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. 2007;35(12):e88.
doi: 10.1093/nar/gkm449. Epub 2007 Jun 18.

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

Silvia Berkner  1 , Dennis Grogan, Sonja-Verena Albers, Georg Lipps
Affiliations

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

Silvia Berkner et al. Nucleic Acids Res. 2007.

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 beta-glycosidase (lacS) gene of S. solfataricus. In addition, we demonstrate that this beta-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.

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Figures

Figure 1.
Figure 1.
Physical maps. (A) Positions of the insertion sites of the E. coli replicon and the pyrEF marker genes for shuttle vectors pA–pN. (B): Conserved features of pRN1: thick arrows: conserved open reading frames, gray area: conserved on the nucleotide level within the pRN family plasmids, black arrows: transcripts. (C) Vector map of the shuttle construct pC. Positions of the restriction sites are (clockwise from the top): HindIII (1849), SacI (2792), HindIII (2833), SacII (5374), NotI (5380), SacI (6349).
Figure 2.
Figure 2.
Analysis of S. acidocaldarius transformants. (A) Southern blot of HindIII digested genomic DNA preparations; the probes complementary to pyrE and a pRN1-fragment (see the Materials and Methods section) were used concurrently. −c: untransformed MR31, A+c: positive control: plasmid pA from E. coli, G+c: separate positive control (plasmid pG from E. coli) because of the additional HindIII restriction site present in pG. (B) Southern blot like in A for controls and cultures transformed with pC and pE after 200 generations of consecutive cultivation. (C) Colony hybridizations for S. acidocaldarius cultures transformed with pC and pE with pRN1 specific probes. (D) Restriction analysis (SacI) of retransformation experiments for all shuttle constructs, o: original plasmid prepared from E. coli, r: retransformed plasmid. (E) Restriction analysis (SacI) of shuttle vectors isolated directly from S. acidocaldarius: o: Plasmid from E. coli, S: plasmid from Sulfolobus (∼40 times more concentrated than from E. coli), r: retransformed plasmid.
Figure 3.
Figure 3.
Growth of transformants. (A) Growth curves for MR31 transformed with pA to pN. (B) Growth curves for the recipient strain MR31 without addition of uracil (U), with the addition of uracil and transformed with pC and pE. (C) Retention of the shuttle vectors under non-selective conditions.
Figure 4.
Figure 4.
Plasmid copy number per cell (triangles) for MR31 transformed with pC (left panel) and pE (right panel) and corresponding growth curves (line).
Figure 5.
Figure 5.
Replication of pJlacS in S. acidocaldarius (A) Southern blot (SacI) of plasmid pJlacS from E. coli, MR31 transformed with pJlacS and untransformed MR31 (−c). The pRN1 specific probe detects the 3.7-kb restriction fragment, the lacS probe detects the 7.3-kb fragment. (B) Retransformation of pJlacS (SacI), +c: pJlacS from E. coli, 1,2,3: retransformants. (C) X-gal test with untransformed MR31 and MR31 transformed with pJlacS (10 min at 75°C). (D) Growth curves for MR31 transformed with pJ (control) and with pJlacS. (E) Reporter gene experiment showing copy numbers of pJlacS per cell and the corresponding β-galactosidase activities.
Figure 6.
Figure 6.
Replication of pJlacS in S. solfataricus (A) Southern blot (SacI) of untransformed PBL2025 (−c), PBL2025 transformed with pJlacS and pJlacS from E. coli (+c). The pRN1-specific probe detects the 3.7-kb restriction fragment, the lacS probe detects the 7.3-kb fragment. (B) Growth curves of untransformed PBL2025 in 0.4% lactose medium, PBL2025 transformed with pJlacS in 0.4% lactose medium and in 0.2% tryptone medium. (C) Copy numper per cell for pJlacS in PBL2025 in 0.4% lactose medium. (D) Retransformation of pJlacS back into E. coli. (E) Plating of PBL2025 transformed with pJlacS after 100 generations of consecutive cultivation on non-selective tryptone and selective lactose plates stained with X-gal.
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