THE MISMATCH REPAIR PROTEIN MUTS CONTROLS POL IV DEPENDENT-MUTAGENESIS INDUCED BY SUBINHIBITORY CONCENTRATIONS OF CIPROFLOXACIN

MARGARA LUCÍA M.; ARGARAÑA CARLOS E.; MONTI MARIELA R.

Congreso; XI Reunión Anual de SAMIGE; 2015

The rapid emergenceof de novo antibiotic resistance upon treatment of bacterial populations withsubinhibitory antibiotic concentrations is a serious concern. It is, therefore,of considerable significance to understand the molecular mechanisms implicatedin the emergence of resistance at low antibiotic concentrations. We previously found that subinhibitory concentrations of thequinolone antibiotic ciprofloxacin (Cip) increase the emergence of Cipresistant mutants of the pathogenic bacteria Pseudomonas aeruginosa. A DNAmicroarray assay revealed that P.aeruginosa genes encoding low fidelity DNA polymerases (Pol), including PolIV, are up-regulated in response to Cip exposure. Another work demonstratedthat subinhibitory concentrations of b-lactam antibiotics induce a Pol IV-dependentmutagenesis in Escherichia coli, P. aeruginosa and Vibrio cholerea. This mutagenesis also depends on thedown-regulation of the mismatch repair (MMR) protein MutS by the small RNA SdsRin E. coli. We here investigatedthe contribution of both MutS and Pol IV factors in the mutagenesis induced by low Cip concentrations. In this sense, we have recently described a novel mechanism by which MutS controls the access toreplication of Pol IV through its interaction with the processivity factor β clamp. Briefly, based on in vitro assays, we demonstrated that MutS inhibits Pol IV association to β clamp, which is absolutely required forthe activity of this Pol. In addition, MutS limits the Pol IV-induced mutagenesis incells growing under normal conditions. Inthis work, we first exposed a mutant P.aeruginosa strain harboring a chromosomal mutSb allele(which encodes a MutS mutant that does not interact with β clamp) andthe parental strain (WT) to low Cip concentrations. Then, mutation ratesof resistance to different antibiotics were determined using a fluctuation test.We observed that mutation rates increased in both strains suggesting that low Cip concentrations inducea global mutagenesis on the P. aeruginosachromosome. However, this mutagenesis was higher in the mutSb strain compared to that observed in the WT strain. To study whetherthis is due to Pol IV activity, we constructed Pol IV-deficient strains bydeleting dinB. Mutations rates were significantly decreased in the mutSβ dinB strain relative to the mutSb strain indicating that Pol IV mutator activity isresponsible of the increased mutagenesis observed in the mutSb background. Finally, and to determine if the MutS expression changed bythe treatment with low Cip concentrations, protein levels were measured by Westernblot assays. MutS expression levels were increased after Cip exposure comparedto untreated cells. Accordingly, the small RNA SdsR sequencewas not detected in the P. aeruginosagenome. Altogether, these findings suggest that Pol IV contribute to Cipinduced mutagenesis in the mutSb strain but its activity is probably limited by MutS in the parentalstrain.