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The Escherichia coli isocitrate dehydrogenase (ICDH) is one of the tricarboxylic acid cycle enzymes, playing key roles in energy production and carbon flux regulation. E. coli ICDH was the first bacterial enzyme shown to be regulated by reversible phosphorylation. However, the effect of lysine acetylation on E. coli ICDH, which has no sequence similarity with its counterparts in eukaryotes, is still unclear. Based on previous studies of E. coli acetylome and ICDH crystal structures, eight lysine residues were selected for mutational and kinetic analyses. They were replaced with acetyllysine by the genetic code expansion strategy or substituted with glutamine as a classic approach. Although acetylation decreased the overall ICDH activity, its effects were different site by site. Deacetylation tests demonstrated that the CobB deacetylase could deacetylate ICDH both in vivo and in vitro , but CobB was only specific for lysine residues at the protein surface. On the other hand, ICDH could be acetylated by acetyl-phosphate chemically in vitro . And in vivo acetylation tests indicated that the acetylation level of ICDH was correlated with the amounts of intracellular acetyl-phosphate. This study nicely complements previous proteomic studies to provide direct biochemical evidence for ICDH acetylation. [ABSTRACT FROM AUTHOR]

Glycolic acid (GA) is an ⍺-hydroxy acid used in cosmetics, packaging, and medical industries due to its excellent properties, especially in its polymeric form. In this study, Escherichia coli was engineered to produce GA from D-xylose by linking the Dahms pathway, the glyoxylate bypass, and the partial reverse glyoxylate pathway (RGP). Initially, a GA-producing strain was constructed by disrupting the xylAB and glcD genes in the E. coli genome and overexpressing the xdh(Cc) from Caulobacter crescentus. This strain was further improved through modular optimization of the Dahms pathway and the glyoxylate bypass. Results for module 1 showed that the rate-limiting step of the Dahms pathway was the xylonate dehydratase reaction, and the overexpression of yagF was sufficient to overcome this bottleneck. Furthermore, the appropriate aldolase gene for module 1 was proven to be yagE. The results also show that overexpression of the lactaldehyde dehydrogenase gene, aldA, is needed to increase the GA production while the overexpression of glyoxylate reductase gene, ycdW, was only essential when the glyoxylate bypass was active. On the other hand, the module 2 enzymes AceA and AceK were vital in activating the glyoxylate bypass, while the RGP enzymes were dispensable. The final strain (GA19) produced 4.57 g/L GA with a yield of 0.46 g/g from D-xylose. So far, this is the highest value achieved for GA production in engineered E. coli through the Dahms pathway. [ABSTRACT FROM AUTHOR]

Two different approaches to activate the glyoxylate bypass in model Escherichia coli K-12 strains for succinate biosynthesis during dual-phase fermentation in minimal glucose media were examined. Inactivation of IclR and FadR, the transcriptional regulators of the aceBAK operon, were insufficient for the involvement of the glyoxylate bypass in anaerobic succinate biosynthesis by strains grown aerobically under glucose-abundant conditions. In contrast, the strains that constitutively expressed the aceEF- lpdA operon coding for the pyruvate dehydrogenase complex could partially synthesise succinate anaerobically via the glyoxylate bypass, even in the presence of intact regulators. The results suggest that the intensive acetyl-CoA formation in the strains constitutively expressing pyruvate dehydrogenase matches the physiological conditions that favour the activation of the glyoxylate bypass. [ABSTRACT FROM AUTHOR]

