Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus
Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus
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DOI：
10.1007/s00792-009-0254-2
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发表时间：
2009-07-01
期刊：
影响因子：
2.9
通讯作者：
She, Qunxin
中科院分区：
文献类型：
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作者：
Deng, Ling;Zhu, Haojun;She, Qunxin
Sulfolobus islandicus正被用作研究古生物生物学、地球生物学和进化的模型。然而，这种有机体没有可用的遗传系统。为了产生适合于遗传分析的岛状链球菌突变体，我们筛选了具有自发pyrEF突变的菌落。获得了一个突变等位基因中仅含有233bp的原始PYRE序列的突变体，并将其作为宿主来缺失β-糖苷酶(Lacs)基因。采用了两种非标记基因缺失方法，即质粒整合和分离，以及标记替换和环出，每种方法都获得了无标记的LacS突变体。在后一种方法中，一种新的重组机制，即标记环化和整合，被证明是可行的，它没有产生设计的缺失突变。随后，Sulfolobus-E.Coli构建穿梭载体，转化后对Delta pyrEF Delta Lacs突变进行基因互补。从而建立了以PYREF和LacS为遗传标记的完整的岛状链霉菌遗传工具。
Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce an S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation. One mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the beta-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out, and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus-E. coli plasmid shuttle vectors were constructed, which genetically complemented Delta pyrEF Delta lacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.