Fig. 7: RutR is enzymatically acetylated by PatZ/YfiQ and YiaC in the histone-like N-terminus and non-enzymatically by acetyl-phosphate.

a Identification of PatZ/YfiQ and YiaC as RutR KATs. Purified RutR was incubated with the KAT enzymes (active/inactive) in presence/absence of acetyl-coenzyme A (Ac-CoA). The samples were analyzed by immunoblotting with anti-AcK AB (IB: AcK). Total protein staining using 2,2,2-trichloroethanol (TCE) was used as loading control. All KATs, except YiaC show an autoacetyltransferase activity (Supplementary Fig. 5a). The KATs PatZ/YfiQ (left panels) and YiaC (right panels) lysine-acetylate RutR. Inactive KATs: PatZ/YfiQ E909A, YiaC Y115A. The results were confirmed in at least three replicates (n ≥ 3). Long exposure served to visualize weak signals (red: oversaturation of signal). Source data are provided as Source Data files. b K7 and K11 in the RutR N-terminal are acetylated enzymatically by KATs PatZ/YfiQ and YiaC as shown by LC-MS/MS. Both can simultaneously acetylate RutR at K7 and K11. Shown are the calculated overall intensities summed up from single peptides. Presented are the means ± standard deviations from three biologically independent experiments (n = 3). a.u.: arbitrary units. black: no acetyl-CoA; orange: no KAT; blue: PatZ/YfiQ; black/grey-shaded: PhnO; pink: RimI; red: YiaC; green: YjaB (Supplementary Data 1). Source data are provided as Source Data files. c PatZ/YfiQ and YiaC act as KATs acetylating RutR exclusively in the N-terminal tail. RutR Δ1-12 is not acetylated by PatZ/YfiQ or YiaC (long exposure) suggesting that the acetylation sites reside in the N-terminal region in RutR. Immunoblotting (IB) was conducted with anti-AcK-AB and total protein staining using TCE served as loading control. One exemplary result is shown from three independent replicates (n = 3). Red indicates oversaturation of signal. Source data are provided as Source Data files. d RutR is non-enzymatically lysine-acetylated by acetyl-phosphate (AcP) conducted at different pH-values and time ranges. All samples were analyzed in immunoblots (IB) stained with anti-AcK AB. TCE staining served as loading control. LC-MS/MS analyzes shows that RutR is acetylated non-enzymatically by AcP at K7, K11, the triple K-motif (19-KKK-21), K52, K95 and K150 (Supplementary Data 2; Supplementary Table 5; Supplementary Fig. 6). One exemplary result out of two replicates is shown (n = 2). Red indicates oversaturation of signal. Source data are provided as Source Data files.