Background: Whilst being closely related to the model actinomycete Streptomyces coelicolor A3(2), S. lividans 66 differs from it in several significant and phenotypically observable ways, including antibiotic production. Previous comparative gene hybridization studies investigating such differences have used low-density (one probe per gene) PCR-based spotted arrays. Here we use new experimentally optimised 104,000 × 60-mer probe arrays to characterize in detail the genomic differences between wild-type S. lividans 66, a derivative industrial strain, TK24, and S. coelicolor M145. Results: The high coverage and specificity (detection of three nucleotide differences) of the new microarrays used has highlighted the macroscopic genomic differences between two S. lividans strains and S. coelicolor. In a series of case studies we have validated the microarray and have identified subtle changes in genomic structure which occur in the Asp-activating adenylation domains of CDA non-ribosomal peptide synthetase genes which provides evidence of gene shuffling between these domains. We also identify single nucleotide sequence inter-species differences which exist in the actinorhodin biosynthetic gene cluster. As the glyoxylate bypass is non-functional in both S. lividans strains due to the absence of the gene encoding isocitrate lyase it is likely that the ethylmalonyl-CoA pathway functions as the alternative mechanism for the assimilation of C2 compounds. Conclusions: This study provides evidence for widespread genetic recombination, rather than it being focussed at 'hotspots', suggesting that the previously proposed 'archipelago model' of genomic differences between S. coelicolor and S. lividans is unduly simplistic. The two S. lividans strains investigated differ considerably in genetic complement, with TK24 lacking 175 more genes than its wild-type parent when compared to S. coelicolor. Additionally, we confirm the presence of bldB in S. lividans and deduce that S. lividans 66 and TK24, both deficient in the glyoxylate bypass, possess an alternative metabolic mechanism for the assimilation of C2 compounds. Given that streptomycetes generally display high genetic instability it is envisaged that these high-density arrays will find application for rapid assessment of genome content (particularly amplifications/deletions) in mutational studies of S. coelicolor and related species. [ABSTRACT FROM AUTHOR]

Escherichia coli isocitrate dehydrogenase (ICDH) can be phosphorylated and dephosphorylated by a single bifunctional protein, isocitrate dehydrogenase kinase/phosphatase (AceK), which is encoded by the aceK gene. In order to investigate the regulatory mechanism of (de)phosphorylation of ICDH by AceK, AceK was successfully cocrystallized in complex with its intact protein substrate, ICDH, in the presence of ATP. The complex crystal was obtained by the hanging-drop vapour-diffusion technique using PEG 300 as a precipitant and magnesium sulfate as an additive. SDS-PAGE analysis of dissolved crystals showed that the crystals contained both AceK and ICDH proteins. The complex crystals diffracted to a resolution of 2.9 Å in space group P63, with unit-cell parameters a = b = 196.80, c = 156.46 Å. [ABSTRACT FROM AUTHOR]

The Escherichia coli aceK gene encodes isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5), a bifunctional protein that phosphorylates and dephosphorylates isocitrate dehydrogenase (IDH), resulting in its inactivation and activation, respectively. This reversible (de)phosphorylation directs isocitrate, an intermediate of the citric acid cycle, to either go through the full cycle or to enter the glyoxylate bypass. In the present study, the AceK protein from E. coli has been purified and crystallized. Three crystal forms were obtained from very similar crystallization conditions. The crystals belong to space groups P41212, P3221 and P212121 and diffracted X-rays to resolutions of 2.9, 3.0 and 2.7 Å, respectively. [ABSTRACT FROM AUTHOR]

Abstract: In order to improve the production of succinate and malate by the filamentous fungus Aspergillus niger the activity of the glyoxylate bypass pathway was increased by over-expression of the isocitrate lyase (icl) gene. The hypothesis was that when isocitrate lyase was up-regulated the flux towards glyoxylate would increase, leading to excess formation of malate and succinate compared to the wild-type. However, metabolic network analysis showed that an increased icl expression did not result in an increased glyoxylate bypass flux. The analysis did show a global response with respect to gene expression, leading to an increased flux through the oxidative part of the TCA cycle. Instead of an increased production of succinate and malate, a major increase in fumarate production was observed. The effect of malonate, a competitive inhibitor of succinate dehydrogenase (SDH), on the physiological behaviour of the cells was investigated. Inhibition of SDH was expected to lead to succinate production, but this was not observed. There was an increase in citrate and oxalate production in the wild-type strain. Furthermore, in the strain with over-expression of icl the organic acid production shifted from fumarate towards malate production when malonate was added to the cultivation medium. Overall, the icl over-expression and malonate addition had a significant impact on metabolism and on organic acid production profiles. Although the expected succinate and malate formation was not observed, a distinct and interesting production of fumarate and malate was found. [Copyright &y& Elsevier]

