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

Berkner S, Grogan D, Albers SV, Lipps G - Nucleic Acids Res. (2007)

Bottom Line: The shuttle vectors do not integrate into the genome and do not rearrange.In addition, we demonstrate that this beta-glycosidase gene could function as selectable marker in S. solfataricus.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Bayreuth, 95440 Bayreuth, Germany.

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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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.
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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.

Mentions: The direction of the insertion in a given region does not influence performance of the vector. The vectors pF, pI and pG, for example, have insertion sites within 15‚ÄČnt of each other. In pG, the pyrEF genes are oriented clockwise, in pI and pF counter clockwise, without detectable effects on plasmid stability or growth (Figure 3). In addition, the growth phenotype of transformed cells is comparable to that of the untransformed recipient strain when supplemented with uracil.Figure 3.


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

Berkner S, Grogan D, Albers SV, Lipps G - Nucleic Acids Res. (2007)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919505&req=5

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.
Mentions: The direction of the insertion in a given region does not influence performance of the vector. The vectors pF, pI and pG, for example, have insertion sites within 15‚ÄČnt of each other. In pG, the pyrEF genes are oriented clockwise, in pI and pF counter clockwise, without detectable effects on plasmid stability or growth (Figure 3). In addition, the growth phenotype of transformed cells is comparable to that of the untransformed recipient strain when supplemented with uracil.Figure 3.

Bottom Line: The shuttle vectors do not integrate into the genome and do not rearrange.In addition, we demonstrate that this beta-glycosidase gene could function as selectable marker in S. solfataricus.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Bayreuth, 95440 Bayreuth, Germany.

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.

Show MeSH
Related in: MedlinePlus