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Molecular Architecture and Assembly Principles of Vibrio cholerae Biofilms

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Science  13 Jul 2012:
Vol. 337, Issue 6091, pp. 236-239
DOI: 10.1126/science.1222981

Biofilms Up Close

Many bacterial infections involve biofilm formation. Cells within a biofilm are significantly more resistant to immune clearance and antibiotics compared to unattached, planktonic cells. Berk et al. (p. 236) applied superresolution optical methods to image living bacteria with nanometer-scale precision as they form a biofilm. Vibrio cholerae biofilms were observed to have three distinct levels of spatial organization: cells, clusters of cells, and collections of clusters. Each cell cluster was wrapped in a flexible, elastic envelope. Several V. cholerae matrix proteins played complementary architectural roles during biofilm development. RbmA provided cell-cell adhesion, Bap1 allowed the developing biofilm to adhere to surfaces, and heterogeneous mixtures of VPS, RbmC, and Bap1 formed the dynamic, flexible, and ordered envelopes that encase the cell clusters.

Abstract

In their natural environment, microbes organize into communities held together by an extracellular matrix composed of polysaccharides and proteins. We developed an in vivo labeling strategy to allow the extracellular matrix of developing biofilms to be visualized with conventional and superresolution light microscopy. Vibrio cholerae biofilms displayed three distinct levels of spatial organization: cells, clusters of cells, and collections of clusters. Multiresolution imaging of living V. cholerae biofilms revealed the complementary architectural roles of the four essential matrix constituents: RbmA provided cell-cell adhesion; Bap1 allowed the developing biofilm to adhere to surfaces; and heterogeneous mixtures of Vibrio polysaccharide, RbmC, and Bap1 formed dynamic, flexible, and ordered envelopes that encased the cell clusters.

Microbes within biofilms are more resistant to antibiotics; immune clearance; and osmotic, acid, and oxidative stresses as compared with planktonic cells (17). Despite advances in identifying the polysaccharide and proteinaceous constituents of the biofilm extracellular matrix, the mechanisms by which these factors yield a mechanically defined and spatially organized biofilm are largely unknown (810). Vibrio cholerae biofilm formation involves the production of Vibrio polysaccharide (VPS) and three matrix proteins (RbmA, RbmC, and Bap1) predicted to contain carbohydrate-binding domains (fig. S1A) (1113). To investigate the molecular mechanisms of biofilm development, we used a V. cholerae rugose variant with increased capacity to form biofilms (11). We inserted Myc, FLAG, and human influenza hemagglutinin epitopes into its genome at the 3′ ends of the rbmA, rbmC, and bap1 genes, respectively (fig. S1B), allowing us to label these matrix proteins in vivo by supplementing the growth medium with corresponding cyanine dye–labeled primary antibodies (Fig. 1).

Fig. 1

V. cholerae biofilm structure. (A) Optical section of biofilm 4 μm above the coverslip. Images are pseudocolored in blue (cells), gray (RbmA), red (RbmC), and green (Bap1). RbmA localizes around and within cell clusters; RbmC and Bap1 encase cell clusters. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 3 μm. (B) Three-dimensional biofilm architecture. Colors are as in (A). (C) Enlargement of the boxed region in (B). The red arrow indicates one cell cluster. The red signal is now rendered partially transparent to allow visualization of cells within an RbmC-containing cluster. (D) Comparison of biofilm architecture formed by rugose (Rg) and ΔrbmA (A-) strains. RbmA is required for cell cluster formation. Scale bars, 2 μm.

We used four-color confocal imaging to validate this labeling strategy and obtain a diffraction-limited overview of biofilm architecture (Fig. 1, A to C, and movie S1). Cells were mainly organized into elongated clusters whose boundaries were defined by three-dimensional (3D) envelopes of the RbmC (red) and Bap1 (green) proteins (Fig. 1C, red arrow). Within the envelope that encases the cell clusters, the relative Bap1 signal was highest in those areas with the least RbmC (Fig. 1A and figs. S5 and S6). Deletion of either RbmC or Bap1 did not impair cluster formation or the resultant architecture of the envelope (Fig. 1D and fig. S7) (11, 14). The cell clusters had a regular width of 2.2 ± 0.3 μm (n = 42 clusters), whereas their length varied from 2 to 8 μm (fig. S8). Each cell within a cluster contacted the cluster boundary and, thus, the interstitial space between clusters, perhaps facilitating nutrient delivery and waste disposal.

However, although Bap1 and RbmC share 47% peptide sequence similarity (11), their spatial distributions differed notably at the interface between the coverslip and the cell clusters (fig. S9). Bap1 was concentrated at the biofilm-surface interface (14), whereas RbmC was absent from the interface (Fig. 1, B and C, and fig. S9). Moreover, a bap1 deletion strain had a more severely altered biofilm phenotype than a rbmC deletion strain (11, 14), all pointing to Bap1 having two separable functions—namely, encasing cell clusters and attaching cells to the surface.

