Transposon mutagenesis in Caulobacter crescentus
Transposon mutagenesis in Caulobacter crescentus
复制标题
新月柄杆菌的转座子诱变
DOI：
10.1128/jb.149.2.620-625.1982
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发表时间：
1982
影响因子：
3.2
通讯作者：
R. H. Croft
中科院分区：
文献类型：
--
作者：
B. Ely;R. H. Croft
转座子Tn5 （Km）和Tn7 （Tp和Sm）通过p型抗生素耐药因子转移到新月茎杆菌。通过每个转座子染色体插入的分离证实了转座子的存在。当C. crescent菌株携带RP4 aphA::Tn7时，引入野生型RP4导致了驻留质粒的丢失。同时选择Kmr和Smr得到了带有Tn7染色体插入的菌落。对1万多个Tn7染色体插入的检查表明没有营养缺陷或运动突变。因此，Tn7似乎在月牙蛾中具有很高的插入特异性。含mu质粒pJB4JI将Tn5转移到月牙蛾，但质粒未维持。对照实验表明，含mu质粒在月牙菌中恢复的频率极低，并且恢复的质粒DNA发生了广泛的缺失。据推测，新月锥虫无法耐受Mu基因组的某些部分。含mu质粒的不稳定性使其成为引入转座子的绝佳载体，我们已经使用pJB4JI分离了Tn5的染色体插入。当几千个这样的插入突变体被检查时，我们发现营养不良和运动突变的频率分别为1%和2%。这些结果表明，Tn5在月牙菇中具有较低的插入特异性，因此可能是一种有用的诱变原，可以获得多种突变表型。
Transposons Tn5 (Km) and Tn7 (Tp and Sm) were transferred to Caulobacter crescentus via P-type antibiotic resistance factors. Transposition was demonstrated by the isolation of chromosomal insertions of each transposon. With C. crescentus strains harboring RP4 aphA::Tn7, the introduction of a wild-type RP4 resulted in the loss of the resident plasmid. Simultaneous selection for Kmr and Smr yielded colonies with chromosomal insertions of Tn7. Examination of over 10,000 chromosomal insertions of Tn7 indicated no auxotrophic or motility mutants. Thus, Tn7 appears to have a high specificity of insertion in C. crescentus. The Mu-containing plasmid pJB4JI transferred Tn5 to C. crescentus, but the plasmid was not maintained. Control experiments showed that recovery of Mu-containing plasmids occurred at very low frequencies in C. crescentus and that the plasmids which were recovered had undergone extensive deletion of plasmid DNA. Presumably, some part of the Mu genome was not tolerated by C. crescentus. The instability of the Mu-containing plasmids makes them excellent vectors for the introduction of transposons, and we have used pJB4JI to isolated chromosomal insertions of Tn5. When several thousand of these insertion mutants were examined, we found auxotrophic and motility mutants at frequencies of 1 and 2%, respectively. These results indicate that Tn5 had a low specificity of insertion in C. crescentus and therefore would be a useful mutagen for obtaining a variety of mutant phenotypes.