Thiel Axel, Michoud Grégoire, Moalic Yann, Flament Didier, Jebbar Mohamed
Université de Bretagne Occidentale, UMR 6197-Laboratoire de Microbiologie des Environnements Extrêmes, Institut Universitaire Européen de la Mer, Plouzané, France.
Appl Environ Microbiol. 2014 Apr;80(7):2299-306. doi: 10.1128/AEM.00084-14. Epub 2014 Jan 31.
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 (ΔpyrF) 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 (ΔpyrF) or UBOCC-3260 (ΔpyrF ΔhisB), 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.
在本研究中，我们开发了一种用于嗜压嗜热栖热菌（Thermococcus barophilus）的基因破坏系统，该系统使用辛伐他汀进行正向选择，5-氟乳清酸（5-FOA）进行负向选择或反选择，以通过单交换和双交换事件获得无标记缺失突变体。构建了携带每个目标基因侧翼区域的破坏质粒，并通过转化导入野生型嗜压嗜热栖热菌MP细胞。最初，获得了一个pyrF缺失突变体作为构建进一步无标记突变体的起点。在UBOCC-3256（ΔpyrF）背景下也构建了hisB基因的缺失突变体，产生了一株对组氨酸营养缺陷的菌株（UBOCC-3260）。将来自嗜压嗜热栖热菌的功能性pyrF或hisB等位基因插入UBOCC-3256（ΔpyrF）或UBOCC-3260（ΔpyrF ΔhisB）的染色体中，实现这些突变体的同源互补。本研究中开发的嗜压遗传工具提供了一种构建具有多种遗传背景菌株的方法，这将有助于对嗜热嗜压古菌进行进一步的遗传学研究。
