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. 2013 May;81(5):1696–1708. doi: 10.1128/IAI.01210-12

Fig 5.

Fig 5

E. faecalis OG1RF ahrC expression at the RNA and protein levels. (A) Gene expression in batch culture. Aliquots of OG1RF cells growing in BHI were collected at the indicated time points and processed for quantitation by serial dilution and plating or for RNA extraction. Cell counts (circles connected by dashed line) correspond to the left y axis. cDNA copy numbers per μl for ahrC (squares) and gelE (triangles), included as a control for a gene known to be expressed in the stationary phase after cells reach an fsr-dependent quorum, correspond with values on the right y axis. Symbols are the mean of n = 3 biological replicates. Error bars represent standard deviations (SD). (B) Gene expression in biofilms. OG1RF planktonic and biofilm cells grown in plastic dishes in TSB-dex were harvested for quantitation and RNA extraction after growth for 6 or 22 h. Values are the means ± SD of n = 5 to 6 biological replicates collected on 2 separate days. Relative expression of ahrC and gelE was calculated by dividing the copies of detected transcript per μl of cDNA by the corresponding CFU/ml count from the harvested biofilm cells. b.d., below detectable limit of the qPCR assay. (C) Protein expression in early exponential and stationary phases. Whole-cell lysates of exponential (E)- or stationary (S)-phase cells were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-AhrC polyclonal antisera. His-tagged AhrC is approximately 18 kDa. The upper band is a cross-reactive protein of unknown identity that was used as a loading control in densitometry measurements, which revealed that the relative amount of AhrC detected in stationary-phase OG1RF lysates was 25% less than the amount detected in exponential-phase lysates. Due to the strong overexpression of AhrC in the ΩahrC-p23ahrCKI strain (labeled “ΩahrC-KI” on the figure for clarity), a shorter exposure of the same blot is shown in the bottom image. The top image was adjusted for brightness and contrast after densitometry in order to make the loading control bands visible for publication.