Genomic DNA was isolated from peripheral blood lymphocytes working with the QIAamp DNA Blood Midi kit (Qiagen, Valencia, CA), in line with the manufacturer’s instructions

d the production of HAQs in four other constitutively expressing RND efflux pump mutants, i.e. in nfxB2, mexZ2, nalC2 and mexL2 mutants. These mutants overproduce MexCD-OprJ, MexXY-OprM, MexABOprM and MexJK-OprM, respectively. We found that none of these other efflux pumps influences HHQ and PQS production. Together, these results indicate that HHQ and PQS are specifically modulated by MexEF-OprN in the MGL01 mexS2 mutant. Expression of the pqsH gene is upregulated in a PA14 mexS2 mutant To understand the reason for the HHQ and PQS imbalance characterizing the mexS2 mutant, the expression patterns of appropriate QS regulators and their related genes were studied using transcriptional and translational reporter fusions. We found that the transcriptional activity of the mvfR promoter is nearly two-times higher in MGL01 than in the wild-type strain. As expected, the transcription of the pqsABCDE operon, which is activated by MvfR, was found to be upregulated as well. The molecular basis explaining this increased expression is unknown. Since mvfR and pqsH are positively controlled by the LasRI QS system, it was interesting to investigate the expression of the lasR and lasI genes in MGL01. The lasI translational reporter fusion revealed that the 3oxo-C12-HSL synthase is half-fold more expressed in MGL01 than in PA14. Accordingly, 3-oxo-C12-HSL concentrations quantified by LC-MS/MS are higher in MGL01 cultures when compared to PA14. In contrast, C4-HSL concentrations are lower in MGL01 cultures. Intriguingly, although PQS, the product of PqsH activity, is produced in lower concentrations in MGL01, mvfR and pqsABCDE are up-regulated in this strain. Thus, it was interesting to verify whether the decrease in PQS is due to a decreased transcription of pqsH in MGL01 strain. This was verified using a pqsH promoter-lacZ transcriptional fusion chromosomally integrated into the wild-type and MGL01 strains, yielding PA14 and MGL01 strains, respectively. Unexpectedly, pqsH expression is upregulated in MGL01 compared to the wild-type. Yet, this was not reflected in a higher production of PQS, although mexS2 mutant produces high concentrations of the PQS precursor, HHQ. translational status of lasRI, mvfR, pqsABCDE and pqsH genes but also by another unidentified mechanism. We thus hypothesized that the activity of the MexEF-OprN efflux pump directly modulates HAQ intracellular concentrations, the fact that could explain the weak PQS synthesis by the mexS2 mutant cells. This was first verified by directly quantifying cell-associated HHQ and PQS molecules in PA14 and MGL01. As shown in E133 figure 4A, proportions of cell-associated HHQ are lower in MGL01 than in the wild-type strain PA14. This result is even more striking when considering that MGL01 is a hyper HHQ producer. Nonetheless, a higher concentration of cell-associated PQS is observed in the wild-type strain PA14, which is consistent with the higher production of PQS in PA14 cultures. In light of these results, we compared ” the intracellular availability of PQS and HHQ for MvfR binding under MexEFOprN overexpression. PQS or HHQ was exogenously added to cultures of an HAQ-deficient PA14 strain, or of its isogenic nfxC mutant, carrying the pqsA-lacZ transcriptional reporter plasmid pGX5. We found that addition of PQS results in a stronger induction from the pqsA promoter “2987731 in MGL02 than in PA14 pqsA2 pqsH2 . This observation is likely to result from the strong activating effect of PQS on the MvfR transcripti