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

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

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

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

<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

Experimental design for competition experiments.

<p>Mixtures of <i>Vibrio fischeri</i> wild type (MJ1) and <i>luxI</i> mutant (MJ211) strains at optical density based starting ratios of 1∶1 or 1∶10 were inoculated (100 µl) into 5 ml of fresh media in triplicate. Each replicate culture was subsequently subcultured daily for ten days and wild type to mutant ratios determined every second day.</p

Impact of <i>luxI</i> deletion on competition in <i>Vibrio fischeri</i>.

<p>Percentage of luminescence colonies plated from mixed <i>V. fischeri</i> MJ1 (wild type) and MJ211 (<i>luxI</i> mutant) cultures subcultured daily for ten days. The starting ratio of MJ1 to MJ211 was either 1∶1 (Panel A) or 1∶9 (Panel B). Regardless of the initial ratio of wild type (MJ1) to mutant (MJ211) cells, the wild type lineage dominated the cultures within days suggesting the <i>luxI</i> gene represents a selective advantage independent of the bioluminescence phenotype. Average values from triplicate cultures are presented. Error bars represent standard deviation.</p

Primers used for sequencing <i>V. fischeri</i> bioluminescence regulatory genes.

a<p>Primers and base numbering based on <i>luxR</i> and <i>luxI</i> (Genbank AF170104.1), ^bPrimers and numbering based on <i>ainR</i> and <i>ainS</i> (Genbank L37404), ^c<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067443#pone.0067443-Dunn1" target="_blank">[38]</a>.</p