Mutation spectra modulation leads to highly inactivated variants of the multidrug efflux pump repressor NfxB in Pseudomonas aeruginosa

LUCIA M MARGARA; CARLOS E ARGARAÑA; MARIELA R MONTI

Congreso; LIII Reunión anual de SAIB - Reunión Conjunta de Sociedades de BioCiencias; 2017

Abstract: MutS maintains the DNA replication fidelity by recognizingmispairs and recruiting factors involved in the Mismatch Repair.In a previous work, we described a new mechanism by which MutSalso contributes to the replication fidelity: regulation of the accessto replication sites of the low fidelity DNA Polymerase (Pol) IV inPseudomonas aeruginosa (PA). We showed that MutS inhibits PolIV association with β clamp, which is absolutely required for Pol IVactivity. In addition, MutS limited Pol IV mutagenic activity in exponentiallygrowing cells. Briefly, we compared mutation rates to resistanceto ciprofloxacin (CipR) and the nfxB mutation spectra in thewild type (WT) strain, the mutSβ strain (harboring a chromosomalmutSb allele which encodes a MutS mutant that does not bind to βclamp) and the Pol IV-deficient strains dinB and mutSβ dinB. Therewere no differences in CipR mutation rates among these strains.However, inactivation of Pol IV did not change nfxB mutation spectrabase substitutions in the mutSβ background. Here, we evaluated thephenotypic consequences of Pol IV action on nfxB. This gene encodesa transcriptional repressor of the MexCD-OprJ multidrug effluxpump, which corresponds to one of the primary antibiotic resistancemechanisms in PA. To test NfxB activity, we integrated on thePA chromosome a reporter fusion of the mexCD-oprJ promoter tothe lux operon. We found that a high proportion of CipR clones in themutSβ strain exhibits lower NfxB activity. These highly inactive variantswere not detected in the mutSβ dinB strain, indicating that PolIV is a key factor for generation of these NfxB versions. As expected,Pol IV had not effect on the generation of nfxB mutants in WT strainsince there were no differences with dinB strain. Remarkably, theseresults suggest that the modulation of mutation spectra could be animportant way to generate highly inactivated NfxB variants.Keywords: nfxB, mutation spectra, MutS, Pol IV.