Structure of phosphorylated-like RssB, the adaptor delivering σs to the ClpXP proteolytic machinery, reveals an interface switch for activation.

In enterobacteria such as Escherichia coli, the general stress response is mediated by σs, the stationary phase dissociable promoter specificity subunit of RNA polymerase. σs is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which is thought to be stimulated by the phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the structure of RssB bound to the IraD anti-adaptor, our new RssB structure with bound beryllofluoride reveals conformational differences and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the interdomain segmented helical linker. These are accompanied by masking of the α4-ß5-α5 (4-5-5) "signaling" face of the RssB receiver domain by its C-terminal domain. Critically, using hydrogen-deuterium exchange mass spectrometry, we identify σs-binding determinants on the 4-5-5 face, implying that this surface needs to be unmasked to effect an interdomain interface switch and enable full σs engagement and hand-off to ClpXP. In activated receiver domains, the 4-5-5 face is often the locus of intermolecular interactions, but its masking by intramolecular contacts upon phosphorylation is unusual, emphasizing that RssB is a response regulator that undergoes atypical regulation.

Regulation of acetate metabolism and coordination with the TCA cycle via a processed small RNA.

Bacterial regulatory small RNAs act as crucial regulators in central carbon metabolism by modulating translation initiation and degradation of target mRNAs in metabolic pathways. Here, we demonstrate that a noncoding small RNA, SdhX, is produced by RNase E-dependent processing from the 3'UTR of the sdhCDAB-sucABCD operon, encoding enzymes of the tricarboxylic acid (TCA) cycle. In Escherichia coli, SdhX negatively regulates ackA, which encodes an enzyme critical for degradation of the signaling molecule acetyl phosphate, while the downstream pta gene, encoding the enzyme critical for acetyl phosphate synthesis, is not significantly affected. This discoordinate regulation of pta and ackA increases the accumulation of acetyl phosphate when SdhX is expressed. Mutations in sdhX that abolish regulation of ackA lead to more acetate in the medium (more overflow metabolism), as well as a strong growth defect in the presence of acetate as sole carbon source, when the AckA-Pta pathway runs in reverse. SdhX overproduction confers resistance to hydroxyurea, via regulation of ackA SdhX abundance is tightly coupled to the transcription signals of TCA cycle genes but escapes all known posttranscriptional regulation. Therefore, SdhX expression directly correlates with transcriptional input to the TCA cycle, providing an effective mechanism for the cell to link the TCA cycle with acetate metabolism pathways.

Acetylation of PhoP K88 Is Involved in Regulating Salmonella Virulence.

The PhoP-PhoQ two-component regulation system of Salmonella enterica serovar Typhimurium is involved in the response to various environmental stresses and is essential for bacterial virulence. Our previous studies showed that acetylation plays an important role in regulating the activity of PhoP, which consequently mediates the change in virulence of S Typhimurium. Here, we demonstrate that a conserved lysine residue, K88, is crucial for the function of PhoP and its acetylation-downregulated PhoP activities. K88 could be specifically acetylated by acetyl phosphate (AcP), and the acetylation level of K88 decreased significantly after phagocytosis of S Typhimurium by macrophages. Acetylation of K88 inhibited PhoP dimerization and DNA-binding abilities. In addition, mutation of K88 to glutamine, mimicking the acetylated form, dramatically attenuated intestinal inflammation and systemic infection of S Typhimurium in the mouse model. These findings indicate that nonenzymatic acetylation of PhoP by AcP is a fine-tuned mechanism for the virulence of S Typhimurium and highlights that virulence and metabolism in the host are closely linked.

Observation of acetyl phosphate formation in mammalian mitochondria using real-time in-organelle NMR metabolomics.

Recent studies point out the link between altered mitochondrial metabolism and cancer, and detailed understanding of mitochondrial metabolism requires real-time detection of its metabolites. Employing heteronuclear 2D NMR spectroscopy and 13C3-pyruvate, we propose in-organelle metabolomics that allows for the monitoring of mitochondrial metabolic changes in real time. The approach identified acetyl phosphate from human mitochondria, whose production has been largely neglected in eukaryotic metabolism since its first description about 70 years ago in bacteria. The kinetic profile of acetyl phosphate formation was biphasic, and its transient nature suggested its role as a metabolic intermediate. The method also allowed for the estimation of pyruvate dehydrogenase (PDH) enzyme activity through monitoring of the acetyl-CoA formation, independent of competing cytosolic metabolism. The results confirmed the positive regulation of mitochondrial PDH activity by p53, a well-known tumor suppressor. Our approach can easily be applied to other organelle-specific metabolic studies.

