Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis
Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis
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DOI：
10.1128/aem.71.7.3889-3899.2005
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发表时间：
2005-07-01
影响因子：
4.4
通讯作者：
Imanaka, T
中科院分区：
文献类型：
--
作者：
Sato, T;Fukui, T;Imanaka, T
我们最近开发了一个基因中断系统的超嗜热古菌Thermococcus kodakaraensis利用pyrF缺陷突变体，KU25，作为宿主菌株和pyrF基因作为选择标记。为了实现用于更先进的体内基因功能分析的多个遗传操作，有必要建立多个宿主标记系统或开发一个标记基因可以重复利用的系统。在本研究中，我们首先构建了一个新的宿主菌株，KU216（Δ pyrF），通过特异性和几乎完全删除内源pyrF通过同源重组。在这种改良的宿主中，不需要考虑由随机诱变引起的未知突变，并且与先前的宿主KU25不同，标记基因和染色体等位基因之间发生非预期重组的可能性很小（如果有的话）。此外，开发了trpE缺失体KW 128（Delta pyrF Delta trpE：pyrF）和trpE基因的新宿主标记组合。该系统使得有可能通过更简单的选择程序分离转化体以及推导转化效率，克服了第一系统的实际缺点。利用该系统还研究了转化条件的影响。最后，我们还建立了一个系统，其中可以通过弹出重组切除反选择pyrF标记来重复利用该标记。内源和外源序列都可以作为标记pyrF侧翼的串联重复序列应用于弹出重组。用弹出策略构建的双缺失突变体KUW 1（Delta pyrF Delta trpE）被证明是pyrF和trpE双标记的有用宿主。同样，也可以构建三重缺失突变体KUWH1（Δ pyrF Δ trpE Δ hisD）。这里开发的转化系统现在提供了在这种超嗜热古菌中进行广泛遗传研究的手段。
We have recently developed a gene disruption system for the hyperthermophilic archaeon Thermococcus kodakaraensis by utilizing a pyrF-deficient mutant, KU25, as a host strain and the pyrF gene as a selectable marker. To achieve multiple genetic manipulations for more advanced functional analyses of genes in vivo, it is necessary to establish multiple host-marker systems or to develop a system in which repeated utilization of one marker gene is possible. In this study, we first constructed a new host strain, KU216 (Delta pyrF), by specific and almost complete deletion of endogenous pyrF through homologous recombination. In this refined host, there is no need to consider unknown mutations caused by random mutagenesis, and unlike in the previous host, KU25, there is little, if any, possibility that unintended recombination between the marker gene and the chromosomal allele occurs. Furthermore, a new host-marker combination of a trpE deletant, KW128 (Delta pyrF Delta trpE:::pyrF), and the trpE gene was developed. This system made it possible to isolate transformants through a more simple selection procedure as well as to deduce the transformation efficiency, overcoming practical disadvantages of the first system. The effects of the transformation conditions were also investigated using this system. Finally, we have also established a system in which repeated utilization of the counterselectable pyrF marker is possible through its excision by pop-out recombination. Both endogenous and exogenous sequences could be applied as tandem repeats flanking the marker pyrF for pop-out recombination. A double deletion mutant, KUW1 (Delta pyrF Delta trpE), constructed with the pop-out strategy, was demonstrated to be a useful host for the dual markers pyrF and trpE. Likewise, a triple deletion mutant, KUWH1 (Delta pyrF Delta trpE Delta hisD), could also be constructed. The transformation systems developed here now provide the means for extensive genetic studies in this hyperthermophilic archaeon.