1 M NaHCO3). OG1RF containing P ebpR ::lacZ (triangle) or P ebpA ::lacZ (square) was grown in air (closed black symbol) or in the presence of 5% CO2/0.1 M NaHCO3 (open orange symbol). B. The ΔebpR mutant containing P ebpR ::lacZ is represented by closed green diamond when grown in air and with open brown diamond when grown in the presence of 5% CO2/0.1 M NaHCO3. To determine whether the selleck chemicals CO2/NaHCO3 effect on ebpA expression was dependent on the presence of ebpR, we tested ebpA expression in an ebpR deletion mutant (TX5514). Using the ebpR deletion mutant (TX5514) containing P ebpA ::lacZ, β-gal production was assessed in air and in the presence

of 5% CO2/0.1 M NaHCO3 and β-gal production remained at the background level in both conditions (Fig. 2B). These results combined with our previously published results [11] indicate that, in air as well as in the presence of 5% CO2/0.1 M NaHCO3, ebpR is important for ebpA expression and that the 5% CO2/0.1 M NaHCO3 effect on ebpA expression level also requires the presence of ebpR. We previously reported that only a fraction of the OG1RF cells were positive for pilus expression by immunofluorescence ([11]). To examine whether the presence of CO2/NaHCO3 affected the amount of pili per cell or the percentage of cells positive for pilus production, we

used flow cytometry. As early as entry into stationary growth phase, a difference in the percentage of pilus positive cell was visible (Fig. 3A) with 53% positive SNS-032 when grown in air Selleck SU5416 compared to 87% positive

when click here grown in the presence of CO2/NaHCO3. The difference in the percentage of positive cells remained in later stages of growth. Specifically, Fig. 3B shows that, at 6 hr, 76% of the cells were positive when grown in air compared to 99% when the cells were grown in the presence of CO2/NaHCO3. The mean fluorescence intensity, between growth conditions and growth phases, remained constant with an average of 268. We also used anti-EbpC antibodies to probe mutanolysin extracts spotted on a dot blot for pilus production. An approximately four-fold increased signal density was observed in cells grown in the presence of CO2/NaHCO3 compared to the cells grown in air (Fig. 3C). Additionally, no signal was detectable under either growth condition in the mutant lacking ebpR, confirming the importance of ebpR for ebpABC expression and pilus production aerobically as well as in the presence of 5% CO2/0.1 M NaHCO3. Figure 3 Detection of EbpC produced by OG1RF, Δ fsrB , and Δ ebpR . A. Flow cytometry analysis of OG1RF grown in air (black) or in the presence of 5% CO2/0.1 M NaHCO3 (green) labeled with an anti-EbpC rabbit polyclonal immune serum and detected with phycoerythrin. The cells were collected at “”T4″”, which corresponds to the entry into stationary growth phase (4 hrs after starting the culture). The percentages between brackets indicate the percentage of positive cells (WinMDI 2.