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首页> 外文期刊>Applied Microbiology >Novel System for Efficient Isolation of Clostridium Double-Crossover Allelic Exchange Mutants Enabling Markerless Chromosomal Gene Deletions and DNA Integration
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Novel System for Efficient Isolation of Clostridium Double-Crossover Allelic Exchange Mutants Enabling Markerless Chromosomal Gene Deletions and DNA Integration

机译：有效分离Clostridium Double-Crossover等位基因交换突变体的新型系统，可实现无标记染色体基因缺失和DNA整合

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Isolation of Clostridium mutants based on gene replacement via allelic exchange remains a major limitation for this important genus. Use of a heterologous counterselection marker can facilitate the identification of the generally rare allelic exchange events. We report on the development of an inducible counterselection marker and describe its utility and broad potential in quickly and efficiently generating markerless DNA deletions and integrations at any genomic locus without the need for auxotrophic mutants or the use of the mobile group II introns. This system is based on a codon-optimized mazF toxin gene from Escherichia coli under the control of a lactose-inducible promoter from Clostridium perfringens . This system is potentially applicable to almost all members of the genus Clostridium due to their similarly low genomic GC content and comparable codon usage. We isolated all allelic-exchange-based gene deletions ( ca_p0167 , sigF , and sigK ) or disruptions ( ca_p0157 and sigF ) we attempted and integrated a 3.6-kb heterologous DNA sequence (made up of a Clostridium ljungdahlii 2.1-kb formate dehydrogenase [ fdh ] gene plus a FLP recombination target [FRT]-flanked thiamphenicol resistance marker) into the Clostridium acetobutylicum chromosome. Furthermore, we report on the development of a plasmid system with inducible segregational instability, thus enabling efficient deployment of the FLP-FRT system to generate markerless deletion or integration mutants. This enabled expeditious deletion of the thiamphenicol resistance marker from the fdh integrant strain as well as the sigK deletion strain. More generally, our system can potentially be applied to other organisms with underdeveloped genetic tools.
机译：基于等位基因交换的基因置换分离梭状芽胞杆菌突变体仍然是该重要属的主要限制。异源抗选择标记的使用可以促进通常罕见的等位基因交换事件的鉴定。我们报告了诱导型反选择标记的发展，并描述了它的实用性和广泛的潜力，可快速有效地在任何基因组基因座上生成无标记的DNA缺失和整合，而无需营养缺陷型突变体或使用II型移动内含子。该系统基于在产气荚膜梭菌的乳糖诱导型启动子的控制下，来自大肠杆菌的密码子优化的mazF毒素基因。该系统可能几乎适用于梭菌属的几乎所有成员，因为它们的基因组GC含量较低，并且密码子使用率相当。我们尝试分离所有基于等位基因交换的基因缺失（ca_p0167，sigF和sigK）或破坏（ca_p0157和sigF），并整合了一个3.6 kb的异源DNA序列（由梭状芽胞杆菌2.1 kb甲酸脱氢酶[fdh组成] ]基因加上FLP重组靶标[FRT]-侧接的甲砜霉素抗性标记）进入丙酮丁醇梭菌染色体。此外，我们报道了具有可诱导的分离不稳定性的质粒系统的发展，从而使FLP-FRT系统的有效部署能够产生无标记的缺失或整合突变体。这使得能够从fdh整合菌株以及sigK缺失菌株中迅速删除对苯甲砜霉素的抗性标记。更笼统地说，我们的系统可以潜在地应用于遗传工具欠发达的其他生物。

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