Adenylate kinase-independent thiamine triphosphate accumulation under severe energy stress in Escherichia coli

Background  

Thiamine triphosphate (ThTP) exists in most organisms and might play a role in cellular stress responses. In E. coli, ThTP is accumulated in response to amino acid starvation but the mechanism of its synthesis is still a matter of controversy. It has been suggested that ThTP is synthesized by an ATP-dependent specific thiamine diphosphate kinase. However, it is also known that vertebrate adenylate kinase 1 catalyzes ThTP synthesis at a very low rate and it has been postulated that this enzyme is responsible for ThTP synthesis in vivo.     

Adenosine thiamine triphosphate accumulates in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells in response to specific conditions of metabolic stress

Background  

E. coli cells are rich in thiamine, most of it in the form of the cofactor thiamine diphosphate (ThDP). Free ThDP is the precursor for two triphosphorylated derivatives, thiamine triphosphate (ThTP) and the newly discovered adenosine thiamine triphosphate (AThTP). While, ThTP accumulation requires oxidation of a carbon source, AThTP slowly accumulates in response to carbon starvation, reaching ~15% of total thiamine. Here, we address the question whether AThTP accumulation in E. coli is triggered by the absence of a carbon source in the medium, the resulting drop in energy charge or other forms of metabolic stress.     

Effect of thiamine phosphates on the activity of regulatory enzymes of the pyruvate dehydrogenase complex

The effect of thiamine triphosphate (ThTP) and thiamine diphosphate (ThDP) on the activity of rat liver pyruvate dehydrogenase complex regulatory enzymes (kinase and phosphatase) was studied in experiments with isolated enzyme preparations. It is shown that ThDP caused a pronounced activation of pyruvate dehydrogenase phosphatase (Ka is equal to 65.0 nM). ThTP inhibits phosphatase competitively against the substrate--the phosphorylated pyruvate dehydrogenase complex. The both thiamine phosphates inhibit the pyruvate dehydrogenase kinase activity almost similarly in concentrations exceeding 10 microM. The physiological significance of the antagonistic action of ThDP and ThTP on the pyruvate dehydrogenase phosphatase activity is discussed.     

Regulation of Staphylococcal Enterotoxin B: Effect of Thiamine Starvation

During the transition between the exponential and stationary phases of growth, there was a rapid accumulation of both cell-associated and extracellular enterotoxin B. Extracellular enterotoxin was synthesized until the cells entered the stationary phase during which cell-bound toxin was not detected. The differential rate of toxin synthesis relative to that of total protein synthesis was greater at pH 7.7 than at 6.0. Addition of glucose decreased the differential rate of toxin synthesis. This decrease was greater at pH 7.7 than at 6.0. Addition of pyruvate decreased the differential rate at pH 7.7 but not at 6.0. Analysis of the nongaseous end products of glucose and pyruvate metabolism showed that conditions which favor the oxidative decarboxylation of pyruvate also favor the repression of toxin synthesis. Elimination of thiamine from the medium prevented the oxidative decarboxylation of pyruvate by Staphylococcus aureus S-6 and partially or completely reversed the repression of toxin synthesis by glucose and pyruvate. In the absence of an added energy source, thiamine starvation caused a decrease in protein synthesis but an increased differential rate of toxin synthesis which was greater at pH 7.7 than at 6.0. In the absence of thiamine, pyruvate was not metabolized but caused a decrease in the rate of protein synthesis. This resulted in a twofold increase in the differential rate of toxin synthesis. Thus, conditions which altered the oxidative decarboxylation of pyruvate or decreased the rate of protein synthesis increased the rate of enterotoxin B synthesis.     

Acetate metabolism in a pta mutant of Escherichia coli W3110: importance of maintaining acetyl coenzyme A flux for growth and survival.

In order to study the physiological role of acetate metabolism in Escherichia coli, the growth characteristics of an E. coli W3100 pta mutant defective in phosphotransacetylase, the first enzyme of the acetate pathway, were investigated. The pta mutant grown on glucose minimal medium excreted unusual by-products such as pyruvate, D-lactate, and L-glutamate instead of acetate. In an analysis of the sequential consumption of amino acids by the pta mutant growing in tryptone broth (TB), a brief lag between the consumption of amino acids normally consumed was observed, but no such lag occurred for the wild-type strain. The pta mutant was found to grow slowly on glucose, TB, or pyruvate, but it grew normally on glycerol or succinate. The defective growth and starvation survival of the pta mutant were restored by the introduction of poly-beta-hydroxybutyrate (PHB) synthesis genes (phbCAB) from Alcaligenes eutrophus, indicating that the growth defect of the pta mutant was due to a perturbation of acetyl coenzyme A (CoA) flux. By the stoichiometric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis. These results suggest that E. coli excretes acetate due to the pyruvate flux from PTS and that any method which alleviates the oversupply of acetyl CoA would restore normal growth to the pta mutant.     

