M I N I R E V I E W Exopolymericsubstances(EPS)from Bacillussubtilis :polymersand genesencoding their synthesis Massimiliano Marvasi1, Pieter T. Visscher2 & Lilliam Casillas Martinez3 1Biology Department, Pontifical Catholic University of Puerto Rico, Ponce, PR, USA 2Center for Integrative Geosciences, University of Connecticut, Storrs, CT, USA and 3Biology Department, University of Puerto Rico in Humacao, Humacao, PR, USA Correspondence: Lilliam Casillas Martinez, Biology Department, University of Puerto Rico in Humacao, Box CUH, Humacao, PR 00791, USA. Tel.: 11 787 850 0000, ext. 9162 fax: 11 787 850 9439 e-mail: lilliam.casillas@upr.edu Received 28 February 2010 revised 16 July 2010 accepted 18 July 2010. Final version published online 23 August 2010. DOI:10.1111/j.1574-6968.2010.02085.x Editor: Simon Silver Keywords EPS Bacillus subtilis biofilm. Abstract Bacterial exopolymeric substances (EPS) are molecules released in response to the physiological stress encountered in the natural environment. EPS are structural components of the extracellular matrix in which cells are embedded during biofilm development. The chemical nature and functions of these EPS are dependent on the genetic expression of the cells within each biofilm. Although some bacterial matrices have been characterized, understanding of the function of the EPS is relatively limited, particularly within the Bacillus genus. Similar gaps of knowledge exist with respect to the chemical composition and specific roles of the macro- molecules secreted by Bacillus subtilis in its natural environment. In this review, the different EPS from B. subtilis were classified into four main functional categories: structural (neutral polymers), sorptive (charged polymers), surface-active and active polymers. In addition, current information regarding the genetic expression, production and function of the main polymers secreted by B. subtilis strains, particularly those related to biofilm formation and its architecture, has been compiled. Further characterization of these EPS from B. subtilis remains a challenge. Introduction Microbial exopolymeric substances (EPS) include a wide diversity of molecules released by microorganisms in their natural environment as well as under laboratory conditions (Flemming et al., 2004 Dupraz & Visscher, 2005 Aguilar et al., 2007). Although initially the term EPS was used to describe extracellular polysaccharides, recent studies have revealed that these matrixes are more complex, including lipopolysaccharides, glycolipids, lipids, proteins or peptides and nucleic acids (Wingender et al., 1999 Decho, 2000). This complex structure comprises the exopolymeric matrix in which cells are embedded, and is also referred to as the biofilm (O���Toole & Ghannoum, 2004). The chemical com- position of the EPS depends on the genetics of the microbial cells and the physicochemical environment in which the biofilm matrix develops (Sutherland, 2001a). Consequently, environmental conditions ultimately dictate the key proper- ties of the biofilms such as porosity, density, water content, charge, sorption and ion exchange properties, hydrophobi- city and mechanical stability (Wingender et al., 1999). Substances associated with exopolymeric matrices have multiple functions. Some serve as signaling molecules or messengers and others are energy and nutrient reserves with an important role in polymer degradation and surface adhesion (O���Toole & Ghannoum, 2004 Decho et al., 2010). Recently, the polyelectrolytic nature of some of these mole- cules has been described with concomitant use in the fabrication of nanowires (Dobrynin, 2008 Lovley, 2008). Although EPS are common to bacteria and critical in cell survival, they are relatively poorly studied, especially with respect to the matrix composition in natural environments (Davey & O���Toole, 2000). In this review, some of the current information on the EPS of Bacillus subtilis is compiled. The role of these molecules within natural environment is also discussed. The focus is on B. subtilis because it is ubiquitous, present in almost all ecosystems and the EPS produced by this organism have significant ecological relevance with respect to cell survival and differentiation within a biofilm (Earl et al., 2008). As shown in Supporting Information, Table S1, a wide variety of EPS are secreted by B. subtilis strains. In the review process, the lack of classification of the FEMS Microbiol Lett 313 (2010) 1���9 c 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved MICROBIOLOGY LETTERS

