Biofilms of BP1470, BP1432, BP1462, BP1531, and BP1532 were grown in flow cells and subjected to fluorescence microscopy. Four time points were selected for each strain; these are printed on top of the respective images. #JQEZ5 randurls[1|1|,|CHEM1|]# At the very top of each column, promoter names are printed. Images were taken at 1,000 fold magnification. The images from Figure 1 were converted into quantitative data by calculating the percent area of the images that were fluorescent. The resulting

expression profile for flhD showed a peak at 12 h (Figure 2A, yellow line, blue triangles). Fluorescence was lowest at 35 h and increased again towards 51 h. We also noticed a small single point peak at 3 h, which is in agreement with the occasional high fluorescence of small

numbers of individual bacteria that was visualized on the images (Figure 1). Since fluorescence from the green fluorescence protein reporter is indicative of flhD expression, we conclude that flhD expression was highest at 12 h, lowest at 35 h, and increased again towards 51 h. Figure 2 Temporal expression of flhD, ompR, rcsB in AJW678 and flhD in the ompR and rcsB mutant strains. A. Fluorescence was quantified as percent area of the images that were fluorescent, averages and standard deviations were determined. The x-axis indicates the time (hours) of biofilm formation. The y-axis indicates the total fluorescence intensity in percent area for the different strains at the different time points. The yellow, black, and blue lines are showing the gene expression profile of BP1470 (AJW678 flhD::gfp), BP1432 (AJW678 ompR::gfp), and BP1462 (AJW678 selleckchem rcsB::gfp), respectively. The red line is the temporal expression profile Florfenicol of BP1531 (flhD::gfp ompR::Tn10), the orange line that of BP1532 (flhD::gfp rcsB::Tn5). The purple line is our housekeeping strain BP1437 which contains the aceK::gfp fusion plasmid. B. Confidence bands were calculated using the loess procedure. Upper and lower lines of each colors are indicating

the highest and the lowest level of the total fluorescence intensity. The color code is identical to A. The temporal expression of ompR, but not rcsB, correlated inversely with that of flhD Expression of the negative regulator of flhD expression, OmpR, exhibited a temporal profile (Figure 1, second column from the left and Figure 2A, black line, blue circles) that was almost the inverse of flhD expression between 21 h and 51 h of biofilm formation. Specifically, ompR expression increased between 21 h and 34 h, while flhD expression decreased. Between 34 and 51 h, ompR expression decreased, while flhD expression increased. Expression of another negative regulator of flhD expression, RcsB, did not correlate with the temporal expression profile for flhD (Figure 1, center column and Figure 2A, blue line, blue diamond’s).