Guanidine hydrochloride denaturation of human serum albumin originates by local unfolding of some stable loops in domain III

Thiamine triphosphate (ThTP) is found in most organisms and may be an intracellular signal molecule produced in response to stress. We have recently cloned the cDNA coding for a highly specific mammalian 25-kDa thiamine triphosphatase. The enzyme was active in all mammalian species studied except pig, although the corresponding mRNA was present. In order to determine whether the very low ThTPase activity in pig tissues is due to the absence of the protein or to a lack of catalytic efficiency, we expressed human and pig ThTPase in E. coli as GST fusion proteins. The purified recombinant pig GST-ThTPase was found to be 2-3 orders of magnitude less active than human GST-ThTPase. Using site-directed mutagenesis, we show that, in particular, the change of Glu85 to lysine is responsible for decreased solubility and catalytic activity of the pig enzyme. Immunohistochemical studies revealed a distribution of the protein in pig brain very similar to the one reported in rodent brain. Thus, our results suggest that a 25-kDa protein homologous to hThTPase but practically devoid of enzyme activity is expressed in pig tissues. This raises the possibility that this protein may play a physiological role other than ThTP hydrolysis.     

Pyruvate formate lyase and acetate kinase are essential for anaerobic growth of Escherichia coli on xylose

During anaerobic growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under anaerobic conditions, confirming the need for the ATP produced by acetate kinase for anaerobic growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H+ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for anaerobic growth of E. coli on xylose due to energetic constraints.     

Effect of amino acid starvation on glucose transport in two archaeal organisms

When protein synthesis is arrested by amino acid starvation, Escherichia coli wild-type strains show stringent control (SC) over stable RNA (sRNA) accumulation as well as a large number of other growth-related processes. One of the events under SC is transport of metabolites. Thus, under amino acid starvation, E. coli fails to accumulate the non-metabolizable glucose analog alpha-methyl-D-glucoside, whereas isogenic relaxed strains continue to take up this glucose analog. Unlike the Bacteria, most wild-type archaeal strains show relaxed control of sRNA accumulation, although a number of stringent strains have been identified. In order to determine whether stringency in the Archaea affects physiological events different from sRNA accumulation, transport of glucose analogs was examined under amino acid starvation in two stringent archaeal strains, Haloferax volcanii and Sulfolobus acidocaldarius. The experiments were performed with 2-deoxy-D-glucose, which was shown to be transported, but metabolized very limitedly. Unlike E. coli, H. volcanii and S. acidocaldarius continued to transport 2-deoxy-D-glucose under amino acid starvation. Thus, in both Archaea glucose analog transport is not under SC, as it is in E. coli.     

Pig tissues express a catalytically inefficient 25-kDa thiamine triphosphatase: Insight in the catalytic mechanisms of this enzyme

Thiamine triphosphate (ThTP) is found in most organisms and may be an intracellular signal molecule produced in response to stress. We have recently cloned the cDNA coding for a highly specific mammalian 25-kDa thiamine triphosphatase. The enzyme was active in all mammalian species studied except pig, although the corresponding mRNA was present. In order to determine whether the very low ThTPase activity in pig tissues is due to the absence of the protein or to a lack of catalytic efficiency, we expressed human and pig ThTPase in E. coli as GST fusion proteins. The purified recombinant pig GST-ThTPase was found to be 2–3 orders of magnitude less active than human GST-ThTPase. Using site-directed mutagenesis, we show that, in particular, the change of Glu85 to lysine is responsible for decreased solubility and catalytic activity of the pig enzyme. Immunohistochemical studies revealed a distribution of the protein in pig brain very similar to the one reported in rodent brain. Thus, our results suggest that a 25-kDa protein homologous to hThTPase but practically devoid of enzyme activity is expressed in pig tissues. This raises the possibility that this protein may play a physiological role other than ThTP hydrolysis.     

