The aggregation of microbial cells in biofilms, and the complex multicellular interactions that ensue, depend on the production of a cohesive extracellular matrix (Figure 1). Bacteria and fungi that build biofilms secrete extracellular polymeric substances (EPS) that form a highly hydrated slime in which cells are embedded and held in dense agglomerations (Branda et al., 2005). Here I will tackle some myths surrounding the biofilm matrix, discuss two areas of inquiry related to EPS that are likely to change our understanding of biofilms over the next few years, and offer a hypothesis.

The biofilm matrix has some of the qualities of a mythological creature. Its basic composition remains in debate. Polysaccharides are widely acknowledged to be important matrix polymers. But proteins are abundant in the extracellular matrix of biofilms, and it is not clear how much these proteins are structural components and how much they serve functions independent of mechanical integrity. DNA has been found in the biofilm matrix and implicated in cohesion (Whitchurch et al., 2002). Can DNA, the genetic master code, double as mortar? A persistent and widely told myth of the biofilm matrix is that the EPS physically excludes antimicrobial agents, a notion refuted by many measurements of diffusion in biofilms (Stewart, 2003). One of the unstated assumptions about the biofilm matrix is that synthesis of a polymer such as a polysaccharide is sufficient to provide a mechanically stable extracellular matrix. This is probably simplistic. Surely the polymer strands must interact to create a cohesive network, in the same way that a net must be knotted if it is to hold a fish. I will return to this hypothesis.

One of the reasons there is so much mystery surrounding the matrix is that it is normally invisible. Hydrated EPS is translucent and all but impossible to image by conventional light microscopy. Imagine trying to examine a thin film of agar or gelatin coated on a glass slide immersed in a basin of water. The watery, clear gel layer would disappear. Conventional electron microscopy often yields images of fibrous strands connecting cells within a biofilm, but these are understood to be collapsed structures arising from dehydration during sample preparation. The good news is that techniques for imaging EPS are advancing. Perhaps the best way to light up the EPS is with a fluoresecently-tagged lectin. Lectins are plant-derived proteins that bind to specific sugar moieties in polysaccharides. When labeled with a fluorophore, such probes can reveal, with excellent specificity, particular constituents of the extracellular matrix (Bockelmann et al., 2002). A wide variety of amine-reactive probes are commercially available and could be useful in visualizing nitrogen-containing EPS (Figure 1). New electron microscopy techniques based on freeze-substitution also hold promise for visualizing, at high resolution, EPS in its native state (Hunter and Beveridge, 2005).

Bioinformatic and genetic approaches are leading to the identification and biochemical characterization of matrix polymers. One recurring theme emerging from this work is the enzymatic modification of polysaccharides by the addition or removal of acyl groups. These simple modifications profoundly alter the physical-chemical properties of the polymer. For example, a final step in the synthesis of alginate made by mucoid strains of Pseudomonas bacteria is the O-acetylation of mannuronic acid residues (Nivens et al., 2001). This addition dramatically increases the viscosity of the alginate. Mutant bacteria incapable of performing the acetylation step are deficient in biofilm formation. An important polysaccharide EPS of staphylococcal biofilms is synthesized by polymerizing N-acetylglucosamine monomers. As the newly made polymer leaves the cell, some of the acetyl groups are enzymatically removed, conferring a positive charge to the polymer (Vuong et al., 2004). Robust biofilm formation depends on this step. The larger message is that specific regulatory and biochemical pathways of EPS production are being illumined.

One of the archetypal models of EPS is the alginate paradigm in which negatively charged polysaccharide strands are cross-linked by positively charged, multivalent cations such as calcium. Here is an alternative cross-linking mechanism that I submit as a hypothesis.

It is increasingly clear that bacteria synthesize multiple extracellular polymeric substances. For example, the matrix of Escherichia coli biofilms has been reported to contain proteinaceous appendages called curli, along with polysaccharides including colanic acid (negatively charged), cellulose, and a polyglucosamine (positively charged). In Staphylococcus epidermidis, the spotlight has long been on the positively-charged polyglucosamine. A recent report highlights a second major constituent of the EPS of this bacterial biofilm, teichoic acid (Sadovskaya et al., 2005). Teichoic acid is a polymer of glycerol phosphate and is highly negatively charged. If a microorganism synthesizes a positively charged polymer and a negatively charged polymer in concert, it has the makings of a strong glue. When polymers of opposite charge interact they form tough structures called polyelectrolyte complexes. A microbe synthesizing these two parts is a bit like a builder with a tube of epoxy resin and a tube of hardener. The microorganism has the potential to control matrix cohesiveness by regulating the amounts of the two polymers synthesized or by adjusting the charge density on each polymer. What this conceptual model has in common with the alginate model is the interaction between separate EPS strands. I suspect that this is a general requirement for matrix cohesion and that insights into biofilm structure and function will emerge as the physical-chemical interactions between extracellular polymers are elucidated.

In comparison to what is known about the cells themselves, very little is known about the biofilm matrix. A critical first step is to learn how to see the matrix. This is underway. The tools of modern molecular biology will unveil the heretofore anonymous slime of biofilm and assign names, pathways, and chemical structures. It remains for physical chemists to describe the interactions between matrix polymers that confer cohesiveness. When these pieces come together, new technologies for shifting biofilm ecology and controlling unwanted biofilms will appear.

Figure 1. Visualization of bacterial cells (green) and an unidentified matrix constituent (red) in a fully hydrated Pseudomonas aeruginosa biofilm. The bacterial cells harbor a green fluorescent protein, and the specimen was counterstained with a red fluorescent Bodipy-succinimidyl ester which reacted with a matrix component.

REFERENCES

Bockelmann, U., W. Manz, T. R. Neu, and U. Szewzyk. 2002. Investigation of lotic microbial aggregates by a combined technique of fluorescent in situ hybridization and lectin-binding analysis. J. Microbiol. Methods 49:75-87.

Branda, S. S., A. Vik, L. Friedman, and R. Kolter. 2005. Biofilms: the matrix revisited. Trends Microbiol. 13:20-26.

Hunter, R. C. and T. J. Beveridge. 2005. High-resolution visualization of Pseudomonas aeruginosa PAO1 biofilms by freeze-substitution transmission electron microscopy. J. Bacteriol. 187:7619-7630.

Nivens, D. E., D. E. Ohman, J. Williams, and M. J. Franklin. 2001. Role of alginate and its O-acetylaction in formation of Pseudomonas aeruginosa microcolonies and biofilms. J. Bacteriol. 183:1047-1057.

Sadovskaya, I., E. Vinogradov, S. Flahaut, G. Kogan, and S. Jabbouri. 2005. Extracellular carbohydrate-containing polymers of a model biofilm-producing strain, Staphylococcus epidermidis RP62A. Infect. Immun. 73:3007-3017.

Stewart, P. S. 2003. Diffusion in biofilms. J. Bacteriol. 185:1485-1491.

Vuong, C., S. Kocianova, J. M. Voyich, Y. Yao, E. R. Fischer, F. R. EdLeo, and M. Otto. 2004. A crucial role for exopolysaccharide modification in bacterial biofilm formation, immune evasion, and virulence. J. Biol. Chem. 279:54881-54886.

Whitchurch, C. B., T. Tolker-Nielsen, P. C. Ragas, and J. S. Mattick. 2002. Extracellular DNA required for bacterial biofilm formation. Science 295:1487.

Biofilm Perspectives are published by the Montana State University Center for Biofilm Engineering and disseminated via www.BiofilmsOnline.com. Copyright © 2006, MSU Center for Biofilm Engineering