Contribution of DNA polymerase IV to mutagenesis under oxidative stress in the opportunistic pathogen Pseudomonas aeruginosa

CASTELL SOFÍA; TUMAS IGNACIO; MONTI MARIELA R

Congreso; LVII Reunión Anual de SAIB; 2021

Translesion DNA polymerases (Pol) function in the bypass of template lesions to relieve stalled replication forks. Effective activity of these Pols require association with the processivity factor β clamp, which dictates their access to sites of DNA synthesis. We reported that the Mismatch Repair protein MutS regulates the access of the error-prone Pol IV to replication sites in Pseudomonas aeruginosa by controlling Pol IV interaction with β clamp. Previous data from our laboratory suggested that Pol IV introduces mutations associated with oxidative damage when MutS cannot regulate Pol IV. These results were obtained by examining the mutation spectra of a reporter gene in the wild type (WT) strain and the mutSβ strain, harboring a chromosomal mutSβ allele which encodes a MutS mutant that does not bind to β clamp, and the Pol IV-deficient strains dinB and mutSβdinB. In the present work, we evaluated the Pol IV mutagenesis under oxidative stress by analyzing a MutT-deficient strain, mutT, where prevention of nucleotide oxidation is impaired. We found a significant increase in the mutation rates to ciprofloxacin, rifampicin and amikacin resistance in the mutT mutSβ (Tβ) strain compared to the mutT strain (T). Pol IV contributed to this increased mutagenesis as the mutT mutSβ dinB (TβD) strain exhibited reduced mutation rates compared to Tβ strain. Conversely, Pol IV-deficiency did not decrease the rate of mutations in the mutT DinB strain (TD) relative to the T strain. Furthermore, the Tβ strain was more resistant to the killing effect of the oxidizing agent paraquat than the T strain. In order to better study the role of Pol IV in the mutagenesis of the P. aeruginosa entire genome, we performed a mutation accumulation (MA) experiment with the T, TD, Tβ and TβD strains. MA lines were initiated by creating replicates of each of the founder strains and propagating lines for 1500 generations through repeated bottlenecks of a single, randomly chosen individual, thereby greatly reducing the effectiveness of selection. MA lines exhibited changes in the colony morphology, pigmentation and fitness with respect to the founder strains. We found that the T MA lines evolved toward lower fitness, an effect that was not observed in the Tβ MA lines, and secondly TβD MA lines produced higher levels of pyocyanin than founder strains. The whole genome of founder strains and each MA line are currently being analyzed by next generation sequencing to evaluate the mutational events that occurred over the time frame of the MA experiment. In conclusion, this work reveals that Pol IV activity and its regulation by MutS significantly impacted mutagenic processes under oxidative stress.