ABSTRACT:
Transposition of the insertion sequence (IS) IS
Ppu12
is actively induced after conjugative interaction. The transposase of this IS can act in
trans
on structures flanked by inverted repeats similar to those of the transposon. Based on that fact, an IS
Ppu12
-based minitransposon, miniUIB, has been constructed in order to biotechnologically exploit the self-regulation of IS
Ppu12
and its increased activity after conjugative interaction. Mobilization of the miniUIB structure into the genome of
Pseudomonas stutzeri
AN10 after conjugative interaction was demonstrated. A single gene, i.e., the kanamycin resistance determinant, or large genetic structures of >12 kb, i.e.,
alkBFGHJKL
and
alkST
operons of
Pseudomonas putida
TF4-1L (GPo1), have been easily integrated in
P. stutzeri
AN10 by an RP4-based delivery system. Therefore, the integration of the
alk
determinants by use of the miniUIB system has extended the biodegradation capabilities of this strain. Plasmid pJOC100, containing the transposase and regulator genes of IS
Ppu12
adjacent 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.