Institute of Cell and Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JH, UK1
Tel: +44 131 650 5331. Fax: +44 131 650 5392. e-mail I.W.Sutherland{at}ed.ac.uk
Keywords: biofilm, exopolysaccharide, gels, viscosity, conformation
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Overview |
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Any study of biofilms must accept that biofilms may develop in an enormous number of environments, and that the structural intricacies of any single biofilm formed under any specific set of parameters may well be unique to that single environment and microflora. The enormous number of microbial species capable of forming biofilms or interacting with others to do so, together with the very great range of polysaccharides produced, gives rise to an infinite number of permutations. In natural conditions, monospecies biofilms are relatively rare; thus most biofilms are composed of mixtures of micro-organisms. This adds to the interspecies and intraspecies interactions and to the general complexity of the macromolecular mixture present.
The exopolysaccharides (EPS) synthesized by microbial cells vary greatly in their composition and hence in their chemical and physical properties. Some are neutral macromolecules, but the majority are polyanionic due to the presence of either uronic acids (D-glucuronic acid being the commonest, although D-galacturonic and D-mannuronic acids are also found) or ketal-linked pyruvate. Inorganic residues, such as phosphate or rarely sulphate, may also confer polyanionic status (Sutherland, 1990 ). A very few EPS may even be polycationic, as exemplified by the adhesive polymer obtained from strains of Staphylococcus epidermidis strains associated with biofilms (Mack et al., 1996
). The composition and structure of the polysaccharides determines their primary conformation. Further, ordered secondary configuration frequently takes the form of aggregated helices. In some of these polymers, the backbone composition of sequences of 1,4-ß- or 1,3-ß-linkages may confer considerable rigidity, as is seen in the cellulosic backbone of xanthan from Xanthomonas campestris. Other linkages in polysaccharides may yield more flexible structures. These can be exemplified by the 1,2-
- or 1,6-
-linkages found in many dextrans. The transition in solution from random coil to ordered helical aggregates is often greatly influenced by the presence or absence of acyl substituents such as O-acetyl or O-succinyl esters or pyruvate ketals (Sutherland, 1997
). In most natural and experimental environments, the EPS will be found in the ordered configurations which are found at lower temperatures and in the presence of salts. The polysaccharides are essentially very long, thin molecular chains with molecular mass of the order of 0·52·0x106 Da, but they can associate in a number of different ways. In several preparations, the polysaccharides have been visualized as fine strands attached to the bacterial cell surface and forming a complex network surrounding the cell. Mayer et al. (1999)
suggested that electrostatic and hydrogen bonds are the dominant forces involved. Ionic interactions may be involved, but more subtle chainchain complex formation in which one macromolecule fits into the other may result in either floc formation or networks which are very poorly soluble in aqueous solvents. Another result may be the formation of strong or weak gels. The polysaccharides can thus form various types of structures within a biofilm. However, in biofilms the polysaccharides do not exist alone but may interact with a wide range of other molecular species, including lectins, proteins, lipids etc., as well as with other polysaccharides. The resultant tertiary structure comprises a network of polysaccharide and other macromolecules, in which cells and cell products are also trapped.
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Are there specific biofilm polysaccharides? |
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The amount of EPS synthesis within the biofilm will depend greatly on the availability of carbon substrates (both inside and outside the cell) and on the balance between carbon and other limiting nutrients. The presence of excess available carbon substrate and limitations in other nutrients, such as nitrogen, potassium or phosphate, will promote the synthesis of EPS. The slow bacterial growth observed in most biofilms would also be expected to enhance EPS production. In organisms such as colanic-acid-producing Es. coli, the production of EPS forms part of the stress response under control of the rpoS gene. A similar response might account for the starvation-specific formation of an adhesive EPS observed in cultures of a marine bacterium, Pseudomonas sp. strain S9 (Wrangstadh et al., 1990 ). Increased EPS synthesis has indeed now been observed as part of a major change in gene expression in the biofilm state for a number of bacterial species. These include both colanic acid production in Es. coli (Prigent-Combaret et al., 1999
) and alginate synthesis in Pseudomonas aeruginosa (Davies & Geesey, 1995
), as well as secretion of a galactoglucan EPS of unknown structure in Vibrio cholerae El Tor (Watnick & Kolter, 1999
). Is it possible that, because of the soluble EPS within the biofilm, localized high osmolarity within the biofilm could be one of the signals? It is known to act as a signal for enhanced transcription of the algD promoter in P. aeruginosa (Berry et al., 1989
) and was earlier shown to enhance colanic acid synthesis.
It is clear from a number of studies that mutants unable to synthesize the EPS are unable to form biofilms (Allison & Sutherland, 1987 ; Watnick & Kolter, 1999
), although they may still attach to surfaces and form micro-colonies to a limited extent. However, in our study of a natural biofilm isolate attaching to glass, most of the EPS- mutant bacteria were seen as well-separated cells; under calcium-limiting conditions, where little EPS was synthesized, the effect was very similar. However, when the bacteria are components of mixed biofilms, the presence of one species producing copious amounts of EPS may enhance the stability of other cell types even if they do not themselves synthesize EPS. Such stabilizing effects were considered by James et al. (1995)
to be commensal interactions. As pointed out by Skillman et al. (1999)
, the proportions of different EPS in mixed biofilms do not necessarily reflect the proportions of the cells present, nor do the EPS contribute equally to the structure and properties of the resulting biofilms.
