In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology
In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology
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DOI：
10.1128/aem.03909-14
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发表时间：
2015-06-01
影响因子：
4.4
通讯作者：
Tolker-Nielsen, Tim
中科院分区：
文献类型：
--
作者：
Fazli, Mustafa;Harrison, Joe J.;Tolker-Nielsen, Tim
新洋葱伯克霍尔德菌是一种新兴的机会致病菌，在免疫功能低下的个体和囊性纤维化患者中引起危及生命的感染，这些感染通常很难治疗，如果不是不可能的话。了解这种新兴病原体毒力的遗传基础对于开发新的治疗方案非常重要。在预测编码毒力决定因子的基因中产生缺失突变是研究发病机制的基础。然而，缺乏用于B的合适的选择性和反选择性标记。cenocepacia，使其基因操作成问题。在这里，我们描述了一个网关兼容的等位基因交换系统的基础上的可逆选择pheS基因和I-SceI归巢核酸内切酶。该系统提供了克隆靶基因同源区的效率，并允许在B中产生精确和未标记的基因缺失。cenocepacia。作为概念的证明，我们通过删除Bcam 1349基因来证明其实用性，该基因编码对生物膜形成重要的环状二GMP（c-di-GMP）响应调节蛋白。
Burkholderia cenocepacia is an emerging opportunistic pathogen causing life-threatening infections in immunocompromised individuals and in patients with cystic fibrosis, which are often difficult, if not impossible, to treat. Understanding the genetic basis of virulence in this emerging pathogen is important for the development of novel treatment regimes. Generation of deletion mutations in genes predicted to encode virulence determinants is fundamental to investigating the mechanisms of pathogenesis. However, there is a lack of appropriate selectable and counterselectable markers for use in B. cenocepacia, making its genetic manipulation problematic. Here we describe a Gateway-compatible allelic exchange system based on the counterselectable pheS gene and the I-SceI homing endonuclease. This system provides efficiency in cloning homology regions of target genes and allows the generation of precise and unmarked gene deletions in B. cenocepacia. As a proof of concept, we demonstrate its utility by deleting the Bcam1349 gene, encoding a cyclic di-GMP (c-di-GMP)-responsive regulator protein important for biofilm formation.