Insulin action on Escherichia coli. Regulation of the adenylate cyclase and phosphotransferase enzymes.

Insulin on Escherichia coli was studied using wild type E. coli B/r and K12 strains and a number of phosphoenolpyruvate phosphotransferase mutants. In vivo, the effects of insulin on the differential rate of tryptophanase synthesis, the rate of alpha-methylglucoside uptake and the rate of growth on glucose were determined in E. coli B/r. In vitro, the effect of insulin on the adenylate cyclase and the phosphotransferase activities was determined using toluenized cell preparations of E. coli B/r, E. coli K12 and phosphotransferase mutant strains. The specificity of insulin action on E. coli was determined using glucagon, vasopressin and somatropin as well as insulin antisera. Results show the specific action of insulin on E. coli, inhibiting tryptophanase induction and adenylate cyclase activity, while stimulating growth on glucose and uptake and phosphorylation of alpha-methylglucoside.     

The Escherichia coli adenylate cyclase complex. Regulation by enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.

The activity of adenylate cyclase of Escherichia coli measured in toluene-treated cells under standard conditions is subject to control by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Sugars such as glucose, which are transported by the PTS, will inhibit adenylate cyclase provided the PTS is functional. An analysis was made of the properties of E. coli strains carrying mutations in PTS proteins. Leaky mutants in the PTS protein HPr are similar to wild-type strains with respect to cAMp regulation; adenylate cyclase activity in toluene-treated cells and intracellular cAMP levels are in the normal range. Furthermore, adenylate cyclase in toluene-treated cells of leaky HPr mutants is inhibited by glucose. In contrast, mutations in the PTS protein Enzyme I result in abnormalities in cAMP regulation. Enzyme I mutants generally have low intracellular cAMP levels. Leaky Enzyme I mutants show an unusual phosphoenolpyruvate-dependent activation of adenylate cyclase that is not seen in Enzyme I⁺ revertants or in Enzyme I deletions. A leaky Enzyme I mutant exhibits changes in the temperature-activity profile for adenylate cyclase, indicating that adenylate cyclase activity is controlled by Enzyme I. Temperature-shift studies suggest a functional complex between adenylate cyclase and a regulator protein at 30 °C that can be reversibly dissociated at 40 °C. These studies further support the model for adenylate cyclase activation that involves phosphoenolpyruvate-dependent phosphorylation of a PTS protein complexed to adenylate cyclase.     

On the regulation of cyclic AMP level in bacteria. II. In vitro regulation of adenylate cyclase activity. Solubilization and reconstitution of a functional membrane-bound adenylate cyclase system responsive to regulation by glucose.

1. The in vitro regulation of the membrane bound adenylate cyclase of Escherichia coli B/r by a variety of carbohydrates and one mammalian hormone was examined. 2. The membrane bound adenylate cyclase was found responsive to regulation by the various growth substrates and to glucagon. 3. Solubilization of the bacterial membrane preparation by a procedure specific for the solubilization of the phosphotransferase enzyme E1 1 to its E1 1 A and E1 1 B subunits was found to be accompanied by the loss of the adenylate cyclase regulation by glucose. 4. Reconstitution of the membrane was found to result in a recovery of the regulative response of the adenylate cyclase to glucose. 5. A model for the intermediate steps in the interaction between glucose and phosphotransferase E1 1 and the adenylate cyclase is discussed.     

The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene

In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIA^Glc, a component of the phosphotransferase system for glucose transport. In strains deficient in EnzymeIIA^Glc, CAMP levels are very low. Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIA^Glc. In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIA^Glc were obtained. The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end. Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIA^Glc. In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished. This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose. Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain.     

Effects of colicins K and E1 on the glucose phosphotransferase system

1. Glycerol-grown cells of Escherichia coli and its mutant uncA, treated with colicin E1 or K, exhibited a several-fold higher level of α-methylglucoside uptake than untreated cells. This stimulation was independent of the carbon source present during the uptake test. In a mutant strain that has elevated levels of α-methylglucoside accumulation the addition of colicin E1 or carbonylcyanide m-chlorophenylhydrazone (CCCP) did not further enhance the uptake.2. Colicins K and E1 decreased the apparent K_m for α-methylglucoside uptake significantly and increased the V about twofold. The exit of the glucoside was severely inhibited by the colicins.3. In the presence of colicins, α-methylglucoside is still accumulated via the phosphoenolpyruvate-phosphotransferase system since no accumulation or phosphorylation occurs in an enzyme I mutant. The colicins increased the relative intracellular concentration of phosphorylated α-methylglucoside, possibly by inhibiting the dephosphorylation reaction, and caused an excretion of this compound.4. The results are interpreted as indicating that energization of the membrane has an inhibitory effect on the phosphotransferase system. Possible modes of action are discussed.     