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What do we know of the structure and properties of the biofilm polysaccharides? |
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Many of these polysaccharides are relatively soluble, and because of their large molecular mass, yield highly viscous aqueous solutions. A few will form weak gels, which dissolve in excess solvent, thus sloughing off the exposed surface of biofilms. Changes may occur when ions are present. Some ions may specifically interact with exposed carboxylic acid groups on the EPS to yield networks of macromolecules which show increased viscosity or even gelation. Various cations may compete for the same binding site, as was shown by Loaëc et al. (1997) ; alternatively, ion binding may be less specific. The ionic radii of the cations may sometimes be important in determining the extent of interaction between the polymer chains and the ultimate extent of aggregation of helices. In most cases, the presence of multivalent cations such as Ca2+ will lead to more extensive formation of ordered helices than monovalent ions, although some polysaccharides resemble kappa carrageenan and reveal aggregates of double helices in the presence of K+.
The EPS contribute directly to the properties of biofilms in that they normally permit considerable amounts of water to be bound. This is not a feature which has been extensively examined. However, polysaccharides such as hyaluronic acid can bind up to 1 kg water (g polysaccharide)-1. It is probable that many of the EPS in biofilms bind lesser quantities whilst some, like bacterial cellulose, mutan or curdlan, manage to exclude most water from their tertiary structure. The EPS will also contribute to the mechanical stability of the biofilms (Mayer et al., 1999 ), enabling them to withstand considerable shear forces. In some polymers, the interaction with ions may yield relatively rigid gels which are less readily deformed by shear, thus producing a much more stable biofilm. Mayer et al. (1999)
suggested that biofilms might indeed represent gel-like structures, but these may be very weak and consequently may be readily destroyed by shear or dissolution of the polysaccharides. It should not be forgotten that a small number of EPS, because of their composition and tertiary structure, might actually be hydrophobic (Neu & Poralla, 1988
). Others possess localized hydrophilic and hydrophobic regions. They thus confer very different properties on the matrices in which they are found and account for the wide differences in properties found in different biofilms.
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What is the relationship of structure to function? |
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Many bacterial EPS possess backbone structures in which there exist sequences of 1,3- or 1,4-ß-linked hexose residues. When such sequences are present, the polymers tend to be much more rigid in structure, less deformable and, in the case of neutral polysaccharides such as mutan or those from some strains of Enterobacter agglomerans, either poorly soluble or effectively insoluble. These EPS molecules may be very robust. The long chains of stiff macromolecules may be present as gels due to the entanglements found within the long chains and also, in some polymers, to the ionic environment (Ross-Murphy & Shatwell, 1993 ; Ross-Murphy, 1995
). The stability of the gel state will depend on the effective polysaccharide concentration, the ionic status and the other macromolecules present. Those EPS molecules, which are effectively in solution, may well dissolve with dilution, thus accounting in part for the observed sloughing off of biofilm material. It has to be remembered that enzymes will also contribute to this (Boyd & Chakrabarty, 1994
). Another aspect, which has received relatively little study, is the possibility of interaction of EPS with proteins and other excreted or surface-associated macromolecules. Either association or segregation may occur.
Other EPS, of which curdlan is an example, form triple helices in which the strands are very firmly held together by hydrogen bonds. Such tight bonding together of the linear molecules may effectively exclude water molecules. Any substituents present may very greatly affect the conformation, as is seen in the commercial product gellan from a Sphingomonas elodea strain. The native, acylated polymer forms weak gels, whereas the deacylated material yields brittle, rigid gels (Chandrasekaran & Thailambal, 1990 ). Similarly, comparison of the polysaccharides from Klebsiella aerogenes K54 and Enterobacter aerogenes XM6 indicates the role of acetyl groups. Both EPS have the same tetrasaccharide repeat units containing D-glucose, L-fucose and D-glucuronic acid in the molar ratio 2:1:1, and both yield viscous aqueous solutions (ONeill et al., 1986
). However, the non-acetylated XM6 polymer presents a highly crystalline structure recognizable by X-ray fibre diffraction (Atkins et al., 1987
), whereas the K54 polymers carrying either 0·5 or 1 acetate per repeat unit are amorphous. Deacetylation of the latter converts its pattern to one similar to XM6. XM6 and deacetylated K54 gel in the presence of various ions, whilst native K54 does not (Nisbet et al., 1984
). Possibly many biofilm EPS form either highly viscous solutions or localized gels, the latter being deformable under shear but recovering to something like their original state after the shear is removed.