Effect of vitamin concentration on the synthesis of lactate, ethanol, pyruvate, and ethyl acetate in cells of the yeast Dipodascus magnusii

In the yeast Dipodascus magnusii, which is auxotrophic for thiamine and biotin, during cultivation on glucose with excessive thiamine concentration, pyruvate metabolism was shown to result in the synthesis of fermentation products, namely, ethanol and, to a lesser extent, lactate. Substantial synthesis of ethyl acetate was also observed under these conditions. Introduction of nicotinic acid (NA) into the medium resulted in time separation of ethanol and lactate production. It was shown that cultivation of the yeast under biotin deficiency resulted in nearly complete suppression of aerobic production of ethanol and cessation of ethyl acetate synthesis, whereas lactate synthesis was activated as early as in the first hours of cultivation. Upon introduction of NA under these conditions, lactate concentration sharply increased. These results show that the combination of thiamine and biotin with other vitamins can stimulate utilization of the pyruvate pool in yeasts towards formation of considerable amounts of lactate, which is typical only of cells of higher eukaryotes and bacteria.     

Thiamine Triphosphate Synthesis in Rat Brain Occurs in Mitochondria and Is Coupled to the Respiratory Chain

In animals, thiamine deficiency leads to specific brain lesions, generally attributed to decreased levels of thiamine diphosphate, an essential cofactor in brain energy metabolism. However, another far less abundant derivative, thiamine triphosphate (ThTP), may also have a neuronal function. Here, we show that in the rat brain, ThTP is essentially present and synthesized in mitochondria. In mitochondrial preparations from brain (but not liver), ThTP can be produced from thiamine diphosphate and P_i. This endergonic process is coupled to the oxidation of succinate or NADH through the respiratory chain but cannot be energized by ATP hydrolysis. ThTP synthesis is strongly inhibited by respiratory chain inhibitors, such as myxothiazol and inhibitors of the H⁺ channel of F₀F₁-ATPase. It is also impaired by disruption of the mitochondria or by depolarization of the inner membrane (by protonophores or valinomycin), indicating that a proton-motive force (Δp) is required. Collapsing Δp after ThTP synthesis causes its rapid disappearance, suggesting that both synthesis and hydrolysis are catalyzed by a reversible H⁺-translocating ThTP synthase. The synthesized ThTP can be released from mitochondria in the presence of external P_i. However, ThTP probably does not accumulate in the cytoplasm in vivo, because it is not detected in the cytosolic fraction obtained from a brain homogenate. Our results show for the first time that a high energy triphosphate compound other than ATP can be produced by a chemiosmotic type of mechanism. This might shed a new light on our understanding of the mechanisms of thiamine deficiency-induced brain lesions.     

Decrease in culturability of Vibrio cholerae caused by glucose.

The culturability of Vibrio cholerae O1 serotype Inaba strain 569B was decreased by the addition of glucose to cell suspensions in starvation media. A similar effect was observed with sucrose, maltose, and fructose. We term this inhibitory effect glucose shock. It was not observed with arabinose or xylose or with carboxylates, such as acetate and pyruvate. No acidification of the medium occurred in the presence of these carbohydrates. Glucose shock was prevented by the addition of nitrogen or phosphorus sources. In the presence of phosphate, the bacterium produced formic acid from glucose. The phenomenon of glucose shock was also observed in V. cholerae O1 serotype Inaba strain RIMD 2203082 but not in strain RIMD 2203088 (O1 Inaba), IID 936 (O1 Ogawa), or RIMD 2214034 (non-O1). The culturability of Escherichia coli, Enterobacter aerogenes, and Listonella anguillarum did not decrease in starvation media with added glucose. Hence, the phenomenon should have ecological significance in determining the distribution of bacteria in marine ecosystems in situations where carbohydrates are abundant, but nitrogen and phosphorus are limiting.     

Effect of vitamin concentration on the synthesis of lactate, ethanol, pyruvate, and ethyl acetate in cells of the yeast Dipodascus magnusii

In the yeast Dipodascus magnusii, which is auxotrophic for thiamine and biotin, during cultivation on glucose with excessive thiamine concentration, pyruvate metabolism was shown to result in the synthesis of fermentation products, namely, ethanol and, to a lesser extent, lactate. Substantial synthesis of ethyl acetate was also observed under these conditions. Introduction of nicotinic acid (NA) into the medium resulted in time separation of ethanol and lactate production. It was shown that cultivation of the yeast under biotin deficiency resulted in nearly complete suppression of aerobic production of ethanol and cessation of ethyl acetate synthesis, whereas lactate synthesis was activated as early as in the first hours of cultivation. Upon introduction of NA under these conditions, lactate concentration sharply increased. These results show that the combination of thiamine and biotin with other vitamins can stimulate utilization of the pyruvate pool in yeasts towards formation of considerable amounts of lactate, which is typical only of cells of higher eukaryotes and bacteria.     

Thiamine status in humans and content of phosphorylated thiamine derivatives in biopsies and cultured cells

Background

Thiamine (vitamin B1) is an essential molecule for all life forms because thiamine diphosphate (ThDP) is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome) remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines.

