For high-temperature stress experiments, log-phase cells were transferred to pre-warmed 50°C tubes and incubated at 50°C for 5 min. For low pH stress experiments, log-phase cells were incubated at

37°C in TMH medium adjusted by adding 2 M HCl to pH 3.0 for 10 min. To test the effect of oxidative stress, the cells were incubated for 10 min in 220 mM H2O2. The bacterial viable count after exposure to the appropriate stresses was determined by pelleting the appropriate dilutions on the BHI agar plates, which were then ACY-1215 incubated at 26°C for 36 h. Macrophage infection assay J774A.1 mouse macrophage cells (6 × 105) were seeded in 24-well tissue culture plates (0.5 ml/well) and maintained in the minimum essential medium (MEM) containing the modified Eagle’s medium (Invitrogen) supplemented selleck with 10% heat-inactivated fetal bovine serum,

2 mM L-glutamine until confluence was achieved at 37°C under 5% CO2. WT and ΔompR were grown in TMH as described above. The cultures were collected and suspended in the MEM medium and then respectively added to cell monolayers in 24-well tissue culture plates at a multiplicity of infection generally of 20:1 (bacteria to macrophages). After incubation at 37°C for 1 h to permit phagocytosis, 6 wells of infected cell monolayers were washed thrice with 1× phosphate-buffered saline (PBS). Afterwards, the number of total macrophage cell-associated bacteria was determined. Cell-associated bacteria were determined by harvesting in 0.5 ml of 0.1% Triton X-100 in 1× PBS. After 10 min, infected cell lysates were collected serially and diluted 10-fold

in PBS; on the other hand, viable bacterial CFU was determined as described above. A second set of 6 infected monolayer wells were washed twice with 1× PBS. MEM medium supplemented with 200 μg/ml gentamicin (Invitrogen) was added to these wells for 1 h to kill extracellular bacteria. The infected monolayers were then lysed and treated as described above to determine the number of intracellular bacteria. Each experiment was repeated three or four times on VE-822 datasheet different days, and each bacteria sample was used to infect at least four wells of macrophage monolayers. Results Non-polar mutation of ompR Given that the coding regions of ompR and envZ overlap in the ompB operon, a partial segment of the coding region of ompR was replaced by the kanamycin Gefitinib manufacturer resistance cassette to generate the ompR mutant (ΔompR). Real-time RT-PCR was performed to assess the ompR mRNA levels in WT, ΔompR, and C-ompR (the complemented mutant). The ompR transcript was lacking in ΔompR, while it was restored in C-ompR relative to WT (data not shown), indicating successful mutation and complementation. To prove the non-polar mutation of ompR, we constructed the pRW50-harboring fusion promoter consisting of a promoter-proximal region of ompF and promoterless lacZ, and then transformed into WT, ΔompR and C-ompR, respectively (Additional file 2).