Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea.
Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea.
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DOI：
10.1093/nar/gkm449
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发表时间：
2007
影响因子：
14.9
通讯作者：
Lipps G
中科院分区：
文献类型：
--
作者：
Berkner S;Grogan D;Albers SV;Lipps G
硫化叶菌属的极端嗜热嗜酸菌是研究得最好的古菌之一，但缺乏小而可靠的质粒载体，这些载体已被证明对操纵和分析其他微生物中的基因非常有用。在这里，我们报告了一系列的成功构建Sulfolobus-Escherichia coli穿梭载体的基础上小多拷贝质粒pRN 1从Sulfolobus islandicus。通过在质粒中包含pyrEF基因来提供合适的尿嘧啶营养缺陷型的选择。穿梭载体不整合到基因组中并且不重排。这些质粒允许基因的功能性过表达，如可以从S.太阳神此外，我们证明该β-糖苷酶基因可以作为选择标记在S。太阳神穿梭质粒在pRN 1内的中断位点不同，并允许我们描绘pRN 1的功能重要区域。orf 56/orf 904操纵子似乎是pRN 1复制所必需的，相反，高度保守的orf 80/plrA基因的中断是耐受的。新的载体系统有望促进硫化叶菌的遗传研究，并具有生物技术用途，例如在嗜温宿主中不容易进行的嗜热酶的过表达或优化。
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.