Protein acetylation dynamics in response to carbon overflow in Escherichia coli

Abstract

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. Since 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.

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

“…Under these growth conditions, the cells acetylate their proteins during stationary phase (1)(2)(3). This glucose-induced acetylation is due primarily to production of acetyl phosphate (1,2), a metabolic intermediate of the acetate fermentation pathway (4).…”

Sirtuin-dependent reversible lysine acetylation of glutamine synthetases reveals an autofeedback loop in nitrogen metabolism

“…In particular, RLA is being increasingly recognized as an important metabolic regulatory mechanism in bacteria. In the last decade, advancements in mass spectrometry (MS) and high-affinity purification of acetylated peptides have accelerated the identification of lysine acetylation sites, and the acetylproteome of several microorganisms has been reported (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). The picture emerging from these studies indicates that lysine acetylation is a conserved regulatory mechanism of metabolism.…”

“…However, at present, our understanding of how the regulation of the acetylation system is integrated with the regulation of genes encoding proteins whose function is controlled by acetylation is limited. More recently, insights into the complexities of such regulatory integration have been reported for Escherichia coli and S. enterica (13,17). Results from studies by Yu et al and Schilling et al suggest that the levels of acetylation are regulated in response to environmental changes (8,13).…”

“…More recently, insights into the complexities of such regulatory integration have been reported for Escherichia coli and S. enterica (13,17). Results from studies by Yu et al and Schilling et al suggest that the levels of acetylation are regulated in response to environmental changes (8,13). Nevertheless, the means by which signals dictate the level of protein acetylation and lysine acylation remain poorly understood.…”

“…In E. coli, two cAMPmediated acetylation signaling pathways have been identified (13,19). The carbon regulator cAMP receptor protein (CRP)-cAMP complex induces the expression of yfiQ, which encodes the Gcn5-like acetyltransferase (GNAT) YfiQ (PatZ, Pka) and thus increases the acetylation level of proteins in response to the intracellular cAMP signal (19).…”