Fig. 1

Enhancement of T3S and colony formation by nitrate. EHEC O157 Sakai wild-type strains were grown in LB containing various concentrations of sodium nitrate under microaerobic conditions at 37 °C. a. Growth of EHEC in the media containing various concentration of sodium nitrate (mM). Cell density was measured by OD₆₀₀ every 1 h. The representative of three independent experiments is shown. b. Secretion of EspB by EHEC. Proteins in culture supernatant (Sup) were collected from 5 h post-inoculation by TCA precipitation and dissolved in SDS-sample buffer as normalized by OD₆₀₀ of culture. Whole cell lysate (Whole) was prepared from 0.5 ml culture by collecting bacteria and dissolved in SDS-sample buffer as normalized by OD₆₀₀. EspB protein was detected by immunoblotting with anti-EspB antibody. c. Microcolony formation on HeLa cells. Bacterial culture at 3 h post-inoculation was used for the infection of HeLa cells. Microcolonies formed at 3.5 h after washing at 1.5 h post-infection were determined by staining cells with Giemsa. A colony with at least 8 bacteria was counted as a microcolony. The average of three independent experiments along with standard error is shown. *: p < 0.05