Kiljunen Saija, Pajunen Maria I, Dilks Kieran, Storf Stefanie, Pohlschroder Mechthild, Savilahti Harri
Division of Genetics and Physiology, Department of Biology, University of Turku, Turku, Finland.
Current address: Department of Biosciences, Division of Biochemistry and Biotechnology, University of Helsinki, Helsinki, Finland.
BMC Biol. 2014 Dec 9;12:103. doi: 10.1186/s12915-014-0103-3.
Archaea share fundamental properties with bacteria and eukaryotes. Yet, they also possess unique attributes, which largely remain poorly characterized. Haloferax volcanii is an aerobic, moderately halophilic archaeon that can be grown in defined media. It serves as an excellent archaeal model organism to study the molecular mechanisms of biological processes and cellular responses to changes in the environment. Studies on haloarchaea have been impeded by the lack of efficient genetic screens that would facilitate the identification of protein functions and respective metabolic pathways.
Here, we devised an insertion mutagenesis strategy that combined Mu in vitro DNA transposition and homologous-recombination-based gene targeting in H. volcanii. We generated an insertion mutant library, in which the clones contained a single genomic insertion. From the library, we isolated pigmentation-defective and auxotrophic mutants, and the respective insertions pinpointed a number of genes previously known to be involved in carotenoid and amino acid biosynthesis pathways, thus validating the performance of the methodologies used. We also identified mutants that had a transposon insertion in a gene encoding a protein of unknown or putative function, demonstrating that novel roles for non-annotated genes could be assigned.
We have generated, for the first time, a random genomic insertion mutant library for a halophilic archaeon and used it for efficient gene discovery. The library will facilitate the identification of non-essential genes behind any specific biochemical pathway. It represents a significant step towards achieving a more complete understanding of the unique characteristics of halophilic archaea.
古菌与细菌和真核生物具有一些基本特性。然而，它们也具有独特的属性，而这些属性在很大程度上仍未得到充分表征。嗜盐栖热菌是一种需氧的中度嗜盐古菌，可在限定培养基中生长。它是研究生物过程的分子机制以及细胞对环境变化反应的优秀古菌模式生物。由于缺乏有效的遗传筛选方法，阻碍了对嗜盐古菌的研究，而这种筛选方法有助于识别蛋白质功能和相应的代谢途径。
在此，我们设计了一种插入诱变策略，该策略将 Mu 体外 DNA 转座与基于同源重组的嗜盐栖热菌基因靶向相结合。我们构建了一个插入突变体文库，其中的克隆含有单个基因组插入片段。从该文库中，我们分离出了色素沉着缺陷型和营养缺陷型突变体，并且各自的插入片段确定了一些先前已知参与类胡萝卜素和氨基酸生物合成途径的基因，从而验证了所用方法的性能。我们还鉴定出了在编码未知或假定功能蛋白质的基因中存在转座子插入的突变体，这表明可以赋予未注释基因新的作用。
我们首次为嗜盐古菌构建了一个随机基因组插入突变体文库，并将其用于高效的基因发现。该文库将有助于识别任何特定生化途径背后的非必需基因。这代表着朝着更全面了解嗜盐古菌独特特性迈出的重要一步。
