Chromosomal integration vectors allowing flexible expression of foreign genes in Campylobacter jejuni.
Chromosomal integration vectors allowing flexible expression of foreign genes in Campylobacter jejuni.
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DOI：
10.1186/s12866-015-0559-5
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发表时间：
2015-10-24
期刊：
影响因子：
4.2
通讯作者：
Linton D
中科院分区：
文献类型：
--
作者：
Jervis AJ;Butler JA;Wren BW;Linton D
空肠弯曲杆菌是人类肠胃炎的一个主要原因，但有有限的知识是如何引起疾病。分子遗传学方法对于研究这种重要病原体的毒力机制至关重要。通过染色体重组在空肠空肠杆菌中表达基因的载体对于插入敲除突变体的遗传互补特别有用，更普遍的是对于特定空肠空肠杆菌宿主背景的基因表达。利用标准克隆技术构建了3个由3个不同的强启动子驱动表达的载体，使其能够整合到空肠梭菌染色体上。在整合到空肠C.染色体上后，用荧光测量和荧光显微镜观察细胞的表达水平。我们已经创建了质粒pCJC1，设计用于重组介导的基因传递到空肠梭菌染色体上。该质粒含有一个具有上游和下游限制性位点的氯霉素耐药盒（cat），两侧是空肠假基因Cj0223的区域。克隆猫基因上游或下游的基因，允许它们随后被引入假基因中的空肠梭菌染色体。基因表达可以由原生基因启动子驱动（如果包含），或者由cat启动子驱动（如果基因克隆在cat的下游，并且转录方向与cat相同）。为了增加空肠c染色体基因的表达，我们分别从空肠c、幽门螺杆菌和白痢幽门螺杆菌的porA、ureI和flaA基因中引入三个较强的启动子，对pCJC1进行了修饰。这些启动子及其相关的核糖体结合位点被克隆到pCJC1上的cat基因上游，形成质粒pCJC2、pCJC3和pCJC4。为了测试它们的有效性，将绿色荧光蛋白（gfp）报告基因插入三个启动子的下游，并将启动子基因融合到空肠梭菌宿主染色体上，定量表达水平。porA启动子的表达产生最高的荧光，flaA的表达产生中等水平的荧光，ureI的表达产生最低的荧光。来自porA启动子的gfp的表达使得HeLa细胞入侵后空肠梭菌细胞内的荧光显微镜可见。所构建的质粒允许空肠梭菌基因在染色体上稳定表达，并且根据所使用的启动子的不同，获得不同的表达水平，使这些质粒成为遗传互补和高水平表达的有用工具。本文的在线版本（doi:10.1186/s12866-015-0559-5）包含补充材料，可供授权用户使用。
Campylobacter jejuni is a major cause of human gastroenteritis yet there is limited knowledge of how disease is caused. Molecular genetic approaches are vital for research into the virulence mechanisms of this important pathogen. Vectors that allow expression of genes in C. jejuni via recombination onto the chromosome are particularly useful for genetic complementation of insertional knockout mutants and more generally for expression of genes in particular C. jejuni host backgrounds. A series of three vectors that allow integration of genes onto the C. jejuni chromosome were constructed by standard cloning techniques with expression driven from three different strong promoters. Following integration onto the C. jejuni chromosome expression levels were quantified by fluorescence measurements and cells visualized by fluorescence microscopy. We have created plasmid, pCJC1, designed for recombination-mediated delivery of genes onto the C. jejuni chromosome. This plasmid contains a chloramphenicol resistance cassette (cat) with upstream and downstream restriction sites, flanked by regions of the C. jejuni pseudogene Cj0223. Cloning of genes immediately upstream or downstream of the cat gene allows their subsequent introduction onto the C. jejuni chromosome within the pseudogene. Gene expression can be driven from the native gene promoter if included, or alternatively from the cat promoter if the gene is cloned downstream of, and in the same transcriptional orientation as cat. To provide increased and variable expression of genes from the C. jejuni chromosome we modified pCJC1 through incorporation of three relatively strong promoters from the porA, ureI and flaA genes of C. jejuni, Helicobacter pylori and Helicobacter pullorum respectively. These promoters along with their associated ribosome binding sites were cloned upstream of the cat gene on pCJC1 to create plasmids pCJC2, pCJC3 and pCJC4. To test their effectiveness, a green fluorescent protein (gfp) reporter gene was inserted downstream of each of the three promoters and following integration of promoter-gene fusions onto the C. jejuni host chromosome, expression levels were quantified. Expression from the porA promoter produced the highest fluorescence, from flaA intermediate levels and from ureI the lowest. Expression of gfp from the porA promoter enabled visualization by fluorescent microscopy of intracellular C. jejuni cells following invasion of HeLa cells. The plasmids constructed allow stable chromosomal expression of genes in C. jejuni and, depending on the promoter used, different expression levels were obtained making these plasmids useful tools for genetic complementation and high level expression. The online version of this article (doi:10.1186/s12866-015-0559-5) contains supplementary material, which is available to authorized users.