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

MARGARA LUCÍA M; ARGARAÑA CARLOS E; MONTI MARIELA R

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

MutS maintains the DNAreplication fidelity by recognizing mispairs and recruiting factors involved inthe Mismatch Repair. In a previous work, we described a new mechanism by whichMutS also contributes to the replication fidelity: regulation of the access toreplication sites of the low fidelity DNA Polymerase (Pol) IV in Pseudomonasaeruginosa (PA). We showed that MutS inhibits Pol IV associationwith β clamp, whichisabsolutely required for Pol IV activity. In addition, MutS limited Pol IVmutagenic activity in exponentially growing cells. Briefly, we comparedmutation rates to resistance to ciprofloxacin (CipR) and the nfxBmutation spectra in the wild type (WT) strain, the mutSβ strain (harboring achromosomal mutSβallele which encodes a MutS mutant that does not bindto β clamp)and the Pol IV-deficient strains dinB and mutSβdinB. There wereno differences in CipR mutation rates among these strains. However,inactivation of Pol IV did not change nfxB mutationspectra in the WT background,but it significantly decreased spontaneous base substitutions in the mutSβ background. Here,we evaluated the phenotypic consequences of Pol IV action on nfxB. Thisgene encodes a transcriptional repressor of the MexCD-OprJ multidrug efflux pump,which corresponds to one of the primary antibiotic resistance mechanisms in PA.To test NfxB activity, we integrated on the PA chromosome a reporter fusion ofthe mexCD-oprJ promoter to the lux operon.We found that a high proportion of CipR clones in the mutSβ strain exhibitslower NfxB activity. These highly inactive variants were not detected in the mutSβdinBstrain, indicating thatPol IV is a key factor for generation of these NfxB versions. As expected, PolIV had not effect on the generation of nfxB mutantsin WT strain since there were no differences with dinB strain.Remarkably, these results suggest that the modulation of mutation spectra couldbe an important way to generate highly inactivated NfxB variants.