Interactions of DNA with Biofilm-Derived Membrane Vesicles

Abstract

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...

Virulence and Immunomodulatory Roles of Bacterial Outer Membrane Vesicles

“…Vesicles from oral bacteria, in particular, promote biofilm formation and colonization (48,71,72,94). Vesicles are abundant in natural biofilms, and OM vesicle surface-bound DNA appears to be an electrostatic, bridging component that is central to membrane vesicle-bacterium-biofilm matrix interactions (120,121). Porphyromonas gingivalis vesicles mediate the coaggregation of the periodontopathogen Tannerella forsythia, and vesicles mediate the interaction/aggregation of staphylococci and Prevotella intermedia (65,71,72).…”

Origin of life: LUCA and extracellular membrane vesicles (EMVs)

“…During the growth the OM 'blebs' outwards and pinches off, forming vesicles (20-250 nm) that are spherical portions of OM with lumenal periplasmic content. Thus, the composition of OMVs reflects components of the OM and periplasm, for example, soluble proteins, integral membrane proteins, lipoproteins and glycolipids (Beveridge 1999;Horstman & Kuehn 2000;Schooling et al 2009). OMVs are potent virulence factors of pathogenic diderm bacteria.…”

Role of Pseudomonas aeruginosa Peptidoglycan-Associated Outer Membrane Proteins in Vesicle Formation

“…OMVs also contain outer membrane proteins and entrap periplasmic components as they are released (16)(17)(18). OMVs have been found to be associated with Gram-negative bacteria growing planktonically and in surface-attached biofilm communities as well as natural environments (11,(19)(20)(21).…”