Development and application of an Insertional system for gene delivery and expression in Campylobacter jejuni
Development and application of an Insertional system for gene delivery and expression in Campylobacter jejuni
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DOI：
10.1128/aem.71.7.4004-4013.2005
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发表时间：
2005-07-01
影响因子：
4.4
通讯作者：
Wren, BW
中科院分区：
文献类型：
--
作者：
Karlyshev, AV;Wren, BW
空肠弯曲杆菌是一种重要的胃肠道病原体，其遗传学研究因缺乏有效的外源遗传信息导入系统而受到阻碍，这是因为为大肠杆菌和其他细菌设计的常用载体不能在弯曲杆菌细胞中维持。此外，弯曲杆菌中的基因表达需要物种特异性启动子的存在。在这项研究中，我们利用了几个保守拷贝的rRNA基因簇插入到染色体的同源重组的各种基因的可用性。rRNA序列的高度保守性意味着该方法可以应用于其他弯曲杆菌菌株。该载体中弯曲杆菌衍生的启动子的存在确保了外源基因在靶细胞中的表达。通过两种弯曲杆菌菌株的突变互补证明了该方法的有效性。此外，我们将该系统用于绿色荧光蛋白（GFP）的引入和表达。表达GFP的弯曲杆菌允许在固定液体培养物中附着于玻璃表面的固着细菌的可视化。该研究表明，附着的细菌含有球形和螺旋形的组合，具有保存活的高度能动细胞的液体通道。我们展示了一种新的通用程序的基因传递和表达，可以作为一种有效的工具来研究这种知之甚少的病原体。这项研究中开发的原理可以更广泛地应用于对遗传分析难治的其他细菌的操作。
The genetic investigation of Campylobacter jejuni, an important gastrointestinal pathogen, has been hampered by the lack of an efficient system for introduction of exogenous genetic information, as commonly used vectors designed for Escherichia coli and other bacteria cannot be maintained in Campylobacter cells. Additionally, gene expression in Campylobacter requires the presence of species-specific promoters. In this study we exploited the availability of several conserved copies of rRNA gene clusters for insertion of various genes into the chromosome by homologous recombination. The high conservation of the rRNA sequences means that the procedure can be applied to other Campylobacter strains. The presence of a Campylobacter-derived promoter in this vector ensures expression of exogenous genes in target cells. The efficiency of the procedure was demonstrated by complementation of mutations in two strains of Campylobacter. In addition, we applied the system for introduction and expression of a green fluorescent protein (GFP). GFP-expressing Campylobacter allowed visualization of sessile bacteria attached to a glass surface in stationary liquid culture. The study demonstrated that the attached bacteria contained an assemblage of coccoid and spiral forms with liquid channels preserving viable highly motile cells. We demonstrate a novel universal procedure for gene delivery and expression that can be used as an efficient tool to study this poorly understood pathogen. The principles developed in this study could be more widely applied for the manipulation of other bacteria that are refractory to genetic analysis.