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REVIEWS The biofilm matrix Hans-Curt Flemming and Jost Wingender Abstract | The microorganisms in biofilms live in a self-produced matrix of hydrated extracellular polymeric substances (EPS) that form their immediate environment. EPS are
  Microorganisms do not live as pure cultures of dis-persed single cells but instead accumulate at interfaces to form polymicrobial aggregates such as films, mats, flocs, sludge or ‘ biofilms ’ (REF. 1) . In most biofilms, the microorganisms account for less than 10% of the dry mass, whereas the matrix can account for over 90%. The matrix is the extracellular material, mostly produced by the organisms themselves, in which the biofilm cells are embedded. It consists of a conglomeration of dif-ferent types of biopolymers — known as extracellular poly meric substances  (EPS) — that forms the scaffold for the three-dimensional architecture of the biofilm and is responsible for adhesion to surfaces and for cohe-sion in the biofilm. The formation of a biofilm allows a lifestyle that is entirely different from the planktonic state. Although “the precise and molecular interactions of the various secreted biofilm matrix polymers … have not been defined, and the contributions of these com-ponents to matrix integrity are poorly understood at a molecular level” (REF. 2) , several functions of EPS have been determined (TABLE 1) , demonstrating a wide range of advantages for the biofilm mode of life.EPS immobilize biofilm cells and keep them in close proximity, thus allowing for intense interactions, including cell–cell communication, and the formation of synergistic microconsortia. Owing to the retention of extracellular enzymes, a versatile external diges-tive system is generated, sequestering dissolved and particulate nutrients from the water phase and allow-ing them to be utilized as nutrient and energy sources. The matrix also acts as a recycling centre by keeping all of the components of lysed cells available. This includes DNA, which may represent a reservoir of genes for hori-zontal gene transfer. EPS can also serve as a nutrient source, although some components of EPS are only slowly biodegradable and, owing to the complexity of EPS, complete degradation of all components requires a wide range of enzymes. The matrix protects organisms against desiccation, oxidizing or charged biocides, some anti biotics and metallic cations, ultraviolet radiation, many (but not all) protozoan grazers and host immune defences. Ecologically, competition and cooperation in the confined space of the EPS matrix lead to a constant adaptation of population fitness.It is unclear whether the matrix confers an ecologi-cal advantage on all cells in the biofilm, in particular those that are furthest from the surface. Simulations of competition in a biofilm revealed a strong evolution-ary benefit for polymer producers at the expense of non-producers, possibly because polymers push the daughter cells of polymer producers closer to oxygen-rich environments 3 .EPS have been called ‘the dark matter of biofilms’ because of the large range of matrix biopolymers and the difficulty in analysing them 4 . EPS can vary greatly between biofilms, depending on the microorganisms present, the shear forces experienced, the temperature and the availability of nutrients. EPS were initially denoted ‘extracellular polysaccharides’ but were renamed, as it became clear that the matrix also contains proteins, nucleic acids, lipids and other biopolymers such as humic substances 1,5 . Extracellular bacterial structures such as flagella , pili  and fimbriae  can also stabilize the matrix 6 . Membrane vesicles  derived from outer membranes of Gram-negative bacteria can contain a range of enzymes and DNA and can alter matrix properties 7 , sometimes acting as ‘killer vesicles’ targeted at competing biofilm organisms. Biofilm Centre, University of Duisburg-Essen, Geibelstrasse 41, D-47057 Duisburg, Germany.Correspondence to H.