Fig. 9: Acetylation in the RutR N-terminus modulates its transcriptional regulator activity in vivo.

E. coli U65 ΔrutR with a genomic P_carA-lacZ fusion was transformed with pRSFDuet-1 empty vector, pRSFDuet-1/rutR, pRSFDuet-1/rutR K7Q, K7R, K11Q, K11R and the double mutants K7Q/K11Q and K7R/K11R as indicated. Cultures were harvested at OD₆₀₀ of app. 0.6 and analyzed in a lacZ reporter assay. Left panel: Cell lysates of the E. coli U65 cells expressing RutR wild-type and the mutants were analyzed by immunoblotting (IB) using an anti-His₆-antibody (IB: αHis). Neither RutR Δ1-12 nor RutR Δ1-12 K52Q were expressed. For the single mutants (red bars) and the double mutants (blue bars) of the N-terminal lysines we observed a reduced expression even when normalizing for the protein loading and/or different optical densities of cultures. To this end, we perform statistical analyzes only between samples of similar expression, i.e. single mutants to each other and double mutants to each other. Positive control for IB: Sirt1_225-664-His₆; negative control: CobB. TCE staining served as loading control. Right panel: transcriptional reporter β-galactosidase assays in the absence (empty vector) and presence of rutR upon ectopic expression of RutR-WT and mutated RutR. As expected, RutR K52Q results in a statistically significant reduction in β-galactosidase activity compared to RutR WT and the empty vecor control (black bars). Comparing the single mutants (red bars) shows no statistically relevant alterations in β-galactosidase activity. Comparison of double mutated K7Q/K11Q with K7R/K11R (blue bars), which show a similar expression level, shows that the double Q acetylation mimicking mutant shows a statistically significant reduction in β-galactosidase activity compared to the double R-mutant. All RutR K to R mutants did not impair β-galactosidase expression suggesting that at K7 and K11 electrostatic quenching is the major molecular mechanism by which acetylation impairs DNA-binding. Experiments were performed in three biologically independent experiments and bars depict means ± standard deviations of determined relative β-galactosidase activity in miller units (n = 3). Statistical significance (*: p ≤ 0.05; ***: p ≤ 0.001; ****: p ≤ 0.0001) was tested using t-tests. Source data are provided as Source Data file.