We previously identified a gene cluster, epa (for enterocococcal polysaccharide antigen), involved in polysaccharide biosynthesis of Enterococcus faecalis and showed that disruption of epaB and epaE resulted in attenuation in translocation, biofilm formation, resistance to polymorphonuclear leukocyte (PMN) killing, and virulence in a mouse peritonitis model. Using five additional mutant disruptions in the 26-kb region between orfde2 and OG1RF_0163, we defined the epa locus as the area from epaA to epaR. Disruption of epaA, epaM, and epaN, like prior disruption of epaB and epaE, resulted in alteration in Epa polysaccharide content, more round cells versus oval cells with OG1RF, decreased biofilm formation, attenuation in a mouse peritonitis model, and resistance to lysis by the phage NPV-1 (known to lyse OG1RF), while mutants disrupted in orfde2 and OG1RF_163 (the epa locus flanking genes) behaved like OG1RF in those assays. Analysis of the purified Epa polysaccharide from OG1RF revealed the presence of rhamnose, glucose, galactose, GalNAc, and GlcNAc in this polysaccharide, while carbohydrate preparation from the epaB mutant did not contain rhamnose, suggesting that one or more of the glycosyl transferases encoded by the epaBCD operon are necessary to transfer rhamnose to the polysaccharide. In conclusion, the epa genes, uniformly present in E. faecalis strains and involved in biosynthesis of polysaccharide in OG1RF, are also important for OG1RF shape determination, biofilm formation, and NPV-1 replication/lysis, as well as for E. faecalis virulence in a mouse peritonitis model.Enterococci are among the major causes of endocarditis and nosocomial infections, with Enterococcus faecalis the most commonly identified species (7,22). The antibiotic resistance of enterococci not only causes difficulty in treatment of enterococcal infections but also appears to promote their survival in hospitalized, antibiotic-exposed individuals, helping to explain their important role in the nosocomial milieu.In order to understand the mechanisms of enterococcal pathogenesis, which may lead to development of alternative approaches to prevent and/or treat enterococcal infections, we and other investigators have identified several enterococcal factors important for virulence, including proteins and polysaccharides (11). Polysaccharides are composed of repeating units of oligosaccharides, are important components of bacterial cell walls, and can be associated with bacterial surfaces through linkage to the cell membrane, peptidoglycan, or by other unknown mechanisms. Polysaccharides also play important roles in bacterial pathogenesis. They have been shown to be important for adherence to and invasion of host tissues (10, 19), for resistance to host defense systems such as phagocytosis (1,2,8,12,24,30), and for induction of host inflammatory responses (14,25). Vaccine potential of surface polysaccharides has been shown for different bacteria, such as pneumococci (20), and antibodies against capsular carbohydrates have been shown to promo...