-C.F. e-mail: doi:10.1038/nrmicro2415 Published online 2 August 2010 Biofilm A loose definition for microbial aggregates that usually accumulate at a solid–liquid interface and are encased in a matrix of highly hydrated EPS. Included in this definition are cell aggregates such as flocs (floating biofilms) and sludge, which are not attached to an interface but which share the characteristics of biofilms. Multispecies biofilms can form stable microconsortia, develop physiochemical gradients, and undergo horizontal gene transfer and intense cell–cell communication, and these consortia therefore represent highly competitive environments. The biofilm matrix Hans-Curt Flemming and Jost Wingender  Abstract | The microorganisms in biofilms live in a self-produced matrix of hydrated extracellular polymeric substances (EPS) that form their immediate environment. EPS are mainly polysaccharides, proteins, nucleic acids and lipids; they provide the mechanical stability of biofilms, mediate their adhesion to surfaces and form a cohesive, three-dimensional polymer network that interconnects and transiently immobilizes biofilm cells. In addition, the biofilm matrix acts as an external digestive system by keeping extracellular enzymes close to the cells, enabling them to metabolize dissolved, colloidal and solid biopolymers. Here we describe the functions, properties and constituents of the EPS matrix that make biofilms the most successful forms of life on earth. REVIEWS NATURE REVIEWS |   MICROBIOLOGY  VOLUME 8 |  SEPTEMBER 2010 |   623 © 20 Macmillan Publishers Limited. All rights reserved10  Extracellular polymeric substances Hydrated biopolymers (including polysaccharides, proteins, nucleic acids and lipids) that are secreted by biofilm cells to encase and immobilize microbial aggregates. These biopolymers are responsible for the macroscopic appearance of biofilms, which are frequently referred to as ‘slime’. Globally, EPS represent a dominant fraction of the reduced-carbon reservoir in soils and in sediments, and suspended aggregates in oceans and freshwater. There, they serve as nutrients and thus play an important part in microbial ecology  8–12 .In this Review, we focus on the role of these matrix components in the architecture of bacterial biofilms, discuss the challenges of isolating EPS and describe the different components of biofilms. BOX 1  provides infor-mation about EPS of other organisms. EPS and biofilm architecture Cells in a biofilm are surrounded by EPS, which consti-tute the immediate environment of these cells. Some EPS, in particular those forming capsules , are associated more closely with cell surfaces than others. The formation and maintenance of structured multicellular microbial communities crucially depend on the production and quantity of EPS 13 . The concentration, cohesion, charge, sorption capacity, specificity and nature of the individual components of EPS, as well as the three-dimensional architecture of the matrix (the dense areas, pores and channels), determine the mode of life in a given biofilm. The resulting biofilm morphology can be smooth and flat, rough, fluffy or filamentous, and the biofilm can also vary in its degree of porosity, having mushroom-like macrocolonies surrounded by water-filled voids. All of these morphologies have the same effect: to transiently immobilize biofilm cells and allow the existence of long-term mixed-species microconsortia, with their interac-tions and gradients; this provides very diverse habitats on a small scale, favouring biodiversity. Table 1 |   Functions of extracellular polymeric substances in bacterial biofilms Function Relevance for biofilmsEPS components involved Adhesion Allows the initial steps in the colonization of abiotic and biotic surfaces by planktonic cells, and the long-term attachment of whole biofilms to surfacesPolysaccharides, proteins, DNA and amphiphilic moleculesAggregation of bacterial cellsEnables bridging between cells, the temporary immobilization of bacterial populations, the development of high cell densities and cell–cell recognitionPolysaccharides, proteins and DNACohesion of biofilmsForms a hydrated polymer network (the biofilm matrix), mediating the mechanical stability of biofilms (often in conjunction with multivalent cations) and, through the EPS structure (capsule, slime or sheath), determining biofilm architecture, as well as allowing cell–cell communicationNeutral and charged polysaccharides, proteins (such as amyloids and lectins), and DNARetention of waterMaintains a highly hydrated microenvironment around biofilm organisms, leading to their tolerance of dessication in water-deficient environments Hydrophilic polysaccharides and, possibly, proteinsProtective barrierConfers resistance to nonspecific and specific host defences during infection, and confers tolerance to various antimicrobial agents (for example, disinfectants and antibiotics), as well as protecting cyanobacterial nitrogenase from the harmful effects of oxygen and protecting against some grazing protozaPolysaccharides and proteinsSorption of organic compoundsAllows the accumulation of nutrients from the environment and the sorption of xenobiotics (thus contributing to environmental detoxification)Charged or hydrophobic polysaccharides and proteinsSorption of inorganic ionsPromotes polysaccharide gel