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首页> 外文期刊>Applied Microbiology >Genetic Manipulations of the Hyperthermophilic Piezophilic Archaeon Thermococcus barophilus
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Genetic Manipulations of the Hyperthermophilic Piezophilic Archaeon Thermococcus barophilus

机译：嗜热嗜热古细菌嗜热球菌嗜热球菌的遗传操纵。

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In this study, we developed a gene disruption system for Thermococcus barophilus using simvastatin for positive selection and 5-fluoroorotic acid (5-FOA) for negative selection or counterselection to obtain markerless deletion mutants using single- and double-crossover events. Disruption plasmids carrying flanking regions of each targeted gene were constructed and introduced by transformation into wild-type T. barophilus MP cells. Initially, a pyrF deletion mutant was obtained as a starting point for the construction of further markerless mutants. A deletion of the hisB gene was also constructed in the UBOCC-3256 (Δ pyrF ) background, generating a strain (UBOCC-3260) that was auxotrophic for histidine. A functional pyrF or hisB allele from T. barophilus was inserted into the chromosome of UBOCC-3256 (Δ pyrF ) or UBOCC-3260 (Δ pyrF Δ hisB ), allowing homologous complementation of these mutants. The piezophilic genetic tools developed in this study provide a way to construct strains with multiple genetic backgrounds that will allow further genetic studies for hyperthermophilic piezophilic archaea.
机译：在这项研究中，我们开发了一种嗜热嗜热球菌的基因破坏系统，使用辛伐他汀进行阳性选择，使用5-氟乳清酸（5-FOA）进行阴性选择或反选择，从而使用单交叉和双交叉事件获得了无标记缺失突变体。构建携带每个靶基因侧翼区域的破坏质粒，并通过转化入野生型嗜热嗜热菌MP细胞而引入。最初，获得pyrF缺失突变体作为构建其他无标记突变体的起点。还在UBOCC-3256（ΔpyrF）背景中构建了hisB基因的缺失，产生了对组氨酸营养缺陷的菌株（UBOCC-3260）。将来自嗜热嗜热菌的功能性pyrF或hisB等位基因插入UBOBC-3256（ΔpyrF）或UBOBC-3260（ΔpyrFΔhisB）的染色体，从而允许这些突变体的同源互补。在这项研究中开发的亲嗜性遗传工具提供了一种构建具有多种遗传背景的菌株的方法，这将允许对超嗜热嗜性古细菌进行进一步的遗传研究。

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