Methodology and Principal Findings

Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP])/([Thiamine]+[ThMP]) ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines.

Conclusions and Significance

The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells, suggesting a hitherto unsuspected link between these compounds and cell division or differentiation.     

Screening of pyruvate-producing yeast and effect of nutritional conditions on pyruvate production

AIMS: To find a yeast strain that can overproduce pyruvate and to investigate the effect of nutrients on pyruvate production. METHODS AND RESULTS: Trichosporon cutaneum PD70, a yeast strain that can overproduce pyruvate, was isolated from shake-flask cultures of 132 yeast strains. Pyruvate was measured by the HPLC or DNP method (see Materials and methods). Pyruvate production reached approximately 30.0 +/- 1.0 g l(-1) in basal fermentation medium. Different nutrient supplements had great effects on pyruvate production. Some of the conditions that gave the highest yield are described. CONCLUSIONS: Exogenous thiamine supplement caused a decrease in pyruvate yield. Some amino acids, such as L-arginine, L-isoleucine and L-valine, caused a minor increase in pyruvate yield. Soybean peptone was the most suitable nitrogen source for pyruvate production. A glucose concentration of 15% in fermentation medium gave the highest yield (34.6 g l(-1)) and the highest yield against consumed glucose (0.429 g g(-1)). SIGNIFICANCE AND IMPACT OF THE STUDY: Nutrients have significant impacts on pyruvate production. As a pyruvate overproducing yeast strain independent of exogenous vitamins or amino acids, T. cutaneum PD70 provides an advantage for commercial pyruvate production.     

Between feast and famine: endogenous inducer synthesis in the adaptation of Escherichia coli to growth with limiting carbohydrates.

Escherichia coli adapted to growth with low carbohydrate concentrations bypassed the requirement for exogenous inducer with at least three well-studied sugar regulons. Induction of mgl and gal genes became independent of added galactose in bacteria approaching stationary phase or during continuous culture with micromolar glucose in the medium. Bacteria became independent of exogenous induction because endogenous galactose and cyclic AMP (cAMP) pools were sufficient for high expression of mgl and gal genes under glucose limitation. Limitation-stimulated induction of mgl was dependent on a functional galETK operon for synthesis of the inducer galactose. Intracellular galactose levels were maximal not during starvation (or slow steady-state growth rates approaching starvation) but at fast growth rates with micromolar glucose. The extent of mgl/gal induction correlated better with inducer availability than with cAMP concentrations under all conditions tested. Endogenous inducer accumulation represents an adaptation to low-nutrient environments, leading to derepression of high-affinity transport systems like Mgl essential for bacterial competitiveness at low nutrient concentrations.     

Studies on ATP

The experiments described in this paper serve as a contribution to the solution of the discrepancies which exist in the assay of ATP:thiamine diphosphate phosphotransferase activity (EC 2.7.4.15), presently in use as a tool for the diagnosis of Leigh's disease (SNE, subacute necrotizing encephalomyelopathy). The results obtained with this phosphotransferase assay can, in part, be explained by the presence of thiamine triphosphate (ThTP) in the preparation of thiamine diphosphate (ThDP) used as a substrate, by the inhibition by ATP of the ThTP phosphohydrolase activity, present in fractions of rat brain homogenates, and by the stimulation by ThDP of the ATPase activity. When [2-¹⁴C-thiazole]thiamine was used for the synthesis of [¹⁴C]ThTP in fractions of rat brain, it was found that after chromatographic separation of thiamine and its phosphates,¹⁴C radio-activity could be demonstrated in the ThTP fractions, even in the absence of an enzyme source. Probably a complex is formed between [¹⁴C]thiamine and a phosphate ester which behaves chromatographically as ThTP. It is concluded that the assay system for the measurement of ThTP synthesis in its present form is, in our hands, not suitable for diagnostic purposes.     

Growth of the haploid phase of the myxomycete Physarum flavicomum in defined minimal medium.

The haploid phase (myxamoebae-swarm cells) of the myxomycete Physarum flavicomum grew readily in chemical defined liquid media. The minimal medium contained salts, glucose, biotin, thiamine, hematin, glycine, L-arginine and L-methionine. Cell yields of 1.4 times 10(7) cells/ml were obtained in this medium in aerobic shake culture. These cells consumed about 35 muliters of oxygen/mg protein-hr in the minimal medium. The morphology of cells maintained in this medium appeared to be "normal". L-valine replaced either glycine or L-methionine in the minimal medium but the growth rates and cell yields were reduced. Growth rates increased in media containing four, seven, or fourteen amino acids.