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...

Flagellar biogenesis and hence motility of Vibrio fischeri depends upon the presence of magnesium. In the absence of magnesium, cells contain few or no flagella and are poorly motile or nonmotile. To dissect the mechanism by which this regulation occurs, we screened transposon insertion mutants for those that could migrate through soft agar medium lacking added magnesium. We identified mutants with insertions in two distinct genes, VF0989 and VFA0959, which we termed mifA and mifB, respectively, for magnesium-dependent induction of flagellation. Each gene encodes a predicted membrane-associated protein with diguanylate cyclase activity. Consistent with that activity, introduction into V. fischeri of medium-copy plasmids carrying these genes inhibited motility. Furthermore, multicopy expression of mifA induced other phenotypes known to be correlated with diguanylate cyclase activity, including cellulose biosynthesis and biofilm formation. To directly test their function, we introduced the wild-type genes on high-copy plasmids into Escherichia coli. We assayed for the production of cyclic di-GMP using two-dimensional thin-layer chromatography and found that strains carrying these plasmids produced a small but reproducible spot that migrated with an R f value consistent with cyclic di-GMP that was not produced by strains carrying the vector control. Disruptions of mifA or mifB increased flagellin levels, while multicopy expression decreased them. Semiquantitative reverse transcription-PCR experiments revealed no significant difference in the amount of flagellin transcripts produced in either the presence or absence of Mg 2؉ by either vector control or mifA-overexpressing cells, indicating that the impact of magnesium and cyclic-di-GMP primarily acts following transcription. Finally, we present a model for the roles of magnesium and cyclic di-GMP in the control of motility of V. fischeri.The limiting step in understanding signal transduction most often is the identification of the environmental signal that induces a physiological change. This has been true for twocomponent signaling (13, 58) and may also be true for the pathways that control the production of cyclic di-GMP (c-di-GMP) (45). This newly appreciated second messenger is synthesized from two GTP molecules by diguanylate cyclases (DGCs). These enzymes, found in numerous and diverse bacterial genomes, are readily identifiable through their signature GGDEF domains (19,39,40,50,53,56). Furthermore, many bacterial species possess multiple proteins with domains that contain this GGDEF domain (for a recent review, see reference 45). Degradation of c-di-GMP is accomplished by phosphodiesterases containing either EAL or HD-GYP domains (8,15,49,52,57). Together, these activities maintain the steady-state concentration of c-di-GMP (46). c-di-GMP first was discovered as a component of the cellulose biosynthesis enzyme complex from Gluconacetobacter xylinus, where it plays a vital role in promoting cellulose biosynthesis (46). c-di-GMP is now known to control exopolys...

Acetyl phosphate (acetyl-P) serves critical roles in coenzyme A recycling and ATP synthesis. It is the intermediate of the Pta-AckA pathway that inter-converts acetyl-coenzyme A and acetate. Acetyl-P also can act as a global signal by donating its phosphoryl group to specific two-component response regulators. This ability derives from its capacity to store energy in the form of a high-energy phosphate bond. This bond, while critical to its function, also destabilizes acetyl-P in cell extracts. This lability has greatly complicated biochemical analysis, leading in part to widely varying acetyl-P measurements. We therefore developed an optimized protocol based on two-dimensional thin layer chromatography that includes metabolic labeling under aerated conditions and careful examination of the integrity of acetyl-P within extracts. This protocol results in greatly improved reproducibility, and thus permits precise measurements of the intracellular concentration of acetyl-P, as well as that of other small phosphorylated molecules.

Magnesium-dependent induction of Vibrio fischeri flagellar (Mif) biogenesis depends upon two diguanylate cyclases, suggesting an inhibitory role for cyclic di-GMP. Here, we report that cells defective for the sugar phosphotransferase system (PTS) exhibited a magnesium-independent phenotype similar to that of mutants of the Mif pathway. Unlike Mif mutants, PTS mutants also were hyperbioluminescent.The second messenger, cyclic AMP (cAMP), is synthesized by adenylate cyclase (AC). In bacteria of the family Enterobacteriaceae (e.g., Escherichia coli), the activity of AC becomes enhanced by its interaction with the phosphorylated version of EIIA Glc (phospho-EIIA Glc ), the glucose-specific IIA component of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) (6,17,18,20). The glucose-specific PTS is composed of three soluble, cytoplasmic proteins (EI, HPr, and EIIA Glc ) and one integral cytoplasmic membrane protein (EIICB Glc ). These proteins sequentially transfer a phosphoryl group from PEP to glucose, with the concomitant transport of the sugar across the membrane (Fig. 1). The presence of glucose in the environment pulls phosphoryl groups through the PTS, ensuring that the system's components remain essentially unphosphorylated; depletion of that glucose results in the accumulation of phospho-EIIA Glc , which activates AC to synthesize cAMP. This cAMP binds cAMP receptor protein (CRP, also known as CAP), which then regulates the transcription of hundreds of genes, including flhDC, the master regulator of the Enterobacteriaceae flagellar regulon (for reviews, see references 6a, 10, 11, 17, and 30).Cyclic di-GMP (c-di-GMP) is a newly appreciated second messenger, apparently unique to bacteria, that modulates diverse cellular processes (for a recent review, see reference 21). First identified as a positive effector of cellulose synthase in Gluconoacetobacter xylinus (reviewed in reference 22), c-di-GMP regulates transition from the motile, planktonic state to sessile, community-based behaviors, such as biofilm development. It tends to inhibit motility, both flagellar and twitching, while enhancing the biosynthesis of capsular components required by developing biofilms. c-di-GMP is synthesized by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE) (21). Together these activities maintain the steadystate concentration of c-di-GMP (22). DGC activity has been associated with the highly conserved GGDEF domain. One PDE activity (to linear di-GMP) has been associated with the highly conserved EAL domain, while a second PDE activity (to two GMPs) has been associated with the HD-GYP domain (21). Finally, a c-di-GMP-binding domain (termed PilZ) was recently reported (2) and shown to inhibit flagellar assembly in E. coli (25).Insights into the control of c-di-GMP production and its targets have come from our investigations of motility in the marine bacterium Vibrio fischeri. This bacterium, found as free-living, motile individuals or as a sessile community in association with the Hawaiia...