Monson Rita, Smith Debra S, Matilla Miguel A, Roberts Kevin, Richardson Elizabeth, Drew Alison, Williamson Neil, Ramsay Josh, Welch Martin, Salmond George P C
Department of Biochemistry, University of Cambridge Cambridge, UK.
Front Microbiol. 2015 Dec 22;6:1442. doi: 10.3389/fmicb.2015.01442. eCollection 2015.
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.
随机转座子诱变是一种强大的技术，用于在许多不同的细菌菌株中生成基因插入文库。在此，我们开发了一种系统，便于在一系列不同的革兰氏阴性细菌菌株中进行随机转座子诱变，这些菌株包括黑胫果胶杆菌、鼠柠檬酸杆菌、粘质沙雷氏菌ATCC39006、普城沙雷氏菌、达旦氏果胶杆菌等等。在这些菌株中分别优化了转座子诱变，并展示了三项研究以证明该系统的有效性。首先，对重要的农业病原菌达旦氏果胶杆菌进行诱变。鉴定并表征了两个蛋白酶产量降低的突变体和一个产生先前未知色素靛蓝素的突变体。其次，对肠杆菌粘质沙雷氏菌ATCC39006进行诱变，鉴定出了不能产生气体囊泡（蛋白质类细胞内细胞器）的突变体。其中一个突变体在对气体囊泡产生至关重要的gvpA1基因内含有一个β-半乳糖苷酶转录融合体。最后，该系统用于对植物相关肠杆菌索氏果胶杆菌MK10中抗真菌、抗卵菌和抗癌聚酮化合物卵霉素A的生物合成基因簇进行诱变。开发的诱变系统便于在转座子切除后轻松生成包含转座子和相邻DNA的复制子，通过测序容易鉴定转座子插入位点。此外，该系统还可以创建以β-半乳糖苷酶或β-葡萄糖醛酸酶作为报告基因的转录融合体，并利用多种耐药标记，以便在单个菌株中产生多个可选择的融合体。这种包含各种转座子的系统具有广泛的用途，可以以许多不同的方式组合。
