Cyclic-AMP (cAMP) assays

Intracellular cAMP levels of EHEC grown under aerobic or anaerobic conditions were determined by using Cyclic AMP EIA Kit (Cayman Chemical, Ann Arbor, MI). Bacteria were grown in 20 ml of LB medium to late logarithmic phase and harvested. The cell pellets were once washed in phosphate buffered saline (PBS) and suspended in 0.5 ml of EIA buffer supplied in the kit, then lysed by sonication. The lysate was centrifuged and the amount of cAMP in the resulting supernatant was quantified using cAMP AChE Tracer, cAMP EIA Antiserum and Ellman's reagent according to the manufacturer protocol. cAMP levels in fraction of the cell lysate were represented as pmol per cells producing 1 μg of protein.