MONITORING OF IN PLATE BACTERIAL MUTAGENESIS USING A LUMINESCENT APPROACH

MORERO NR; ARGARAÑA CE

Congreso; XLVII Reunión Anual de SAIB; 2011

Rifampicin resistance assay is the most used method to determine mutation rates in bacteria. Although it is a useful technique in a wide range of bacterial species, it has several limitations such as lack of representation of frameshift mutations and the difficulty to examine mutant emergence along with incubation time. Here we present a novel strategy to assay mutation rate and spectrum based on a luminescent reporter adapted from the mini-Tn7 vector, which inserts specifically in the attTn7 chromosomal site of P. aeruginosa. In our construction, the promoter of P. aeruginosa MexCD-OprJ efflux pump (Pcd) was cloned upstream of lux operon in the pUC18-mini-Tn7-Gm-lux vector. Pcd promoter is endogenously repressed by NfxB protein, and we showed that many types of mutations occurring in this protein (including frameshifts) release the expression of the controlled operon. The expression of lux operon provides a strong luminescent phenotype which can be visualized on colonies directly from the plates and time-monitored. We also describe an application of this reporter strategy by comparing the emergence of NfxB mutants on ciprofloxacin containing plates from P. aeruginosa WT strain compared to the recA- and lexA- deficient strains, in order to investigate the participation of the SOS response and RecA-dependent recombination in the mutagenic effect of sub-lethal doses of ciprofloxacin.