5). Five hundred microliters of each donor culture was mixed with the same volume of recipient and then centrifuged at 16 000 g for 1 min. The bacterial pellet was spread on a BHI plate at 30 °C for 2 h. Cells on the BHI plates were harvested using a loop and resuspended in 2.5 mL of LDK378 price BHI, and then 50 and 100 μL were plated on BHI plates containing 200 μg mL−1 of streptomycin and 7.5 μg mL−1 of chloramphenicol. The plates were
incubated at 30 °C overnight and then at 37 °C. After conjugation, deletion of sigB in the L. monocytogenesΔsigB mutant was confirmed by PCR. Listeria monocytogenes strains carrying the reporter gene fusion were grown to the mid-exponential growth phase in BHI broth at 180 r.p.m. and 37 °C, followed by a 1 : 25 dilution into fresh BHI broth. To induce cell wall stress, vancomycin (final concentration of 2 μg mL−1) was added during the early exponential growth phase (OD600 nm=0.3). β-Galactosidase assays were performed as described by Miller (1972). All samples were XL765 concentration collected at the indicated times by centrifugation for 1 min at 16 000 g at room temperature. Cells were then washed with Z buffer (16.1 g of Na2HPO4·7H2O, 5.5 g of NaH2PO4·H2O, 0.75 g of KCl and 0.246 g of MgSO4·7H2O,
L−1). Permeabilization was performed using SDS and chloroform, followed by vigorous vortexing for 30 s and incubation at 37 °C with o-nitrophenyl β-d-galactopyranoside as a substrate. The reaction was stopped by the addition
of 0.5 mL of 1 M Na2CO3, after which samples were centrifuged to remove cellular interference. Absorbances were then read at 420 nm and protein levels were determined using Bio-Rad protein assay reagent Unoprostone (Bio-Rad, Hercules, CA). Specific activity was defined as ΔA420 nm× 1000 min−1 mg−1 of protein. Cells were harvested 40 min after vancomycin (final concentration of 2 μg mL−1) treatment by centrifugation at 3500 g for 5 min. Cells were washed twice with phosphate-buffered saline (pH 7) solution. Pellets were suspended in 2 mL of disintegration buffer [7.8 g of NaH2PO4, 7.1 g of Na2HPO4, 0.247 g of MgSO4·7H2O and protease inhibitor mix (Amersham Biosciences, Piscataway, NJ)], followed by sonication on ice for 5 min at 1-min intervals. Unbroken cells were separated by centrifugation at 3500 g for 10 min. The supernatant was collected and the protein concentration was measured using the Bio-Rad protein assay reagent (Bio-Rad). Coomassie-blue staining and in-gel tryptic digestion were performed as reported previously (Park et al., 2009). Briefly, protein bands were excised from coomassie-stained gels and destained by incubation in 75 mM ammonium bicarbonate/40% ethanol (1 : 1). Disulfide bonds were reduced by 5 mM dithiothreitol/25 mM ammonium bicarbonate, followed by alkylation with 55 mM iodoacetamide at room temperature for 30 min.