CONTRIBUTION OF DNA POLYMERASE IV TO MUTAGENESIS UNDER OXIDATIVE STRESS IN THE OPPORTUNISTIC PATHOGEN Pseudomonas aeruginosa

CASTELL SOFIA; TUMAS IGNACIO NICOLAS; MONTI MARIELA ROXANA

Evento virtual

Congreso; XVI Annual Meeting of the Argentinean Society for General Microbiology (SAMIGE); 2021

SAIB-SAMIGE

Translesion DNA polymerases (Pol) function in the bypass of template lesions to relieve stalled replication forks. The effectiveactivity of these Pols requires association with the processivity factor β clamp, which dictates their access to sites of DNAsynthesis. We reported that the Mismatch Repair protein MutS regulates the access of the error-prone Pol IV to replicationsites in Pseudomonas aeruginosa by controlling Pol IV interaction with β clamp. Previous data from our laboratory suggestedthat Pol IV introduces mutations associated with oxidative damage when MutS cannot regulate Pol IV. These results wereobtained by examining the mutation spectra of a reporter gene in the wild type (WT) strain and the mutSβstrain, harboring achromosomal mutSβallele which encodes a MutS mutant that does not bind to β clamp, and the Pol IV-deficient strains dinBand mutSβdinB. In the present work, we evaluated the Pol IV mutagenesis under oxidative stress by analyzing a MutT-deficientstrain, mutT, where prevention of nucleotide oxidation is impaired. We found a significant increase in the mutation rates tociprofloxacin, rifampicin, and amikacin resistance in the mutT mutSβ(Tβ) strain compared to the mutT strain (T). Pol IVcontributed to this increased mutagenesis as the mutT mutSβ dinB (TβD) strain exhibited reduced mutation rates compared toTβ strain. Conversely, Pol IV-deficiency did not decrease the rate of mutations in the mutT DinB strain (TD) relative to the Tstrain. 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 mutationaccumulation (MA) experiment with the T, TD, Tβ, and TβD strains. MA lines were initiated by creating replicates of each ofthe founder strains and propagating lines for 1500 generations through repeated bottlenecks of a single, randomly chosenindividual, 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, aneffect that was not observed in the Tβ MA lines, and secondly, TβD MA lines produced higher levels of pyocyanin thanfounder strains. The whole genome of founder strains and each MA line are currently being analyzed by next-generationsequencing to evaluate the mutational events that occurred over the time frame of the MA experiment. In conclusion, this workreveals that Pol IV activity and its regulation by MutS significantly impacted mutagenic processes under oxidative stress.