1. Volume 26, Issue 2, Pages 195-206 (January 2016)
Auto Poisoning of the Respiratory Chain by a Quorum-Sensing-Regulated Molecule Favors Biofilm Formation and Antibiotic Tolerance 
Ronen Hazan, Yok Ai Que, Damien Maura, Benjamin Strobel, Paul Anthony Majcherczyk, Laura Rose Hopper, David J. Wilbur, Teri N. Hreha, Blanca Barquera, Laurence G. Rahme 
Current Biology 
Volume 26, Issue 2, Pages (January 2016)
DOI: /j.cub
Copyright © 2016 Elsevier Ltd Terms and Conditions

2. Figure 1
P. aeruginosa Autolysis in Liquid Cultures Is an MvfR-Dependent Process Controlled by the Quorum-Sensing-Regulated Molecule HQNO
(A–F and H) Culture growth curves (OD600nm). Absorbance was recorded every 15 min for 48 hr. Curves are color-coded according to either strains or compound concentrations and match the related legends. Results are representative of at least triplicate experiments.
(A) In contrast to PA14 (positive control), mvfR–, pqs operon mutants pqsA–, pqsBC–, and pqsD–, as well a pqsL– cells do not lyse.
(B) Image of 48 hr P. aeruginosa cultures. In contrast to PA14 WT cultures, pqsA– cultures do not lyse unless HQNO is added.
(C) Pharmacologic inhibition of MvfR and pqs operon activity. The anthranilic acid analog 6-CABA [32] and the recently developed MvfR inhibitor, M64 [33], abolishes PA14 autolysis.
(D) The pqsE– and pqsH– mutants lyse similar to PA14, which together with (B) and (C), suggest the involvement of an mvfR–-regulated small molecule in the autolysis process.
(E) Unlike HHQ or PQS, the exogenous addition of HQNO to pqsA– cultures restores autolysis.
(F and H) HQNO restores autolysis in pqsA– (F) and pqsL– (H) cultures in a dose-dependent manner, with maximum autolysis occurring at a concentration similar to that typically observed in PA14 cultures.
(G) Typical concentrations of the major MvfR-regulated secreted small molecules measured in the supernatants of PA14 cultures at specified time points.
Autolysis is conserved. It occurs in many P. aeruginosa strains, including isolates (Figures S1A and S1B) and in various growth conditions (Figures S1C and S1D).
Current Biology  , DOI: ( /j.cub )
Copyright © 2016 Elsevier Ltd Terms and Conditions

3. Figure 2
The HQNO-Mediated Decrease in OD600nm Absorbance Is Due to Cell Death
(A) The colony forming units of 24 and 48 hr cultures reveal massive loss of cell viability in PA14 and HQNO-treated pqsA– cultures, compared to pqsA– cultures (p < 0.05, unpaired t test with Bonferroni correction).
(B) The relative levels of eDNA were measured by qPCR of the P. aeruginosa genes rpoD and ldhD in cell-free supernatants at 17, 24, and 31 hr of PA14 (black), pqsA– (red), and HQNO-treated pqsA– (green) cultures. The results were calibrated for each condition to levels measured at 17 hr.
Current Biology  , DOI: ( /j.cub )
Copyright © 2016 Elsevier Ltd Terms and Conditions

4. Figure 3
HQNO-Dependent Autolysis Is Due to Inhibition of the Qi Site of Cytochrome bc1 Complex
(A) A rendering of the structure of the bc1 complex (respiratory chain complex III) together with its major active sites, relevant inhibitors (upper panel, adapted with permission from and structure of its operon in the PA14 strain (bottom panel).
(B) Schematic representation of the aerobic respiratory chain of Pseudomonas aeruginosa. The role of the bc1 complex in ROS production is highlighted, and its activity in various growth stages is shown in Table S1.
(C–E) Growth curves are plotted as in Figure 1.
(C) pqsA– cells grown in Luria media (LM) were treated with various concentrations of the bc1 complex Qi site inhibitor antimycin A (blue), using PA14 (black), untreated pqsA cells (red), and HQNO-treated pqsA– cells (green) as controls.
(D) Mutants in the cytochrome b and the Rieske genes of bc1 complex operon did not lyse and were insensitive to HQNO treatment.
(E and F) The cytochrome bc1 complex, cytochrome Qo site inhibitor, myxothiazol, prevented autolysis in both PA14 (E), and in HQNO-treated pqsA– (F), cells. PA14, pqsA– cultures with and without HQNO served as positive and negative controls, respectively.
Current Biology  , DOI: ( /j.cub )
Copyright © 2016 Elsevier Ltd Terms and Conditions

5. Figure 4
HQNO-Induced Autolysis Requires the Generation of Reactive Oxygen Species
(A) Relative amounts of ROS detected from the decay of the EPR signal of the stable radical TEMPO normalized to bacterial density (Figure S1A) in pre-lysis (24 hr), during (31 hr), and post-lysis (48 hr and 60 hr) from cultures of PA14 (black), pqsA– (red), or HQNO-treated pqsA– (green). The differences between pqsA–and the other two samples in 24, 31, 48, and 60 hr are statistically significant (p < 0.05, unpaired t test with Bonferroni correction). These results were validated by H2DCFDA fluorescence dye (Figure S2B).
(B–F) Growth of PA14 (C and D), pqsA– (B), or HQNO-treated pqsA– (E and F) cultures followed a similar pattern, as shown in Figure 1. Prior to the start of autolysis, oxidative agent paraquat (B), the antioxidant glutathione (C and E), or L-cysteine (D and F) was added (arrow), and the cultures were further incubated for up to 48 hr. The colors of the curves match the font of the concentrations (mM) of the added compounds.
(G) The levels of cellular glutathione are reduced at the onset of the stationary phase in PA14 (black), pqsA– (red), and HQNO-treated pqsA– (green) cultures. The connected lines show growth, and the dashed lines represent relative glutathione levels.
Current Biology  , DOI: ( /j.cub )
Copyright © 2016 Elsevier Ltd Terms and Conditions