Control of acetyl phosphate-dependent phosphorylation of the response regulator CiaR by acetate kinase in Streptococcus pneumoniae.

The two-component regulatory system CiaRH of Streptococcus pneumoniae affects a large variety of physiological processes including ß-lactam resistance, competence development, maintenance of cell integrity, bacteriocin production, but also host colonization and virulence. The response regulator CiaR is active under a wide variety of conditions and the cognate CiaH kinase is not always needed to maintain CiaR activity. Using tetracycline-controlled expression of ciaR and variants, acetyl phosphate was identified in vivo as the alternative source of CiaR phosphorylation in the absence of CiaH. Concomitant inactivation of ciaH and the acetate kinase gene ackA led to very high levels of CiaR-mediated promoter activation. Strong transcriptional activation was accompanied by a high phosphorylation status of CiaR as determined by Phos-tag gel electrophoresis of S. pneumoniae cell extracts. Furthermore, AckA acted negatively upon acetyl phosphate-dependent phosphorylation of CiaR. Experiments using the Escherichia coli two-hybrid system based on adenylate cyclase reconstitution indicated binding of AckA to CiaR and therefore direct regulation. Subsequent in vitro CiaR phosphorylation experiments confirmed in vivo observations. Purified AckA was able to inhibit acetyl phosphate-dependent phosphorylation. Inhibition required the presence of ADP. AckA-mediated regulation of CiaR phosphorylation is the first example for a regulatory connection of acetate kinase to a response regulator besides controlling acetyl phosphate levels. It will be interesting to see if this novel regulation applies to other response regulators in S. pneumoniae or even in other organisms.

Global Lysine Acetylation in Escherichia coli Results from Growth Conditions That Favor Acetate Fermentation.

Lysine acetylation is thought to provide a mechanism for regulating metabolism in diverse bacteria. Indeed, many studies have shown that the majority of enzymes involved in central metabolism are acetylated and that acetylation can alter enzyme activity. However, the details regarding this regulatory mechanism are still unclear, specifically with regard to the signals that induce lysine acetylation. To better understand this global regulatory mechanism, we profiled changes in lysine acetylation during growth of Escherichia coli on the hexose glucose or the pentose xylose at both high and low sugar concentrations using label-free mass spectrometry. The goal was to see whether lysine acetylation differed during growth on these two different sugars. No significant differences, however, were observed. Rather, the initial sugar concentration was the principal factor governing changes in lysine acetylation, with higher sugar concentrations causing more acetylation. These results suggest that acetylation does not target specific metabolic pathways but rather simply targets accessible lysines, which may or may not alter enzyme activity. They further suggest that lysine acetylation principally results from conditions that favor accumulation of acetyl phosphate, the principal acetate donor in E. coliIMPORTANCE Bacteria alter their metabolism in response to nutrient availability, growth conditions, and environmental stresses using a number of different mechanisms. One is lysine acetylation, a posttranslational modification known to target many metabolic enzymes. However, little is known about this regulatory mode. We investigated the factors inducing changes in lysine acetylation by comparing growth on glucose and xylose. We found that the specific sugar used for growth did not alter the pattern of acetylation; rather, the amount of sugar did, with more sugar causing more acetylation. These results imply that lysine acetylation is a global regulatory mechanism that is responsive not to the specific carbon source per se but rather to the accumulation of downstream metabolites.

Heterologous phosphoketolase expression redirects flux towards acetate, perturbs sugar phosphate pools and increases respiratory demand in Saccharomyces cerevisiae.

