The biofilm matrix contributes to the chemistry, structure, and function of biofilms. Biofilm-derived membrane vesicles (MVs) and DNA, both matrix components, demonstrated concentration-, pH-, and cationdependent interactions. Furthermore, MV-DNA association influenced MV surface properties. This bears consequences for the reactivity and availability for interaction of matrix polymers and other constituents.The biofilm matrix contributes to the chemistry, structure, and function of biofilms and is crucial for the development of fundamental biofilm properties (46,47). Early studies defined polysaccharides as the matrix component, but proteins, lipids, and nucleic acids are all now acknowledged as important contributors (7, 15). Indeed, DNA has emerged as a vital participant, fulfilling structural and functional roles (1,5,6,19,31,34,36,41,43,44). The phosphodiester bond of DNA renders this polyanionic at a physiological pH, undoubtedly contributing to interactions with cations, humic substances, fine-dispersed minerals, and matrix entities (25,41,49).In addition to particulates such as flagella and pili, membrane vesicles (MVs) are also found within the matrices of gram-negative and mixed biofilms (3,16,40). MVs are multifunctional bilayered structures that bleb from the outer membranes of gram-negative bacteria (reviewed in references 4, 24, 27, 28, and 30) and are chemically heterogeneous, combining the known chemistries of the biofilm matrix. Examination of biofilm samples by transmission electron microscopy (TEM) has suggested that matrix material interacts with MVs ( Fig. 1). Since MVs produced in planktonic culture have associated DNA (11,12,13,20,21,30,39,48), could biofilm-derived MVs incorporate DNA (1, 39, 40, 44)? MATERIALS AND METHODSBiofilm growth and isolation and purification of matrix and MVs. Pseudomonas aeruginosa PAO1 and green fluorescent protein-tagged PAO1 (17) biofilms were grown using the agar plate model (40). Matrix was isolated (40) and sequentially filtered through 0.22-, 0.45-, and 1.2-m cellulose acetate filters and the filtrate collected. Absence of cells was confirmed by plating 100-l aliquots (18 h at 37°C; trypticase soy agar; n ϭ 3) and TEM of whole-mount preparations (see below). Matrix for characterization was dialyzed (24 h at 4°C; Spectrapor regenerated cellulose dialysis membranes [molecular mass cutoff, 3,000 Da]; Fisher). Particulate components were harvested by ultracentrifugation (125,000 ϫ g for 1.5 h at 5°C; Beckman Ti45 rotor), the pellets resuspended in 30% (vol/vol) Optiprep (Sigma), and the components separated on Optiprep density gradients (2) at 0% (1 ml), 18% (1 ml), 20% (1 ml), 22.5% (3 ml), 25% (3 ml), 27.5% (3 ml), 30% (3 ml), and 50% (1 ml) (vol/vol). All Optiprep solutions contained 10 mM HEPES, 0.85% (wt/vol) NaCl (pH 7.4). Samples were centrifuged to equilibrium (100,000 ϫ g for 16 h at 5°C; Beckman SW28.1 rotor) and fractionated (200-l aliquots) and whole mounts assessed by TEM (see below). MV-containing fractions were combined, washed twice in water (1...