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How do biofilm polysaccharides interact? |
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EPS may also interact with protein molecules and micelles, i.e. effectively with microbial cells and their associated surface proteins within the biofilm matrix, as well as with soluble proteins, including enzymes. The two types of biopolymer may interact in different ways. They may be associative, but are more commonly segregative or incompatible. In the latter example, the EPS concentration near the protein molecule is reduced; there may even be phase separation into polysaccharide-rich and protein-rich phases. Polysaccharides may also adsorb onto more than one protein surface to cause coacervation (Tuinier, 1999 ). The effect of such polymerpolymer interactions on biofilm structure and stability has not yet received attention.
As yet, relatively little is known about the interactions which occur between biofilm EPS and enzymes (Sutherland, 1999b ). Whilst proteases would certainly affect those proteins which interact with EPS within biofilms, polysaccharases and polysaccharide lyases can have a much greater effect. A wide range of highly specific polysaccharases and polysaccharide lyases are available from lysates of EPS-producing bacteria, following infection with virulent bacteriophage (Sutherland, 1999a
; Hughes et al., 1998a
). In experimental systems, it is clear that the effect of any enzyme degrading any one EPS will depend on the other EPS and microbial cells present in the biofilm. Thus in a Pseudomonas fluorescens/En. agglomerans mixed biofilm, addition of an alginate lyase released many of the P. fluorescens cells from the surface whilst attached cell numbers of the enteric species increased (V. Morton & I. W. Sutherland, unpublished results). Hughes et al. (1998b)
observed a rapid decline in the number of attached En. agglomerans cells following the addition of a phage-induced polysaccharide depolymerase. This was also confirmed by scanning electron microscopy. Using GFP-labelled En. agglomerans cells, Skillman et al. (1999)
noted that treatment with either the polysaccharase or a protease reduced the adhesion of these bacteria to a monolayer of Klebsiella pneumoniae cells. This indicated roles for both the polysaccharide and proteins in the adhesion process and suggests that considerable differences can be expected when different combinations of microbial species are examined.
Some bacteria secrete esterases with wide specificity; these can remove acyl groups from bacterial polymers as well as from other esters (Cui et al., 1999 ). Such enzymes could alter the physical properties of a biofilm structure, either locally or to a greater extent. Deacylation of the bacterial polysaccharide succinoglycan improved pseudoplasticity in aqueous solution as well as increasing the cooperativity of the orderdisorder transition (Ridout et al., 1997
). On the other hand, deacylation of some polysaccharides may lead to loss of any ordered conformation (Villain-Simonnet et al., 2000
). Other polysaccharides, such as XM6 and gellan, can readily form gels when freed of acyl substituents (Sutherland, 1997
); this would lead to strengthening of portions of biofilms containing such polymers.
Many bacteria are capable of synthesizing and excreting surfactants, some of which, such as emulsan, resemble lipopolysaccharides (LPS), whilst rhamnolipids are products of Pseudomonas spp. Al-Tahhan et al. (2000) pointed out that even very low levels of a rhamnolipid biosurfactant could render the cell surface more hydrophobic, causing loss of LPS in the process. It has also been suggested that biosurfactants might be involved in the horizontal transfer of exopolymer from one bacterial species to another (Osterreicher-Ravid et al., 2000
). This could take place much more efficiently within the matrix of a biofilm where cells are in close proximity to each other. The production of these biosurfactants also enables the component cells within biofilms to solubilize and utilize substrates which would otherwise be inaccessible.
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Do the biofilm polysaccharides offer any protection to the cells within the biofilm? |
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As any biofilm is unlikely to comprise a single type of EPS, the effect of enzyme action will depend on whether its substrate plays a major role in maintaining the biofilm structure. Thus, Skillman et al. (1999) observed that in biofilms composed of mixed enteric species, hydrolysis of one EPS caused greater destruction of the biofilm than did removal of the other. This would indicate that, as in cell walls, certain polymers may provide a fairly rigid scaffolding onto or into which other polymers attach to fill the interstices. In oral biofilms, many of the component bacteria are capable of synthesizing several different EPS, including dextrans (
-D-glucans) and levans (ß-D-fructans). In addition, both dextranases and fructan hydrolases may be secreted. Little is known of the effects such polysaccharide hydrolases have on oral biofilms, but recent studies on regulation of expression of the fructan-degrading enzyme in Streptococcus mutans may start to provide an insight (Burne et al., 1999
). The effects of polysaccharases released by senescent bacterial cells have also not been studied.
The environments in which biofilms are found vary greatly. In some, the aqueous milieu is effectively stagnant with no shear exerted on the biofilm and its components. Others, including oral biofilms, are subjected to repeated, and sometimes high, shear forces. These will inevitably displace or destroy sections of the biofilm. Where the shear is constant, the eventual structure will again be different. As pointed out by Ross-Murphy & Shatwell (1993) , large deformations lead to rupture of strong gels similar to agarose, whereas weak gels like xanthan will recover and can even flow during the shear. Both types of behaviour can be expected from biofilm EPS. This was observed in mixed species biofilms studied by Stoodley et al. (1999b)
. Under laminar flow, roughly circular micro-colonies were separated by water channels, whereas in turbulent flow, filamentous streamers were seen with ripple-like structures after prolonged growth. Unfortunately, although cell counts for the different component species were determined, the nature and composition of the polysaccharides was not analysed.
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Conclusion |
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