Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase.

Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion.     

Studies of regulatory metabolism in Moniezia expansa: general considerations.

The activities of selected enzymes in the branched metabolic pathway to succinate or lactate were determined in cytosol and mitochondrial fractions. The enzymes of lowest activity in the cytosol, and thus possibly regulatory, are phosphofructokinase and pyruvate kinase. Malic enzyme activity could scarcely be detected in either compartment; phosphoenolpyruvate carboxykinase and malate dehydrogenase occur in both. The end products of metabolism are succinate and lactate; under anaerobic conditions lactate production increases whereas succinate production shows a small decrease. The presence of glucose in the medium does not influence the change, but causes an increase in total endproduct accumulation. Levels of metabolic intermediates in worms incubated aerobically and anaerobically are presented, and ‘cross-over’ plots and calculations of apparent equilibrium constants identify hexokinase, phosphofructokinase and pyruvate kinase as regulatory. Under aerobic conditions a large increase in the size of the malate pool is observed suggesting that the depression of lactate production is produced by its inhibitory effect on pyruvate kinase. Adenine nucleotide levels are maintained whether or not the worm is incubated under anaerobic conditions.     

Properties of an inducible C 4 -dicarboxylic acid transport system in Bacillus subtilis

The transport of the tricarboxylic acid cycle C(4)-dicarboxylic acids was studied in both the wild-type strain and tricarboxylic acid cycle mutants of Bacillus subtilis. Active transport of malate, fumarate, and succinate was found to be inducible by these dicarboxylic acids or by precursors to them, whereas glucose or closely related metabolites catabolite-repressed their uptake. l-Malate was found to be the best dicarboxylic acid transport inducer in succinic dehydrogenase, fumarase, and malic dehydrogenase mutants. Succinate and fumarate are accumulated over 100-fold in succinic dehydrogenase and fumarase mutants, respectively, whereas mutants lacking malate dehydrogenase were unable to accumulate significant quantities of the C(4)-dicarboxylic acids. The stereospecificity of this transport system was studied from a comparison of the rates of competitive inhibition of both succinate uptake and efflux in a succinate dehydrogenase mutant by utilizing thirty dicarboxylic acid analogues. The system was specific for the C(4)-dicarboxylic acids of the tricarboxylic acid cycle, neither citrate nor alpha-ketoglutarate were effective competitive inhibitors. Of a wide variety of metabolic inhibitors tested, inhibiors of oxidative phosphorylation and of the formation of proton gradients were the most potent inhibitors of transport. From the kinetics of dicarboxylic acid transport (K(m) approximately 10(-4) M for succinate or fumarate in succinic acid dehydrogenase and fumarase mutants) and from the competitive inhibition studies, it was concluded that an inducible dicarboxylic acid transport system mediates the entry of malate, fumarate, or succinate into B. subtilis. Mutants devoid of alpha-ketoglutarate dehydrogenase were shown to accumulate both alpha-ketoglutarate and glutamate, and these metabolites subsequently inhibited the transport of all the C(4)-dicarboxylic acids, suggesting a regulatory role.     

Molybdenum cofactor negative mutants of Escherichia coli use citrate anaerobically

Anaerobically, Escherichia coli cannot grow using either glycerol or citrate as sole carbon and energy source. However, it has been reported that a mixture of glycerol and citrate will support growth. We have found that wild-type strains of E. coli K-12 do not grow on glycerol plus citrate anaerobically. However, growth eventually occurs due to the frequent appearance of mutants. We found that such Cit+ mutants were defective in anaerobic respiration with nitrate or trimethylamine-N-oxide and were chlorate resistant (i.e. molybdenum cofactor deficient). Conversely, well characterized mutants in any of chlA, B, D, E, G and N were also able to use citrate anaerobically. No anaerobic growth differences between wild type and chl mutants were observed either with fermentable sugars or with glycerol plus fumarate or glycerol plus tartrate. Citrate lyase was induced anaerobically by citrate and repressed by glucose in both wild type strains and chl mutants. Furthermore, levels of citrate lyase, fumarate reductase, malate dehydrogenase, fumarase and alcohol dehydrogenase were similar in both types of strains under anaerobic conditions. It is conceivable that a functioning molybdenum cofactor prevents use of citrate by keeping citrate lyase in the inactive form.     

Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis

Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli. The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E. coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively. Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns. The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme. In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH. Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.     

