Fig. 4: Applying the genetic code expansion concept in vivo in E. coli to elucidate that RutR AcK52 affects its transcriptional regulator activity.

a Acetylation mimetic mutations K52R and K52Q are imperfect to study RutR K52-acetylation. Left panel: SDS-PAGE gel shows a similar expression level of RutR wild-type and the K52Q and K52R mutants. Right panel: transcriptional reporter β-galactosidase assays in the absence and presence of rutR as well as upon ectopic expression of non-acetylated and mutated RutR. At OD₆₀₀ = 0.6 cells were analyzed by lacZ reporter assay. Experiments were performed in three biologically independent experiments (n = 3). Bars depict means ± standard deviations of determined miller units. Statistical significance (**: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001) was tested using t-tests. Source Data are provided as Source Data files. b Adjustment of similar protein levels of non-acetylated RutR and acetylated RutR AcK52. E. coli U65 ΔrutR P_carA-lacZ was transformed with pRSFDuet-1/rutR or pRSFDuet-1/rutRK52_amber, respectively. At OD₆₀₀ = 0.6 cultures were harvested and 30 μg of proteins from cell lysates were analyzed in immunoblots (IB), probed with anti-His₆-AB, and related to total protein staining with TCE. Source Data are provided as Source Data files. c Acetylation of K52 in RutR impairs β-galactosidase expression. Transcriptional reporter β-galactosidase assays were conducted upon ectopic expression of non-acetylated and acetylated RutR. E. coli U65 ΔrutR P_carA-lacZ was transformed with pRSFDuet1 empty, pRSFDuet1/rutR, or pRSFDuet-1/rutRK52_amber as indicated. Cultures were harvested at OD₆₀₀ = 0.6 and analyzed in lacZ reporter assays. Bars depict means ± standard deviations of determined miller units. Experiments were performed in three biologically independent experiments (n = 3) and statistically analyzed using t-tests (**: p ≤ 0.01; ***: p ≤ 0.001). Source Data are provided as Source Data files. d Gene expression analysis by qRT-PCR upon ectopic expression or non-acetylated and acetylated RutR. E. coli U65 ΔrutR P_carA-lacZ was transformed with pRSFDuet-1 empty vector (black bars), pRSFDuet-1/rutR (red bars), or pRSFDuet-1/rutRK52_amber (blue bars) as indicated. Significance (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001) level was tested between ΔCt values of the indicated samples and ΔCt values of the corresponding 16S-rRNA control using t-tests. Experiments were performed in three biologically independent experiments (n = 3). Source Data are provided as Source Data file.