Protein acetylation dynamics in response to carbon overflow in Escherichia coli

• Published in: Molecular microbiology Vol. 98; no. 5; pp. 847 - 863
• Main Authors: Schilling, Birgit, Christensen, David, Davis, Robert, Sahu, Alexandria K., Hu, Linda I., Walker‐Peddakotla, Arti, Sorensen, Dylan J., Zemaitaitis, Bozena, Gibson, Bradford W., Wolfe, Alan J.
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01-12-2015
• Subjects: Acetates - metabolism; Acetylation; Acetyltransferases - genetics; Acetyltransferases - metabolism; Carbon; Carbon - metabolism; Carbon Cycle - genetics; E coli; Enzymes; Escherichia coli - genetics; Escherichia coli - growth & development; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Gene Expression Regulation, Bacterial; Glucose; Glucose - metabolism; Lysine - metabolism; Metabolic Networks and Pathways; Protein Processing, Post-Translational; Proteins; Proteomics
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/mmi.13161

Summary
In Escherichia coli, acetylation of proteins at lysines depends largely on a non‐enzymatic acetyl phosphate‐dependent mechanism. To assess the functional significance of this post‐translational modification, we first grew wild‐type cells in buffered tryptone broth with glucose and monitored acetylation over time by immunochemistry. Most acetylation occurred in stationary phase and paralleled glucose consumption and acetate excretion, which began upon entry into stationary phase. Transcription of rprA, a stationary phase regulator, exhibited similar behavior. To identify sites and substrates with significant acetylation changes, we used label‐free, quantitative proteomics to monitor changes in protein acetylation. During growth, both the number of identified sites and the extent of acetylation increased with considerable variation among lysines from the same protein. As glucose‐regulated lysine acetylation was predominant in central metabolic pathways and overlapped with acetyl phosphate‐regulated acetylation sites, we deleted the major carbon regulator CRP and observed a dramatic loss of acetylation that could be restored by deleting the enzyme that degrades acetyl phosphate. We propose that acetyl phosphate‐dependent acetylation is a response to carbon flux that could regulate central metabolism.
Using immunochemistry and label‐free, quantitative proteomics, we monitored protein lysine acetylation over time in response to glucose supplementation. The resultant data supports the hypothesis that carbon flux in excess of central metabolic capacity (overflow metabolism) causes acetyl phosphate‐dependent acetylation and the hypothesis that the major carbon regulator CRP regulates this process by facilitating the synthesis of acetyl phosphate.