Abstract
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.
Highlights
IntroductionTransposable elements have greatly assisted our understanding of eukaryotic and prokaryotic genetics
Since their initial discovery, transposable elements have greatly assisted our understanding of eukaryotic and prokaryotic genetics
Antibiotics and supplements were added at the following concentrations: ampicillin, 100 μg ml−1; chloramphenicol, 50 μg ml−1; tetracycline, 15 μg ml−1; kanamycin, 15 μg ml−1 (E. coli strain β2163) and 50 μg ml−1 (S. plymuthica A153, D. solani MK10 and sp. 39006 (S39006)); erythromycin, 200 μg ml−1, and 2-6-diaminopimelic acid (DAPA), 300 μM
Summary
Transposable elements have greatly assisted our understanding of eukaryotic and prokaryotic genetics. Plasmid-Transposon Mutagenesis in Enterobacteria or bacteriophage genome, driving genetic evolution and contributing to the spread of antibiotic resistance gene clusters between different bacteria (Jimenez and Davies, 1980; Berg and Berg, 1983). Engineered mobile elements (transposons) have been used in genetic analysis to generate random insertions within the chromosome of a target organism (Berg and Berg, 1983) and insertion of a transposon near, or within, a gene can alter or destroy its function. Transposons can jump between any genetic elements in their bacterial hosts: chromosome, plasmids or phage genomes (Berg and Berg, 1983). When expression of the transposase protein is decoupled from the transposon, a system can be engineered to generate immobile single transposon insertions in target DNA of interest
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