Sidorenko Julia, Jatsenko Tatjana, Kivisaar Maia
Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, 23 Riia Street, 51010, Tartu, Estonia.
Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, 23 Riia Street, 51010, Tartu, Estonia.
Mutat Res. 2017 Mar;797-799:26-37. doi: 10.1016/j.mrfmmm.2017.03.005. Epub 2017 Mar 16.
Sublines of the major P. aeruginosa reference strain PAO1 are derivatives of the original PAO1 isolate, which are maintained in laboratories worldwide. These sublines display substantial genomic and phenotypic variation due to ongoing microevolution. Here, we examined four sublines, MPAO1, PAO1-L, PAO1-DSM and PAO1-UT, originated from different laboratories, and six DNA polymerase-deficient mutants from the P. aeruginosa MPAO1 transposon library for their employment in elucidation of DNA damage repair and tolerance mechanisms in P. aeruginosa. We found that PAO1 subline PAO1-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 PAO1 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, MPAO1 was the only subline with the functional MexEF-OprN multidrug efflux system. Active efflux through MexEF-OprN rendered MPAO1 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 MPAO1, while were easily detectable in other sublines. Finally, the frequencies of spontaneous and MMS-induced Rif mutations were also significantly lower in MPAO1 in comparison to the PAO1 sublines with impaired MexEF-OprN efflux system. The MexEF-OprN-attributed differences were also observed between MPAO1 and MPAO1-derived transposon mutants from the two-allele transposon mutant collection. Thus, the accumulating mutations and discordant phenotypes of the PAO1 derivatives challenge the reproducibility and comparability of the results obtained with different PAO1 sublines and also limit the usage of the MPAO1 transposon library in DNA damage tolerance and mutagenesis studies.
铜绿假单胞菌主要参考菌株PAO1的亚系是原始PAO1分离株的衍生物，在世界各地的实验室中保存。由于持续的微进化，这些亚系表现出显著的基因组和表型变异。在这里，我们研究了来自不同实验室的四个亚系，MPAO1、PAO1-L、PAO1-DSM和PAO1-UT，以及来自铜绿假单胞菌MPAO1转座子文库的六个DNA聚合酶缺陷突变体，以阐明铜绿假单胞菌的DNA损伤修复和耐受机制。我们发现PAO1亚系PAO1-UT存在一个大的缺失，包括DNA损伤诱导的imuA-imuB-imuC操纵子（PA0669-PA0671），这在几个物种的诱变中都有涉及。此外，导致MexEF-OprN外排系统功能变异的基因变化在很大程度上导致了铜绿假单胞菌PAO1亚系之间的表型不一致。具体来说，我们在mexT基因中鉴定出多个突变，该基因编码mexEF-oprN基因的转录调节因子，在mexF中也有突变，并且这些基因完全缺失。在四个测试的亚系中，MPAO1是唯一具有功能性MexEF-OprN多药外排系统的亚系。通过MexEF-OprN的主动外排使MPAO1对氯霉素和环丙沙星具有高度抗性。此外，在4-NQO诱导的DNA损伤耐受中，专门的DNA聚合酶IV和核苷酸切除修复（NER）的功能在MPAO1中似乎被掩盖，而在其他亚系中很容易检测到。最后，与MexEF-OprN外排系统受损的PAO1亚系相比，MPAO1中自发和MMS诱导的利福平抗性突变频率也显著降低。在MPAO1和来自双等位基因转座子突变体库的MPAO1衍生转座子突变体之间也观察到了MexEF-OprN引起的差异。因此，PAO1衍生物积累的突变和不一致的表型挑战了使用不同PAO1亚系获得的结果的可重复性和可比性，也限制了MPAO1转座子文库在DNA损伤耐受和诱变研究中的应用。
