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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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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).
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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).

Mentions: In principle, shuttle vectors can be constructed from two plasmids that replicate in different hosts simply by fusing them at two points that preserve all the important functions of each plasmid. However, in the case of pRN1, it was not clear which ORFs or intergenic regions may be important for successful replication in Sulfolobus hosts. We therefore used transposition to generate pRN1 constructs interrupted at a number of different sites without regard to the location or its sequence context. From the initial transposition mixture, 13 distinct insertion points were chosen for further development, which included addition of the pyrEF genes of S. solfataricus as selectable marker (Figure 1, Table 1). In addition to providing more chances for a successful construct, this unbiased approach allowed us to evaluate possible differences in the performance of the vector constructs in Sulfolobus. This would provide some of the first functional data regarding which of the conserved open reading frames are important for plasmid replication and maintenance.Figure 1.


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)

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

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

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).
Mentions: In principle, shuttle vectors can be constructed from two plasmids that replicate in different hosts simply by fusing them at two points that preserve all the important functions of each plasmid. However, in the case of pRN1, it was not clear which ORFs or intergenic regions may be important for successful replication in Sulfolobus hosts. We therefore used transposition to generate pRN1 constructs interrupted at a number of different sites without regard to the location or its sequence context. From the initial transposition mixture, 13 distinct insertion points were chosen for further development, which included addition of the pyrEF genes of S. solfataricus as selectable marker (Figure 1, Table 1). In addition to providing more chances for a successful construct, this unbiased approach allowed us to evaluate possible differences in the performance of the vector constructs in Sulfolobus. This would provide some of the first functional data regarding which of the conserved open reading frames are important for plasmid replication and maintenance.Figure 1.

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