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

Abstract

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)...

Predatory activity of Myxococcus xanthus outer-membrane vesicles and properties of their hydrolase cargo

“…2(a, b) shows M. xanthus cells surrounded by myxovesicles of 50-100 nm diameter, some of which are apparently broken, while others remain intact. Occasionally mxOMVs appeared to be linked to the cell surface by a tether, as reported elsewhere (Palsdottir et al, 2009), and vesicles could also appear densely stained, implying that mxOMVs are right-side out and filled with cargo (Fig. 2a, b).…”

“…Images of mxOMVs have been published previously (Kahnt et al, 2010;Palsdottir et al, 2009;Whitworth, 2011); however, no images have yet captured mxOMV production. To visualize mxOMV production, a culture of M. xanthus was embedded in agar, and cross-sections were imaged by electron microscopy.…”

Gliding Motility Revisited: How Do the Myxobacteria Move without Flagella?

“…4A) (127). PilQ belongs to a large family of integral outer membrane secretin proteins that are essential for both pilus biogenesis and TFP-mediated motility (93,126 (96).…”

All in the Family: Kin Contact Leads to Outer Membrane Exchange

“…In 2009, Palsdottir et al (12) described small (30-to 60-nm) OMVs in the extracellular matrix of M. xanthus biofilm material. Electron tomography revealed that OMVs were tethered to each other and to the outer membrane.…”