Accurate quantification of site-specific acetylation stoichiometry reveals the impact of sirtuin deacetylase CobB on the E. coli acetylome

We applied SD-SILAC to determine absolute acetylation stoichiometry in exponentially-growing and stationary-phase wild type and sirtuin deacetylase CobB-deficient E. coli. We further assayed stoichiometry in phophotransacetylase (pta) and acetate kinase (ackA) mutant strains in the presence and absence of the sirtuin inhibitor nicotinamide. Comparison to relative quantification under the same conditions validated our stoichiometry estimates at hundreds of sites, demonstrating the accuracy of our method. We measured acetylation stoichiometry at 3,669 unique sites and found that the vast majority of acetylation occurs at very low stoichiometry (median 0.04%). Manipulations that cause increased nonenzymatic acetylation by acetyl-phosphate (AcP), such stationary-phase arrest and deletion of ackA, resulted in globally increased acetylation stoichiometry. Similar to sirtuin deacetylase 3 (SIRT3) in mitochondria, CobB suppressed acetylation to lower than median stoichiometry at hundreds of sites in WT, pta, and ackA cells. These results suggest an evolutionarily conserved function of sirtuin deacetylases in suppressing low stoichiometry acetylation.