The mismatch repair protein MutS controls PolIV-dependent mutagenesis induced by sub-inhibitory concentrations of cyprofloxacin

MARGARA LUCÍA M; ARGARAÑA CARLOS E; MONTI MARIELA R

Córdoba

Congreso; XI Congreso Argentino de Microbiología General; 2015

SAMIGE

The rapid emergence of de novo antibiotic resistance upon treatment of bacterial populations with subinhibitory antibiotic concentrations is a serious concern. It is, therefore, of considerable significance to understand the molecular mechanisms implicated in the emergence of resistance at low antibiotic concentrations. We previously found that subinhibitory concentrations of the quinolone antibiotic ciprofloxacin (Cip) increase the emergence of Cip resistant mutants of the pathogenic bacteria Pseudomonas aeruginosa. A DNA microarray assay revealed that P. aeruginosa genes encoding low fidelity DNA polymerases (Pol), including Pol IV, are up-regulated in response to Cip exposure. Another work demonstrated that subinhibitory concentrations of Beta-lactam antibiotics induce a Pol IV-dependent mutagenesis in Escherichia coli, P. aeruginosa and Vibrio cholerea. This mutagenesis also depends on the down-regulation of the mismatch repair (MMR) protein MutS by the small RNA SdsR in E. coli. We here investigated the 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 to replication of Pol IV through its interaction with the processivity factor Beta-clamp. Briefly, based on in vitro assays, we demonstrated that MutS inhibits Pol IV association to clamp, which is absolutely required for the activity of this Pol. In addition, MutS limits the Pol IV-induced mutagenesis in cells growing under normal conditions. In this work, we first exposed a mutant P. aeruginosa strain harboring a chromosomal mutSallele (which encodes a MutS mutant that does not interact with Beta-clamp) and the parental strain (WT) to low Cip concentrations. Then, mutation rates of resistance to different antibiotics were determined using a fluctuation test. We observed that mutation rates increased in both strains suggesting that low Cip concentrations induce a global mutagenesis on the P. aeruginosa chromosome. However, this mutagenesis was higher in the mutSstrain compared to that observed in the WT strain. To study whether this is due to Pol IV activity, we constructed Pol IV-deficient strains by deleting dinB. Mutations rates were significantly decreased in the mutSbeta dinB strain relative to the mutSbeta strain indicating that Pol IV mutator activity is responsible of the increased mutagenesis observed in the mutSbetabackground. Finally, and to determine if the MutS expression changed by the treatment with low Cip concentrations, protein levels were measured by Western blot assays. MutS expression levels were increased after Cip exposure compared to untreated cells. Accordingly, the small RNA SdsR sequence was not detected in the P. aeruginosa genome. Altogether, these findings suggest that Pol IV contribute to Cip induced mutagenesis in the mutSstrain but its activity is probably limited by MutS in parental strain.