Extracellular DNA (eDNA) is an important component of the biofilm matrix. We show that removal of eDNA from Gram-positive bacteria reduces initial adhesion to and aggregation of bacteria on surfaces. Thermodynamic analyses indicated that eDNA introduces favorable acid-base interactions, explaining the effect of eDNA on aggregation and adhesion to the surface.Extracellular polymeric substances in bacterial biofilms are composed of polysaccharides, proteins, and extracellular DNA (eDNA) (6). eDNA released by autolysis (2,3,10,11,12) acts as an adhesive (13) and strengthens biofilms (14). In Staphylococcus epidermidis 1457, autolysin E (encoded by atlE) induces production of eDNA, and a strain lacking AtlE (a ⌬atlE mutant) formed significantly less biofilm (11).Bacterial adhesion and aggregation are mediated by nonspecific long-range attractive Lifshitz-Van der Waals forces and electrostatic and acid-base interactions, as well as by proteinspecific interactions, as a localized corollary of the abovementioned forces (1,8).Initial adhesion to substratum surfaces and aggregation of bacteria are important steps in biofilm formation, but the role of eDNA in these processes is unclear. Therefore, we investigated the effect of naturally occurring eDNA on the initial adhesion and surface aggregation of several Gram-positive bacteria. The mechanism by which eDNA affects the adhesion and surface aggregation of two model strains (S. epidermidis 1457 and the ⌬atlE mutant) was analyzed by a surface thermodynamic approach.The strains listed in Table 1 were grown on blood agar at 37°C, except Streptococcus mutans LT11, which was grown on brain heart infusion (BHI) agar and incubated in 5% CO 2 at 37°C. Ten-milliliter precultures in tryptone soya broth for staphylococci, or BHI for S. mutans LT11, were used to inoculate 200-ml main cultures in the same media. After 16 h of growth, cultures were washed in phosphate-buffered saline (PBS: 150 mM NaCl-10 mM potassium phosphate, pH 6.8) and sonicated on ice for 3 ϫ 10 s at 30 W (5) to remove aggregates. Finally, bacteria were resuspended in PBS to a density of 3 ϫ 10 8 ml Ϫ1 . To remove eDNA, bacterial suspensions were treated with DNase I in the presence of 10 mM MgCl 2 for 45 min at 37°C and subsequently washed twice with PBS.Glass or dimethyldichlorosilane (DDS)-coated glass microscope slides, possessing a hydrophilic or hydrophobic surface, respectively, were placed in the bottom of a parallel-plate flow chamber (5). Bacterial adhesion and surface aggregation at a shear rate of 16 s Ϫ1 was monitored for 60 min by microscopy. Photographic images were used to calculate the initial deposition rate (j 0 ), the total number of bacteria adhering per unit area at time t, and the degree of surface aggregation. The area, in terms of pixel number, occupied by a single attached bacterium was different for each strain and determined by image analysis in order to calculate the number of bacteria present in an aggregate. The percentage of adhering bacteria in large aggregates (Ͼ5 bacteria) was c...

Bacterial adhesion and biofilm formation are both dependent on the production of extracellular polymeric substances (EPS) mainly composed of polysaccharides, proteins, lipids, and extracellular DNA (eDNA). eDNA promotes biofilm establishment in a wide range of bacterial species. In Pseudomonas aeruginosa eDNA is major component of biofilms and is essential for biofilm formation and stability. In this study we report that production of pyocyanin in P. aeruginosa PAO1 and PA14 batch cultures is responsible for promotion of eDNA release. A phzSH mutant of P. aeruginosa PAO1 that overproduces pyocyanin displayed enhanced hydrogen peroxide (H2O2) generation, cell lysis, and eDNA release in comparison to its wildtype strain. A ΔphzA-G mutant of P. aeruginosa PA14 deficient in pyocyanin production generated negligible amounts of H2O2 and released less eDNA in comparison to its wildtype counterpart. Exogenous addition of pyocyanin or incubation with H2O2 was also shown to promote eDNA release in low pyocyanin producing (PAO1) and pyocynain deficient (PA14) strains. Based on these data and recent findings in the biofilm literature, we propose that the impact of pyocyanin on biofilm formation in P. aeruginosa occurs via eDNA release through H2O2 mediated cell lysis.

Bacteria adhere to natural and engineered surfaces and develop into mature biofilms encased in self-produced extracellular polymeric substances (EPSs). EPS consists of polysaccharides, proteins, metabolites and extracellular DNA (eDNA). Extracellular DNA release by bacteria is mediated by both quorum-sensing (QS)-dependent and -independent mechanisms. Quorum-sensing-independent mechanisms are responsible for basal levels of eDNA release, whereas QS-dependent mechanisms control the production of prophages, phenazines and proteins involved in cell lysis and subsequent release of elevated amounts of eDNA. Extracellular DNA binds with other biopolymers such as polysaccharides, proteins or metabolites like phenazines, thereby providing structural integrity to EPS. Extracellular DNA promotes attractive acid-base interactions between bacterial cells and between bacteria and surfaces. It therefore plays an essential structural role in stabilising biofilms and protecting bacterial cells from physical and chemical challenges. Accordingly, with current knowledge, it becomes clear that targeting and destroying eDNA in bacterial EPS is a promising strategy for treatment of bacterial-associated infections in a medical context and biofilm control on surfaces to prevent biocorrison in an engineering context. In contrast, the addition of DNA can be applied to engineering of biofilms for beneficial purposes such as remediation of environmental pollutants and electricity or fuel production in bioelectrochemical systems or bioreactors.

Bacterial resistance to conventional antibiotics necessitates the identification of novel leads for infection control. Interference with extracellular phenomena, such as quorum sensing, extracellular DNA integrity and redox active metabolite release, represents a new frontier to control human pathogens such as Pseudomonas aeruginosa and hence reduce mortality. Here we reveal that the extracellular redox active virulence factor pyocyanin produced by P. aeruginosa binds directly to the deoxyribose-phosphate backbone of DNA and intercalates with DNA nitrogenous base pair regions. Binding results in local perturbations of the DNA double helix structure and enhanced electron transfer along the nucleic acid polymer. Pyocyanin binding to DNA also increases DNA solution viscosity. In contrast, antioxidants interacting with DNA and pyocyanin decrease DNA solution viscosity. Biofilms deficient in pyocyanin production and biofilms lacking extracellular DNA show similar architecture indicating the interaction is important in P. aeruginosa biofilm formation.