0) or a low (5.5) pH. The bacteria grew at pH 9.0, but biofilm formation was abrogated, especially

in the presence of 5% serum. Interestingly, at pH 5.5, biofilm formation was significantly greater in TSB+5% serum than at a neutral pH. EAP was still required for biofilm formation at pH 5.5, but Nptase was not required (Fig. 2). This effect may be due to alterations in the charge properties of extracellular proteins and a subsequent alleviation of the requirement for Nptase to anchor EAP to the bacterial cell surface. To confirm the role for EAP and Nptase in biofilm formation in the presence of serum, we transduced the eap and nptase deletion mutations to an additional Protein Tyrosine Kinase inhibitor strain of S. aureus, 10833, and complemented the mutations in trans by cloning the genes into an IPTG-inducible plasmid. One millimolar of IPTG was sufficient to restore

biofilm formation in the presence of serum (Supporting Information, Fig. S1), and this concentration complemented the expression of the genes as demonstrated by RT-PCR (Fig. 3) and by phosphatase assay (Fig. S2). Strain 10833 was a weaker biofilm former Ku-0059436 supplier than SA113, but, nonetheless, the deletion mutations had a significant effect on biofilm-forming activity in the presence of 5% serum (Fig. 4). While the eap and nptase deletion mutants were defective for biofilm formation in TSB containing 5% serum, complementation of the genes restored the phenotype, confirming that the eap and nptase mutations were responsible for the effect (Fig. 4). The finding that EAP only played a role in the presence of serum suggested that serum proteins such as fibronectin and fibrinogen, which have been shown to bind to EAP (Palma et al., 1999), could contribute to the formation of a biofilm on polystyrene. The role for Nptase in biofilm formation is likely due to its ability to dock EAP to the bacterial cell surface. In sum, these results indicate Ceramide glucosyltransferase that EAP and Nptase contribute to biofilm formation in the presence of 5% human serum. The effect of serum suggests a role for EAP not only for

aggregation and adherence to host tissues in vivo but also for biofilm formation during infection as well. Intravenous catheters and other inserted synthetic medical devices are exposed to blood components and extracellular matrix proteins that are recognized by EAP. Therefore, EAP may play an important role in the formation of a biofilm on these surfaces. The pH of the growth medium played a role as well, in that low pH augmented biofilm formation in the presence of serum and alleviated the requirement for Nptase. While pH 5.5 is not physiologically relevant, this finding suggests that the charge properties of extracellular bacterial proteins and possibly of serum proteins are important in the process of EAP-mediated biofilm formation.