Volume 51, Issue 2, Pages 265-272 (July 2013) Acetyl-Phosphate Is a Critical Determinant of Lysine Acetylation in E. coli  Brian T. Weinert, Vytautas Iesmantavicius, Sebastian A. Wagner, Christian Schölz, Bertil Gummesson, Petra Beli, Thomas Nyström, Chunaram Choudhary  Molecular Cell  Volume 51, Issue 2, Pages 265-272 (July 2013) DOI: 10.1016/j.molcel.2013.06.003 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 E. coli Acetylation Is Globally Affected by Acetyl-Phosphate Formed through Glycolysis or Acetate Assimilation (A) Model of the box plot representation of a SILAC ratio distribution. (B) SILAC ratios for acetylated proteins (ac-K proteins), acetylation sites (ac-K sites), acetylation sites normalized for differences in protein abundance (ac-K sites∗), and unmodified corresponding peptides (CPs) are shown. Number of quantified proteins, sites, or peptides is indicated in parenthesis. See also Figure S2. (C) Anti-acetyllysine immunoblot of EP BL21 cells (EP), growth-arrested (GA) cells without glucose (GA -G), growth-arrested cells with glucose (GA +G), and cells grown with 0.2% 2-deoxy-D-glucose (GA +2DG). (D) Increasing concentrations of acetate (0.25%, 1.0%, and 2.5%) were added to an early EP culture of BL21 cells in lysogeny broth (LB) media and analyzed by anti-acetyllysine immunoblot at the indicated times after the addition of acetate. (E) Model depicting the formation of acetate from glycolysis; key enzymes involved in the interconversion of acetyl-CoA and acetate are shown in red. (F) Acetylation induced by growth arrest with glucose (GA +G) or without glucose (GA −G) is compared by anti-acetyllysine immunoblot for wild-type (WT) and the indicated mutant strains. (G) WT EP and mutant E. coli strains (grown on glucose-containing media) were growth arrested in the presence of 1% acetate, and acetylation was analyzed by anti-acetyllysine immunoblot at the indicated periods of time. (H) Box plots compare the SILAC ratio distributions of unmodified peptides (unmod.) and acetylation sites (acetyl) in the indicated strains compared to WT EP cells. Numbers of peptides analyzed are indicated in parenthesis. Asterisk indicates statistical significance p < 2.2e−16 by Wilcoxen test. See also Figures S3E–S3H. Molecular Cell 2013 51, 265-272DOI: (10.1016/j.molcel.2013.06.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Acetyl-Phosphate Acetylates Protein Lysine Residues In Vitro (A) The line chart shows the intensity of acetylated peptides from purified AdK that was treated with the indicated concentrations of AcP. Peptides containing the indicated acetylated (ac) lysine (K) residues are indicated in the legend. See also Figure S5A. (B) Graph showing the median SILAC ratio of unmodified peptides (black) and acetylation sites (blue) from AcP-treated proteins compared to untreated proteins. The number of peptides or sites quantified in each experiment is shown in parenthesis. See also Figure S5C. (C) The AcP concentration relative to exponential phase (EP) BW25113 cells (Time = 0) is shown; error bars indicate standard deviation of triplicate measurements. Time (hr) indicates hours after growth arrest in the presence of glucose. BW25113, BW25113 ptaΔ, and BW25113 ackAΔ cells were analyzed from cells harvested during mid-exponential phase growth. (D) Anti-acetyllysine immunoblot of the samples shown in (C). Molecular Cell 2013 51, 265-272DOI: (10.1016/j.molcel.2013.06.003) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 CobB Suppresses Acetylation at a Subset of Sites in Both Growing and Growth-Arrested Cells (A) Histogram showing the SILAC ratio distribution of acetylation sites in EP cobBΔ cells compared to EP WT cells. Sites with elevated acetylation (>2-fold) in cobBΔ cells are defined as CobB regulated and are colored red. All other sites are defined as unregulated and are colored blue. Numbers of quantified sites (n) in each category are indicated. (B) The fraction of CobB-regulated (red) and unregulated (blue) acetylation sites occurring within 10 amino acids (<10 aa) or 20 amino acids (<20 aa) of either the N or C terminus of a protein is shown. Significance (p) was calculated by Fisher’s exact test. (C) The fraction of acetylation sites occurring on proteins associated with the indicated gene ontology (GO) terms is shown. Significance (p) was calculated by Fisher’s exact test. (D) Box plots showing the SILAC ratio distribution of unregulated and CobB-regulated acetylation sites quantified in WT SP (WT-SP) and cobBΔ SP (cobBΔ-SP) cells compared to WT-EP cells. Numbers of sites quantified are shown in parenthesis. Asterisk indicates statistical significance p < 2.2e−16 by Wilcoxen test. (E) Graph showing median SILAC ratio of CobB-regulated (red) and unregulated (blue) acetylation sites from AcP-treated proteins compared to untreated proteins. Asterisk indicates statistical significance p < 2.2e−16 by Wilcoxen test. See also Figure S6. Molecular Cell 2013 51, 265-272DOI: (10.1016/j.molcel.2013.06.003) Copyright © 2013 Elsevier Inc. Terms and Conditions