The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene

In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIA^Glc, a component of the phosphotransferase system for glucose transport. In strains deficient in EnzymeIIA^Glc, CAMP levels are very low. Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIA^Glc. In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIA^Glc were obtained. The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end. Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIA^Glc. In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished. This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose. Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain.     

A possible role of adenylate cyclase in the longterm dietary regulation of insulin secretion from rat islets of Langerhans

1. Adenylate cyclase activity and patterns of insulin release in response to various concentrations of glucose were determined in islets of Langerhans isolated from starving, fed, or glucose-loaded rats. 2. Basal and glucagon-stimulated activities of adenylate cyclase were lower in islets from starved than from fed rats. The minimum glucose concentration required for stimulation of insulin secretion was higher, whereas the maximum secretory response to glucose was lower, in islets from starved than from fed rats. 3. Adenylate cyclase activity in islets of Langerhans obtained from fed rats loaded with glucose by intermittent intravenous or intraperitoneal injections over 5h was significantly higher than that seen in islets from normal fed rats. Islets obtained from glucose-loaded rats required a lower glucose concentration for stimulation of insulin secretion and attained a higher maximal response to glucose stimulation than those derived from fed rats. 4. Incubation in vitro of islets isolated from normal fed rats, for periods of 1 to 24h in the presence of high concentrations of glucose resulted in an activation of adenylate cyclase that occurred progressively from 2 to 7h and which was maintained during 24h of incubation. The increase of adenylate cyclase activity in isolated islets incubated for 4h in the presence of glucose was not prevented by addition of cycloheximide or actinomycin D. Galactose or 2-deoxyglucose was ineffective in increasing adenylate cyclase activity, and pyruvate (20mm) was less effective than glucose. 5. It is suggested that glucose or a glucose metabolite may exert long-term effects on islet cell adenylate cyclase.     

Mutation of the ptsG Gene Results in Increased Production of Succinate in Fermentation of Glucose by Escherichia coli

Escherichia coli NZN111 is blocked in the ability to grow fermentatively on glucose but gave rise spontaneously to a mutant that had this ability. The mutant carries out a balanced fermentation of glucose to give approximately 1 mol of succinate, 0.5 mol of acetate, and 0.5 mol of ethanol per mol of glucose. The causative mutation was mapped to the ptsG gene, which encodes the membrane-bound, glucose-specific permease of the phosphotransferase system, protein EIICB^glc. Replacement of the chromosomal ptsG gene with an insertionally inactivated form also restored growth on glucose and resulted in the same distribution of fermentation products. The physiological characteristics of the spontaneous and null mutants were consistent with loss of function of the ptsG gene product; the mutants possessed greatly reduced glucose phosphotransferase activity and lacked normal glucose repression. Introduction of the null mutant into strains not blocked in the ability to ferment glucose also increased succinate production in those strains. This phenomenon was widespread, occurring in different lineages of E. coli, including E. coli B.     

Subcellular localization and some properties of the adenylate cyclase activity of the yeast, Saccharomyces cerevisiae

Saccharomyces cerevisiae was grown in the presence of 5% (w.v) Glucose and converter to protoplasts. The total particulate material obtained from lysed protoplasts was fractionated by sucrose density gradient ultracentrifugation and the distribution of adenylate cyclase throughout the gradient determined. Adenylate cyclase activity was found to be larger associated whith intracellular particulate fractions. Little activity was found in the plasma membrane-rich fraction.The adenylate cyclase activity was found to be inhibited by F^?, pyrophosphate and aminophylline, whereas glucagon, 5-hydroxytryptamine and concanavalin A were without effect.The enzymic activity appeared to be modulated by “catabolite repressors” (glucose, fructose and α-methylglucoside) as well as by acetate. A possible role for adenylate cyclase in regulating the levels of cyclic AMP in the cell during glucose repression is suggested.     