6. Figure 5 HQNO-Induced Autolysis Is Associated with Membrane Damage
(A and B) Membrane potential is altered during autolysis. Cells were stained with DiOC2(3) at various time points. (A) The ratio between red and green fluorescence was calculated using population mean intensities. (B) Analysis using red fluorescence parameters reveals a shift of PA14 and HQNO-treated pqsA– cells, but not of untreated pqsA– cells from the non-lysing (NL) to the lysing (L) gates, with relative fluorescence intensities of 38–107 and 7–28, respectively. The populations at each time point are overlayed. Flow cytometric data were collected with log amplification.
(C) The ratio between cells counted in the NL compared to the L.
(D) Live/dead staining using propidium iodide dye at 24, 31, and 48 hr, for PA14, pqsA, and HQNO-treated pqsA cultures. Bacteria with intact membranes stain green, while bacteria with damaged membranes stain red.
(E) The percentage of green cells per microscopic field. In all sub figures, black denotes WT, red is pqsA mutant and green is pqsA mutant treated with HQNO. (C and E) The differences between pqsA– and the other two samples in 48 and 72 hr time points are statistically significant (p < 0.05, unpaired t test with Bonferroni correction).
Current Biology  , DOI: ( /j.cub )
Copyright © 2016 Elsevier Ltd Terms and Conditions

7. Figure 6
P. aeruginosa Autolysis Can Be Beneficial to the Cell Population by Promoting Biofilm Formation and Increasing Antibiotic Tolerance via eDNA Release
(A and B) HQNO promotes biofilm formation via the release of eDNA. (A) Biofilm biomass quantified by crystal violet staining. pqsA cells make less biofilm than PA14 (p < 0.001) and HQNO addition to pqsA rescues biofilm (p < 0.001, unpaired t test with Bonferroni correction). (B) Effect of DNase I on the biofilm viable cell concentration assessed by CFU measurements. pqsA cells make less biofilm than PA14 (p < 0.05) and HQNO addition to pqsA rescues biofilm (p < 0.05, unpaired t test with Bonferroni correction). The addition of DNase in PA14 or pqsA + HQNO reduces biofilm (p < and p < 0.01, respectively), whereas addition to pqsA does not (p < 0.01, unpaired t test with Bonferroni correction). (C and D) HQNO is promoting antibiotic tolerance in biofilm via the release of eDNA.
(C) Survival fraction of biofilm cells exposed to various concentrations of the antibiotic meropenem. The survival fraction represents the ratio between biofilm viable cells after and before antibiotic treatment. pqsA biofilm is more sensitive than PA14 to 1, 10, or 100 μg/ml Meropenem (p < 0.015, p < and p < 0.005, respectively) and HQNO addition reduces pqsA sensitivity (p < 0.05, p < 0.01, or p < 0.5, respectively, unpaired t test with Bonferroni correction).
(D) Effect of DNase I on the survival fraction of biofilm cells exposed to meropenem (100 μg/ml). The addition of DNase in PA14 or pqsA + HQNO increases biofilm sensitivity to meropenem (p < 0.01 and p < 0.6, respectively), whereas addition to pqsA does not (p < 0.005, unpaired t test with Bonferroni correction). Error bars show mean ± SEM of at least three replicates. No differences in minimal inhibitory concentration (MIC) (0.25 mg/l) were found between parental and isogenic pqsA– mutant.
Current Biology  , DOI: ( /j.cub )
Copyright © 2016 Elsevier Ltd Terms and Conditions

8. Figure 7
Similarities between HQNO-Mediated Cell Autolysis and Mitochondrial MPT Pore Formation
(A) Model for HQNO-mediated P. aeruginosa autolysis. HQNO production depends on the PqsABCD enzymes controlled by the hydroxyquinolone quorum-sensing transcription regulator MvfR and on PqsL controlled by the homoserine-lactone quorum-sensing regulator LasR (1). Secreted HQNO diffuses into the environment and signals neighboring cells to inhibit the Qi site of cytochrome bc1 complex (2). This inhibition induces a ROS burst, which is not counteracted by glutathione, since reduction of glutathione gene expression occurs at the onset of stationary growth (3). ROS-induced membrane damage results in cell autolysis and death (4). As a consequence, nutrients and eDNA are released into the medium to promote planktonic growth/survival of remaining cells (5), as well as biofilm formation and antibiotic tolerance (6).
(B and C) The MPT inhibitor cyclosporine A [48] on P. aeruginosa autolysis. PA14 (Figure 7B) and HQNO-treated pqsA– cells (Figure 7C) were grown to early stationary phase and treated with cyclosporine A. Cyclosporine A reduced P. aeruginosa autolysis in a dose-dependent manner in both PA14 and pqsA–-HQNO-treated cells.
Current Biology  , DOI: ( /j.cub )
Copyright © 2016 Elsevier Ltd Terms and Conditions