Study of the involvement of PolIV, ImuB and DnaE2 error-prone DNA Polymerases in UV-induced and stationary-phase mutagenesis in Pseudomonas aeruginosa

FELIZIANI S; LUJÁN AM; MOYANO AJ; SMANIA AM

Villa Carlos Paz

Congreso; VI Congreso Argentino de Microbiología General de la SAMIGE; 2009

Stress-induced mutagenesis is an active process employed by bacteria to adapt and survive to different stresses. This mechanism requires SOS-regulated error-prone DNA polymerases which are able to bypass lesions allowing the continuation of DNA replication. It has been reported that several bacterial species possess these specialized DNA polymerases such as PolIV and ImuB, which belong to the Y-family, and DnaE2 which is related to the catalytic subunit DnaE of Pol III. However, in the opportunistic pathogen Pseudomonas aeruginosa these polymerases remain poorly investigated. In this study, we raised a question about the involvement of PolIV, ImuB and DnaE2 in P. aeruginosa stress-induced mutagenesis. For this purpose, we constructed P. aeruginosa PAO1 dinB, imuB and dnaE2 single deletion mutants and investigated their role in UV-induced mutagenesis. After UV irradiation, PAO1 strain showed a 2.6 fold increase (P=0.038) in the mutation frequency compared to untreated controls, confirming that P. aeruginosa displays a subtle UV-induced mutator phenotype. PolIV deficiency resulted in levels of UV-induced mutagenesis similar to PAO1 (ratio 3.6; P=0.019), suggesting that this polymerase was not involved in the mutagenic repair of UV lesions. In contrast, imuB and dnaE2 strains did not show a statistically significant increment in their mutation frequencies (ratio 0.83, P=0.09; ratio 0.72, P=0.29 respectively), indicating that in P. aeruginosa UV-induced mutagenesis might depend on both ImuB and DnaE2. We further investigated the role of these polymerases in the stationary-phase mutagenesis. For this, we employed a test system to study starvation-induced mutagenesis which was previously characterized in P. putida. This system consists in a set of plasmids containing the reporter gene pheA, which encodes for a phenol monooxygenase and whose expression allows phenol degradation. These plasmids were engineered to harbor loss-of-function mutations in the coding region of pheA. Thus, this system allows the detection of -1 deletions and base substitutions that revert the engineered alterations and permit bacteria to grow using phenol as the sole carbon source. Results obtained from incubating PAO1 cells that carried the test-plasmids on minimal phenol selective plates, confirmed that, as previously observed for P. putida, P. aeruginosa displays stationary-phase mutagenesis. We are currently testing the dinB, imuB and dnaE2 strains in order to study the role of PolIV, ImuB and DnaE2 in stationary-phase mutagenesis and consequently to contribute to a better understanding of the mutational mechanisms operating in P. aeruginosa.