Emergence of the P2 Phenotype in Pseudomonas aeruginosa PAO1 Strains Involves Various Mutations in mexT or mexF

Abstract

We recently demonstrated that Pseudomonas aeruginosa PAO1 undergoes a pronounced phenotypic change when introduced into the intestines of rats during surgical injury. Recovered strains displayed a specific phenotype (termed the P2 phenotype) characterized by altered pyocyanin production, high collagenase activity, high swarming motility, low resistance to chloramphenicol, and increased killing of Caenorhabditis elegans compared to the inoculating strain (termed the P1 phenotype). The aims of this study were to characterize the differences between the P. aeruginosa P1 and P2 phenotypes in quorum sensing and competitiveness. We then determined the presence of the P2 phenotype among PAO1 strains from various laboratories. Results demonstrated that P2 cells display accelerated growth during early exponential phase and early activation of quorum-sensing systems and overcome the growth of P1 cells in a mixed population. Among eight PAO1 strains obtained from different laboratories, four exhibited the P2 phenotype. Of 27 mutants analyzed from the P. aeruginosa MPAO1 transposon library, 25 displayed P2 phenotypes. The P2 phenotype in both cases correlated with a lack of expression of mexE or mexF due to mutations in mexT and mexF genes. In summary, strains possessing the P2 phenotype are distributed among PAO1 strains commonly used across a variety of research laboratories. Genetically, they are characterized by various mutations in mexT or mexF. Stable genetic mutations that change phenotypes can arise as microbes adapt to their environment to maximize propagation. This ecologically dependent shift in phenotype has been documented in many bacterial species and in diverse contexts (1, 2). Recently, we demonstrated that the MPAO1 strain of Pseudomonas aeruginosa became transformed to (or selected for) a more virulent phenotype when present in the colon of rats subjected to preoperative radiation followed by colon resection and anastomosis (3). The strain recovered from the anastomotic tissue (termed MPAO1-P2, i.e., having the P2 phenotype) exhibited high collagenase activity, swarming ability, increased pyocyanin production in liquid medium, and increased tissue-destroying capacity compared to the initial strain (termed MPAO1-P1, having the P1 phenotype). Comparative genomic sequencing analysis revealed that MPAO1-P2 harbored a single-nucleotide polymorphism (SNP) in the mexT gene that results in a truncated and nonfunctional MexT protein. Transformation of mexT-P1 into MPAO1-P2 reverted the strain back to the P1 phenotype. The phenotypes of P. aeruginosa that are similar to P1 and P2 have been previously described (4-7). In the cited studies, the wild-type PAO1 strain harbored an 8-bp insertion in mexT that results in a nonfunctional MexT protein and a phenotype similar to P2. Its derivative mutant that was selected by norfloxacin (referred to as a nfxC-type mutant) had a functional MexT protein and was characterized by attenuated pyocyanin production (5), high resistance to chloramphenicol (6), and absence of swarming ...

Pseudomonas aeruginosa LysR PA4203 Regulator NmoR Acts as a Repressor of the PA4202 nmoA Gene, Encoding a Nitronate Monooxygenase

“…We next decided to delete the nmoR gene from another PAO1 strain (the PAO1 UK strain from the University of Nottingham). This strain was described as the C-PAO1 strain by Luong et al (37). Confirming the results of Luong et al, both the wild type and the nmoR mutant had a 2-bp deletion, at positions 905 and 906 of mexT, resulting in a frameshift (see Fig.…”

“…Despite the fact that only one binding site for the NmoR regulator was found in the genome of PAO1, the deletion of the nmoR gene had a profound effect on the expression of many genes, as shown by the microarray experiment results presented in Table 3 and in Table S1 in the supplemental material. A recent publication highlighted the differences between PAO1 strains in different laboratories (37). The strain used in this study clearly has the P1 phenotype, characterized by the absence of an 8-bp insertion in the mexT gene.…”

“…The strain used in this study clearly has the P1 phenotype, characterized by the absence of an 8-bp insertion in the mexT gene. Our strain corresponds to the D-PAO1 strain described by Luong et al (37), which is characterized by the presence of a functional MexT LysR regulator and a nonfunctional MexEF-OprN pump. It is interesting that all the described MexT-dependent genes (PA1970, PA2486, mexE, PA2759, PA3229, PA4623, and PA4881) were highly expressed in the wild type but were expressed at very low levels in the ⌬nmoR mutant, although both strains were confirmed to have an intact mexT gene.…”

De Novo Synthesis of Phosphorylated Triblock Copolymers with Pathogen Virulence-Suppressing Properties That Prevent Infection-Related Mortality

“…In this set of experiments, we used an MPAO1-P2 strain that produces a higher amount of pyocyanin compared to that of the MPAO1-P1 strain. 16 In our preliminary experiments, we found that supplementation of media with iron increases pyocyanin production in this nutrient-limited DCM media. Therefore, we supplemented DCM with 2 μ M Fe 3+ (1 μ M Fe 2 (SO 4 ) 3 ).…”

Construction of prcK and prcR Mutant Strains of Lactobacillus paracasei HD1.7 and the Impact on the Production of Paracin 1.7

“…[7][8][9][10] Insertional inactivation has been the main method applied to G + bacteria. 11 In this method, the flanking sequences of the exogenous DNA imported into host cells and of the target gene in the chromosome of host cells are homologous.…”