Figure 1.
Extracellular DNA inhibits planktonic growth by rapidly killing Pseudomonas aeruginosa.
Growth of PAO1 in (A) LB or (B) BM2 media in cultures supplemented with % (w/v) extracellular salmon sperm DNA, as indicated. Growth (OD600) was measured every 20 minutes, over 20 h. (C) Overnight cultures of PAO1::p16slux were washed and 5×107 cfu resuspended in sodium phosphate buffer (25 mM, pH 7.4). Resuspended cells were treated with varying concentrations of salmon sperm DNA, as indicated, and luminescence was measured in cps (counts per second) over time, as a measure of viability. Cells were also resuspended in buffer +0% (w/v) DNA as a negative control. Data is expressed as percentage survival relative to the untreated control. For experiments 1A–C the mean of three replicate experiments is represented. The standard deviation, omitted for clarity, was not greater than +/−10% the mean. (D) The loss of viability of PAO1::p16Slux, following 2% (w/v) DNA treatment, was confirmed by stamping of cultures at indicated time points post-treatment on LB agar. Cells resuspended in buffer in the absence of DNA remained viable. Two replicate wells are shown for each condition.
Figure 2.
Extracellular DNA induces membrane perturbation, cell lysis, DNA release and death.
(A) Membrane integrity was monitored by visualizing DNA, EDTA or buffer treated P. aeruginosa producing mCherry fluorescent lipoproteins in either the IM or OM. The release of OMVs (white arrow) and genomic DNA strands (grey arrow) into the extracellular milieu following cell lysis in response to DNA or EDTA treatment was demonstrated by (B) PI staining and (C) semi-quantitative PCR which detects P. aeruginosa genomic DNA but not salmon sperm DNA. Cells were treated with 2% (w/v) salmon sperm DNA, 2 mM EDTA, or buffer alone (negative control), pelleted and 1 µl of supernatent used as a template for semi-quantitative PCR. PCR controls included 2% (w/v) salmon sperm DNA (primer specificity) and a negative and positive PCR control. The scale bar equals 2.5 microns.
Figure 3.
DNA induces cell death by acting as a cation chelator.
(A) Killing assays indicate relative protection provided by cations following pre-incubation of salmon sperm DNA with cations (25 mM Mg2+; 6.25 mM Ca2+; 6.25 Mn2+; 3.125 mM Zn2+) in Hepes buffer (50 mM, pH 7.4). (B) Restoration of PAO1 growth in BM2 supplemented with 1.5% (w/v) DNA following addition of excess individual cations (10 mM Mg2+, 10 mM Ca2+, 0.5 mM Mn2+, 2.5 mM Zn2+). Each experiment was performed at least five times and representative curves are shown. Standard deviations for each experiment were not greater than +/−10% of the value shown.
Figure 4.
Extracellular DNA induces PA3553 gene expression in planktonic cultures.
Effects of (A) salmon sperm DNA, (B) salmon sperm DNA in the presence of excess (5 mM) Mg2+ (C) sonicated P. aeruginosa PAO1 genomic DNA and (D) DNAsed salmon sperm DNA on the expression of the PA3553::lux transcriptional fusion in planktonic cultures. Gene expression was normalized to growth for each condition and CPS/OD600 values are presented. Each growth experiment was performed at least five times and representative curves are shown. (D) Buffer control indicates that this sample was treated identically to the DNAsed DNA sample except for the addition of DNAseI enzyme. Standard deviations for each experiment were not greater than +/−10% of the value shown.
Figure 5.
Visualization of DNA as a component of peg-adhered biofilms.
(A) The extracellular stain DDAO demonstrated DNA is a component of PAO1 biofilms cultivated in BM2 on pegs (40× magnification). (B) PI staining indicates the presence of DNA as a mesh-like pattern in 1 day-old biofilms (right panel) (40× magnification). The scale bar equals 10 microns. Images presented are representative of triplicate experiments.
Figure 6.
Extracellular DNA induces PA3553 gene expression in peg-adhered biofilms.
(A) Adhesion of PAO1 and PA3553::lux to polystyrene pegs assessed by crystal violet staining and OD600 measurement at 24 h in cultures supplemented with salmon sperm DNA, as indicated. (B) Gene expression from PA3553::lux was monitored in peg-adhered biofilms at 24 h in DNA supplemented conditions, as indicated. CPS values were normalized to the number of peg-adherent cells (OD600 of CV staining). Bars in (A) and (B) represent the average values obtained from eight pegs and the error bars represent the standard deviations.
Figure 7.
Multiple cations are bound by DNA and repress the induction of PA3553 gene expression.
(A) Elemental analysis of cations in buffer after size-exclusion centrifugation to remove DNA illustrates percentage bound to DNA. Values presented represent the % (w/v) bound to DNA relative to the total amount of cation added. The negative control indicated relates to the concentration of Mg2+ that bound to the column in the absence of DNA. (B) The influence of excess Mg2+, Ca2+, Mn2+ or Zn2+ on PA3553 gene expression when grown in BM2 media with low (20 µM) Mg2+. Cations are added at concentrations of 5 mM, 5 mM, 1 mM and 2.5 mM, respectively. A representative curve from triplicate experiments is shown. Standard deviations for each experiment were not greater than +/−10% of the value shown.
Table 1.
Influence of extracellular DNA on antibiotic resistance in biofilms.
Table 2.
DNA-induced resistance of biofilms requires both cultivation and challenge under cation-limiting conditions.