title: Random and cyclical deletion of large DNA segments in the genome of Pseudomonas putida.
abstract: Cumulative site-directed mutagenesis is of limited suitability for the global analysis of the gene functions in the microbe's cellular network. In order to simplify and stabilize the genome of the soil bacterium Pseudomonas putida, we developed a recyclable three-step excision method based on the combination of customized mini-transposons and the FLP-FRT site-specific recombination system. To demonstrate the powerful potential of these tools, we first established insertion mutant libraries that allow users to study gene functions with respect either to phenotypic characteristics (single insertions) or to their involvement in predicted networks (double insertions). Based on these libraries, we generated as a proof-of-principle, single-deletion mutants lacking ~4.1% of the genome (~3.7% of the gene repertoire). A cyclical application of the method generated four double-deletion mutants of which a maximum of ~7.4% of the chromosome (~6.9% of the gene count) was excised. This procedure demonstrates a new strategy for rapid genome streamlining and gain of new insights into the molecular interactions and regulations.
authors.affiliation: Systems and Synthetic Biology Group, Helmholtz-Centre for Infection Research, Braunschweig, Germany.
authors.forename: Audrey
authors.initials: A
authors.lastname: Leprince
authors.forename: Víctor
authors.initials: V
authors.lastname: de Lorenzo
authors.forename: Petra
authors.initials: P
authors.lastname: Völler
authors.forename: Mark W J
authors.initials: MW
authors.lastname: van Passel
authors.forename: Vitor A P
authors.initials: VA
authors.lastname: Martins dos Santos
mesh_terms: D004247:DNA / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D016958:Pseudomonas putida / Q000235:genetics*; D017384:Sequence Deletion
chemical_list: D004247:DNA; D004254:DNA Nucleotidyltransferases