The effect of calcium on somatostatin inhibition of insulin release and cyclic AMP production in mouse pancreatic islets

The effect of somatostatin on glucose-induced insulin secretion and cyclic AMP accumation in isolated islets from obese, hyperglycemic ob/ob mice was studied in a microperifusion system. The normal biphasic pattern of insulin release as well as the inhibitory pattern of insulin release produced by somatostatin (0.5–1 μg/ml) was matched by similar changes in the intracellular concentration of cyclic AMP. When islets were stimulated by glucose (3 mg/ml) plus 3-isobutyl-1-methylxanthine (0.1 mM), somatostatin (0.5 μg/ml) failed to inhibit insulin secretion or cyclic AMP formation in the second phase whereas in the first phase both parameters were significantly reduced by somatostatin (0.5 μg/ml). In batch-type incubations it was shown that addition of excess calcium (to 6 mM) reversed this inhibition. In the second phase calcium potentiated the (glucose + 3-isobutyl-1-methylxanthine)-stimulated insulin secretion without affecting the cyclic AMP production. This potentiation was inhibited by somatostatin (0.1 μg/ml). Somatostatin (1 μg/ml) inhibited adenylate cyclase activity in islet homogenates. No effect of somatostatin on islet glucose utilization could be demonstrated.The results indicate a dual action of somatostatin in the inhibition of insulin release, one involving the islet adenylate cyclase and one affecting the islet uptake of calcium.     

Study of the Functional Organization of a Novel Adenylate Cyclase Signaling Mechanism of Insulin Action

In this study we continued decoding the adenylate cyclase signaling mechanism that underlies the effect of insulin and related peptides. We show for the first time that insulin signal transduction via an adenylate cyclase signaling mechanism, which is attended by adenylate cyclase activation, is blocked in the muscle tissues of the rat and the mollusk Anodonta cygnea in the presence of: 1) pertussis toxin, which impairs the action of the inhibitory GTP-binding protein (G_i); 2) wortmannin, a specific blocker of phosphatidylinositol 3-kinase; and 3) calphostin C, an inhibitor of different isoforms of protein kinase C. The treatment of sarcolemmal membrane fraction with cholera toxin increases basal adenylate cyclase activity and decreases the sensitivity of the enzyme to insulin. We suggest that the stimulating effect of insulin on adenylate cyclase involves the following stages of hormonal signal transduction cascade: receptor tyrosine kinase → G_iprotein (βγ) → phosphatidylinositol 3-kinase → protein kinase C (ζ?) → G_sprotein → adenylate cyclase → cAMP.     

Transductional construction of aspartase-hyperproducing strain of Escherichia coli B

Two aspartase-overproducing mutants of Escherichia coli B were characterized. Strain EAPc7 had a mutation enhancing aspartase formation in the region of aspartase gene. This mutation did not affect catabolite repression by aspartase. Strain EAPc244 showed a high cAMP content and an elevated adenylate cyclase activity. This mutation was closely linked to the ilv locus and caused the release of catabolite repression for various catabolite repression-sensitive enzymes, resulting in overproduction of adenylate cyclase. This mutation was transduced to an Ile⁻ strain derived from strain EAPc7 using the Ile⁺ selective marker. The constructed strain AT202, having the above 2 mutations, produced about 3-fold and 18-fold more aspartase than did the 2 parent strains and the wild-type strain, respectively, when cultured in the medium used for industrial production of aspartase. Strain AT202 maintained stably high aspartase activity after 30 cell generations. On the other hand, in E. coli K-12 harboring the aspA⁺ recombinant plasmid pYT471 (pBR322-aspA⁺), the activity decreased to the E. coli K-12 level. Hence, strain AT202 is more advantageous for industrial production of l-aspartic acid than cells harboring the aspA⁺-recombinant plasmid pYT471.     

Involvement of the glucose enzymes II of the sugar phosphotransferase system in the regulation of adenylate cyclase by glucose in Escherichia coli.

The nature of the interaction of glucose with toluene-treated cells of Escherichia coli leading to inhibition of adenylate cyclase was examined by the use of analogues. Those analogues with variations of the substituents about carbon atoms 1 or 2 (e.g. alpha-methylglucoside or 2-deoxyglucose) are inhibitory, and they are also substrates of the phosphoenolpyruvate-dependent sugar phosphotransferase system. Analogues with changes in other parts of the molecule (e.g. 3-O-methylglucose or galactose), L-glucose and several disaccharides and pentoses, do not inhibit adenylate cyclase and are not substrates of the phosphotransferase system. This correlation suggests some functional relationship between the adenylate cyclase and phosphotransferase systems. Further studies were done with mutants defective in glucose enzymes II of the phosphotransferase system (designated GPT and MPT); these two activities are measured by phosphorylation of alpha-methyl-glucoside and 2-deoxyglucose, respectively. The wild-type parent phosphorylates both analogues, and both inhibit adenylate cyclase. In the GPT- mutant, alpha-methylglucoside does not inhibit adenylate cyclase and is not phosphorylated, while 2-deoxyglucose is inhibitory and phosphorylated. In the GPT- MPT- double mutant, adenylate cyclase activity is present, but neither alpha-methylglucoside nor 2-deoxyglucose inhibits adenylate cyclase, and neither sugar is phosphorylated. These studies demonstrate that glucose inhibition of adenylate cyclase in toluene-treated cells requires an interaction of this sugar with either the GPT or mpt enzyme II of the phosphotransferase system.     

