Insertion-Duplication Mutagenesis of Neisseria: Use in Characterization of DNA Transfer Genes in the Gonococcal Genetic Island

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.

我们构建了适用于淋病奈瑟菌（Neisseria gonorrhoeae）插入重复诱变（insertion-duplication mutagenesis，IDM）的质粒。该诱变方法的优势在于，将质粒转化至淋病奈瑟菌前仅需单次克隆步骤。克隆至该质粒的染色体DNA可通过单交换（坎贝尔型）重组事件，靶向同源位点插入宿主染色体。其中2种载体携带带有强启动子的红霉素抗性基因ermC，且其转录方向可使RNA聚合酶的转录延伸至克隆插入片段内，因此此类质粒可用于构建对下游基因转录几乎无极性效应的插入突变。除优化后的ermC基因外，该载体还携带2份奈瑟菌属DNA摄取序列，以提升转化过程中的DNA高频摄取效率。通过使用不同长度的染色体DNA插入片段，我们证实即便小型插入片段也可通过该方法实现靶向插入突变，且插入突变可在淋病奈瑟菌染色体中稳定维持。我们已利用IDM技术在淋病奈瑟菌遗传岛（gonococcal genetic island，GGI）的2个基因中构建敲除突变，并通过染色体步移法克隆了GGI的其他区域。对traG与traH突变株的表型表征结果表明，其编码蛋白参与新型IV型分泌系统介导的DNA分泌过程。