Protein acetylation dynamics in response to carbon overflow in Escherichia coli.

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.

Acetylation of the response regulator RcsB controls transcription from a small RNA promoter.

Nε-lysine acetylation was recently discovered on many bacterial proteins that function in diverse cellular processes. Thus, many questions remain unanswered. For example, what mechanisms regulate lysine acetylation? Does acetylation affect physiology? To help answer these questions, we studied the Escherichia coli response regulator and transcription factor RcsB, which is reported to be acetylated in vitro. To characterize RcsB acetylation, we monitored transcription from the rprA promoter, which requires RcsB. The conventional view is that RcsB is activated by phosphorylation through either the Rcs phosphorelay or acetyl phosphate. We affirmed that rprA transcription requires phosphorylated RcsB and showed that acetyl-phosphate (AcP) is a phosphoryl group donor to RcsB. However, a mutant that accumulates AcP (ackA) exhibited a reduction in rprA transcription instead of the predicted increase. rprA transcription also diminished in the cobB mutant, which lacks the only known E. coli protein deacetylase. This suggests the existence of an inhibitory mechanism that involves lysine acetylation, a supposition supported by the observation that RcsB isolated from the ackA or cobB mutant was hyperacetylated. Finally, we used a genetic approach to identify an AckA- and CobB-sensitive lysine (Lys-154) that controls RcsB activity. We propose that acetylation inhibits RcsB activity and that some of this inhibition acts through the acetylation of Lys-154.