Acetylation of the Response Regulator RcsB Controls Transcription from a Small RNA Promoter

Abstract

c N-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.

Regulation, Function, and Detection of Protein Acetylation in Bacteria

“…A K154Q acetyl-mimic mutation supported this function (50). Another site, Lys180, was found to be acetylated by YfiQ and deacetylated by CobB in vitro (97,98), but these observations have not been supported in vivo (97).…”

“…As the positively charged lysine residues can normally interact with the negatively charged phosphate groups on DNA, the presence of acetylation inhibits their DNA binding. For example, RcsB, which regulates capsule and flagellar synthesis in E. coli, is acetylated nonenzymatically in vivo at K154, and this event inhibits DNA binding (97). A K154Q acetyl-mimic mutation supported this function (50).…”

“…Large-scale quantitative studies later revealed that acetylation of K291 is acetylphosphate sensitive (59), likely representing the mechanism of acetylation, and this acetylation event is indeed glucose induced (106). The CTD of RpoA makes direct contacts with DNA elements and transcription factors (107,108), providing a potential mechanism by which acetylation can rapidly alter transcription in response to environmental cues (97,104,105,(107)(108)(109). In B. subtilis, an RNA helicase (CshA) that associates with RNAP is required for glucose induction of SigX-and SigM-dependent genes (110).…”

Interaction of the RcsB Response Regulator with Auxiliary Transcription Regulators in Escherichia coli

“…The interaction of RcsB with the auxiliary proteins alters the DNA binding specificity because the DNA-binding motifs of RcsB homodimers and heterodimers with RcsA, BglJ, and MatA are similar in one half-site, which is presumably RcsB-bound, whereas the DNA sequence of the other half-site varies (9,12,18,23,49). Note that mutation of RcsB residue Lys-180 in the helix-turn-helix motif renders RcsB and its heterodimers inactive and that acetylation of this residue inhibits RcsB DNA binding (50,51).…”

Acylation of Biomolecules in Prokaryotes: a Widespread Strategy for the Control of Biological Function and Metabolic Stress

“…Acetylation of RcsB decreases its ability to bind DNA, an effect that is reversed by incubation of RcsB Ac with the sirtuin EcCobB and NAD ϩ (149). More recently reported work did not find direct evidence that EcPka acetylates RcsB in E. coli, leaving the identity of the acetyltransferase that modifies RcsB in these bacteria unknown (205).…”