Figure 5.

Role of intact NorV in NO‐mediated anaerobic growth inhibition, NO level in bacterial cells and Shiga toxin production in EHEC under anaerobic conditions. (a) Gene structure of the norV in the deleted norV‐type wild EHEC EDL933 and the intact norV‐replacement mutant EHEC EVm. (b) Comparison of NO‐mediated anaerobic growth inhibition between the deleted norV‐type EDL933 and the intact norV‐type EVm. EHEC strains were cultured in LB medium containing various concentrations of DETA‐NONOate (DETA/NO) for 18 hr at 37°C under anaerobic conditions. The optical density at 600 nm (OD ₆₀₀) was measured. Data are the means ± standard deviations of values from five experiments. *p < 0.01. (c) Comparison of the NO level in bacterial cells in medium between the deleted norV‐type EDL933 and the intact norV‐type EVm. EHEC strains harboring the NO reporter plasmid pRPL3 were cultured in LB medium containing various concentrations (200 or 400 μmol/L for EDL933; 200, 400 or 800 μmol/L for EVm) of DETA/NO for 18 hr at 37°C under anaerobic conditions. Relative light units (RLU) and the number of bacteria were measured by a luminometer and bacteria plate counts (cfu), respectively. Data are the means ± standard deviations of values from three experiments. (d, e) NO level in the deleted norV‐type EDL933 within macrophages and the concentration of the NO metabolite NO ⁻ ₂ in the culture medium of infected macrophages. The EHEC strain harboring the NO reporter plasmid pRPL3 was added to the monolayer of RAW264.7 cells, and incubated for 20 min (0 hr). The medium was changed to include 100 μg/ml gentamicin. After 2 hr, the cells were washed, and the medium was changed to include 12 μg/ml gentamicin with (black) or without (white) 4 mM NOS inhibitor, L‐NMMA. The infected monolayers were either lysed or further incubated. The RLU and number of surviving bacteria were determined by luminometry and bacteria plate counts (cfu) (d). The concentrations of NO metabolite NO ⁻ ₂ in medium were determined by Griess assay (e). Data are the means ± standard deviations of values from three experiments. *p < 0.01. (f) Comparison of NO‐enhanced Shiga toxin production between the deleted norV‐type EDL933 and the intact norV‐type EVm. EHEC strains were cultured in LB medium containing various concentrations of DETA/NO for 18 hr at 37°C under anaerobic conditions. EHEC strains were fractionated into culture supernatant fractions and cell‐associated fractions. A 0.2 μg sample of each protein of the cell‐associated fraction was analyzed by Immunoblot analysis using anti‐Stx1 antiserum and anti‐RNA α antibody as an internal control. Each volume, which corresponds to 0.2 μg of protein of the cell‐associated fraction, of the supernatant fraction was analyzed by Immunoblot analysis using anti‐Stx2 antiserum.