Herring Christopher D, Blattner Frederick R
Laboratory of Genetics, University of Wisconsin-Madison, 445 Henry Mall, Madison, WI 53706, USA.
J Bacteriol. 2004 May;186(9):2673-81. doi: 10.1128/JB.186.9.2673-2681.2004.
The essential genes of microorganisms encode biological functions important for survival and thus tend to be of high scientific interest. Drugs that interfere with essential functions are likely to be interesting candidates for antimicrobials. However, these genes are hard to study genetically because knockout mutations in them are by definition inviable. We recently described a conditional mutation system in Escherichia coli that uses a plasmid to produce an amber suppressor tRNA regulated by the arabinose promoter. This suppressor was used here in the construction of amber mutations in seven essential E. coli genes. Amber stop codons were introduced as "tagalong" mutations in the flanking DNA of a downstream antibiotic resistance marker by lambda red recombination. The drug marker was removed by expression of I-SceI meganuclease, leaving a markerless mutation. We demonstrate the method with the genes frr, gcpE, lpxC, map, murA, ppa, and rpsA. We were unable to isolate an amber mutation in ftsZ. Kinetics of cell death and morphological changes were measured following removal of arabinose. As expected given the wide range of cellular mechanisms represented, different mutants showed widely different death curves. All of the mutations were bactericidal except the mutation in gcpE, which was bacteriostatic. The strain carrying an amber mutation in murA was by far the most sensitive, showing rapid killing in nonpermissive medium. The MurA protein is critical for peptidoglycan synthesis and is the target for the antibiotic fosfomycin. Such experiments may inexpensively provide valuable information for the identification and prioritization of targets for antibiotic development.
微生物的必需基因编码对生存至关重要的生物学功能，因此往往具有很高的科学研究价值。干扰必需功能的药物很可能是有吸引力的抗菌候选药物。然而，这些基因很难通过遗传学方法进行研究，因为根据定义，它们的敲除突变是不可存活的。我们最近在大肠杆菌中描述了一种条件突变系统，该系统使用质粒产生受阿拉伯糖启动子调控的琥珀抑制tRNA。在此，这种抑制子被用于构建大肠杆菌七个必需基因中的琥珀突变。通过λ红重组，在下游抗生素抗性标记的侧翼DNA中引入琥珀终止密码子作为“伴随”突变。通过表达I-SceI巨核酸酶去除药物标记，留下无标记突变。我们用frr、gcpE、lpxC、map、murA、ppa和rpsA基因演示了该方法。我们无法分离ftsZ中的琥珀突变。去除阿拉伯糖后，测量细胞死亡动力学和形态变化。鉴于所代表的细胞机制范围广泛，不同的突变体显示出差异很大的死亡曲线。除gcpE中的突变是抑菌的外，所有突变都是杀菌的。携带murA琥珀突变的菌株是迄今为止最敏感的，在非允许培养基中显示出快速死亡。MurA蛋白对肽聚糖合成至关重要，是抗生素磷霉素的作用靶点。此类实验可能以低成本为抗生素开发靶点的鉴定和优先级排序提供有价值的信息。