formation, ion exchange, mineral formation and the accumulation of toxic metal ions (thus contributing to environmental detoxification)Charged polysaccharides and proteins, including inorganic substituents such as phosphate and sulphateEnzymatic activityEnables the digestion of exogenous macromolecules for nutrient acquisition and the degradation of structural EPS, allowing the release of cells from biofilmsProteinsNutrient sourceProvides a source of carbon-, nitrogen- and phosphorus-containing compounds for utilization by the biofilm communityPotentially all EPS componentsExchange of genetic informationFaciliates horizontal gene transfer between biofilm cellsDNA Electron donor or acceptorPermits redox activity in the biofilm matrixProteins (for example, those forming pili and nanowires) and, possibly, humic substancesExport of cell componentsReleases cellular material as a result of metabolic turnoverMembrane vesicles containing nucleic acids, enzymes, lipopolysaccharides and phospholipidsSink for excess energyStores excess carbon under unbalanced carbon to nitrogen ratiosPolysaccharidesBinding of enzymesResults in the accumulation, retention and stabilization of enzymes through their interaction with polysaccharidesPolysaccharides and enzymes EPS, extracellular polymeric substances. REVIEWS 624 |  SEPTEMBER 2010 |  VOLUME 8 © 20 Macmillan Publishers Limited. All rights reserved10  Humic substance A component of the natural organic matter in soil and water enviroments. Humic substances are mixtures of compounds that are formed by limited degradation and transformation of dead organic matter and that are resistant to complete biodegradation. They can be divided into three main fractions: humic acids, fulvic acids and humin. They usually include phenolic and polyaromatic compounds (containing peptide and carbohydrate moieties with carboxylic substituents), providing the acidic character. Flagellum A long, thin, helically shaped bacterial appendage that provides motility. A flagellum consists of several components and moves by rotation, much like a propeller. The motor is anchored in the cytoplasmic membrane and the cell wall. Pilus A bacterial surface structure that is similar to a fimbria but is typically a longer structure, and that is present on the cell surface in one or two copies. Pili can be receptors for bacteriophages and also facilitate genetic exchange between bacterial cells during conjugation. Type IV pili mediate twitching motility, which is a flagella-independent form of bacterial translocation over surfaces, and can be involved in biofilm development. The use of microelectrodes (to monitor oxygen levels, for example) revealed spatial heterogeneity in biofilms on a micrometre scale 14   (FIG. 1) . On the basis of staining with lectins  and imaging with confocal laser scanning microscopy to differentiate various EPS com-ponents and biofilm organisms, it was concluded that the EPS matrix provides a physical structure that segre-gates microdomains 15 . These regions harbour different biochemical environments that are enzymatically mod-ified in response to changing conditions. For further investigation of the matrix architecture, a reliable alloca-tion of the binding sites of lectins is crucial. Chemical analyses can possibly be put into a spatial context by combining confocal laser scanning microscopy and Raman microscopy 16   (BOX 2) . The architecture of biofilms is influenced by many factors, including hydrodynamic conditions, concen-tration of nutrients, bacterial motility and intercellu-lar communication as well as exopolysaccharides and proteins, as demonstrated by the altered morphology of biofilms produced by mutants lacking components of EPS. For example, exopolysaccharides of Vibrio chol-erae 17  and colanic acid of Escherichia coli 18  are involved in the formation of a three-dimensional biofilm archi-tecture. The Bacillus subtilis  biofilm matrix consists of an exopolysaccharide and the secreted protein TasA, both of which are required for the structural integrity of the matrix and the development of biofilm architecture in the form of fruiting body-like structures 19 . During aggre-gation of the soil bacterium  Myxococcus xanthus , the polysaccharide component of the extracellular matrix forms a scaffold within the fruiting-body structure 20 . One of the best studied exopolysaccharides involved in biofilm formation is alginate in the biofilms of mucoid strains of the opportunistic pathogen Pseudomonas aeruginosa 21,22 . Alginate is not essential for P. aeruginosa  biofilm formation 23 , but it has a notable effect on bio-film architecture when it is present. Under conditions in which alginate