Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO, 65212, USA.
BMC Microbiol. 2018 Oct 24;18(1):158. doi: 10.1186/s12866-018-1319-0.
Transposon mutagenesis is highly valuable for bacterial genetic and genomic studies. The transposons are usually delivered into host cells through conjugation or electroporation of a suicide plasmid. However, many bacterial species cannot be efficiently conjugated or transformed for transposon saturation mutagenesis. For this reason, temperature-sensitive (ts) plasmids have also been developed for transposon mutagenesis, but prolonged incubation at high temperatures to induce ts plasmid loss can be harmful to the hosts and lead to enrichment of mutants with adaptive genetic changes. In addition, the ts phenotype of a plasmid is often strain- or species-specific, as it may become non-ts or suicidal in different bacterial species.
We have engineered several conditional suicide plasmids that have a broad host range and whose loss is IPTG-controlled. One construct, which has the highest stability in the absence of IPTG induction, was then used as a curable vector to deliver hyperactive miniTn5 transposons for insertional mutagenesis. Our analyses show that these new tools can be used for efficient and regulatable transposon mutagenesis in Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. In P. aeruginosa PAO1, we have used this method to generate a Tn5 insertion library with an estimated diversity of ~ 10, which is ~ 2 logs larger than the best transposon insertional library of PAO1 and related Pseudomonas strains previously reported.
We have developed a number of IPTG-controlled conditional suicide plasmids. By exploiting one of them for transposon delivery, a highly efficient and broadly useful mutagenesis system has been developed. As the assay condition is mild, we believe that our methodology will have broad applications in microbiology research.
转座子诱变在细菌遗传和基因组研究中非常有价值。转座子通常通过自杀质粒的接合或电穿孔导入宿主细胞。然而，许多细菌物种不能有效地进行接合或转化，以进行转座子饱和诱变。出于这个原因，也开发了温度敏感（ts）质粒用于转座子诱变，但长时间在高温下孵育以诱导 ts 质粒丢失可能对宿主有害，并导致具有适应性遗传变化的突变体富集。此外，质粒的 ts 表型通常是菌株或物种特异性的，因为它在不同的细菌物种中可能变得非 ts 或自杀。
我们已经设计了几种具有广泛宿主范围且其丢失受 IPTG 控制的条件性自杀质粒。然后，使用一种在没有 IPTG 诱导时稳定性最高的构建体作为可治愈的载体，用于递送超活性 miniTn5 转座子进行插入诱变。我们的分析表明，这些新工具可用于在大肠杆菌、鲍曼不动杆菌和铜绿假单胞菌中进行高效和可调节的转座子诱变。在铜绿假单胞菌 PAO1 中，我们使用该方法生成了一个 Tn5 插入文库，估计多样性约为 ~10，比以前报道的 PAO1 和相关假单胞菌菌株的最佳转座子插入文库大约 2 个对数级。
我们开发了几种 IPTG 控制的条件性自杀质粒。通过利用其中一种进行转座子传递，开发了一种高效且广泛适用的诱变系统。由于该测定条件温和，我们相信我们的方法将在微生物学研究中具有广泛的应用。
