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.