producers form structurally heterogene-ous biofilms, non-mucoid strains develop flat and more homogeneous biofilms (FIG. 2a–c) .Acetyl groups are common substituents of exopoly-saccharides, and they increase the adhesive and cohe-sive properties of EPS and alter biofilm architecture. The modification of alginate with acetyl groups strongly influ-ences the aggregation of bacteria into microcolonies and determines the structurally heterogeneous architecture of mature biofilms 21,22   (FIG. 2e,f) . Biofilm architecture can also be strongly influenced by the interaction of anionic EPS, containing carboxylic groups, with multivalent cations. For example, Ca 2+  can form a bridge between polyanionic alginate molecules, stimulating the devel-opment of thick and compact biofilms with increased mechanical stability  24   (FIG. 2d) . Isolation of EPS The identification of EPS components depends on the isolation method used. However, efficient EPS isola-tion is challenging, particularly for EPS from environ-mental biofilms, which can contain an immense range of components that each require different extraction methods. In a mixed-species biofilm, many members of the microbial community contribute their own (and often specific) EPS that then merge into a complex mix-ture 11  and remain in the matrix even after their pro-ducers have died or left the biofilm. Furthermore, it is next to impossible to quantitatively isolate EPS from a given biofilm, because some of the EPS fraction remains bound to the bacteria, and because the isolation pro-cedure damages cells, causing intracellular material to leak into the matrix.There is no universal EPS isolation method — the extraction procedure has to be adapted to the specific type of biofilm under investigation. Centrifugation, filtration, heating, blending, sonication, and treatment with complexing agents and with ion exchanger resins have been described 25,26 , and the use of sodium hydrox-ide has even been reported 27 , although this method almost certainly leads to contamination with cytoplas-mic components. One popular method uses a cation exchanger resin 28 , which removes the cations that bridge the negatively charged groups of the polysaccharide and protein moieties of EPS. Alginate from P. aeruginosa  is comprised solely of    uronic acids, which are not found inside the cells and can therefore be used as EPS mark-ers during isolation 29 . The presence of intracellular enzymes, such as glucose-6-phosphate 1-dehydrogenase (G6PD, also known as Zwf), indicates contamination with cellular components. Following extraction, a com-mon concentration step is to precipitate solubilized EPS by adding ethanol or acetone 11 ; however, this method primarily precipitates polysaccharides, leading to an underestimation of the other components of EPS.Common EPS isolation techniques inherently select for water-soluble EPS and lose insoluble EPS, including cellulose, which is an important constituent of the matri-ces of many bacteria. Cellulose plays an important part in biofilm-related infections caused by Escherichia coli , Klebsiella pneumoniae , Enterobacter spp.,  Citrobacter spp . and Salmonella enterica subsp.  enterica serovar Typhimurium 6,30–32 . Isolation of cellulose requires harsh conditions, such as treatment with acetic acid and nitric acid at 95 °C 3 . Exopolysaccharides Polysaccharides are a major fraction of the EPS matrix 28,29 . Most are long molecules, linear or branched, with a molecular mass of 0.5 × 10 6  daltons to 2 × 10 6  daltons. Box 1 | Extracellular polymeric substances from fungi, algae and archaea Extracellular polymeric substances (EPS) are not unique to bacteria. Some of the most abundant EPS producers are microalgae (in particular, diatoms) 103 . Microalgal EPS play important parts in the stabilization of sediments 104  and the entrainment of sand 105 , but they are also involved in marine fouling. The green alga Penium margaritaceum has been shown to produce large amounts of EPS (predominantly polysaccharides 106,107 ) that, in turn, support the growth of heterotrophic bacteria which use EPS as a substrate. Fungi (yeasts and moulds) also produce EPS. Examples are certain Candida  spp. 108  that produce EPS which are involved in the processes of flocculation, adhesion and biofilm formation 109 . The archaeon Sulfolobus solfataricus  produces polysaccharides in response to adhesion 110 ; other than this, there is surprisingly little information about the EPS matrices of archaea. REVIEWS NATURE REVIEWS |   MICROBIOLOGY  VOLUME 8 |  SEPTEMBER 2010 |   625 © 20 Macmillan Publishers Limited. All rights reserved10  CH 2 CH 2 OHCOO – COO –– OOCCH 2 OHCH 2 CH 2 