INTRODUCTION: Phosphoketolases (Xfpk) are a non-native group of enzymes in yeast, which can be expressed in combination with other metabolic enzymes to positively influence the yield of acetyl-CoA derived products by reducing carbon losses in the form of CO2. In this study, a yeast strain expressing Xfpk from Bifidobacterium breve, which was previously found to have a growth defect and to increase acetate production, was characterized. RESULTS: Xfpk-expression was found to increase respiration and reduce biomass yield during glucose consumption in batch and chemostat cultivations. By cultivating yeast with or without Xfpk in bioreactors at different pHs, we show that certain aspects of the negative growth effects coupled with Xfpk-expression are likely to be explained by proton decoupling. At low pH, this manifests as a reduction in biomass yield and growth rate in the ethanol phase. Secondly, we show that intracellular sugar phosphate pools are significantly altered in the Xfpk-expressing strain. In particular a decrease of the substrates xylulose-5-phosphate and fructose-6-phosphate was detected (26% and 74% of control levels) together with an increase of the products glyceraldehyde-3-phosphate and erythrose-4-phosphate (208% and 542% of control levels), clearly verifying in vivo Xfpk enzymatic activity. Lastly, RNAseq analysis shows that Xfpk expression increases transcription of genes related to the glyoxylate cycle, the TCA cycle and respiration, while expression of genes related to ethanol and acetate formation is reduced. The physiological and transcriptional changes clearly demonstrate that a heterologous phosphoketolase flux in combination with endogenous hydrolysis of acetyl-phosphate to acetate increases the cellular demand for acetate assimilation and respiratory ATP-generation, leading to carbon losses. CONCLUSION: Our study shows that expression of Xfpk in yeast diverts a relatively small part of its glycolytic flux towards acetate formation, which has a significant impact on intracellular sugar phosphate levels and on cell energetics. The elevated acetate flux increases the ATP-requirement for ion homeostasis and need for respiratory assimilation, which leads to an increased production of CO2. A majority of the negative growth effects coupled to Xfpk expression could likely be counteracted by preventing acetate accumulation via direct channeling of acetyl-phosphate towards acetyl-CoA.

Evaluation of Natural and Synthetic Phosphate Donors for the Improved Enzymatic Synthesis of Phosphate Monoesters.

Undesired product hydrolysis along with large amounts of waste in form of inorganic monophosphate by-product are the main obstacles associated with the use of pyrophosphate in the phosphatase-catalyzed synthesis of phosphate monoesters on large scale. In order to overcome both limitations, we screened a broad range of natural and synthetic organic phosphate donors with several enzymes on a broad variety of hydroxyl-compounds. Among them, acetyl phosphate delivered stable product levels and high phospho-transfer efficiency at the lower functional pH-limit, which translated into excellent productivity. The protocol is generally applicable to acid phosphatases and compatible with a range of diverse substrates. Preparative-scale transformations using acetyl phosphate synthesized from cheap starting materials yielded multiple grams of various sugar phosphates with up to 433 g L-1 h-1 space-time yield and 75% reduction of barium phosphate waste.

Acetyl Phosphate as a Primordial Energy Currency at the Origin of Life.

Metabolism is primed through the formation of thioesters via acetyl CoA and the phosphorylation of substrates by ATP. Prebiotic equivalents such as methyl thioacetate and acetyl phosphate have been proposed to catalyse analogous reactions at the origin of life, but their propensity to hydrolyse challenges this view. Here we show that acetyl phosphate (AcP) can be synthesised in water within minutes from thioacetate (but not methyl thioacetate) under ambient conditions. AcP is stable over hours, depending on temperature, pH and cation content, giving it an ideal poise between stability and reactivity. We show that AcP can phosphorylate nucleotide precursors such as ribose to ribose-5-phosphate and adenosine to adenosine monophosphate, at modest (~2%) yield in water, and at a range of pH. AcP can also phosphorylate ADP to ATP in water over several hours at 50 °C. But AcP did not promote polymerization of either glycine or AMP. The amino group of glycine was preferentially acetylated by AcP, especially at alkaline pH, hindering the formation of polypeptides. AMP formed small stacks of up to 7 monomers, but these did not polymerise in the presence of AcP in aqueous solution. We conclude that AcP can phosphorylate biologically meaningful substrates in a manner analogous to ATP, promoting the origins of metabolism, but is unlikely to have driven polymerization of macromolecules such as polypeptides or RNA in free solution. This is consistent with the idea that a period of monomer (cofactor) catalysis preceded the emergence of polymeric enzymes or ribozymes at the origin of life.

Biochemical Basis of E. coli Topoisomerase I Relaxation Activity Reduction by Nonenzymatic Lysine Acetylation.

