The Intracellular Concentration of Acetyl Phosphate in Escherichia coli Is Sufficient for Direct Phosphorylation of Two-Component Response Regulators

Abstract

Acetyl phosphate, the intermediate of the AckA-Pta pathway, acts as a global signal in Escherichia coli. Although acetyl phosphate clearly signals through two-component response regulators, it remains unclear whether acetyl phosphate acts as a direct phospho donor or functions through an indirect mechanism. We used two-dimensional thin-layer chromatography to measure the relative concentrations of acetyl phosphate, acetyl coenzyme A, ATP, and GTP over the course of the entire growth curve. We estimated that the intracellular concentration of acetyl phosphate in wild-type cells reaches at least 3 mM, a concentration sufficient to activate two-component response regulators via direct phosphoryl transfer.Bacterial cells must respond properly to diverse external and internal cues, a process that requires global signaling. The ideal global signal is metabolically inexpensive, short-lived, indicative only of significant changes, and capable of effecting the coordinated regulation of diverse cellular processes. Several lines of evidence suggest that the small molecule acetyl phosphate (acetyl-P) can serve as such a global signal.Acetyl-P is the high-energy, acid/base-labile intermediate of the reversible Pta-AckA pathway (Fig. 1). This pathway interconverts coenzyme A (HS-CoA), ATP, and acetate with acetyl coenzyme A (acetyl-CoA), ADP, and orthophosphate (P i ) (9, 43). The reversibility of this pathway permits both acetyl-CoA synthesis (acetate activation) and acetate evolution (acetogenesis). During acetogenesis, Pta synthesizes acetyl-P and HSCoA from acetyl-CoA and P i , while AckA generates ATP from acetyl-P and ADP. Simultaneously, AckA produces acetate, which cells excrete into the environment. Thus, the steadystate concentration of acetyl-P depends upon the rate of its formation catalyzed by Pta and the rate of its degradation catalyzed by AckA (for reviews, see references 45 and 59).Acetyl-P has been proposed to act as a global regulator by direct phosphorylation of response regulators (RRs) of the family of two-component signal transduction (2CST) pathways (34, 55). The simplest of these 2CST pathways consists of a histidine kinase (HK) and an RR. Using ATP as its phosphoryl donor, the HK autophosphorylates a conserved histidine residue. In turn, the RR autophosphorylates a conserved aspartate residue, using its phosphorylated cognate HK as its phosphoryl donor. Experiments performed in vitro have clearly demonstrated that acetyl-P can donate its phosphoryl group to purified RRs but not to HKs (for reviews, see references 52 and 57). This ability arises from its capacity to store energy. Acetyl-P possesses a larger ⌬G°of hydrolysis (Ϫ43.3 kJ/mol) than ATP possesses (Ϫ30.5 kJ/mol in complex with Mg 2ϩ ). This difference forms the basis for the role of acetyl-P in generating ATP (for a review, see reference 59).Although acetyl-P can function as a phosphoryl donor in vitro, its ability to function in vivo as a global signal has remained essentially unproven, despite a wealth of seemingly supportive data (for a r...

A pivotal role for the response regulator DegU in controlling multicellular behaviour

“…Consistent with this suggestion, DegU of L. monocytogenes can be phosphorylated by acetyl phosphate in vitro and alterations in the level of acetyl phosphate in vivo control motility and biofilm formation (Gueriri et al, 2008a). As the level of acetyl phosphate varies with both growth condition and growth phase (Klein et al, 2007;Wolfe, 2005) it is possible that DegU functions as a read-out of the metabolic status of L. monocytogenes.…”

Inhibition of Acetyl Phosphate-dependent Transcription by an Acetylatable Lysine on RNA Polymerase

“…1B), which has been shown to function in vitro as a phosphoryl donor to CpxR (6), and has been proposed to do likewise in vivo (5,14). If AcP could function as the phosphoryl donor, then the cpxR1 ackA double mutant (strain AJW3827; Table 1), which accumulates AcP (12,16), should respond to the addition of glucose in a Asp-51-dependent manner. In contrast, we predicted that the cpxR1 pta ackA triple mutant (strain AJW3875; Table 1), which cannot synthesize AcP (12, 16), would not respond to exogenous glucose regardless of the status of Asp-51.…”

“…One such behavior is the CpxA-independent induction of cpxP transcription that occurs when glucose is added to tryptone-based growth media (8,14,15). If a behavior strictly depends on AcP to donate its phosphoryl group to a response regulator, then an ackA mutant, which accumulates AcP (16), should elicit behavior opposite to that exhibited by a pta mutant, which cannot synthesize AcP (16). Thus, if glucose-induced cpxP transcription depended strictly on AcP, then an ackA mutant should respond more robustly to glucose than its wild-type (WT) parent, whereas the pta (or pta ackA) mutant should respond more poorly.…”

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

“…Under these conditions, the cultures enter stationary phase when the glucose is depleted. We also knew that the concentration of acetyl phosphate peaks during exponential growth in unbuffered TB without glucose supplementation (6) and during growth in MOPS (morpholinepropanesulfonic acid) minimal medium with glucose as the sole carbon source (7). Under both of these growth conditions, acetyl phosphate is undetectable by the time the cultures reach stationary phase.…”