A family of kinetic models has been developed that takes into account available experimental information on the regulation of ace operon expression in Escherichia coli. This has allowed us to study and analyze possible versions of regulation of the ace operon and to test their possibilities. Based on literature analysis, we found that there is an ambiguity of properties of IclR (main repressor of ace operon). The main aspect of this ambiguity are two different forms of IclR purified from E. coli K strain and different coeffector sets for IclR purified from E. coli K and B strains. It has been shown that the full-length form of IclR is physiologically relevant and that IclR truncation is a result of purification of the protein from E. coli K strains. We also found that the IclR protein purified from E. coli B strain carries two coeffector binding sites. Using model-developed levels of steady state aceBAK expression against physiological ranges of coeffectors, concentration has been predicted. [ABSTRACT FROM AUTHOR]

During aerobic growth of Escherichia coli on acetate as sole source of carbon and energy, the organism requires the operation of the glyoxylate bypass enzymes, namely isocitrate lyase (ICL) and the anaplerotic enzyme malate synthase (MS). Under these conditions, the glyoxylate bypass enzyme ICL is in direct competition with the Krebs cycle enzyme isocitrate dehydrogenase (ICDH) for their common substrate and although ICDH has a much higher affinity for isocitrate, flux of carbon through ICL is assured by virtue of high intracellular level of isocitrate and the reversible phosphorylation/inactivation of a large fraction of ICDH. Reversible inactivation is due to reversible phosphorylation catalysed by ICDH kinase/phosphatase, which harbours both catalytic activities on the same polypeptide. The catalytic activities of ICDH kinase/phosphatase constitute a moiety conserved cycle, require ATP and exhibit ‘zero-order ultrasensitivity’. The structural gene encoding ICDH kinase/phosphatase (aceK) together with those encoding ICL (aceA) and MS (aceB) form an operon (aceBAK; otherwise known as the ace operon) the expression of which is intricately regulated at the transcriptional level by IclR, FadR, FruR and IHF. Although ICDH, an NADP+-dependent, non-allosteric dimer, can be phosphorylated at multiple sites, it is the phosphorylation of the Ser-113 residue that renders the enzyme catalytically inactive as it prevents isocitrate from binding to the active site, which is a consequence of the negative charge carried on phosphoserine 113 and the conformational change associated with it. The ICDH molecule readily undergo domain shifts and/or induced-fit conformational changes to accommodate the binding of ICDH kinase/phosphatase, the function of which has now been shown to be central to successful adaptation and growth of E. coli and related genera on acetate and fatty acids. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Escherichia coli changes its metabolism in response to environmental circumstances, and metabolic adaptations are evident in hungry bacteria growing slowly in glucose-limited chemostats. The role of isocitrate lyase (AceA) was examined in E. coli growing under glucose limitation. AceA activity was elevated in a strain-dependent manner in the commonly used E. coli K-12 laboratory strains MG1655 and MC4100, but an aceA disruption surprisingly increased fitness under glucose limitation in both strains. However, in bacteria adapted to limiting glucose in long-term chemostats, mutations outside aceA changed its role from a negative to a positive influence. These results suggest that a recently proposed pathway of central metabolism involving the glyoxylate cycle enzymes is redundant in wild-type bacteria, but may take on a beneficial role after context adaptation. Interestingly, the aceA gene sequence did not alter during prolonged selection, so mutations in unidentified genes changed the metabolic context of unaltered AceA from a negative to a positive influence in bacteria highly adapted to limiting glucose. [Copyright &y& Elsevier]

The glyoxylate bypass genes aceA1 (isocitrate lyase 1, ICL1), aceA2 (isocitrate lyase 2, ICL2) and aceB1 (malate synthase, MS1) of Ralstonia eutropha HF39 were cloned, sequenced and functionally expressed in Escherichia coli. Interposon-mutants of all three genes (ΔaceA1, ΔaceA2 and ΔaceB1) were constructed, and the phenotypes of the respective mutants were investigated. Whereas R. eutropha HF39ΔaceA1 retained only 19% of ICL activity and failed to grow on acetate, R. eutropha HF39ΔaceA2 retained 84% of acetate-inducible ICL activity, and growth on acetate was not retarded. These data suggested that ICL1 is in contrast to ICL2 induced by acetate and specific for the glyoxylate cycle. R. eutropha HF39ΔaceB1 retained on acetate as well as on gluconate about 41–42% of MS activity and exhibited retarded growth on acetate, indicating the presence of a second hitherto not identified MS in R. eutropha HF39. Whereas in R. eutropha HF39ΔaceA1 and R. eutropha HF39ΔaceA2 the yields of poly(3-hydroxybutyric acid), using gluconate as carbon source, were significantly reduced, R. eutropha HF39ΔaceB1 accumulated the same amount of this polyester from gluconate as well as from acetate as R. eutropha HF39. [Copyright &y& Elsevier]

