A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria.
A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria.
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DOI：
10.3389/fmicb.2015.01442
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发表时间：
2015
影响因子：
5.2
通讯作者：
Salmond GP
中科院分区：
文献类型：
--
作者：
Monson R;Smith DS;Matilla MA;Roberts K;Richardson E;Drew A;Williamson N;Ramsay J;Welch M;Salmond GP
随机转座子突变是一种强大的技术，用于在许多不同的细菌菌株中产生基因插入文库。在这里，我们开发了一个促进随机转座子突变的系统，在一系列不同的革兰氏阴性细菌菌株中，包括腐败杆菌，轮状柠檬酸杆菌，沙雷氏菌。ATCC39006、普氏沙雷氏菌、达丹提迪克亚等。在这些菌株中的转座子诱变都进行了优化，并提出了三项研究，以显示该系统的有效性。首先，对重要的农业病原菌大丹进行了诱变。鉴定和鉴定了两个突变株和一个突变株，其中两个突变株显示了较低的蛋白酶产量，一个突变株产生了以前不知名的色素--青黛。其次是肠杆菌，沙雷氏菌。对ATCC39006进行诱变，筛选出不能产生气泡的突变株，即胞内蛋白质细胞器。其中之一在基因gvpA1内含有β-半乳糖苷酶转录融合，这对气泡的产生是必不可少的。最后，利用该系统对植物伴生肠杆菌Dikeya solani MK10中合成的抗真菌、抗卵菌和抗癌多酮基因簇进行了突变。突变系统的开发是为了在切除后简单地产生包含转座子和相邻DNA的复制子后，通过测序来容易地识别转座子插入位点。此外，该系统还可以创建以β-半乳糖苷酶或β-葡萄糖苷酶为报告基因的转录融合，并利用各种耐药标记，从而在单个菌株中产生多个可选择的融合。这种由各种转座子组成的系统具有广泛的实用性，可以通过许多不同的方式组合。
Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.