5) Five hundred microliters of each donor culture was mixed with

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.

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