Expanding the repertoire of gene tools for precise manipulation of the Clostridium difficile genome: allelic exchange using pyrE alleles.
Expanding the repertoire of gene tools for precise manipulation of the Clostridium difficile genome: allelic exchange using pyrE alleles.
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DOI：
10.1371/journal.pone.0056051
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Minton NP
中科院分区：
文献类型：
--
作者：
Ng YK;Ehsaan M;Philip S;Collery MM;Janoir C;Collignon A;Cartman ST;Minton NP
在分子水平上修改基本生物过程的复杂遗传工具在阐明艰难梭菌的分子发病机制中是关键的，艰难梭菌是医疗保健相关疾病的主要原因。在这里，我们已经开发出一个有效的程序，使精确的修改C。艰难梭菌基因组通过基于pyrE的等位基因交换。通过在非流行性菌株630Δ E1中的三个基因（spo 0A、cwp 84和mtlD）和流行性PCR核糖体型027菌株R20291中的两个基因（spo 0A和cwp 84）中创建框内缺失，证明了该方法的稳健性和可靠性。该系统依赖于使用等位基因偶联交换（ACE）初始创建pyrE缺失突变体，其是尿嘧啶营养缺陷型的并且对氟乳清酸（FOA）具有抗性。这使得能够在FOA的存在下通过使用来自生孢梭菌的异源pyrE等位基因作为反选择/负选择标记的等位基因交换来随后修饰靶基因。在修饰靶基因后，使用ACE使所产生的菌株快速恢复为尿嘧啶原养型，从而允许在PyrE精通背景中表征突变体表型。至关重要的是，失活基因的野生型拷贝可以使用ACE伴随pyrE等位基因的校正引入基因组中。这使得互补研究可以在适当的基因剂量下进行，而不是使用多拷贝自主质粒。“校正”方法的快速性（5-7天）使得pyrE −菌株成为诱变研究的有吸引力的宿主。
Sophisticated genetic tools to modify essential biological processes at the molecular level are pivotal in elucidating the molecular pathogenesis of Clostridium difficile, a major cause of healthcare associated disease. Here we have developed an efficient procedure for making precise alterations to the C. difficile genome by pyrE-based allelic exchange. The robustness and reliability of the method was demonstrated through the creation of in-frame deletions in three genes (spo0A, cwp84, and mtlD) in the non-epidemic strain 630Δerm and two genes (spo0A and cwp84) in the epidemic PCR Ribotype 027 strain, R20291. The system is reliant on the initial creation of a pyrE deletion mutant, using Allele Coupled Exchange (ACE), that is auxotrophic for uracil and resistant to fluoroorotic acid (FOA). This enables the subsequent modification of target genes by allelic exchange using a heterologous pyrE allele from Clostridium sporogenes as a counter-/negative-selection marker in the presence of FOA. Following modification of the target gene, the strain created is rapidly returned to uracil prototrophy using ACE, allowing mutant phenotypes to be characterised in a PyrE proficient background. Crucially, wild-type copies of the inactivated gene may be introduced into the genome using ACE concomitant with correction of the pyrE allele. This allows complementation studies to be undertaken at an appropriate gene dosage, as opposed to the use of multicopy autonomous plasmids. The rapidity of the ‘correction’ method (5–7 days) makes pyrE − strains attractive hosts for mutagenesis studies.
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