High-Resolution Visualization of<i>Pseudomonas aeruginosa</i>PAO1 Biofilms by Freeze-Substitution Transmission Electron Microscopy

Hunter, Ryan C.; Beveridge, Terry J.

doi:10.1128/jb.187.22.7619-7630.2005

Cited by 92 publications

(80 citation statements)

References 66 publications

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“…These may in turn interact to form higher-order structures, e.g., lattices of intertwined molecules, or particulate structures such as pili (fimbriae), flagella, phage, and membrane vesicles (MVs), all of which have been reported within biofilms (8,10,18,21,27,38,57,62). Yet, in spite of the tremendous effort to understand the contribution of matrix chemistry to matrix properties, our current knowledge of the ultrastructure of the matrix is still limited (32).…”

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confidence: 99%

Membrane Vesicles: an Overlooked Component of the Matrices of Biofilms

2006

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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.

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Membrane Vesicles: an Overlooked Component of the Matrices of Biofilms

2006

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“…While a variety of biofilms have been studied in depth by optical imaging approaches, only a few have been faithfully preserved and visualized at high resolution by transmission electron microscopy (3,22,25,26,45,48,49). A recent ultrastructural study of Pseudomonas aeruginosa biofilms shows that high-pressure freezing/freeze-substitution results in superior preservation (22).…”

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“…Bacteria in biofilms exhibit protein expression patterns different from those of planktonic bacteria (41). Within a biofilm, bacteria are typically embedded in an extracellular polymeric substance (EPS) matrix (7,17,43) that protects the microbial community members from desiccation, from phagocytosis, and, in the case of human pathogens, from the host immune system (8).While a variety of biofilms have been studied in depth by optical imaging approaches, only a few have been faithfully preserved and visualized at high resolution by transmission electron microscopy (3,22,25,26,45,48,49). A recent ultrastructural study of Pseudomonas aeruginosa biofilms shows that high-pressure freezing/freeze-substitution results in superior preservation (22).…”

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“…A recent ultrastructural study of Pseudomonas aeruginosa biofilms shows that high-pressure freezing/freeze-substitution results in superior preservation (22). Unlike conventional sample preparation, millisecond cryofixation and low-temperature dehydration protocols minimize artifacts from chemical fixation, extraction, and aggregation (15,16,19,20,22).Conventional two-dimensional (2D) imaging provides x,y positional information but cannot resolve features along the z direction. Conventional 2D projection imaging of thin (ϳ70-to 100-nm) sections results in the superposition of ϳ5 to 20 layers of protein along the electron path, obscuring molecular details.…”

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Three-Dimensional Macromolecular Organization of CryofixedMyxococcus xanthusBiofilms as Revealed by Electron Microscopic Tomography

et al. 2009

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Despite the fact that most bacteria grow in biofilms in natural and pathogenic ecosystems, very little is known about the ultrastructure of their component cells or about the details of their community architecture. We used high-pressure freezing and freeze-substitution to minimize the artifacts of chemical fixation, sample aggregation, and sample extraction. As a further innovation we have, for the first time in biofilm research, used electron tomography and three-dimensional (3D) visualization to better resolve the macromolecular 3D ultrastructure of a biofilm. This combination of superb specimen preparation and greatly improved resolution in the z axis has opened a window in studies of Myxococcus xanthus cell ultrastructure and biofilm community architecture. New structural information on the chromatin body, cytoplasmic organization, membrane apposition between adjacent cells, and structure and distribution of pili and vesicles in the biofilm matrix is presented.Bacteria are usually found concentrated at solid-liquid interfaces, where they colonize natural and man-made surfaces in community-like structures termed bacterial biofilms (6). Bacteria in biofilms exhibit protein expression patterns different from those of planktonic bacteria (41). Within a biofilm, bacteria are typically embedded in an extracellular polymeric substance (EPS) matrix (7,17,43) that protects the microbial community members from desiccation, from phagocytosis, and, in the case of human pathogens, from the host immune system (8).While a variety of biofilms have been studied in depth by optical imaging approaches, only a few have been faithfully preserved and visualized at high resolution by transmission electron microscopy (3,22,25,26,45,48,49). A recent ultrastructural study of Pseudomonas aeruginosa biofilms shows that high-pressure freezing/freeze-substitution results in superior preservation (22). Unlike conventional sample preparation, millisecond cryofixation and low-temperature dehydration protocols minimize artifacts from chemical fixation, extraction, and aggregation (15,16,19,20,22).Conventional two-dimensional (2D) imaging provides x,y positional information but cannot resolve features along the z direction. Conventional 2D projection imaging of thin (ϳ70-to 100-nm) sections results in the superposition of ϳ5 to 20 layers of protein along the electron path, obscuring molecular details. True 3D visualization requires electron tomographic data acquisition and 3D reconstruction, upon which both intracellular and extracellular features are resolved. In this way, cellular organelles, molecular machines, and macromolecular complexes can be visualized in their native microenvironment. To date, electron tomographic 3D reconstruction of a bacterial biofilm has not been reported.Myxococcus xanthus is a well-studied gram-negative soil scavenger and predator (21), and it serves as a model system for biofilm formation. M. xanthus moves over surfaces either by adventurous (A) motility or social (S) motility (for a review, see reference 18)...

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“…However, the cell envelope was visibly affected, with undulations of the outer membrane giving the periplasm an irregular appearance. It has been suggested that traditional techniques of specimen preparation for TEM can induce features such as membrane artifacts and the condensing of cytoplasmic material (Hunter & Beveridge, 2005). Some of the artifacts previously described include irregular width of the periplasmic space, poorly defined plasma membrane and undulations of the cell envelope (Hobot et al, 1984).…”

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confidence: 99%