Insertion-duplication mutagenesis of Neisseria:: Use in characterization of DNA transfer genes in the gonococcal genetic island
Insertion-duplication mutagenesis of Neisseria:: Use in characterization of DNA transfer genes in the gonococcal genetic island
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DOI：
10.1128/jb.183.16.4718-4726.2001
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发表时间：
2001-08-01
影响因子：
3.2
通讯作者：
Dillard, JP
中科院分区：
文献类型：
--
作者：
Hamilton, HL;Schwartz, KJ;Dillard, JP
我们创造了用于淋病奈瑟菌插入-复制突变(IDM)的质粒。这种诱变方法的优点是，在转化为淋球菌之前只需要一个克隆步骤。克隆到质粒中的染色体DNA通过单交叉(坎贝尔型)重组事件直接插入到同源部位的染色体中。其中两个载体含有红霉素抗性基因ermC，具有强大的启动子，并且定向转录将进入克隆的插入片段。因此，这些质粒可以用来产生在下游基因转录上有效无极性的插入片段。除了改进的ermC外，该载体还包含两个拷贝的新序列DNA摄取序列，以促进转化过程中高频DNA摄取。使用不同的染色体DNA插入大小，我们已经确定，通过这种方法，即使是很小的插入也可以针对插入突变，并且插入稳定地保持在淋球菌染色体中。我们已经使用IDM在淋球菌遗传岛(GGI)的两个基因中创建了敲除，并通过染色体行走程序克隆了GGI的其他区域。TRAG和TRAH突变体的表型特征表明，编码的蛋白质通过一种新的IV型分泌系统在DNA分泌中发挥作用。
We created plasmids for use in insertion-duplication mutagenesis (IDM) of Neisseria gonorrhoeae. This mutagenesis method has the advantage that it requires only a single cloning step prior to transformation into gonococci. Chromosomal DNA cloned into the plasmid directs insertion into the chromosome at the site of homology by a single-crossover (Campbell-type) recombination event. Two of the vectors contain an erythromycin resistance gene, ermC, with a strong promoter and in an orientation such that transcription will proceed into the cloned insert. Thus, these plasmids can be used to create insertions that are effectively nonpolar on the transcription of downstream genes. In addition to the improved ermC, the vector contains two copies of the neisserial DNA uptake sequence to facilitate high-frequency DNA uptake during transformation. Using various chromosomal DNA insert sizes, we have determined that even small inserts can target insertion mutation by this method and that the insertions are stably maintained in the gonococcal chromosome. We have used IDM to create knockouts in two genes in the gonococcal genetic island (GGI) and to clone additional regions of the GGI by a chromosome-walking procedure. Phenotypic characterization of traG and traH mutants suggests a role for the encoded proteins in DNA secretion by a novel type IV secretion system.