In Pseudomonas aeruginosa, quorum sensing constitutes a highly complex cell-to-cell communication system that, along with the cognate acylhomoserine lactone signals and regulators LasR and RhlR, modulates the production of virulence factors and a wide range of metabolic functions. In a previous paper, the authors reported that mismatch repair disruption in P. aeruginosa results in the spontaneous and reproducible emergence of defined morphological colony variants after a relatively short period of cultivation in an aerated rich medium, in contrast to the non-mutator parental strain, which does not display any kind of diversification under identical incubation conditions. One of the morphotypical variants, mS2, emerges at a high frequency and displays differences in virulence traits that could be regulated by major quorum-sensing regulators. The present study shows that mutS mS2 variants had defective LasR function due to simple but different point mutations along the lasR gene sequence, indicating that LasR inactivation is the main cause of mS2 phenotypic diversification. Moreover, it was determined that a non-functional LasR would confer a selective advantage in the late stationary phase, since viability was notably higher for mS2. Interestingly, in all mS2 variants analysed, no sequence alterations were found in the gacA and rhlR genes, suggesting that the selective pressures for GacA/RhlR and LasR were not the same and differed from those in other Pseudomonas species, which, when incubated in nutrient-rich liquid stationary-phase cultures, show specific high instability in the gacA-gacS genes.
MutS is part of the bacterial mismatch repair system that corrects point mutations and small insertions/deletions that fail to be proof-read by DNA polymerase activity. In this work it is shown that the disruption of the P. aeruginosa mutS gene generates the emergence of diverse colony morphologies in contrast with its parental wild-type strain that displayed monomorphic colonies. Interestingly, two of the mutS morphotypes emerged at a high frequency and in a reproducible way and were selected for subsequent characterization. One of them displayed a nearly wild-type morphology while the other notably showed, compared with the wild-type strain, increased production of pyocyanin and pyoverdin, lower excretion of LasB protease and novel motility characteristics, mainly related to swarming. Furthermore, it was reproducibly observed that, after prolonged incubation in liquid culture, the pigmented variant consistently emerged from the mutS wild-type-like variant displaying a reproducible event. It is also shown that these P. aeruginosa mutS morphotypes not only displayed an increase in the frequency of antibiotic-resistant mutants, as described for clinical P. aeruginosa mutator isolates, but also generated mutants whose antibiotic-resistant levels were higher than those measured from spontaneous resistant mutants derived from wild-type cells. It was also found that both morphotypes showed a decreased cytotoxic capacity compared to the wild-type strain, leading to the emergence of invasive variants. By using mutated versions of a tetracycline resistance gene, the mutS mutant showed a 70-fold increase in the reversion frequency of a +1 frameshift mutation with respect to its parental wild-type strain, allowing the suggestion that the phenotypical diversity generated in the mutS population could be produced in part by frameshift mutations. Finally, since morphotypical diversification has also been described in clinical isolates, the possibility that this mutS diversification was related to the high frequency hypermutability observed in P. aeruginosa CF isolates is discussed.
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