Recruiting alternative glucose utilization pathways for improving succinate production

The phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) of Escherichia coli was usually inactivated to increase PEP supply for succinate production. However, cell growth and glucose utilization rate decreased significantly with PTS inactivation. In this work, two glucose transport proteins and two glucokinases (Glk) from E. coli and Zymomonas mobilis were recruited in PTS^? strains, and their impacts on glucose utilization and succinate production were compared. All PTS^? strains recruiting Z. mobilis glucose facilitator Glf had higher glucose utilization rates than PTS^? strains using E. coli galactose permease (GalP), which was suggested to be caused by higher glucose transport velocity and lower energetic cost of Glf. The highest rate obtained by combinatorial modulation of glf and glk _{E. coli} (2.13 g/L?h) was 81 % higher than the wild-type E. coli and 30 % higher than the highest rate obtained by combinatorial modulation of galP and glk _{E. coli} . On the other hand, although glucokinase activities increased after replacing E. coli Glk with isoenzyme of Z. mobilis, glucose utilization rate decreased to 0.58 g/L?h, which was assumed due to tight regulation of Z. mobilis Glk by energy status of the cells. For succinate production, using GalP led to a 20 % increase in succinate productivity, while recruiting Glf led to a 41 % increase. These efficient alternative glucose utilization pathways obtained in this work can also be used for production of many other PEP-derived chemicals, such as malate, fumarate, and aromatic compounds.     

Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methanol auxotrophs have proved successful, these studies focused on scarce and expensive co-substrates. Here, we engineered E. coli for methanol-dependent growth on glucose, an abundant and inexpensive co-substrate, via deletion of glucose 6-phosphate isomerase (pgi), phosphogluconate dehydratase (edd), and ribose 5-phosphate isomerases (rpiAB). Since the parental strain did not exhibit methanol-dependent growth on glucose in minimal medium, we first achieved methanol-dependent growth via amino acid supplementation and used this medium to evolve the strain for methanol-dependent growth in glucose minimal medium. The evolved strain exhibited a maximum growth rate of 0.15 h⁻¹ in glucose minimal medium with methanol, which is comparable to that of other synthetic methanol auxotrophs. Whole genome sequencing and ¹³C-metabolic flux analysis revealed the causative mutations in the evolved strain. A mutation in the phosphotransferase system enzyme I gene (ptsI) resulted in a reduced glucose uptake rate to maintain a one-to-one molar ratio of substrate utilization. Deletion of the e14 prophage DNA region resulted in two non-synonymous mutations in the isocitrate dehydrogenase (icd) gene, which reduced TCA cycle carbon flux to maintain the internal redox state. In high cell density glucose fed-batch fermentation, methanol-dependent acetone production resulted in 22% average carbon labeling of acetone from ¹³C-methanol, which far surpasses that of the previous best (2.4%) found with methylotrophic E. coli Δpgi. This study addresses the need to identify appropriate co-substrates for engineering synthetic methanol auxotrophs and provides a basis for the next steps toward industrial one-carbon bioprocessing.     

Maximum yield of foreign lipase in Escherichia coli HB101 limited by duration of protein expression

When a microbial lipase was overexpressed in Escherichia coli HB101, the expression kinetics as represented by the expression rate, duration, and maximum yield of lipase were studied. Lipase synthesis, controlled by the tac promoter, continued for about 4h after IPTG induction. The duration of the expression phase was similar, irrespective of expression rate and yield, which were manipulated by using α-methyl glucose (α-MG), a competitive inhibitor of glucose. By measuring the specific oxygen uptake rate, specific CO₂ evolution rate, specific glucose uptake rate, intracellular protease level and the acetate concentration in the culture, the limited duration of the expression phase was found to be caused by metabolic stress arising from the rapid and massive production of the foreign protein under the strong promoter. Neither the total cell number nor the number of living cells increased substantially after induction, whereas the optical density of the culture gradually increased. The duration of the expression phase was reduced to less than 2 h by the addition of menadione, a redox cycling agent, seemingly due to an acceleration of the energetic flow of the host cells after induction. In contrast, the duration of the expression phase was extended to 8 h in the glucose-starved condition, although the maximum expression yield was much lower than that in the glucose-surplus condition. Therefore, it was suggested that the expression rate after induction determined the maximum expression yield of the foreign lipase gene in E. coli HB101 because of the restrained capacity of foreign protein production.     