Isolation and characterization of yeast mutants defective in intermediary carbon metabolism and in carbon catabolite derepression

Summary Yeast mutants deficient in the constitutive ADHI (adc1) were used for the isolation of mutants with deficiencies of the intermediary carbon metabolism, and of mutants defective in carbon catabolite derepression. Mutants were recognized by their inability to grow on YEP-glycerol and/or on ethanol synthetic complete medium. They were either defective in isocitrate lyase (icl1), succinate dehydrogenase (sdh1), or malate dehydrogenase (mdh1, mdh2), mdh-mutants could not uniformely be appointed to one of the known MDH isozymes. Homozygous mdh and sdh1 diploids are unable to sporulate.Three gene loci could be identified by mutants pleiotropically defective in many or all of the enzymes tested. In ccr1 mutants, derepression of isocitrate lyase, fructose-1,6-diphosphatase, ADHII and possibly of the cytoplasmic MDH is prevented, whereas the mitochondrial TCA-cycle enzymes, succinate dehydrogenase and malate dehydrogenase, are not significantly affected. CCR2 and CCR3 have quite similar action spectra. Both genes are obviously necessary for derepression of all enzymes tested. It could be shown that ccr1, ccr2 and ccr3 mutants are not respiratory deficient.     

The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli.

Mutants of Escherichia coli K12 have been isolated that grow on media containing pyruvate of proline as sole carbon sources despite the presence of 10 or 50 mM-sodium fluoroacetate. Such mutants lack either acetate kinase [ATP: acetate phosphotransferase; EC 2.7.2.1] or phosphotransacetylase [acetyl-CoA: orthophosphate acetyltransferase; EC 2.3.1.8] activity. Unlike wild-type E. coli, phosphotransacetylase mutants do not excrete acetate when growing aerobically or anaerobically on glucose; their anaerobic growth on this sugar is slow. The genes that specify acetate kinase (ack) and phosphotransacetylase (pta) activities are cotransducible with each other and with purF and are thus located at about min 50 on the E. coli linkage map. Although Pta- and Ack- mutants are greatly impaired in their growth on acetate, they incorporate [2-14C]acetate added to cultures growing on glycerol, but not on glucose. An inducible acetyl-CoA synthetase [acetate: CoA ligase (AMP-forming); EC 6.2.1.1] effects this uptake of acetate.     

Regulation of the dicarboxylic acid part of the citric acid cycle in Bacillus subtilis.

The regulation of alpha-ketogluterate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase, and malic enzyme has been studied in Bacillus subitilis. The levels of these enzymes increase rapidly during late exponential phase in a complex medium and are maximal 1 to 2 h after the onset of sporulation. Regulation of enzyme synthesis has been studied in the wild type and different citric acid cycle mutants by adding various metabolites to the growth medium. Alpha-ketoglutarate dehydrogenase is induced by glutamate or alpha-ketoglutarate; succinate dehydrogenase is repressed by malate; and fumarase and malic enzyme are induced by fumarate and malate, respectively. The addition of glucose leads to repression of the citric acid cycle enzymes whereas the level of malic enzyme is unaffected. Studies on the control of enzyme activities in vitro have shown that alpha-ketoglutarate dehydrogenase and succinate dehydrogenase are inhibited by oxalacetate. Enzyme activities are also influenced by the energy level, expressed as the energy charge of the adenylate pool. Isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malic enzyme are inhibited at high energy charge values, whereas malate dehydrogenase is inhibited at low energy charge. A survey of the regulation of the citric acid cycle in B.subtilis, based on the present work and previously reported results, is presented and discussed.     

Membrane enzymes associated with the dissimilation of some citric acid cycle substrates and production of extracellular oxidation products in chemostat cultures of Pseudomonas fluorescens

