Use of the promoter fusion transposon Tn5 lac to identify mutations in Bordetella pertussis vir-regulated genes.
Use of the promoter fusion transposon Tn5 lac to identify mutations in Bordetella pertussis vir-regulated genes.
复制标题
使用启动子融合转座子 Tn5 lac 鉴定百日咳博德特氏菌 vir 调节基因的突变。
DOI：
10.1128/iai.57.9.2674-2682.1989
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发表时间：
1989
影响因子：
3.1
通讯作者：
Meidl,JJ
中科院分区：
文献类型：
--
作者：
Weiss,AA;Melton,AR;Walker,KE;Andraos-Selim,C;Meidl,JJ
使用转座子 Tn5 lac 鉴定了毒力相关因子缺陷的百日咳博德特氏菌突变体。 Tn5 lac 是 Tn5 的衍生物，可生成 β-半乳糖苷酶的启动子融合体。通过选择卡那霉素抗性突变体来鉴定百日咳博德特氏菌 vir 调节基因中的 Tn5 lac 插入，这些突变体在 vir 调节基因表达时表达 β-半乳糖苷酶，但在 vir 调节基因关闭时不表达。已鉴定出 vir 调节基因中的 14 种不同突变。两种突变体缺乏丝状血凝素的产生，两种突变体缺乏腺苷酸环化酶毒素和溶血素的产生，一种突变体缺乏皮肤坏死毒素的产生。一个插入位置与百日咳毒素基因相邻，但突变体产生了百日咳毒素。其余八个突变体的表型尚未确定，但突变体似乎在 69,000 道尔顿外膜蛋白（凝集原 3）或荚膜的产生方面没有缺陷。筛选任一菌毛基因中的突变被证明是有问题的，因为发现亲本菌株以高频率从菌毛状态转变为非菌毛状态，这表明菌毛在其他细菌中的亚稳态表达。我们使用针对菌毛序列特异的 30 聚体进行 Southern 印迹分析。没有观察到由于 Tn5 lac 的 12 kilobase 导致的预计大小增加的条带，这表明这些基因都没有发生突变。 Southern印迹分析还显示，八个未识别突变中有七个映射到不同的限制性片段，这表明它们可能缺乏多达七个不同的基因。
Mutants of Bordetella pertussis deficient in virulence-associated factors were identified by using the transposon Tn5 lac. Tn5 lac is a derivative of Tn5 which generates promoter fusions for beta-galactosidase. Tn5 lac insertions in the vir-regulated genes of B. pertussis were identified by selecting for kanamycin-resistant mutants that expressed beta-galactosidase when the vir-regulated genes were expressed but not when the vir-regulated genes were turned off. Fourteen different mutations in vir-regulated genes were identified. Two mutants were deficient in the production of the filamentous hemagglutinin, two mutants were deficient in the production of adenylate cyclase toxin and hemolysin, and one mutant was deficient in the production of dermonecrotic toxin. One insertion mapped adjacent to the pertussis toxin gene, but the mutant produced pertussis toxin. The phenotypes of the remaining eight mutants were not determined, but the mutants did not appear to be deficient in the production of the 69,000-dalton outer membrane protein (agglutinogen 3) or the capsule. Screening for mutations in either of the fimbrial genes proved to be problematic since the parental strain was found to switch from a fimbriated to a nonfimbriated state at a high frequency, which was suggestive of the metastable expression of pili in other bacteria. We used Southern blot analysis with a 30-mer specific for the fimbrial sequences. No bands with the predicted increase in size due to the 12 kilobases from Tn5 lac were observed, which suggests that none of these genes were mutated. Southern blot analysis also revealed that seven of the eight unidentified mutations mapped to different restriction fragments, which suggests that they could be deficient in as many as seven different genes.