that this β-glycosidase gene could function as selectable marker in S. solfataricus. The shuttle plasmids differ in their interruption sites within pRN1 and allowed us to delineate functionally important regions of pRN1. The orf56/orf904 operon appears to be essential for pRN1 replication, in contrast interruption of the highly conserved orf80/plrA gene is tolerated. The new vector system promises to facilitate genetic studies of Sulfolobus and to have biotechnological uses, such as the overexpression or optimization of thermophilic enzymes that are not readily performed in mesophilic hosts.

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

“…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).…”

VapC6, a ribonucleolytic toxin regulates thermophilicity in the crenarchaeote Sulfolobus solfataricus

“…In all samples, the parental wild-type strain was still about 10-fold more resistant to heat killing than the complemented mutant. This difference may arise from increased vapBC6 gene dosage due to use of the low, but not single-copy vector, pRN1 (Berkner et al 2007), for construction of the complementation plasmid (pBN1096). Overproduction of toxins are well known to be toxic (Christensen-Dalsgaard and Gerdes 2006;Daines et al 2007), therefore increased vapBC6 gene dosage may also be deleterious and compromise survival during heat shock.…”

“…into the NotI site. Plasmid pBN1080 was derived from pBN1074 (Berkner et al 2007) by replacment of lacS with malA at the SacII site.…”

Isolation and Characterization of Pyrimidine Auxotrophs from the Hyperthermophilic Archaeon Sulfolobus acidocaldarius DSM 639

“…The pC is a shuttle vector which carries the replication origin from the pRN1 plasmid in S. islandicus REN1H1, and E. coli origin and selectable marker from pBluescript [4]. The vector contains the pyrEF genes for uracil selection in the pyrEF deletion mutant.…”

“…Nevertheless, various genetic tools and methods have been reported especially in halophilic archaea [7,19]. For Sulfolobus species, several genetic systems such as high efficiency transformation technique, construction of shuttle vector, and isolation of gene disruption mutant have been published in the past 10 years, but it remains many problems to overcome the genetic studies of Sulfolobus [4,5,10].…”