The relaxation activity of E. coli topoisomerase I is required for regulation of global and local DNA supercoiling. The in vivo topoisomerase I enzyme activity is sensitive to lysine acetylation⁻deacetylation and can affect DNA supercoiling and growth as a result. Nonenzymatic lysine acetylation by acetyl phosphate has been shown to reduce the relaxation activity of E. coli topoisomerase I. In this work, the biochemical consequence of topoisomerase I modification by acetyl phosphate with enzymatic assays was studied. Results showed that noncovalent binding to DNA and DNA cleavage by the enzyme were reduced as a result of the acetylation, with greater effect on DNA cleavage. Four lysine acetylation sites were identified using bottom-up proteomics: Lys13, Lys45, Lys346, and Lys488. The Lys13 residue modified by acetyl phosphate has not been reported previously as a lysine acetylation site for E. coli topoisomerase I. We discuss the potential biochemical consequence of lysine acetylation at this strictly conserved lysine and other lysine residues on the enzyme based on available genetic and structural information.

Efficient multi-enzyme-catalyzed CDP-choline production driven by an ATP donor module.

Cytidine diphosphate choline (CDP-choline) has been applied for treating acute craniocerebral injury and allowing recovery of consciousness after brain surgery. In this study, an acetate kinase (ACK)/acetyl phosphate system was used to supply ATP and combined with Escherichia coli-overexpressed CMP kinase (CMK), NDP kinase (NDK), choline phosphate cytidylyltransferase (CCT), and choline kinase (CKI) to produce CDP-choline from CMP and choline chloride. Within 1 h, 49 mM CDP-choline was produced, for a molar yield of 89.9 and 68.4 % based on CMP and choline chloride, respectively; the utilization efficiency of energy (UEE) was 79.5 %. Acetyl phosphate, sodium acetate, and CTP inhibited the reaction when the concentration exceeded 18.5, 600, and 30 mM, respectively. This inhibition could be overcome by controlling the rate of acetyl phosphate, CMP addition or using KOH instead of NaOH to regulate the pH in fed-batch transformation. After 24 h, the maximum titer was 124.1 ± 2.7 mM, the productivity was 5.1 ± 0.1 mM l-1 h-1, the molar yield to CMP and choline chloride were 83.8 and 63.7 %, respectively, and the UEE was 58.2 %. This high yield and productivity of CDP-choline through biocatalysis suggest future application at the industrial scale.

Bacterial protein acetylation: new discoveries unanswered questions.

Nε-acetylation is emerging as an abundant post-translational modification of bacterial proteins. Two mechanisms have been identified: one is enzymatic, dependent on an acetyltransferase and acetyl-coenzyme A; the other is non-enzymatic and depends on the reactivity of acetyl phosphate. Some, but not most, of those acetylations are reversed by deacetylases. This review will briefly describe the current status of the field and raise questions that need answering.

On the potential roles of phosphorus in the early evolution of energy metabolism.

Energy metabolism in extant life is centered around phosphate and the energy-dense phosphoanhydride bonds of adenosine triphosphate (ATP), a deeply conserved and ancient bioenergetic system. Yet, ATP synthesis relies on numerous complex enzymes and has an autocatalytic requirement for ATP itself. This implies the existence of evolutionarily simpler bioenergetic pathways and potentially primordial alternatives to ATP. The centrality of phosphate in modern bioenergetics, coupled with the energetic properties of phosphorylated compounds, may suggest that primordial precursors to ATP also utilized phosphate in compounds such as pyrophosphate, acetyl phosphate and polyphosphate. However, bioavailable phosphate may have been notably scarce on the early Earth, raising doubts about the roles that phosphorylated molecules might have played in the early evolution of life. A largely overlooked phosphorus redox cycle on the ancient Earth might have provided phosphorus and energy, with reduced phosphorus compounds potentially playing a key role in the early evolution of energy metabolism. Here, we speculate on the biological phosphorus compounds that may have acted as primordial energy currencies, sources of environmental energy, or sources of phosphorus for the synthesis of phosphorylated energy currencies. This review encompasses discussions on the evolutionary history of modern bioenergetics, and specifically those pathways with primordial relevance, and the geochemistry of bioavailable phosphorus on the ancient Earth. We highlight the importance of phosphorus, not only in the form of phosphate, to early biology and suggest future directions of study that may improve our understanding of the early evolution of bioenergetics.

Metabolic Engineering for Overproduction of Colanic Acid in Escherichia coli Mutant with Short Lipopolysaccharide.

