FIGURE 5.

Regulation of RNR expression during biofilm formation. (A) GFP-based gene reporter assay of P. aeruginosa cells growing as a static biofilm. The induction factor is expressed as the quotient of the fluorescence units measured from one strain at one point in time relative to the corresponding value measured at the first time point (3 h of culture). Each strain was monitored between 3 and 72 h of biofilm growth. Wild-type RNR promoters (PnrdA, PnrdJ, and PnrdD) are represented in continuous lines, and mutant RNR promoters (PnrdJ and PnrdD carrying a mutant version of the putative Anr/Dnr-box identified) are plotted in dotted lines. A promoterless GFP in pETS130 plasmids was used as a negative control, and the PoprF promoter in pETS193 plasmids was used as a positive control for anaerobic induction of gene expression. (B) Fold change in P. aeruginosa PW1965 Δdnr PnrdA, PnrdJ, and PnrdD promoter transcription was determined through real time PCR in 4-day-old cells grown as a static biofilm and compared with transcription in 16-h-old planktonic cells, both of which were cultured under aerobic conditions. The gap gene was used as an internal standard. The results shown represent the mean of three independent experiments ± standard deviation.