Figure 1.
Pyocyanin production in P. aeruginosa PA14 strains and effect of DNase I treatment in PA14 aggregation.
(A) Shows production of pyocyanin in planktonic culture of PA14 DKN370 and wildtype (indicated by green colour) and lack of pyocyanin production in ΔphzA-G 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 ΔphzA-G 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), ΔphzA-G: before DNase I treatment (closed triangle), after DNase I treatment (open triangle).
Figure 2.
Influence of pyocyanin and DNase I treatment in aggregation of P. aeruginosa PA14.
The % reduction in optical density after 60 min due to 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 % of aggregation in comparison to DNase I treated (DKN370 and wildtype) and ΔphzA-G strain (regardless of DNase I treatment) respectively.
Figure 3.
Effect of DNA addition in aggregation of P. aeruginosa PA14.
The % reduction in optical density after 60 min due to aggregation of PA14 wildtype and ΔphzA-G strains before and after addition of 1 µg/ml of final concentration of exogenous DNA isolated from P. aeruginosa cultures. Error bars represents standard deviations from the mean (n = 3). Asterisks indicate statistically significant (p<0.05) differences in % of aggregation in comparison to DNase I treated wildtype strain.
Figure 4.
Size analysis of P. aeruginosa PA14 strains.
(A to C) Hydrodynamic diameter of PA14 wildtype and ΔphzA-G strains, grown over 1 to 3 days respectively and effect of DNase I treatment on it. (D) Represents the change in reduction in hydrodynamic diameter of wildtype and ΔphzA-G after DNase I treatment. Error bars represents standard deviations from the mean (n = 5). Asterisks and hash indicate statistically significant (p<0.05) differences in hydrodynamic diameter in comparison to DNase I treated wildtype and ΔphzA-G strain (regardless of DNase I treatment) respectively. Dollar indicates statistically significant (p<0.05) differences in reduction in hydrodynamic diameter between wildtype (before and after DNase I treatment) and ΔphzA-G (before and after DNase I treatment) respectively.
Figure 5.
Pyocyanin binding with DNA responsible for thick slimy EPS formation.
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 P. aeruginosa PA14 strains (above panel). The slimy eDNA constituted EPS degraded completely after DNase I treatment in wildtype strain where as the pyocyanin deficient mutant ΔphzA-G does not show any effect on DNase I treatment (below panel).
Figure 6.
Pyocyanin intercalation with DNA.
(A) Fluorescence emission spectra of ethidium bromide before and after DNA addition. (B) Decrease in intensity of emission spectra of ethidium bromide-DNA in presence and absence of pyocyanin. (C) Showing structure and colour of pyocyanin at acidic and neutral pH. (D and E) Showing colour change in DNA pellet from white to greenish blue in neutral pH and pink under acidic pH, after treated with pyocyanin and incubated at room temperature for overnight, indicates pyocyanin binded with DNA and the reappearance of white colour of DNA by simple washing of coloured DNA pellet with water resulted in removal of pyocyanin stain (Fig. 6D and E).
Table 1.
Contact angles of P. aeruginosa PA14 strains before and after DNase I treatment using water, formamide and diiodomethane and their surface free energy components: Lifshitz-Van der Waals (γLW), acid-base (γAB), electron-donating (γ−) and electron-accepting (γ+) based on their contact angle values.
Figure 7.
Interfacial free energy of aggregation of P. aeruginosa PA14 strains.
(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 ΔphzA-G strain regardless of DNase I treatment respectively.
Figure 8.
Difference in P. aeruginosa cell surface.
(A and B) Schematic showing changes in PA14 wildtype and ΔphzA-G cell surface respectively before and after DNase I treatment. (A) Natural production of pyocyanin by wildtype strain facilitates EPS (mostly constitute of eDNA) binding to P. aeruginosa cell wall and removal of eDNA by DNase I treatment subsequently removes EPS from its cell wall. (B) The mutant ΔphzA-G deficient in pyocyanin production could not facilitate eDNA/EPS binding with P. aeruginosa cell wall, therewith no effect of DNase I treatment on its cell wall.