Regulation of Arabinose and Xylose Metabolism in Escherichia coli

Bacteria such as Escherichia coli will often consume one sugar at a time when fed multiple sugars, in a process known as carbon catabolite repression. The classic example involves glucose and lactose, where E. coli will first consume glucose, and only when it has consumed all of the glucose will it begin to consume lactose. In addition to that of lactose, glucose also represses the consumption of many other sugars, including arabinose and xylose. In this work, we characterized a second hierarchy in E. coli, that between arabinose and xylose. We show that, when grown in a mixture of the two pentoses, E. coli will consume arabinose before it consumes xylose. Consistent with a mechanism involving catabolite repression, the expression of the xylose metabolic genes is repressed in the presence of arabinose. We found that this repression is AraC dependent and involves a mechanism where arabinose-bound AraC binds to the xylose promoters and represses gene expression. Collectively, these results demonstrate that sugar utilization in E. coli involves multiple layers of regulation, where cells will consume first glucose, then arabinose, and finally xylose. These results may be pertinent in the metabolic engineering of E. coli strains capable of producing chemical and biofuels from mixtures of hexose and pentose sugars derived from plant biomass.The transporters and enzymes in many sugar metabolic pathways are conditionally expressed in response to their cognate sugar or a downstream pathway intermediate. While the induction of these pathways in response to a single sugar has been studied extensively (28), far less is known about how these pathways are induced in response to multiple sugars. One notable exception is the phenomenon observed when bacteria are grown in the presence of glucose and another sugar (10, 15). In such mixtures, the bacteria will often consume glucose first before consuming the other sugar, a process known as carbon catabolite repression (27). The classic example of carbon catabolite repression is the diauxic shift seen in the growth of Escherichia coli on mixtures of glucose and lactose, where the cells first consume glucose before consuming lactose. When the cells are consuming glucose, the genes in the lactose metabolic pathway are not induced, thus preventing the sugar from being consumed. A number of molecules participate in this regulation, including the cyclic AMP receptor protein (CRP), adenylate cyclase, cyclic AMP (cAMP), and EIIA from the phosphoenolpyruvate:glucose phosphotransferase system (PTS) (33). In addition to lactose, the metabolic genes for many other sugars are subject to catabolite repression by glucose in E. coli (27). While the preferential utilization of glucose is well known, it is an open question whether additional hierarchies exist among other sugars.Recently, substantial effort has been directed toward developing microorganisms capable of producing chemicals and biofuels from plant biomass (1, 34, 42). After glucose, l-arabinose and d-xylose are the next most abundant sugars found in plant biomass. Therefore, a key step in producing various chemicals and fuels from plant biomass will be the engineering of strains capable of efficiently fermenting these three sugars. However, one challenge concerns catabolite repression, which prevents microorganisms from fermenting these three sugars simultaneously and, as a consequence, may decrease the efficiency of the fermentation process. E. coli cells will first consume glucose before consuming either arabinose or xylose. As in the case of lactose, the genes in the arabinose and xylose metabolic pathways are not expressed when glucose is being consumed. In addition to glucose catabolite repression, a second hierarchy, between arabinose and xylose, appears to exist. Kang and coworkers have observed that the genes in the xylose metabolic pathway were repressed when cells were grown in a mixture of arabinose and xylose (21). Hernandez-Montalvo and coworkers also observed that E. coli utilizes arabinose before xylose (19). While a number of strategies exist for breaking the glucose-mediated repression of arabinose and xylose metabolism (8, 16, 19, 31), none exist for breaking the arabinose-mediated repression of xylose metabolism. Moreover, little is known about this repression beyond the observations made by these researchers.In this work, we investigate how the arabinose and xylose metabolic pathways are jointly regulated. We demonstrate that E. coli will consume arabinose before consuming xylose when it is grown in a mixture of the two sugars. Consistent with a mechanism involving catabolite repression, the genes in the xylose metabolic pathway are repressed in the presence of arabinose. We found that this repression is AraC dependent and is most likely due to binding by arabinose-bound AraC to the xylose promoters, with consequent inhibition of gene expression.