main EPS from B. subtilis was noticed. It is often unclear whether a particular polymer under investigation is pro- duced by all wild-type strains of B. subtilis or is unique to a particular isolate. Several hundred wild-type B. subtilis strains have been collected to date, only some of which have the potential to produce different types of EPS. One caveat in these studies is that strains able to secrete polymeric substances are not genetically characterized and those ge- netically characterized are defective in EPS production. For example, B. subtilis 168 is the most studied type strain, is used in many laboratories and industrial processes and is an excellent candidate for genetic studies. It is easy to trans- form, it grows under planktonic conditions, its genome has been sequenced (Kunst et al., 1997) and its proteome has been characterized (Wolff et al., 2007). Unfortunately, B. subtilis 168 produces only a few antibiotics and it is defective or attenuated in EPS production (Stein et al., 2004 Aguilar et al., 2007). Several of the biosynthetic pathways are not functional because of the domestication processes (i.e. mutations that allow easier genetic manipulations coupled with repeated growth under artificial settings). The B. subtilis 168 strain derives from X-ray mutations of the original Marburg strain (Burkholder & Giles, 1947 Chu et al., 2006 Earl et al., 2007). In contrast, various other B. subtilis wild-type strains produce numerous peptide anti- biotics as well as abundant EPS (Stein, 2005). In this review, EPS described are specifically matched with the actual Bacillus strains responsible for its production (Table S1). Main characteristics of EPS by B. subtilis EPS produced by wild-type B. subtilis strains grown under controlled laboratory conditions exhibit a wide range of sizes (with molecular weights ranging from 0.57 to 128 kDa) and chemical compositions (i.e. neutral polysaccharides, charged polymers, amphiphilic molecules and proteins) (Priest, 1977 Lin et al., 1999 Omoike & Chorover, 2004). Fourier-transformed infrared spectroscopy studies of cell- bound and ���free��� EPS (in aqueous phase) from B. subtilis ATCC7003 grown in Luria broth showed that the composi- tion of the functional groups of the matrix depends on the cell growth phase (e.g. exponential vs. stationary) (Omoike & Chorover, 2004). Greater amounts of free EPS (relative to cell-bound EPS) are produced during the stationary phase. Quantification of the types of macromolecules within these matrices indicated that proteins and carbohydrates are the major constituents of EPS by mass, with protein levels increasing in free EPS as growth proceeded from the exponential to the stationary phase (Omoike & Chorover, 2004). More detailed investigations are needed to explore differences in the abundance and composition of the pro- teins, acidic groups and sugars of the biofilms of Bacillus grown under specific conditions. Additional knowledge of the chemical composition and three-dimensional architec- ture of the biofilms will aid in solving practical problems in industrial and medical applications and will also help in the classification of EPS based on function. Categories of EPS produced by B. subtilis according to function Flemming et al. (2007) proposed seven categories of EPS: structural, sorptive, surface-active, active, informative, re- dox-active and nutritive EPS. However, only four of these classes occur in molecules identified in B. subtilis: the categories include structural, sorptive, surface-active and active EPS (Table S1). Structural EPS refer to molecules such as neutral polysaccharides, which serve as architectural components in the matrix, facilitating water retention and cell protection. Sorptive EPS are composed of charged polymers, whose function is sorption to other charged molecules involved in cell���surface interactions. Surface- active EPS are molecules with an amphiphilic behavior. These molecules, with different chemical structures and surface properties, are involved in biofilm formation and sometimes possess antibacterial or antifungal activities. The active EPS group is the most diverse group and includes all extracellular proteins produced by B. subtilis. Only those enzymes required for biofilm formation and architecture are discussed. Structural EPS (neutral polysaccharides) Structural EPS are mainly composed of neutral polysacchar- ides that lend structure to the exopolymeric matrix. These exopolysaccharides are formed in the biofilm matrix of many bacterial species for example Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, Klebsiella pneumo- niae and Enterobacter aerogenes (Morikawa et al., 2006 Ryder et al., 2007). However, only a few studies report the isolation and identification of exopolysaccharides from B. subtilis. The best-studied exopolysaccharide produced by B. sub- tilis is levan type I and II. Levan type I consists of b-2,6- linked D-fructose units, whereas type II is a fructose polymer with a glucose residue linked to the terminal fructose by a-glycoside bond. Levan can be synthesized outside the cell following the extrusion of the extracellular enzyme levansu- crase (Abdel-Fattah et al., 2005 El-Refai et al., 2009). Fur- ther details on levansucrase extrusion and induction are included in the section describing active EPS. Levan is widely distributed and produced by various plants and microor- ganisms including B. subtilis strains 327UH, ISS3119, QB112 and Pseudomonas sp. (Yamamoto et al., 1985 Pereira et al., 2001 Shida et al., 2002). In Pseudomonas, it has been suggested that levan forms a capsule protecting against the attack of bacteriophages and also helps prevent cell FEMS Microbiol Lett 313 (2010) 1���9 c 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 2 M. Marvasi et al.