Fig. 10: The NAD⁺-dependent sirtuin CobB deactylates RutR at all functionally important sites but with different efficiency (a) and model for regulation of RutR function by lysine acetylation (b).

a CobB deacetylases RutR at all functionally important sites: AcK7, AcK11, AcK7/11 (right panel), AcK52, AcK62 (left panels). CobB also catalyzes deacetylation of RutR AcK21 within the basic patch (19-KKK-21). Immunoblotting (IB) using anti-AcK-AB (IB: AcK) was used to detect RutR deacetylation. TCE staining served as loading control. 10 mM NA is sufficient to inhibit CobB-catalyzed deacetylation at RutR AcK21, AcK52, and AcK62 but 50 mM NA is needed to inhibit deacetylation at AcK7, AcK11, and AcK7/11 (Supplementary Fig. 9A). One exemplary out of two biological replicates is shown (n = 2). Source data are provided as Source Data file. b Time-course experiment showing efficient deacetylation of RutR AcK7, AcK11 and double-acetylated AcK7/11 and less efficient deacetylation at AcK52. After 2 min (red bars), RutR is more than 25% deacetylated at AcK7, AcK11, and at AcK7/11, while after 120 min AcK52 is 50% deacetylated (blue bars) compared to the sample at 0 min (black bars). The experiment was performed in three/four biologically independent experiments for RutR AcK7/RutR AcK52 (n = 3)/RutR AcK11/RutR AcK7/11 (n = 4). Bars depict means ± standard deviations (n ≥ 3). Source data are provided as Source Data files. c We propose a model after which RutR is enzymatically lysine-acetylated in the unstructured N-terminal tail at K7 and K11 by the KATs PatZ/YfiQ and YiaC (1). Acetylation in the RutR N-terminal tail modulates the association and dissociation to dsDNA. Acetylation in the N-terminal tail can be efficiently reversed by CobB. The positively-charged N-terminal tail of RutR allows an electrostatic steering to the negatively-charged sugar-phosphate backbone of dsDNA. Accumulation of acetyl-phosphate results in non-enzymatic acetylation of RutR at K7, K11, and at K52 (2). K52-and K62-acetylation abrogates binding to dsDNA and can also be reversed by CobB. (3) RutR DNA-binding can be modulated by uracil (Ura) binding to the LBD. Switching-off RutR DNA-binding by lysine acetylation contributes to the activation of transcription of the rutAG operon. RutR acetylation at K52 and K7/K11 indirectly impairs the transcription of the carAB operon. Overall, the RutR lysine acetylation state is regulated enzymatically and non-enzymatically. This enables the adaptation of RutR transcriptional regulator activity to the cellular metabolic state.