(A) and (B) Live or heat – killed E. coli K12 (A) or Salmonella SE2472 (B) were spun down and incubated JQ1 purchase at 37°C in fresh LB supplemented with 10 μM ATP. Culture supernatant from live bacteria was supplemented with ATP to 10 μM. ATP depletion by bacteria cells or culture supernatant was measured by the residual ATP level in culture medium after various culture periods of incubation at 37°C. The residual ATP levels were plotted against the incubation period. (C) and (D)
Free and cell-associated ATP in E. coli (C) or Salmonella (D) culture incubated with S35-α-ATP or P32-γ-ATP. The relative levels of radioactivity in culture supernatant and bacterial cells were determined and plotted against the incubation period. Each experiment was performed BGB324 manufacturer three times and results are from a representative experiment. Since bacterial cells instead of culture supernatant deplete ATP (Figure 5A and B), we reasoned that the reduction of ATP level in the culture supernatant could be due to hydrolysis or degradation of ATP at the bacterial cell surface. Alternatively, ATP level can become lower due to an uptake by bacteria although no ATP transporter or uptake system has been reported in bacteria. To explore the fate of the extracellular ATP, we incubated bacteria with 35S -α-ATP and quantified the radioactivity in the culture supernatant and bacterial pellet. ATP transported back into bacteria should be detected
by cell-associated radioactivity whether it remains as ATP or is hydrolyzed subsequently into ADP or AMP. Stationary
phase cultures of Salmonella and E. coli were spun down and resuspended in fresh LB broth supplemented with 32S-α-ATP. After various periods of incubation, bacteria were spun down, washed, and selleck compound the radioactivity was measured in the culture supernatant or in the bacterial cell pellet. Virtually all radioactivity remained in the culture supernatant and very little radioactivity was detected in bacterial cell pellet of Salmonella or E. coli (Figure 5C and D). We next tested if the extracellular ATP was used in kinase reactions to phosphorylate proteins and other cell surface components. ATP depletion assay was carried out using 32P -γ-ATP as described above for 32S-α-ATP. Quantitation of radioactivity in the culture supernatant and bacterial pellet showed that radioactivity was present almost exclusively in the culture supernatant (Figure 5C and D). This suggests that ATP was most likely hydrolyzed or degraded by bacteria on their surface and was not transported into bacteria or used for phosphorylating bacterial components. Extracellular ATP enhanced stationary survival of E. coli and Salmonella The presence of the extracellular ATP in bacterial cultures was unexpected since it likely represents a loss of the valuable small molecule to bacteria. The extracellular ATP could be an unavoidable cost to bacterial respiration or could be beneficial to bacteria in some aspects.