Fig 1.
The AQ biosynthetic pathway and pqs genes.
Schematic representation of the AQ biosynthetic pathway and the pqs and phn genes in P. aeruginosa PAO1 and the isogenic ∆4AQ and ∆5AQ mutants. Main elements of the pqs QS system (HHQ, PQS, HQNO, PqsE, and PqsR) are in bold face. The PA number is indicated below the genes according to the Pseudomonas Genome Database [13]. Solid grey arrows represent biosynthesis; dashed grey arrows represent information flow; solid black arrow indicates activation (+); black T-line indicates negative regulation (-).
Fig 2.
Venn diagrams showing the number of genes controlled by HHQ, PQS, and PqsE in P. aeruginosa Δ4AQ, and the overlap between the regulons.
Table 1.
Selected genes whose transcription is controlled by HHQ, PQS and/or PqsE.
Fig 3.
Functional classes of PqsE and PQS controlled genes.
Histograms representing the distribution of (A) PqsE-controlled and (B) PQS-controlled genes according to their functional classification. Functional classes are from the Pseudomonas Genome Database [13].
Fig 4.
Validation of the microarray data by Real Time PCR.
Relative mRNA levels of the genes indicated quantified by Real Time PCR in the P. aeruginosa ∆4AQ strain grown in LB supplemented with 1 mM IPTG to induce PqsE expression (light-grey bars), or with 40 μM PQS (white bars), HHQ (dark-grey bars), or HQNO (black bars), with respect to the same strain grown in LB. The average of two independent analyses each performed on three technical replicates is shown with standard deviations.
Fig 5.
Interplay of HHQ, PQS, PqsR and iron in controlling PpqsA and PpqsR activity.
Maximal promoter activity quantified in the indicated strains carrying the transcriptional fusions PpqsA::lux (A and B) or PpqsR::lux (C and D). Strains were grown in LB or in LB supplemented with 40 μM HHQ, PQS or 3-NH2-PQS, as indicated below the graphs, in the absence (white bars) or presence (grey bars) of 100 μM FeCl3. Diamonds indicate the pyoverdine levels in the absence (white diamonds) or in the presence (grey diamonds) of 100 μM FeCl3. Promoter activity and pyoverdine level are reported as Relative Light Units (RLU) and OD405, respectively, normalized to cell density (OD600). The average of three independent experiments is reported with standard deviations.
Fig 6.
Effect of iron on the ability of PQS to stimulate PpqsA activity.
Maximal PpqsA promoter activity measured in the P. aeruginosa ∆4AQ strain carrying the transcriptional fusion PpqsA::lux, grown in LB (white bars) or in LB supplemented with 40 μM HHQ (light-grey bars) or 40 μM PQS (dark-grey bars), and FeCl3 at the concentration indicated below the graph. White diamonds indicate the pyoverdine level in the supernatants of cultures grown in the presence of PQS with or without FeCl3. Promoter activity and pyoverdine are reported as Relative Light Units (RLU) and OD405, respectively, normalized to cell density (OD600). The average of three independent experiments is reported with standard deviation.
Fig 7.
Schematic representation of the pqs QS system in P. aeruginosa.
The core of the pqs QS system is composed of the pqsABCDE-phnAB operon and the pqsR gene. Proteins coded by the pqsABCDE-phnAB operon synthesize HHQ that binds to and activates PqsR. The PqsR-HHQ complex promotes PpqsA activity, thus increasing HHQ and PqsE levels. Notably, the PpqsA promoter is the only target of the PqsR-HHQ complex. Apart from its contribution to HHQ biosynthesis, PqsE influences the P. aeruginosa transcriptome via a still uncharacterized AQ-independent pathway(s). In this way, PqsE up-regulates the expression of genes involved in virulence factor production, biofilm development, and antibiotic resistance. Conversely, PqsE down-regulates PpqsA activity, AQ production and the expression of genes involved in denitrification and T6SS. The pqsH and pqsL genes are required for PQS and HQNO biosynthesis, respectively. HQNO did not affect the P. aeruginosa transcriptome, and probably contributes to environmental competition due to its cytochrome inhibitory activity. PQS chelates iron triggering the iron-starvation response and increasing the transcription of virulence factor genes coding for virulence factors such as pyoverdine, ExoS toxin and AprX protease. Moreover, PQS down-regulates genes involved in denitrification. Most of the regulatory effects exerted by PQS are PqsR-independent, since the PqsR-PQS (or PqsR-HHQ) complex only promotes PpqsA activity. However, PQS also increases PpqsA and PpqsR expression via a PqsR-independent pathway(s) that is unrelated to the iron-starvation response, but is inhibited in the presence of high-iron concentrations. Dotted grey arrows indicate gene expression; solid grey arrows represent biosynthesis; solid black arrow indicates PqsR-dependent activation (+); dashed black arrows indicate PqsR-independent activation (+); black T-line indicates negative regulation (-); dashed grey arrows represent information flow.