Johnston D M, Cannon J G
Department of Microbiology and Immunology, CB#7290, 804 Mary Ellen Jones Building, University of North Carolina, Chapel Hill, NC 27599, USA.
Gene. 1999 Aug 5;236(1):179-84. doi: 10.1016/s0378-1119(99)00238-3.
The pathogenesis of infections caused by Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, can be studied using experimental infection of human male volunteers. The desire to avoid introducing new antibiotic resistance markers into strains to be used in human experimental infection has complicated the construction of genetically defined mutants in which expression of potential virulence factors is inactivated. To facilitate construction of such mutants, we have used a two-step mutagenesis strategy that allows for gene replacements without introducing new selectable markers into the final strain. The method uses a two-gene cassette containing both a selectable marker (ermC') and a counterselectable marker (rpsL). The cassette is cloned into the gene of interest and used to replace the wild-type gene on the chromosome by allelic exchange. A second transformation replaces the cassette-containing version of the gene with an engineered version with an unmarked deletion or other mutation. The rpsL gene of Escherichia coli functioned for the counterselection in the gonococcus, albeit with low efficiency. To improve the efficiency of the counterselection, we cloned the gonococcal rpsL gene and incorporated it into the cassette. This technique has been successful in creating defined mutants for human challenge, and also circumvents the limitation in the number of different selectable markers that are useful in Neisseria species.
淋病奈瑟菌是性传播疾病淋病的病原体，可通过对人类男性志愿者进行实验性感染来研究其所致感染的发病机制。由于不想在用于人类实验性感染的菌株中引入新的抗生素抗性标记，构建使潜在毒力因子表达失活的基因定义突变体变得复杂。为便于构建此类突变体，我们采用了两步诱变策略，该策略可在不向最终菌株中引入新的选择标记的情况下进行基因替换。该方法使用一个双基因盒，其中包含一个选择标记（ermC'）和一个反选择标记（rpsL）。将该盒克隆到感兴趣的基因中，并通过等位基因交换用于替换染色体上的野生型基因。第二次转化用具有无标记缺失或其他突变的工程化版本替换含盒的基因版本。大肠杆菌的rpsL基因在淋球菌中起反选择作用，尽管效率较低。为提高反选择效率，我们克隆了淋球菌rpsL基因并将其纳入盒中。该技术已成功创建用于人类挑战的定义突变体，并且还规避了在奈瑟菌属中有用的不同选择标记数量的限制。
