Ongoing evolution of Pseudomonas aeruginosa PAO1 sublines complicates studies of DNA damage repair and tolerance

Sublines of the major P. aeruginosa reference strain PA01 are derivatives of the original PA01 isolate, which are maintained in laboratories worldwide. These sublines display substantial genomic and phenotypic variation due to ongoing microevolution. Here, we examined four sublines, MPA01, PA01-L, PAO1-DSM and PA01-UT, originated from different laboratories, and six DNA polymerase-deficient mutants from the P. aeruginosa MPA01 transposon library for their employment in elucidation of DNA damage repair and tolerance mechanisms in P. aeruginosa. We found that PA01 subline PA01-UT carries a large deletion encompassing the DNA damage inducible imuA-imuB-imuC cassette (PA0669-PA0671), which is implied in mutagenesis in several species. Furthermore, the genetic changes leading to variation in the functionality of the MexEF-OprN efflux system contributed largely to the phenotypic discordance between P. aeruginosa PA01 sublines. Specifically, we identified multiple mutations in the mexT gene, which encodes a transcriptional regulator of the mexEF-oprN genes, mutations in the mexF, and complete absence of these genes. Of the four tested sublines, MPA01 was the only subline with the functional MexEF-OprN multidrug efflux system. Active efflux through MexEF-OprN rendered MPA01 highly resistant to chloramphenicol and ciprofloxacin. Moreover, the functions of specialized DNA polymerase IV and nucleotide excision repair (NER) in 4-NQO-induced DNA damage tolerance appeared to be masked in MPA01, while were easily detectable in other sublines. Finally, the frequencies of spontaneous and MMS-induced Rif(r) mutations were also significantly lower in MPA01 in comparison to the PA01 sublines with impaired MexEF-OprN efflux system. The MexEF-OprN-attributed differences were also observed between MPA01 and MPA01-derived transposon mutants from the two-allele transposon mutant collection. Thus, the accumulating mutations and discordant phenotypes of the PA01 derivatives challenge the reproducibility and comparability of the results obtained with different PA01 sublines and also limit the usage of the MPA01 transposon library in DNA damage tolerance and mutagenesis studies. (C) 2017 Elsevier B.V. All rights reserved.