Laboratory-scale sequencing batch reactors exhibiting enhanced biological phosphorus removal (EBPR) operated at different influent phosphorus/chemical oxygen demand (COD) ratios were analyzed to evaluate possible anaerobic sources of reducing equivalents. Assuming anaerobic glycogen degradation was the sole anaerobic reducing equivalent source, an anaerobic phase carbon balance showed that glycogen-accumulating metabolism (GAM)-dominated systems were nearly carbon-balanced, but that polyphosphate-accumulating metabolism (PAM)-dominated systems had end-anaerobic phase carbon deficits. An anaerobic-phase reducing equivalent balance showed a reducing equivalent excess for the GAM-dominated systems and a deficit for the PAM-dominated systems, suggesting that glycogen degradation was not the sole reducing equivalent source for PAM. Reducing equivalent balances showed that metabolic models including complete anaerobic tricarboxylic acid (TCA) cycle activity, partial TCA cycle activity, and the glyoxylate bypass could provide the reducing equivalents required in PAM. Metabolic precursors produced in glycolysis, the TCA cycle, or modified versions of the TCA cycle could allow anaerobic growth and account for the PAM carbon deficits. The importance of considering both PAM and GAM activity in evaluating EBPR metabolic models was illustrated. [ABSTRACT FROM AUTHOR]

The aceA and glcB genes, encoding isocitrate lyase (ICL) and malate synthase, respectively, are not in an operon in many bacteria, including Pseudomonas aeruginosa, unlike in Escherichia coli. Here, we show that expression of aceA in P. aeruginosa is specifically upregulated under H2O2-induced oxidative stress and under iron-limiting conditions. In contrast, the addition of exogenous redox active compounds or antibiotics increases the expression of glcB. The transcriptional start sites of aceA under iron-limiting conditions and in the presence of iron were found to be identical by 5' RACE. Interestingly, the enzymatic activities of ICL and isocitrate dehydrogenase had opposite responses under different iron conditions, suggesting that the glyoxylate shunt (GS) might be important under iron-limiting conditions. Remarkably, the intracellular iron concentration was lower while the iron demand was higher in the GS-activated cells growing on acetate compared to cells growing on glucose. Absence of GS dysregulated iron homeostasis led to changes in the cellular iron pool, with higher intracellular chelatable iron levels. In addition, GS mutants were found to have higher cytochrome c oxidase activity on iron-supplemented agar plates of minimal media, which promoted the growth of the GS mutants. However, deletion of the GS genes resulted in higher sensitivity to a high concentration of H2O2, presumably due to iron-mediated killing. In conclusion, the GS system appears to be tightly linked to iron homeostasis in the promotion of P. aeruginosa survival under oxidative stress.

The glyoxylate shunt (GS) is a two-step metabolic pathway (isocitrate lyase, aceA; and malate synthase, glcB) that serves as an alternative to the tricarboxylic acid cycle. The GS bypasses the carbon dioxide-producing steps of the tricarboxylic acid cycle and is essential for acetate and fatty acid metabolism in bacteria. GS can be up-regulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important but poorly explored roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, aceA and glcB are not in an operon, unlike in Escherichia coli In P. aeruginosa, we explored relationships between GS genes and growth, transcription profiles, and biofilm formation. Contrary to our expectations, deletion of aceA in P. aeruginosa improved cell growth under conditions of oxidative and antibiotic stress. Transcriptome data suggested that aceA mutants underwent a metabolic shift toward aerobic denitrification; this was supported by additional evidence, including up-regulation of denitrification-related genes, decreased oxygen consumption without lowering ATP yield, increased production of denitrification intermediates (NO and N2O), and increased cyanide resistance. The aceA mutants also produced a thicker exopolysaccharide layer; that is, a phenotype consistent with aerobic denitrification. A bioinformatic survey across known bacterial genomes showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. This trend is consistent with the hypothesis that the GS plays a previously unrecognized role in allowing bacteria to tolerate oxidative stress., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)