Colanic acid is a major exopolysaccharide existing in most Enterobacteriaceae when exposed to an extreme environment. Colanic acid possesses excellent physical properties and biological activities, which makes it a candidate in the food and healthcare market. Previous strategies for colanic acid overproduction in E. coli mainly focus on removing the negative regulator on colanic acid biosynthesis or overexpressing the rcsA gene to up-regulate the cps operon. In this study, modifications in metabolic pathways were implemented in E. coli mutant strains with shortened lipopolysaccharides to improve colanic acid production. First, ackA was deleted to remove the byproduct acetate and the effect of accumulated acetyl-phosphate on colanic acid production was investigated. Second, 11 genes responsible for O-antigen synthesis were deleted to reduce its competition for glucose-1-phosphate and UDP-galactose with colanic acid production. Third, uppS was overexpressed to supply lipid carriers for synthesizing a colanic acid repeat unit. Colanic acid production in the final engineered strain WZM008/pTrcS reached 11.68 g/L in a 2.0 L bioreactor, 3.54 times the colanic acid production by the WQM001 strain. The results provide insights for further engineering E. coli to maximize CA production.

Influence of Microbial Metabolites on the Nonspecific Permeability of Mitochondrial Membranes under Conditions of Acidosis and Loading with Calcium and Iron Ions.

Mitochondrial dysfunction is currently considered one of the main causes of multiple organ failure in chronic inflammation and sepsis. The participation of microbial metabolites in disorders of bioenergetic processes in mitochondria has been revealed, but their influence on the mitochondrial membrane permeability has not yet been studied. We tested the influence of various groups of microbial metabolites, including indolic and phenolic acids, trimethylamine-N-oxide (TMAO) and acetyl phosphate (AcP), on the nonspecific permeability of mitochondrial membranes under conditions of acidosis, imbalance of calcium ions and excess free iron, which are inherent in sepsis. Changes in the parameters of the calcium-induced opening of the mitochondrial permeability transition pore (MPTP) and iron-activated swelling of rat liver mitochondria were evaluated. The most active metabolites were indole-3-carboxylic acid (ICA) and benzoic acid (BA), which activated MPTP opening and swelling under all conditions. AcP showed the opposite effect on the induction of MPTP opening, increasing the threshold concentration of calcium by 1.5 times, while TMAO activated swelling only under acidification. All the redox-dependent effects of metabolites were suppressed by the lipid radical scavenger butyl-hydroxytoluene (BHT), which indicates the participation of these microbial metabolites in the activation of membrane lipid peroxidation. Thus, microbial metabolites can directly affect the nonspecific permeability of mitochondrial membranes, if conditions of acidosis, an imbalance of calcium ions and an excess of free iron are created in the pathological state.

Bacterial protein acetylation and its role in cellular physiology and metabolic regulation.

Protein acetylation is an evolutionarily conserved posttranslational modification. It affects enzyme activity, metabolic flux distribution, and other critical physiological and biochemical processes by altering protein size and charge. Protein acetylation may thus be a promising tool for metabolic regulation to improve target production and conversion efficiency in fermentation. Here we review the role of protein acetylation in bacterial physiology and metabolism and describe applications of protein acetylation in fermentation engineering and strategies for regulating acetylation status. Although protein acetylation has become a hot topic, the regulatory mechanisms have not been fully characterized. We propose future research directions in protein acetylation.

Acetylation of Response Regulator Protein MtrA in M. tuberculosis Regulates Its Repressor Activity.

MtrA is an essential response regulator (RR) protein in M. tuberculosis, and its activity is modulated after phosphorylation from its sensor kinase MtrB. Interestingly, many regulatory effects of MtrA have been reported to be independent of its phosphorylation, thereby suggesting alternate mechanisms of regulation of the MtrAB two-component system in M. tuberculosis. Here, we show that RR MtrA undergoes non-enzymatic acetylation through acetyl phosphate, modulating its activities independent of its phosphorylation status. Acetylated MtrA shows increased phosphorylation and enhanced interaction with SK MtrB assessed by phosphotransfer assays and FRET analysis. We also observed that acetylated MtrA loses its DNA-binding ability on gene targets that are otherwise enhanced by phosphorylation. More interestingly, acetylation is the dominant post-translational modification, overriding the effect of phosphorylation. Evaluation of the impact of MtrA and its lysine mutant overexpression on the growth of H37Ra bacteria under different conditions along with the infection studies on alveolar epithelial cells further strengthens the importance of acetylated MtrA protein in regulating the growth of M. tuberculosis. Overall, we show that both acetylation and phosphorylation regulate the activities of RR MtrA on different target genomic regions. We propose here that, although phosphorylation-dependent binding of MtrA drives its repressor activity on oriC and rpf, acetylation of MtrA turns this off and facilitates division in mycobacteria. Our findings, thus, reveal a more complex regulatory role of RR proteins in which multiple post-translational modifications regulate the activities at the levels of interaction with SK and the target gene expression.

