Genetic tools for the piezophilic hyperthermophilic archaeon Pyrococcus yayanosii
Genetic tools for the piezophilic hyperthermophilic archaeon Pyrococcus yayanosii
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嗜压超嗜热古菌 Pyrococcus yayanosii 的遗传工具
DOI：
10.1007/s00792-014-0705-2
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发表时间：
2015-01-01
期刊：
影响因子：
2.9
通讯作者：
Xu, Jun
中科院分区：
文献类型：
--
作者：
Li, Xuegong;Fu, Ling;Xu, Jun
超嗜热菌Pyrococcus yayanosii CH 1是迄今为止在古细菌域或任何域中的所有非嗜冷菌中获得的唯一需要高压的微生物。在这项研究中，我们开发了一个遗传操作系统的P. yayanosii后，首先分离出一个faculitarum piezophilic衍生菌株，命名为P. yayanosii A1。3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶基因在菌株P. yayanosii A1中过表达，并被证明赋予宿主细胞对辛伐他汀的抗性。此外，使用辛伐他汀作为选择标记物，通过同源重组破坏了弥野毕赤酵母A1的内源pyrF，从而产生了另外的宿主菌株弥野毕赤酵母A2（ΔpyrF）。通过掺入pyrF-sim（R）盒构建无标记基因破坏载体，所述盒使得能够组合使用辛伐他汀抗性用于正选择和5-FOA用于反选择。通过删除弥野假单胞菌菌株A1的碳-氮水解酶证明了这种通用破坏系统的实用性。这些结果表明，各种遗传工具，现在已经到位，以研究未知的基因功能和P. yayanosii的嗜压适应的分子机制。
The hyperthermophile Pyrococcus yayanosii CH1 is the only high-pressure-requiring microorganism obtained thus far within the archaea domain or among all non-psychrophiles in any domain. In this study, we developed a genetic manipulation system for P. yayanosii after first isolating a facultatively piezophilic derivative strain, designated P. yayanosii A1. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene was overexpressed in strain P. yayanosii A1 and was demonstrated to confer host cell resistance against simvastatin. Furthermore, using simvastatin as a selection marker, the endogenous pyrF of P. yayanosii A1 was disrupted through homologous recombination, thus generating the additional host strain P. yayanosii A2 (ΔpyrF). A markerless gene disruption vector was constructed by incorporating a pyrF-sim (R) cassette that enables the combined use of simvastatin resistance for positive selection and 5-FOA for counter selection. The utility of this versatile disruption system was demonstrated by deleting the carbon-nitrogen hydrolase of P. yayanosii strain A1. These results demonstrate that a variety of genetic tools are now in place to study unknown gene function and the molecular mechanisms of piezophilic adaptation in P. yayanosii.