The qRT-PCR and relative transcription

of genes were analyzed as described previously (Wang et al., 2009). Brucella melitensis strains were grown overnight in TSB medium with aeration at 37 °C. For each strain, three 1-mL aliquots of cultures in TSB medium (initial OD600 nm 0.05) were incubated at 37 °C with shaking in a 24-well plate containing an insert plate with a porous membrane (diameter, 1.0 μm) (BD Falcon). After 24 h, bacteria were fixed for 20 min with 4% paraformaldehyde, and plates were centrifuged for 10 min at 1500 g. Membranes were cut and dehydrated for 5 min in 25%, 50%, 75%, 95% and 100% ethanol at room temperature. They were finally prepared by critical-point drying, mounted on an aluminum stub and covered with a thin layer of gold (20–30 nm). Examination JQ1 purchase was carried out with a scanning electron microscope (Hitachi S450). Exopolysaccharide was stained as follows: bacteria in a middle logarithmic-phase culture (OD600 nm 1.0) were fixed with 4% paraformaldehyde for 20 min before staining. For detection of polysaccharides, 1 mL of 0.05% calcofluor white (fluorescent whitener 28; Sigma) was added to 0.1 mL of paraformaldehyde-fixed cells. Visualization was accomplished using an epifluorescence microscope (Olympus

IX71). The susceptibilities of Brucella strains to polymyxin B (Sigma) were determined hypoxia-inducible factor pathway following a protocol described previously (Martinez de Tejada et al., 1995) with modifications. Brucella melitensis strains were cultured for 72 h on TSA. Then, bacterial suspensions of approximately 1 × 104 CFU mL−1 were prepared in phosphate-buffered saline and 100-μL aliquots were mixed with different concentrations of DNA ligase 100 μL polymyxin B (the final concentrations in the wells were 2000, 1000, 500 and 250 μg mL−1, respectively) and cultured in 96-well plates. After a 1-h incubation at 37 °C in a 5% CO2 atmosphere, a 50-μL aliquot of each well was serially diluted and spread in triplicate on TSA plates for CFU

determination. The results were expressed as the mean±SD of three assays. All the results represent the averages from at least three separate experiments. The sensitivity of Brucella strains to hydrogen peroxide, high-salinity or high-osmolarity stresses was determined as follows: B. melitensis strains inoculated into TSB medium were grown to the early logarithmic phase (OD600 nm 0.6) at 37 °C. To determine the effect of high-salinity or high-osmolarity stress on B. melitensis, the log-phase cells were incubated at 37 °C for 20 min in the presence of NaCl (1.5 M) or sorbitol (1.5 M). To test the effect of oxidative stress, the cells were incubated for 30 min in 440 mM H2O2. After the treatment, the survival percent of the bacteria was determined as above. All the results represent the averages from at least three separate experiments. Previously, we compared proteome differences between BM and BMΔvirB in GEM4, a which strongly induces virB.