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. 2014 Feb;16(2):598-610.
doi: 10.1111/1462-2920.12187. Epub 2013 Jul 15.

Bacterial social networks: structure and composition of Myxococcus xanthus outer membrane vesicle chains

Jonathan P Remis  1 , Dongguang Wei, Amita Gorur, Marcin Zemla, Jessica Haraga, Simon Allen, H Ewa Witkowska, J William Costerton, James E Berleman, Manfred Auer
Affiliations

Bacterial social networks: structure and composition of Myxococcus xanthus outer membrane vesicle chains

Jonathan P Remis et al. Environ Microbiol. 2014 Feb.

Abstract

The social soil bacterium, Myxococcus xanthus, displays a variety of complex and highly coordinated behaviours, including social motility, predatory rippling and fruiting body formation. Here we show that M. xanthus cells produce a network of outer membrane extensions in the form of outer membrane vesicle chains and membrane tubes that interconnect cells. We observed peritrichous display of vesicles and vesicle chains, and increased abundance in biofilms compared with planktonic cultures. By applying a range of imaging techniques, including three-dimensional (3D) focused ion beam scanning electron microscopy, we determined these structures to range between 30 and 60 nm in width and up to 5 μm in length. Purified vesicle chains consist of typical M. xanthus lipids, fucose, mannose, N-acetylglucosamine and N-acetylgalactoseamine carbohydrates and a small set of cargo protein. The protein content includes CglB and Tgl outer membrane proteins known to be transferable between cells in a contact-dependent manner. Most significantly, the 3D organization of cells within biofilms indicates that cells are connected via an extensive network of membrane extensions that may connect cells at the level of the periplasmic space. Such a network would allow the transfer of membrane proteins and other molecules between cells, and therefore could provide a mechanism for the coordination of social activities.

