Pyocyanin mediated H₂O₂ generation and cells lysis.

<p>Increased H₂O₂ production as recorded in bacterial cell free supernatant in PAO1 mutant <i>phzSH</i> (A) and in PA14 wildtype (B) compared with strains producing less pyocyanin. The <i>P. aeruginosa</i> PAO1 (C, E and G) and PA14 (D, F and H) strains producing more pyocyanin (PAO1 <i>phzSH</i> mutant and PA14 wildtype) displayed accelerated decreases in optical density as cultures aged compared with pyocyanin deficient strains. Error bars represent standard deviation from the mean (n = 3). Asterisks indicate statistically significant (p<0.05) differences in H₂O₂ absorbance in comparison to the PAO1 wildtype and PA14 <i>phzA-G</i> mutant (A and B) and percentage of cells alive in comparison to max. cell number (E and F).Dashed line indicates the average decrease in percentage of cell number due to H₂O₂ generation in PAO1 <i>phzSH</i> and PA14 wildtype strains (G and H).</p

eDNA release in <i>P. aeruginosa</i> in response to exogenous supernatant or pyocyanin addition.

<p>(A) <i>P. aeruginosa</i> PAO1 wildtype grown for 24 h in the presence of supernatant from a 72 h old grown bacterial cell free supernatant of PAO1 wildtype and <i>phzSH</i> strains whereas and (B) <i>P. aeruginosa</i> PA14 Δ<i>phzA-G</i> grown in the presence of PA14 Δ<i>phzA-G</i> and wildtype strains. Both PAO1 wildtype and PA14 Δ<i>phzA-G</i> were also grown in the presence of LB broth. PAO1 wildtype (C) and PA14 Δ<i>phzA-G</i> (D) grown for 24 h as a function of concentration of pyocyanin. Error bars represent standard deviation from the mean (n = 3). Asterisks indicate statistically significant (p<0.05) differences in eDNA concentration in comparison to LB (A and B) and 0 µM pyocyanin (C and D). A hash indicates a statistically significant (p<0.05) difference in eDNA concentration in comparison to the wildtype (A) and Δ<i>phzA-G</i> mutant (B).</p

Production of pyocyanin and eDNA release in <i>P. aeruginosa</i> PAO1 strains.

<p>Pyocyanin absorbance (A) and eDNA concentration (B) in supernatants of <i>P. aeruginosa</i> PAO1 wildtype and <i>phzSH</i> mutant over time. Error bars represents standard deviations from the mean (n = 3). Asterisks indicate statistically significant (p<0.05) differences in absorbance and eDNA concentration in comparison to the PAO1 wildtype.</p

Schematic represents quorum sensing regulated expression of genes encoding phenazine and pyocyanin production.

<p><i>P. aeruginosa</i> synthesizes Acylated Homoserine Lactones (AHLs) as their primary quorum sensing signaling molecules; AHLs further control the production of the secondary Pseudomonas Quinone Signaling (PQS) molecule. PQS regulates the synthesis of phenazine-1-carboxylic acid (PCA) through a set of primary phenazine producing genes <i>phzA1-G1</i> and <i>phzA2-G2.</i> PCA then converts into the derivative pyocyanin via <i>phzM</i>.</p

<i>P. aeruginosa</i> strains used in this study and their relevant phenazine producing characteristics.

<p>+ produce <i>basal level of pyocyanin</i>. ++ produce <i>elevated amount of pyocyanin</i>.</p

Production of pyocyanin and eDNA release in <i>P. aeruginosa</i> PA14 strains.

<p>(A) Pyocyanin absorbance (A) and eDNA concentration (B) in supernatants of <i>P. aeruginosa</i> PA14 wildtype and Δ<i>phzA-G</i> mutant over time. Error bars represent standard deviation from the mean (n = 3). Asterisks indicate statistically significant (p<0.05) differences in absorbance and eDNA concentrationin comparison to the mutant strain Δ<i>phzA-G</i>.</p

Exogenous H₂O₂ addition results in a decrease in bacterial cell number and increased eDNA release.

<p>(A) Dose dependent H₂O₂ treatment shows a significant increase in eDNA concentration in bacterial cell free supernatant for PAO1 wildtype especially after 16 and 24 h incubation with 1% H₂O₂. (B) The PA14 Δ<i>phzA-G</i> mutant showed significant increases in eDNA at all H₂O₂ concentration after 16 and 24 h incubation. (C-F) Decrease in bacterial cell number and decrease in cell number (%) due to cell lysis mediated by H₂O₂ treatment. Error bars represent standard deviation from the mean (n = 3). Asterisks indicate a statistically significant (p<0.05) difference in eDNA concentration in comparison to 0% concentration H₂O₂ treatment (A and B) and in number of bacterial cells in comparison to the max. cell number (×10⁸/ml) as a function of time (C and D) and percentage of cells alive in comparison to the max. cell number (E and F).</p

Interfacial free energy of aggregation of <i>P. aeruginosa</i> PA14 strains.

<p>(A to C) Components: Lifshitz-Van der Waals (LW ΔG) and acid-base (AB ΔG) and total interfacial free energy (Total ΔG) of aggregation of PA14 strains before and after DNase I treatment. Error bars represents standard deviations from the mean (n = 3). Asterisks and hash indicate statistically significant (p<0.05) differences in the free energy of aggregation in comparison to DNase I treated wildtype and Δ<i>phzA-G</i> strain regardless of DNase I treatment respectively.</p

Pyocyanin binding with DNA responsible for thick slimy EPS formation.

<p>Photographic image showing formation of bacterial pellet and thick slimy eDNA constituted EPS (only on wildtype strain) formation after harvesting and centrifugation of 3 day old planktonic culture of <i>P. aeruginosa</i> PA14 strains (above panel). The slimy eDNA constituted EPS degraded completely after DNase I treatment in wildtype strain where as the pyocyanin deficient mutant Δ<i>phzA-G</i> does not show any effect on DNase I treatment (below panel).</p

Pyocyanin production in <i>P. aeruginosa</i> PA14 strains and effect of DNase I treatment in PA14 aggregation.

<p>(A) Shows production of pyocyanin in planktonic culture of PA14 DKN370 and wildtype (indicated by green colour) and lack of pyocyanin production in Δ<i>phzA-G</i> strain even over 3 days growth period. (B) Represents an example of raw data showing decrease in absorbance at 600 nm OD due to aggregation of PA14 DKN370, wildtype and Δ<i>phzA-G</i> over 60 minutes. DKN370: before DNase I treatment (closed square), after DNase I treatment (open square), wildtype: before DNase I treatment (closed circle), after DNase I treatment (open circle), Δ<i>phzA-G</i>: before DNase I treatment (closed triangle), after DNase I treatment (open triangle).</p