, 2001), and comX (∆SMcomX) (Li et al., 2002) were used in this study. The comS (∆SMcomS) and the comR (∆SMcomR) mutants were constructed using a nonpolar ligation

PCR mutagenesis method described previously (Lau et al., 2002). Streptococcus mutans strains were grown at 37 °C with 5% CO2 in either Todd-Hewitt broth (Becton Dickinson, MD) containing 0.3% yeast extract (Difco Laboratories) (THYE), or chemically defined medium (CDM) described previously (Mashburn-Warren et al., 2010). Erythromycin Regorafenib price and spectinomycin were used as needed at concentrations of 10 μg mL−1 and 1 mg mL−1, respectively. sXIP and synthetic CSP (sCSP) peptides were synthesized using F-MOC chemistry (Advanced Protein Technology Centre, Hospital for Sick Kids, Toronto, ON, Canada). Stock concentrations of 1 μM of sXIP and 0.4 mM sCSP were prepared

in DMSO and water, respectively. Growth kinetics were monitored using an automated growth reader (Bioscreen C; Labsystems, Finland) as previously described (Senadheera et al., 2007). Overnight cells grown in THYE were pelleted, washed, and resuspended in phosphate buffered saline (1× PBS). The resuspended culture was diluted 1 : 50 using prewarmed THYE or CDM and grown to an OD600 ~ 0.1. Next, 1 μg mL−1 of the donor plasmid DNA (pDL277; specR) (LeBlanc et al., 1992) was added to 1-mL aliquots of the culture in the presence or absence of CSP (0.4 μM) or XIP (10 μM), and samples were incubated for 90 min. For XIP, control cultures containing 1% DMSO GW-572016 molecular weight were utilized. After incubation, cultures were serially diluted and plated on THYE plates with and without antibiotics. TF was calculated as transformant colony-forming units (CFUs) divided by the total number of viable CFUs, times 100. Overnight cultures in THYE were pelleted, washed, resuspended in sterile 1× PBS, and diluted 1 : 50 using warm THYE or CDM. Each suspension was supplemented with either 2 μM CSP or 10 μM XIP. Cultures without peptides Megestrol Acetate or containing 1% DMSO

were used as controls. All cultures were grown to an OD600 ~ 0.8, at which point, the cells were gently sonicated on ice and used for viability assays. Cells were serially diluted, plated on THYE agar, and CFUs counted. Results standardized using cellular dry weight. These standardized values were then used to calculate the percentage survival by dividing the standardized number of viable cells after treatment by the standardized total number of cells without peptide, times 100. Overnight cultures of UA159 in THYE were pelleted, washed, resuspended in sterile 1× PBS, and diluted 1 : 20 using warm CDM. The subcultures were allowed to grow to an OD600 of 0.4, after which they were split into two where one was exposed to 1% DMSO and the other to 10 μM XIP. Cultures were further incubated, and samples were taken at varying time points (0, 1, 2, 3, 4, and 5 h) after exposure to XIP, gently sonicated, serially diluted, and plated on THYE plates for CFU determination.