Figure 4.

Effect of NO on Stx2 production in wild and hmpA‐deficient mutant EHEC strains under aerobic conditions. The deleted norV‐type wild EDL933 and the deleted norV‐type hmpA‐deficient EH grown overnight were diluted with LB broth containing various concentrations of DETA‐NONOate (DETA/NO) and grown statically for 18 hr at 37°C under aerobic conditions. (a) The optical density at 600 nm (OD ₆₀₀) was determined. NO level in the deleted norV‐type wild EDL933 in medium. EHEC strains harboring the NO reporter plasmid pRPL3 were cultured in LB medium containing a various concentrations of DETA/NO for 18 hr at 37°C under aerobic 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. *p < 0.01. (b) EHEC strains were fractionated into the supernatant fraction and cell‐associated fractions. They were then analyzed by Immunoblot analysis using anti‐Stx2 antiserum and anti‐RNA α antibody, respectively. The relative amounts of Stx2 were quantified by densitometry and normalized to internal control RNA α. The inductions of hmpA in wild‐type EHEC were analyzed by real‐time qRT‐PCR. Data are the means ± standard deviations of values from five experiments. *p < 0.01