A new system to analyze recombination in Gram-negative bacteria: example Pseudomonas aeruginosa

BORGOGNO MARÍA V.; MONTI MARIELA R.; ARGARAÑA CARLOS E.

Congreso; XLVII Reunión Anual de SAIB; 2011

The DNA mismatch repair system (MRS) is an evolutionary^conserved pathway that besides of its function in mutation^avoidance, inhibits the recombination between divergent DNA^sequences by interacting with mismatches in the recombination^intermediates. The aim of this study was to construct a system to^analyze recombination between identical (homologous) or^partially divergent (homeologous) DNA sequences in Gramnegative^bacteria. This system, constructed in a broad host-range^plasmid, contained two non-functional 3´- and 5´- truncated lacZ^genes sharing an overlapping region either 100% or 95% identical,^followed by a gentamicin resistance gene. The two lacZ genes were^separated by a spacer region which included a transcriptional^terminator. A single recombination event re-constituted a^functional lacZ gene and allowed the expression of the gentamicin^resistance gene. To analyze the performance of this system, we^transferred the homologous and homeologous recombination^systems to the opportunistic pathogen Pseudomonas aeruginosa.^DNA divergence between the two lacZ genes reduced the^frequency of recombination by 1000-fold. In addition, inactivation^of the MRS proteins MutS or MutL specifically increased the^frequency of homeologous recombination. Finally, the^recombination process was characterized by restriction and^sequencing analysis of the recombinant products.