coli During a study on the role of bacterial physiological properties in the Type III secretion of Salmonella, we carried out experiments to measure the ATP levels in bacterial cells and used the culture supernatant as a negative control. Some culture supernatant samples unexpectedly displayed readily detectable signals in the ATP assay. We proceeded to determine if the ATP in the culture supernatant was due to a bacterial contamination of the culture supernatant. Salmonella cultures were grown at 37°C for 3 hours to the early Y-27632 nmr log phase or overnight to the stationary phase and the cultures were spun down. The culture supernatant from each sample was transferred

to a fresh tube and an aliquot was filtered through a 0.22 μm filter. ATP levels were determined

in both filtered and unfiltered supernatant of the same culture and results were compared. ATP was detected in the supernatant of both early log and stationary phase cultures and filtration did not reduce the ATP levels (Figure 1). The ATP level in the supernatant of the stationary phase culture was just above the detection level (at approximately 1 nM), while the ATP level in the supernatant from the early log phase culture was noticeably higher at over 10 nM (Figure 1). Figure 1 ATP is present in the bacterial culture supernatant and the extracellular ATP is not due to bacteria contamination. Overnight culture of Salmonella strain SE2472 was diluted 1:100 in LB and cultured at 37°C for 3 hours with shaking to reach Crizotinib molecular weight early log phase. The overnight (stationary) and 3 hour (early log phase) cultures were spun down. An aliquot of each culture supernatant was filtered through a 0.22 μm filter to remove any residual bacteria. ATP levels in the filtered (hatched bars) or unfiltered culture supernatant (open bars) were measured. Results are the average of 3 assays and error bars represent standard deviations. Next we tested if the extracellular ATP is only present in specific strains of Salmonella such as the clinical isolate SE2472 we used in the initial analysis.

We tested a collection of clinical strains of Salmonella serovar Enteritidis (11 isolates) and Typhimurium (17 isolates), Amino acid laboratory strains of E. coli K12 MG1655 and BW25113, and clinical strains of E. coli O157:H7 (2 isolates) (Table 1). Overnight culture of each bacterial strain was diluted 1:100 in fresh LB broth and cultured for 3 hours at 37°C with shaking. The ATP level in the culture supernatant was determined (Figure 2). The results showed that various bacterial strains displayed different levels of ATP in the culture supernatant; nevertheless extracellular ATP was detected in all isolates (Figure 2). These results raised a possibility that extracellular ATP is indeed present in the culture supernatant during growth.