In contrast to RbmC and Bap1, we detected RbmA throughout the biofilm (Fig. 1, A to C) (14). Strains lacking RbmA have reduced colony corrugation and are less resistant to detergent treatment (12) but can still adhere to surfaces. Surprisingly, deletion of rbmA caused loss of cell ordering into clusters and associated RbmC-Bap1 envelopes, although both of these proteins were clearly present within the biofilm (Fig. 1D and fig. S7). Thus, Bap1 appears to help the biofilm to adhere to surfaces, RbmC and Bap1 appear to encapsulate cell clusters, and RbmA appears to participate in cell-cell adhesion (movies S2 to S6) (11, 12, 14)

To further test these hypotheses and learn how biofilms assemble, we imaged living biofilms as they developed from a single founder cell into mature biofilms (Fig. 2A and fig. S10). We followed matrix protein secretion and organization with a continuous in situ immunostaining approach (15) in which labeled primary antibodies were added to the growth medium (Fig. 2A). At the time of initial attachment, individual founder cells did not have detectable RbmA, RbmC, and Bap1 on their surface. The first matrix protein to appear postattachment was RbmA, which accumulated at discrete sites on the cell surface. After the first cell division, the newly formed daughter cell remained attached to the founder cell, unlike in planktonic cells, where the two cells quickly separate (Fig. 2A).

Fig. 2

Time-lapse CLSM imaging of V. cholerae biofilm development and cluster formation. (A) Expression and subsequent distribution of matrix proteins followed by time-lapse CLSM using continuous direct immunostaining. Cell outlines (bright field) are gray; RbmA, Bap1, and RbmC are shown in blue, green, and red, respectively. Scale bars, 2 μm. (B) Bright-field biofilm image and corresponding fluorescent channel surface plots of Bap1, RbmA, and RbmC obtained 4.5 hours postinoculation. Fluorescence intensity is color-coded according to the color scale bar. Bap1 spread from a central point corresponding to the founder cell position, whereas RbmA and RbmC were more homogenously distributed through the biofilm cells. Scale bar, 3 μm. (C) Gradual expansion of the RbmC-containing cluster tracked by time-lapse CLSM. Scale bars, 1 μm. (D) Inability to produce VPS (VPS-) prevents retention of daughter cells, as well as accumulation of RbmA and RbmC and also blocks biofilm formation. Scale bars, 3 μm.

Bap1 then appeared at the junction between the two cells and also on the substrate near the cells (Fig. 2A). Bap1 gradually accumulated radially over distances of tens of micrometers from its initial location on or near the founder cell (Fig. 2, A and B). The founder cell and its immediate environment had notably more Bap1 than the rest of the biofilm for the entire 6.5-hour duration of the experiment. The radially symmetrical distribution of Bap1 relative to the founder cell suggests that Bap1 is continuously secreted into solution by the founder cell and other early members of the young biofilm, after which it accumulates on nearby surfaces (Fig. 2B and fig. S11).

The third matrix protein, RbmC, first appeared after 90 min at discrete sites on the cell surface. Later in biofilm development, the RbmC-Bap1 envelopes formed and then grew by expansion in all directions, with the size of the RbmC-Bap1 envelope doubling within three cell divisions to accommodate the new cell mass (Fig. 2C). Biofilm formation thus involves the temporally sequenced and spatially heterogeneous secretion of matrix proteins, which may have complementary architectural functions—namely, retention of daughter cells after division by RbmA, surface functionalization by Bap1, and encapsulation of the cell clusters by RbmC/Bap1.

Next, we investigated how the RbmC and Bap1 matrix proteins interact with VPS during biofilm formation. VPS is a polysaccharide thought to form a polymeric network that gives mechanical continuity to the biofilm (8, 1619). V. cholerae cells lacking either VPS (VPS–) or all three matrix proteins (ABC–) were unable to form 3D biofilms (fig. S12). The parent strain biofilm phenotype could be recovered by coculturing VPS– and ABC– strains, showing that heterologous provision of these four materials is sufficient to restore normal biofilm formation (figs. S1A and S12). VPS– cells could stick to surfaces, but subsequently produced daughter cells did not accumulate and were instead lost in the growth medium (Fig. 2D and movie S5) (20). Although RbmA, RbmC, and Bap1 proteins were synthesized (figs. S13 and S14), they did not accumulate on the surface of VPS– cells (Fig. 2D). Bap1 was detected on the substrate near the founder cell (Fig. 2D), as expected if Bap1’s main function is to adhere to diverse substrates and tether the biofilm (14). Thus, VPS is required for accumulation of the RbmA, RbmC, and Bap1 on the cell surface, which, in turn, is needed for formation of mature biofilms (13, 14).

Because VPS was required for accumulation of matrix proteins on the cell surface, we wondered whether the opposite was also true. We directly stained the VPS with a Cy3-labeled wheat germ agglutinin (WGA), which recognizes N-acetylglucosamine sugars in the VPS (21). RbmC was essential for sustained incorporation of VPS throughout V. cholerae biofilms (fig. S15A). Without RbmC, there were occasional bright dots of VPS within the colony but at a much lower density than in the parent strain biofilm (fig. S15A). Thus, sustained retention of VPS is codependent with retention of RbmC (Fig. 2D and fig. S15). The VPS staining also confirmed that the RbmC/Bap1 envelopes contained VPS, as expected (Fig. 1A and fig. S15B).