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Figures

Figure 1
Figure 1. SEM imaging time course of biofilm and fruiting body formation
M. xanthus cells were grown in submerged culture on silicon wafers..Samples were removed and examined with SEM at 0 h (a-c), 24 h (d-f) and 48 h (g-i). Over this time frame, cells are observed to go from small dispersed clumps to large aggregates. Peritrichous membrane appendages are observed on cells at all time points. Scale bars represent 10 μm (d,g), 2 μm (a), 1 μm (b,e,h), 0.5 μm (c,i), and 0.2 μm (f).
Figure 2
Figure 2. 3D FIB/SEM imaging of the biofilm structure of M. xanthus reveals the prevalence of intercellular appendages
M. xanthus biofilms were grown at a surface-air interface for 4 days at 32 C, embedded in resin and imaged with the macromolecular resolution of 3D FIB/SEM imaging of an ~300 cubic μm total volume. The FIB/SEM image reveals the abundance of vesicle chains and tubes (a-d). An ~500 nm slice through the reconstructed 3D volume reveals that cells are often linked to neighboring cells through membrane connections (a-b). Such connections can be vesicle chain-like (a) or tubular (b) and these membrane-based structures mediate cell-to-cell connectivity. Surface rendering of the 3D volume (c-d) shows the high frequency of such connections (shown in red) between neighboring cells (shown in yellow) in an ~8 μm thick volume of M. xanthus biofilm.
Figure 3
Figure 3. Ultrastructure analysis of vesicle chains
The vesicle chains are seen as long extracellular appendages (a) when living cells are stained with the lipid bilayer dye FM4-64 and visualized by epifluorescence light microscopy. These lipid structures are resolved as vesicle chains/tubes (b and c) when cryofixed and visualized by TEM. Vesicle chains appear to be single membrane structures with regular size and shape (b, white line inset) are distinct from pili (b, dotted line inset) and lack the double membrane structure of the cell (b, solid black line inset). The lumen of the vesicle chains can be seen to be continuous in some cases (c, white line inset). Fast-frozen, artifact-free, preparations (d) show each chain made up of vesicles of similar size, whereas vesicle size can differ between distinct chains. A 1 nm thin slice through a 3D tomogram of a HPF/FS, resin-embedded sample (e) and a negatively stained whole-mount projection image (f) both reveal a continuous lumen inside the constricted vesicle chains. Scale bars represent 5 um (a), 50 nm (b, c) 100 nm (d, e and f).
Figure 4
Figure 4. Compositional analysis of vesicle fractions
Cell-free vesicle chain preparations were obtained and confirmed by negative staining (a). The purified vesicle fraction contains both vesicle chains and free vesicles. The vesicle fraction was subjected to analysis for total carbohydrate and lipid content. Carbohydrate analysis by Fluorophore Assisted Carbohydrate Electrophoresis (FACE) (b) indicates Mannose and Fucose are abundant neutral sugars in vesicle preparations (Lane 2, controls lane 1) while amine sugars, N-acetylgalactosamine and N-acetlyglucosamine (Lane 3) were detected in addition to several unknown amine sugars when vesicle fractions were analyzed for acetylated sugars. Lipid composition was determined by Fatty Acid Methyl Esterification (FAME) analysis (c) indicating that 15- and 17-length carbon chains predominate, and characteristic myxobacteria lipids such as 16:1 w5c were also detected. (d) A vesicle chain with (arrows) hair-like densities covering it are observed when planktonic cells are imaged in thin vitreous ice using whole cell cryo-TEM, these structures likely represent LPS.
Figure 5
Figure 5. Vesicle chains are enriched under biofilm growth conditions
Vesicle chains were purified from planktonic cell cultures or biofilm communities grown on nutrient rich agar plates. After cell mass normalization we quantified FM4-64 intensity to determine membrane structures concentration (a). Aliquots of purified vesicle chains from both planktonic cell cultures (b) and biofilms (c) were examined by whole mount TEM to exclude cell membrane contamination. Negative staining of individual M. xanthus cells fresh from culture solution reveals that such cells rarely show vesicle chains (d), whereas cells resuspended from biofilms frequently show many vesicle chains (e). Vesicle chains are frequently observed in M. xanthus biofilms as revealed by TEM imaging of ultrathin biofilm sections (f).
Figure 6
Figure 6. Functional analysis of vesicle chains
M. xanthus cells labeled with FM4-64 were added to agar coated slides and time-lapse phase contrast microscopy (a-c) was used to track cell movement. After a total of 180 seconds, cells were imaged by fluorescence microscopy (d) to visualize vesicle chains. Arrows point to two examples of motile cells, showing that the presence or absence of a detectable vesicle chain has no bearing on cell movement. SDS-PAGE separation followed by Mass Spectrometry was used to ID protein content with known cell-cell transferred Tgl and CglB proteins specifically identified at their expected sizes (29 and 45 kD) in the vesicle fraction (e). M. xanthus cells and vesicle fractions were mixed with mCherry-labeled E. coli in a predation assay to determine if the vesicle fraction mediates lysis of E. coli cells (f-i). Quantification of viable E. coli by fluorescence, indicates the vesicle fraction has partial lytic activity (“open circles”) relative to E. coli only (“closed circles”) and E. coli incubated with M. xanthus (“X”) (f). E. coli lysis was also observed by light microscopy after 24 h incubation of E. coli cells on agar plates alone (g) with M. xanthus cells (h) and with vesicle fraction (i).
Figure 7
Figure 7. Model for the role of vesicles and vesicle chains in M. xanthus biofilms
We propose that cells maintain connections and possibly exchange signals through vesicles and other extracellular appendages. These structures are covered by a carbohydrate, possibly LPS, which may play a role in cell-cell recognition and intercellular material transfer. Free vesicles provide a diffusion free mechanism of signal transmission, while vesicle chains mediate direct intercellular contacts, creating a tightly-knit multicellular community.
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References

    1. Bajaj Chandrajit, Yu Zeyun, Auer Manfred. Volumetric Feature Extraction and Visualization of Tomographic Molecular Imaging. J Struct Biol. 2003 Nov;144(1-2):132–143. - PubMed
    1. Berleman James E, Kirby John R. Multicellular Development in Myxococcus xanthus Is Stimulated by Predator-prey Interactions. J Bacteriol. 2007 Aug;189(15):5675–5682. doi:10.1128/JB.00544-07. - PMC - PubMed
    1. Berleman James E, Kirby John R. Deciphering the Hunting Strategy of a Bacterial Wolfpack. FEMS Microbiol Rev. 2009 Sep;33(5):942–957. doi:10.1111/j.1574-6976.2009.00185.x. - PMC - PubMed
    1. Costerton JW, Montanaro L, Arciola CR. Bacterial Communications in Implant Infections: a Target for an Intelligence War. Int J Artificial Organs. 2007 Sep;30(9):757–763. - PubMed
    1. Davies DG, Parsek MR, Pearson JP, Iglewski BH, Costerton JW, Greenberg EP. Science. 5361. Vol. 280. New York, N.Y.: Apr 10, 1998. The Involvement of Cell-to-cell Signals in the Development of a Bacterial Biofilm. pp. 295–298. - PubMed

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