MiniUIB, a Novel Minitransposon-Based System for Stable Insertion of Foreign DNA into the Genomes of Gram-Negative and Gram-Positive Bacteria

Abstract

ABSTRACTTransposition of the insertion sequence (IS) ISPpu12is actively induced after conjugative interaction. The transposase of this IS can act intranson structures flanked by inverted repeats similar to those of the transposon. Based on that fact, an ISPpu12-based minitransposon, miniUIB, has been constructed in order to biotechnologically exploit the self-regulation of ISPpu12and its increased activity after conjugative interaction. Mobilization of the miniUIB structure into the genome ofPseudomonas stutzeriAN10 after conjugative interaction was demonstrated. A single gene, i.e., the kanamycin resistance determinant, or large genetic structures of >12 kb, i.e.,alkBFGHJKLandalkSToperons ofPseudomonas putidaTF4-1L (GPo1), have been easily integrated inP. stutzeriAN10 by an RP4-based delivery system. Therefore, the integration of thealkdeterminants by use of the miniUIB system has extended the biodegradation capabilities of this strain. Plasmid pJOC100, containing the transposase and regulator genes of ISPpu12adjacent to the miniUIB structure, was constructed in order to extend the host range of this biotechnologically useful genetic tool to other model and real-world bacteria. The effectiveness of the system for random mutagenesis in a phylogenetic wide range of bacteria and for the insertion of novel functions has been demonstrated, even in successive steps.