Extracellular DNA induces cell death by membrane perturbation and cell lysis 
DNA is a highly anionic polymer due to the phosphates in the deoxyribose backbone.
This property, in combination with the fast-killing observed in response to extracellular DNA led us to hypothesize that addition of exogenous DNA resulted in the loss of membrane integrity through cation chelation, in a manner similar to that observed with the known cation chelator EDTA [58].
The OM of P. aeruginosa contains a 20:1 ratio of Mg2+:Ca2+ cations [59], which bind to and stabilize LPS in the outer leaflet of the OM [58].
EDTA treatment of cells resulted in chelation and removal of divalent cations from the OM, leading to disruption of the OM [58].
To determine the effect of DNA on membrane integrity, microscopic analysis in response to lethal concentrations of DNA and relevant controls was performed.
Lipoproteins are lipid-modified proteins anchored in the outer leaflet of the IM or the inner leaflet of the OM.
P. aeruginosa cells producing mCherry fluorescent membrane-anchored lipoproteins (lipoChFP) that are localized to either the OM or IM [60],[61] were used as markers of OM and IM integrity.
LipoChFP-labelled P. aeruginosa cells showed dramatic membrane perturbations when exposed to 2% (w/v) DNA, but showed uniform membrane staining patterns in untreated cells (Fig 2A).
The OM perturbations in DNA-exposed cells included regions of patchy fluorescence and the release of OMVs, while the IM perturbations were visualized simply as patchy and irregular regions of membrane fluorescence (Fig 2A).
EDTA, the known cation chelator caused comparable IM and OM perturbations as those observed in cells exposed to extracellular DNA.
Propidium iodide (PI) stains extracellular DNA and DNA in dead cells.
PI staining was observed in cells exposed to DNA and EDTA, confirming that this treatment was lethal (Fig 2B).
PI staining also revealed the presence of long strands of genomic DNA, presumably as a consequence of the loss of membrane integrity, cell lysis and release of cytoplasmic contents, including DNA (Fig 2B).
The DNA released by lysed cells formed a mesh-like coating surrounding and connecting individual bacterial cells (Fig 2B).
Degradation of these strands by DNAse treatment of lysed cells confirmed that these fibres were composed of DNA (Fig S1).
Pseudomonas specific semi-quantitative PCR (qPCR) was also performed to confirm that the DNA released following DNA or EDTA treated cells was in fact genomic DNA from P. aeruginosa (Fig 2C).
Buffer treated control cells showed intense green staining with syto9 (indicating viability) and a lack of PI staining (indicating no dead/dying cells or DNA release) (Fig S1).