OHOHCOO – Ca 2+ OHCH 2 OHElectrostatic attractive forcesIonicattractive forcesRepulsive forcesprevent collapsingHydrogen bonding van der Waalsinteractions  + + + ++ + + + +----------     PolysaccharideProteinDNA abcd Fimbria A filamentous structure composed of one or a few proteins that extends from the surface of a cell and can have diverse functions. Fimbriae are involved in attachment to both animate and inanimate surfaces and in the formation of pellicles and biofilms. They assist in the disease process of some pathogens, such as S. enterica , Neisseria gonorrhoea  and Bordetella  pertussis. Membrane vesicle A vesicle that is formed from the outer membrane of Gram-negative bacteria, is secreted from the cell surface and contains extracellular enzymes and nucleic acids. These vesicles may represent mobile elements in the EPS matrix. Capsule A discrete polysaccharide (sometimes also protein) layer that is firmly attached to the surface of a bacterial cell, closely surrounding it, in contrast to less compact, amorphous slime that is shed into the more distant extracellular environment. Lectin A protein or glycoprotein of plant, animal or microbial srcin that binds to carbohydrates with a characteristic specificity. Fluorescently labelled lectins can be used as probes to investigate EPS composition, enabling the microscopic in situ detection of EPS and their distribution in biofilms. Raman microscopy A spectroscopic technique based on inelastic light scattering (Raman scattering) of monochromatic laser light in the near-ultraviolet range, revealing vibrational, rotational and other low-frequency modes in a system. The technique is used for the analysis of chemical bonds and is suitable for very small volumes, allowing spectra and chemical information to be obtained for the molecules present in that volume. Several polysaccharides have been visualized by elec-tron microscopy as fine strands that are attached to the cell surface and form complex networks. Microscopic techniques in combination with specific carbohydrate staining using fluorescently labelled lectins or antibod-ies (BOX 2) , as well as biochemical analyses for inde-pendent verification, have demonstrated the ubiquity of matrix polysaccharides not only in biofilms from natural marine, freshwater and soil environments and from man-made water systems, but also in biofilms associated with chronic infections in humans and in pure-culture experimental biofilms. In recent years, exopolysaccharides from an extensive range of bacterial species from diverse environments have been isolated and characterized 33 .Several exopolysaccharides are homopolysaccharides, including the sucrose-derived glucans and fructans pro-duced by the streptococci in oral biofilms, and cellulose formed by Gluconacetobacter xylinus ,  Agrobacterium tumefaciens , Rhizobium spp. and    various species from the Enterobacteriaceae 6  and Pseudomonadaceae families 29 . However, most exopolysaccharides are Figure 1 |  The extracellular polymeric substances matrix at different dimensions. a | A model of a bacterial biofilm attached to a solid surface. Biofilm formation starts with the attachment of a cell to a surface. A microcolony forms through division of the bacterium, and production of the biofilm matrix is initiated. Other bacteria can then be recruited as the biofilm expands owing to cell division and the further production of matrix components. b | The major matrix components — polysaccharides, proteins and DNA — are distributed between the cells in a non-homogeneous pattern, setting up differences between regions of the matrix. c  | The classes of weak physicochemical interactions and the entanglement of biopolymers that dominate the stability of the EPS matrix 47 . d  | A molecular modelling simulation of the interaction between the exopolysaccharide alginate (right) and the extracellular enzyme lipase (left) of Pseudomonas aeruginosa  in aqueous solution. The starting structure for the simulation of the lipase protein was obtained from the Protein Data Bank 117 . The coloured spheres represent 1,2-dioctylcarbamoyl-glycero-3- O -octylphosphonate in the lipase active site (which was present as part of the crystal structure), except for the green sphere, which represents a Ca 2+  ion. The aggregate is stabilized by the interaction of the positively charged amino acids arginine and histidine (indicated in blue) with the polyanionic alginate. Water molecules are not shown. Image courtesy of H. Kuhn, CAM-D Technologies, Essen, Germany. REVIEWS 626 |  SEPTEMBER 2010 |  VOLUME 8 © 20 Macmillan Publishers Limited. All rights reserved10
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