Pelicic V, Reyrat J M, Gicquel B
Unité de Génétigue Mycobactérienne, CNRS URA 1300, Institut Pasteur, Paris, France.
Mol Microbiol. 1996 Jun;20(5):919-25. doi: 10.1111/j.1365-2958.1996.tb02533.x.
The expression of sacB, the Bacillus subtilis gene encoding levansucrase, is lethal to mycobacteria in the presence of 10% sucrose. In this study, we describe the use of sacB as a marker for positive selection of gene-replacement events into Mycobacterium smegmatis. A sucrose counter-selectable suicide plasmid was used to deliver an inactivated copy of the pyrF gene (pyrF::K(m)) into the M. smegmatis genome. Only uracil auxotroph clones, resulting from replacement of the endogenous pyrF allele, survived in a one-step selection on plates containing kanamycin and 10% sucrose. This demonstrated that selection on sucrose against the maintenance of the vector bearing the sacB gene is 100% efficient, enabling the positive selection of allelic-exchange mutants. Two-step selection is also feasible; it was used to construct unmarked pyrF mutants in which the gene was inactivated by a frameshift mutation. This method of generating unmarked, directed mutations is rapid and simple, making it a powerful tool for the genetic characterization of mycobacteria.
枯草芽孢杆菌编码果聚糖蔗糖酶的基因sacB的表达，在10%蔗糖存在的情况下对分枝杆菌具有致死性。在本研究中，我们描述了使用sacB作为正向选择标记，将基因替换事件导入耻垢分枝杆菌。使用一种蔗糖反向选择自杀质粒将pyrF基因的失活拷贝（pyrF::K(m)）导入耻垢分枝杆菌基因组。只有那些因内源性pyrF等位基因被替换而产生的尿嘧啶营养缺陷型克隆，在含有卡那霉素和10%蔗糖的平板上进行一步筛选时能够存活。这表明针对携带sacB基因的载体进行蔗糖筛选的效率为100%，能够实现等位基因交换突变体的正向选择。两步筛选也是可行的；它被用于构建无标记的pyrF突变体，其中该基因通过移码突变而失活。这种产生无标记定向突变的方法快速且简单，使其成为分枝杆菌遗传特征分析的有力工具。
