The PCR product was blunt-end cloned into the SmaI site of pWKS30 (Wang & Kushner, 1991) to form pYSCN. The ΔyscN mutant was made electrocompetent as previously described (Conchas & Carniel, 1990) and transformed with either vector alone or pYSCN. Transformants were selected by growth on LB Lennox agar with ampicillin (50 μg mL−1). The effect of growth on the parental, selleckchem ΔyscN mutant, and pLcr− strains by incubation at 37 °C with induction of the low calcium response (Straley et al., 1993) was monitored by optical density (OD620 nm). Strains were initially grown overnight at 28 °C in HI broth at 150 r.p.m. They were then diluted to an OD of approximately

0.05 in 50 mL of HI broth supplemented with either CaCl2 or MOX. The cultures were grown for 1 h at 28 °C and then elevated to 37 °C. At hourly intervals, the OD was determined. All growth curves were

performed in triplicate. Yersinia pestis strains were grown in HI broth at 28 °C for 8 h and then diluted 1/20 in HI broth containing MOX. The fresh cultures were grown for 1 h at 28 °C, switched to 37 °C, and grown overnight. The cultures were then pelleted, supernatants collected, and pellets washed. The pellets were then suspended in water with MPBio Lysing Matrix B (MP Biomedicals, Solon, Osimertinib molecular weight OH), bead beat for 40 s with a FastPrep FP120 Cell Disrupter (Thermo Fisher Scientific, Pittsburg, PA), chilled on ice, bead beat again for 40 s, microfuged for 5 m, and then filtered.

The preparation was then sterility checked by plating a portion of the sample on sheep blood agar plates. The Y. pestis supernatants and uninoculated HI broth + MOX were concentrated by passage through a centrifugal filter device (Amicon Ultra-10K; Millipore, Billerica, MA), heat fixed (95 °C for 30 min), and sterility checked as described above. The protein concentrations from the samples were determined using the BCA Protein Assay Kit (Pierce, Rockford, IL) as per the manufacturer’s recommendations. GABA Receptor Samples (approximately 1 μg in 2 μL) were spotted onto PVDF membrane. The membranes were blocked with 7% skim milk in PBS containing 0.1% Tween 20 (PBST). Monoclonal antibody to LcrV (DiMezzo et al., 2009) was used at a dilution of 1 : 5000 in PBST, and secondary rabbit anti-mouse horseradish peroxidase labeled antibody was used at a dilution of 1 : 5000. Reactions were visualized using 4-chloronaphthol/3,3′-diaminobenzidine (Thermo Fisher Scientific, Rockford, IL). The Y. pestis strains were prepared for mouse vaccinations or challenges as previously described (Anderson et al., 1996), except that the bacteria were suspended in 10 mM potassium phosphate buffered saline (KPBS) solution rather than HI broth. To demonstrate loss of virulence and complementation of the mutant, groups of 10 naïve female Swiss–Webster mice were challenged s.c. with CO92 wild type, ΔyscN, ΔyscN + pWKS30, or ΔyscN + pYSCN at 0.2 mL aliquots.