Ultrastructural and electron energy‐loss spectroscopic analysis of an extracellular filamentous matrix of an environmental bacterial isolate

Böckelmann, Uta; Lünsdorf, Heinrich; Szewzyk, Ulrich

doi:10.1111/j.1462-2920.2007.01325.x

Cited by 15 publications

(9 citation statements)

References 27 publications

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“…Freeze-dry electron microscopy and CLSM revealed that E. faecalis produces a DNase I-sensitive fibrous network during development of biofilms under hydrodynamic conditions. This filamentous network is reminiscent of a stable fibrous eDNA network described for the aquatic gram-negative bacterium strain F8 (4,5). The formation of extracellular fibrous DNA networks has also been reported for biofilms of nontypeable Haemophilus influenzae formed in vivo (27).…”

Section: Discussionsupporting

confidence: 57%

Contribution of Autolysin and Sortase A during Enterococcus faecalis DNA-Dependent Biofilm Development

et al. 2009

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Biofilm production is a major attribute of Enterococcus faecalis clinical isolates. Although some factors, such as sortases, autolysin, and extracellular DNA (eDNA), have been associated with E. faecalis biofilm production, the mechanisms underlying the contributions of these factors to this process have not been completely elucidated yet. In this study we define important roles for the major E. faecalis autolysin (Atn), eDNA, and sortase A (SrtA) during the developmental stages of biofilm formation under static and hydrodynamic conditions. Deletion of srtA affects the attachment stage and results in a deficiency in biofilm production. Atn-deficient mutants are delayed in biofilm development due to defects in primary adherence and DNA release, which we show to be particularly important during the accumulative phase for maturation and architectural stability of biofilms. Confocal laser scanning and freeze-dry electron microscopy of biofilms grown under hydrodynamic conditions revealed that E. faecalis produces a DNase I-sensitive fibrous network, which is important for biofilm stability and is absent in atn-deficient mutant biofilms. This study establishes the stage-specific requirements for SrtA and Atn and demonstrates a role for Atn in the pathway leading to DNA release during biofilm development in E. faecalis.Biofilms are bacterial communities encased within an extracellular matrix composed of carbohydrates, proteins, and nucleic acids (10). They are an ideal environment for exchange of genetic materials, such as genes encoding virulence factors and antibiotic resistance determinants, among bacteria within a community (61, 76), while allowing the flow of water and nutrients, as well as ions and various small molecules, to bacteria within the community (8) and providing a protective shield against antibiotics, antimicrobial substances, and phagocytosis (33, 80). Development of a biofilm is a complex multistage process. It is initiated by primary adhesion of the bacteria to a substratum, which is followed by the formation of microcolonies and production of an exopolysaccharide matrix, and it finally culminates with the formation of a three-dimensional (3D) multicellular mature structure (48). Bacterial biofilms are important medically because they have been associated with the pathogenesis of chronic and device-related persistent infections, such as cystic fibrosis, urinary tract infections, and endocarditis (11).Enterococcus faecalis is a gram-positive bacterium that is a commensal of the gastrointestinal tract of healthy individuals. However, it is also an important opportunistic pathogen that is responsible for a wide variety of nosocomial infections, including endocarditis, urinary tract infections, and bacteremia (14,17,23,44). E. faecalis accounts for approximately 65 to 80% of all enterococcal nosocomial infections (16). The ability of E. faecalis to adhere to and develop biofilms on medical devices, such as intravascular and urinary catheters (25,26,36,37,72), is thought to contribute to its pathogenes...

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Section: Discussionsupporting

confidence: 57%

Contribution of Autolysin and Sortase A during Enterococcus faecalis DNA-Dependent Biofilm Development

et al. 2009

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“…The mechanism by which bacteria bind eDNA has not been clearly established though microscopic studies on an aquatic Gammaproteobacterium showed that eDNA formed a defined structure of interconnecting microfilaments between cells (Bockelmann et al 2006(Bockelmann et al , 2007. Possible mediators of this interaction are the multifunctional Type IV pili which are associated with functional properties that involve binding of DNA, including natural competence and transformation (Aas et al 2002;van Schaik et al 2005).…”

Section: Introductionmentioning

confidence: 97%

Extracellular DNA and Type IV pili mediate surface attachment by Acidovorax temperans

Heijstra

Pichler

Liang

et al. 2009

Antonie van Leeuwenhoek

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Extracellular DNA can play a structural role in the microbial environment. Here evidence is presented that an environmental isolate of Acidovorax temperans utilises extracellular DNA for intercellular and cell-surface attachment and that Type IV pili and electrostatic interactions play a role in this interaction. Preliminary attempts to isolate and purify extracellular polysaccharides from A. temperans strain CB2 yielded significant amounts of DNA raising the question of whether this molecule was present as a structural component in the extracellular matrix. The role of DNA in attachment was indicated by experiments in which the addition of DNase to liquid medium inhibited the attachment of Acidovorax to glass wool. A Tn5 insertional mutant, lacking Type IV pili, was unable to initiate attachment. Addition of DNase caused rapid detachment of bound cells, but no detachment occurred when proteinase, RNase or inactivated DNase were used. Addition of MgCl(2) also caused significant detachment, supporting the possible mechanistic role of electrostatic interactions in the attachment process. Although attachment was apparent in early to mid-log phase growth, surprisingly DNA was not detected in the culture supernatant until late stationary phase and coincided with an appreciable loss of cell viability. This suggests that during log-phase growth attachment is mediated by eDNA that is released in low quantities and/or is highly localised within the extracellular matrix and also that stationary phase DNA release through widespread cell lysis may be a separate and unrelated event.

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“…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).…”

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confidence: 99%

Interactions of DNA with Biofilm-Derived Membrane Vesicles

2009

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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...

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Ultrastructural and electron energy‐loss spectroscopic analysis of an extracellular filamentous matrix of an environmental bacterial isolate

Cited by 15 publications

References 27 publications

Contribution of Autolysin and Sortase A during Enterococcus faecalis DNA-Dependent Biofilm Development

Contribution of Autolysin and Sortase A during Enterococcus faecalis DNA-Dependent Biofilm Development

Extracellular DNA and Type IV pili mediate surface attachment by Acidovorax temperans

Interactions of DNA with Biofilm-Derived Membrane Vesicles

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