Natural Competence in the Hyperthermophilic Archaeon Pyrococcus furiosus Facilitates Genetic Manipulation: Construction of Markerless Deletions of Genes Encoding the Two Cytoplasmic Hydrogenases
Natural Competence in the Hyperthermophilic Archaeon Pyrococcus furiosus Facilitates Genetic Manipulation: Construction of Markerless Deletions of Genes Encoding the Two Cytoplasmic Hydrogenases
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DOI：
10.1128/aem.02624-10
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发表时间：
2011-04-01
影响因子：
4.4
通讯作者：
Westpheling, Janet
中科院分区：
文献类型：
--
作者：
Lipscomb, Gina L.;Stirrett, Karen;Westpheling, Janet
在试图开发一种将DNA引入激烈火球菌的方法时，我们在野生型群体中发现了一种天然且有效地胜任DNA摄取的变体。在基因组中构建pyrF基因缺失突变体，并且该菌株的组合转化和重组频率允许通过使用线性DNA的直接选择进行标记替换。我们已经证明了使用这种菌株，命名为COM 1，进行遗传操作。使用遗传选择和反选择尿嘧啶生物合成的基础上，我们产生了单和双缺失突变体的两个基因簇编码的两个细胞质氢化酶。COM 1菌株将为开发更复杂的遗传工具提供基础，从而可以对这种重要的超嗜热菌进行研究和代谢工程。
In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear DNA. We have demonstrated the use of this strain, designated COM1, for genetic manipulation. Using genetic selections and counterselections based on uracil biosynthesis, we generated single- and double-deletion mutants of the two gene clusters that encode the two cytoplasmic hydrogenases. The COM1 strain will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering of this important hyperthermophile.