Metabolic intermediate acetyl phosphate modulates bacterial virulence via acetylation.

Accumulating evidence indicates that bacterial metabolism plays an important role in virulence. Acetyl phosphate (AcP), the high-energy intermediate of the phosphotransacetylase-acetate kinase pathway, is the major acetyl donor in E. coli. PhoP is an essential transcription factor for bacterial virulence. Here, we show in Salmonella typhimurium that PhoP is non-enzymatically acetylated by AcP, which modifies its transcriptional activity, demonstrating that the acetylation of Lysine 102 (K102) is dependent on the intracellular AcP. The acetylation level of K102 decreases under PhoP-activating conditions including low magnesium, acid stress or following phagocytosis. Notably, in vitro assays show that K102 acetylation affects PhoP phosphorylation and inhibits its transcriptional activity. Both cell and mouse models show that K102 is critical to Salmonella virulence, and suggest acetylation is involved in regulating PhoP activity. Together, the current study highlights the importance of the metabolism in bacterial virulence, and shows AcP might be a key mediator.

Contributions to nucleosome dynamics in chromatin from interactive propagation of phosphorylation/acetylation and inducible histone lysine basicities.

The effect of phosphorylation on the basicities of amines in histone H3 peptides and their acetylation kinetics is probed with a mild chemical acetylating agent. Phosphorylation of Ser-10 lowers the rate of chemical acetylation of Lys-9, Lys-14, and Lys-18 by methyl acetyl phosphate in that order consistent with a higher pKa of these Lys residues induced by phosphorylation; basicities increase up to 3 pKa units as a function of distance from Ser-10 phosphate. Enzymic acetylation of Lys residues with high pKa values in nucleosomes is also expected to be enhanced by phosphorylation, consistent with the known mechanism involving binding of protonated amines to N-acetyltransferases; fetal hemoglobin has a related linkage of increased basicity at a specific site, its acetylation, and a resulting decrease in subunit interaction strength. In the absence of a phosphate on Ser-10, the amines of Lys-9, Lys-14, and Lys-18 have lowered pKa values. Chemical acetylation of glycine and glycinamide have analogous kinetic profiles to the histone peptides but the phosphate inductive effect in histone H3 is more potent since the linkage between phosphorylation and acetylation is propagated with a range extending 9-10 amino acids in either direction from the phosphorylation site enhancing protonation of amino groups. We conclude that lysine amine basicities in histone tails are not static but inducible and variable due to a dynamic and immediate interaction between phosphorylation/acetylation that may contribute to inactive heterochromatin by compaction through such Ser phosphate-Lys amine electrostatic interactions and their relaxation by acetylation in euchromatin.

Contributions of SpoT Hydrolase, SpoT Synthetase, and RelA Synthetase to Carbon Source Diauxic Growth Transitions in Escherichia coli.

During the diauxic shift, Escherichia coli exhausts glucose and adjusts its expression pattern to grow on a secondary carbon source. Transcriptional profiling studies of glucose-lactose diauxic transitions reveal a key role for ppGpp. The amount of ppGpp depends on RelA synthetase and the balance between a strong SpoT hydrolase and its weak synthetase. In this study, mutants are used to search for synthetase or hydrolase specific regulation. Diauxic shifts experiments were performed with strains containing SpoT hydrolase and either RelA or SpoT synthetase as the sole source of ppGpp. Here, the length of the diauxic lag times is determined by the presence of ppGpp, showing contributions of both ppGpp synthetases (RelA and SpoT) as well as its hydrolase (SpoT). A balanced ppGpp response is key for a proper adaptation during diauxic shift. The effects of one or the other ppGpp synthetase on diauxic shifts are abolished by addition of amino acids or succinate, although by different mechanisms. While amino acids control the RelA response, succinate blocks the uptake of the excreted acetate via SatP. Acetate is converted to Acetyl-CoA through the ackA-pta pathway, producing Ac-P as intermediate. Evidence of control of the ackA-pta operon as well as a correlation between ppGpp and Ac-P is shown. Finally, acetylation of proteins is shown to occur during a diauxic glucose-lactose shift.