Extracellular DNA exerts bactericidal activity through cation chelation-mediated disruption of the bacterial outer membrane.

<p>(<b>A</b>) Survival analysis of 1 × 10⁷ CFU <i>P. aeruginosa</i> PAO1 coincubated with 0.125% (w/v) DNA or DNA pretreated with DNase I, PTase or 5 mM Mg²⁺. Bacterial counts were performed before (0) and after two hours treatment with DNA (2). Results are representative of three independent experiments. Error bars represent the standard deviation (SD) from eight replicates. (<b>B</b>) High, medium and low concentrations of DNase (430kU, 43kU, 4.3kU), PTase (50 U, 10 U, 1 U) or excess Mg²⁺ (5 mM, 500 μM, 5 μM) leads to increased levels of protection from killing with 0.15% DNA (w/v). (<b>C</b>) Visualization of the outer membrane integrity of <i>P. aeruginosa</i> PAO1::OM-lipoChFP expressing an outer membrane-localized mCherry fluorescent lipoprotein [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004593#ppat.1004593.ref038" target="_blank">38</a>] immediately after 2% (w/v) DNA-exposure. Insets represent increased magnification of presented micrographs. Scale bar: 10 μM. (<b>D</b>) Quantification of ChFP-rich OMV generation in the field of view from 6 representative images generated from bacteria-DNA coincubation as described in (<b>C</b>) or DNA pretreated with DNase, PTase and Mg²⁺. Error bars represent SD from 6 fields of view. (<b>E</b>) Flow cytometry of DNA-exposed <i>P. aeruginosa</i> PAO1 using SYTO9-PI dual staining as a measure of membrane-compromised bacteria [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004593#ppat.1004593.ref028" target="_blank">28</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004593#ppat.1004593.ref032" target="_blank">32</a>]. 2.5 × 10⁷ CFU <i>P. aeruginosa</i> PAO1 were exposed to 0.0125% DNA alone or pretreated as in (<b>A</b>) then immediately analyzed by the collection of positive events (N = 50 000) by BD LSRII. Numbers in corners represent the % of 50 000 events that fall into each quadrant gate. (<b>F</b>) Quantification of membrane-compromised, PI-stained <i>P. aeruginosa</i> PAO1 as measured by flow cytometry. Mean percent PI stained was derived from the average of three replicates (each with N = 50 000 for each plot) in each exposure condition as in (<b>E</b>). *** denotes a significant difference between the control and 0.125% DNA sample. ### and # denote a statistically significant difference, P<0.01 and P<0.05, respectively, between DNA alone sample and pretreated samples. Two-tailed student t-tests were performed to test for significant differences.