Three-dimensional biofilm development requires a specific, mutually interdependent series of protein/VPS synthesis, secretion, capture, and cross-linking steps. However, the ~200-nm spatial resolution of confocal laser scanning microscopy (CLSM) (22, 23) was insufficient to directly visualize these developmental intermediates. We thus constructed a multicolor 3D superresolution imaging apparatus using stochastic optical reconstruction microscopy (2328) with a localization precision of 19, 21, and 42 nm in x, y, and z (full width at half maximum) (fig. S16). As before, we added labels to the growth medium and imaged living biofilms. With the use of a Cy3-WGA reagent, VPS was first detected at several discrete sites on the cell surface at time t = 15 min postattachment (Fig. 3A, white arrows). Over the next 2 hours, the number of VPS spots, as well as their intensity, increased slowly. At t = 60 min postattachment, 3D superresolution images of VPS organization showed that the polymer was primarily organized into 50- to 200-nm diameter spheroids protruding away from the cell surface (Fig. 3B, white arrow). It appears that VPS is progressively extruded from the cell as a flexible polymer that, like all relaxed flexible polymers, adopts an isotropic, spherical configuration.

Fig. 3

Exopolysaccharide secretion, initial organization, and molecular architecture of V. cholerae biofilms. (A) Time-lapse CLSM images of VPS (green) production and secretion in V. cholerae cells during biofilm formation. Fluorescent images of VPS are merged with bright-field images of cells. White arrows indicate VPS. Scale bar, 2 μm. (B) Three-dimensional superresolution image of a single V. cholerae cell. The white arrow indicates a ball-like structure of VPS on the surface of the V. cholerae cell early in biofilm formation. Color corresponds to height: –300 nm (violet) to +300 nm (red). (C) Three-dimensional two-color superresolution image (200-nm z-section) of a rugose variant biofilm showing molecular organization of VPS (red) and RbmC (green) around cell clusters. Cells were counterstained with DAPI (white). (D) Enlarged boxed region in (C) showing organization of cells within the VPS/RbmC-enclosed cluster. Individual cells were outlined (light blue) for clarity. (E) Enlarged boxed region in (D) as it appears in conventional, diffraction-limited microcopy, showing unresolved VPS and RbmC signals. (F) Superresolution image of the same region in (E), showing distribution of RbmC and the VPS polymers in a biofilm matrix. (G) Enlarged boxed region in (F). White symbols indicate the center of a Gaussian fit to each localization events.

Pseudomonas aeruginosa biofilms have been reported to self-heal within minutes after mechanical disruption beyond their yield point, implying that relatively transient interactions are responsible for maintaining the P. aeruginosa matrix (29). How could such recovery be possible if the VPS (or Psl in P. aeruginosa) were irreversibly cross-linked by matrix proteins such as RbmC? We used two-color 3D superresolution imaging to visualize the organization of VPS and RbmC within a biofilm (15). The superresolution microscope has wide dynamic range and can detect single VPS and RbmC molecules. VPS and RbmC were not homogeneously distributed within the mature biofilm, but both matrix components were confined to the envelopes encasing the cell clusters and to the interstitial space between clusters (Fig. 3, C and D). The mechanism(s) by which bacteria achieve such spatial segregation of materials within the biofilm and, thus, generate a matrix architecture with submicrometer features are unknown. Moreover, most RbmC signal was not uniformly distributed within the VPS matrix (Fig. 3, E to G). Hence, RbmC and VPS may have homophilic (RbmC-RbmC or VPS-VPS) and heterophilic (RbmC-VPS) interactions, where RbmC may act as a reversible cross-linker of VPS. VPS organization must also be dynamic; otherwise, the cells could not sharply repartition RbmC and VPS into the envelopes and interstitial spaces (Fig. 3D).

We used a matrix-labeling strategy to observe in real time as V. cholerae biofilms develop with single-protein and single-polymer precision, revealing assembly principles and intermediates. Cells organize into clusters within the biofilm, and the mature biofilm is a composite of these clusters. An envelope composed of VPS, Bap1, and RbmC encloses these clusters, and RbmA is required for their formation. The VPS/Bap1/RbmC envelope is structured on the molecular level by an unknown mechanism(s) and is capable of reforming, stretching, and expanding to accommodate cell growth.

Supplementary Materials

www.sciencemag.org/cgi/content/full/337/6091/236/DC1

Materials and Methods

Figs. S1 to S17

Tables S1 and S2

References (3040)

Movies S1 to S6

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. Acknowledgments: We thank B. Huang for providing image processing software and D. J. Wozniak, J. H. D. Cate, X. Nan, A. Arkin, and A. Yildiz for critical evaluation of the manuscript. This work is supported by the NSF [grant PHY-0647161 (J.L.)] and the NIH [grants AI055987 (F.H.Y.) and GM096450 and GM068518 (X.Z.)]. X.Z. is a HHMI Investigator. J.L. acknowledges support from the U.S. Department of Energy Office of Basic Energy Sciences (FWP SISGRKN) and Lawrence Berkeley National Laboratory.
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