A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria.

摘要: Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop system facilitating random range Gram-negative strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, plymuthica, Dickeya dadantii and more. Transposon was optimized each these strains three studies are presented show the efficacy this system. Firstly, important agricultural pathogen D. mutagenized. Two mutants that showed reduced protease production one mutant producing previously cryptic pigment, indigoidine, were identified characterized. Secondly, enterobacterium, ATCC39006 mutagenized incapable gas vesicles, proteinaceous intracellular organelles, identified. One contained β-galactosidase transcriptional fusion within gene gvpA1, essential for vesicle production. Finally, mutate biosynthetic clusters antifungal, anti-oomycete anticancer polyketide, oocydin A, plant-associated solani MK10. The developed allow easy identification insertion sites by sequencing, after facile generation replicon encompassing adjacent DNA, post-excision. Furthermore, can also create fusions with either or β-glucuronidase as reporters, exploits variety drug resistance markers so multiple selectable be generated single strain. This various transposons has wide utility combined ways.