The matrix helps define the architecture and infrastructure of biofilms and also contributes to their resilient nature. Although many studies continue to define the properties of both gram-positive and gram-negative bacterial biofilms, there is still much to learn, especially about how structural characteristics help bridge the gap between the chemistry and physical aspects of the matrix. Here, we show that membrane vesicles (MVs), structures derived from the outer membrane of gram-negative bacteria, are a common particulate feature of the matrix of Pseudomonas aeruginosa biofilms. Biofilms grown using different model systems and growth conditions were shown to contain MVs when thin sectioned for transmission electron microscopy, and mechanically disrupted biofilms revealed MVs in association with intercellular material. MVs were also isolated from biofilms by employing techniques for matrix isolation and a modified MV isolation protocol. Together these observations verified the presence and frequency of MVs and indicated that MVs were a definite component of the matrix. Characterization of planktonic and biofilm-derived MVs revealed quantitative and qualitative differences between the two and indicated functional roles, such as proteolytic activity and binding of antibiotics. The ubiquity of MVs was supported by observations of biofilms from a variety of natural environments outside the laboratory and established MVs as common biofilm constituents. MVs appear to be important and relatively unacknowledged particulate components of the matrix of gram-negative or mixed bacterial biofilms.

The asymmetric outer membrane of Gram-negative bacteria contains lipopolysaccharides (LPSs) which contribute significantly to the bacterium's surface properties and play a crucial role in regulating membrane permeability. We report on neutron diffraction studies performed on aligned, self-assembled bilayers of Na-, Ca-, and Mg-salt forms of LPS isolated from Pseudomonas aeruginosa PAO1. From the one-dimensional neutron scattering length density profiles we find that water penetrates Ca2+-LPS bilayers to a lesser extent than either Na+- or Mg2+-LPS bilayers. This differential water penetration could have implications as to how small molecules permeate the outer membrane of Gram-negative bacteria and, possibly, how nonlamellar phases are formed.

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...