Enzyme activities forming extracellular products from succinate, fumarate, and malate were examined using washed cell suspensions of Pseudomonas fluorescens from chemostat cultures. Membrane-associated enzyme activities (glucose, gluconate, and malate dehydrogenases), producing large accumulations of extracellular oxidation products in carbon-excess environments, have previously been found in P. fluorescens. Investigations carried out here have demonstrated the presence in this microorganism of a malic enzyme activity which produces extracellular pyruvate from malate in carbon-excess environments. Although the three membrane dehydrogenase enzymes decrease significantly in carbon-limited chemostat cultures, malic enzyme activity was found to increase fourfold under these conditions. The regulation of malate dehydrogenase and malic enzyme by malate or succinate was similar. Malate dehydrogenase increased and malic enzyme decreased in carbon-excess cultures. The opposite effect was observed in carbon-limited cultures. When pyruvate or glucose was used as the carbon source, malate dehydrogenase was regulated similarly by the available carbon concentration, but malic enzyme activity producing extracellular pyruvate was not detected. While large accumulations of extracellular oxalacetate and pyruvate were produced in malate-excess cultures, no extracellular oxidation products were detected in succinate-excess cultures. This may be explained by the lack of detectable activity for the conversion of added external succinate to extracellular fumarate and malate in cells from carbon-excess cultures. In cells from carbon-limited (malate or succinate) cultures, very active enzymes for the conversion of succinate to extracellular fumarate and malate were detected. Washed cell suspensions from these carbon-limited cultures rapidly oxidized added succinate to extracellular pyruvate through the sequential action of succinate dehydrogenase, fumarase, and malic enzyme. Succinate dehydrogenase and fumarase activities producing extracellular products were not detected in cells from chemostat cultures using pyruvate or glucose as the carbon source. Uptake activities for succinate, malate, and pyruvate also were found to increase in carbon-limited (malate or succinate) and decrease in carbon-excess cultures. The role of the membrane-associated enzymes forming different pathways for carbon dissimilation in both carbon-limited and carbon-excess environments is discussed.     

Anaerobic growth defects resulting from gene fusions affecting succinyl-CoA synthetase in Escherichia coli K12

Summary Insertion of the fusion-generating phage Mud1 (Ap, lacZ) yielded two similar isolates, DC511 and DC512, which were unable to grow aerobically on acetate or alphaketoglutarate but which could use succinate, malate, fumarate, glycerol, and various sugars. These mutants were unable to grow anaerobically on most sugars unless provided with methionine, lysine, and delta-aminolevulinic acid, all of which require succinyl-CoA for their synthesis. The insertions of both mutants mapped at 17 min, in the suc operon. Enzyme assays indicated a lack of succinyl-CoA synthetase; however, full activity of the alpha-ketoglutarate dehydrogenase was retained. Beta-galactosidase expression by strains containing these gene fusions was reduced under anaerobic conditions. In aerobically grown cultures, both fusions were induced about fivefold in the presence of acetate. This type of regulation would be expected of a Krebs cycle enzyme.     

Effect of the carbon source and cyclic AMP on isocitrate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase in Klebsiella pneumoniae C3

When strain C3 of Klebsiella pneumoniae is grown on a minimal medium with excess glucose, isocitrate dehydrogenase, malate dehydrogenase, and succinate dehydrogenase specific activities increase in the last period of the exponential growth phase and in the beginning of the stationary phase. Glucose exhaustion does not alter the development of malate dehydrogenase and succinate dehydrogenase, but specific activities are higher than those obtained with excess glucose. In contrast, glucose exhaustion can be correlated with a decrease of isocitrate dehydrogenase specific activity in the stationary phase. Induction of strain C3 isocitrate dehydrogenase by glucose in complex medium and repression by cAMP in mineral medium were observed. Glucose induction and the NADP/NADPH ratio are suggested as regulatory mechanisms controlling isocitrate dehydrogenase synthesis in the Enterobacteriaceae, but the former appears to be restricted to some Klebsiella strains.     

Proteins of the Inner Membrane of Escherichia coli: Changes in Composition Associated with Anaerobic Growth and Fumarate Reductase Amber Mutation

The inner membrane fractions of Escherichia coli grown anaerobically and aerobically were isolated, and their proteins were compared by electrophoresis in polyacrylamide gels. To maximimize the differences between the preparations, the anaerobic cultures were grown on complex medium with added glucose, but glucose was omitted from the aerobic cultures to prevent catabolite repression. The pattern of bands in the two types of preparation differed considerably, and changes in approximately 20 components were observed. In particular, the band identified as succinate dehydrogenase in aerobic preparations was greatly reduced in anaerobic preparations. Mutants lacking fumarate reductase were isolated, and inner membrane preparations of an frd amber mutant were deficient in a major component of 75,000 daltons and possibly a minor one of 87,500 daltons. The former was also present in greater amounts in anaerobic preparations and could represent a fumarate reductase subunit.     

Inactivation of glycerol dehydrogenase of Klebsiella pneumoniae and the role of divalent cations.

Anaerobically induced NAD-linked glycerol dehydrogenase of Klebsiella pneumoniae for fermentative glycerol utilization was reported previously to be inactivated in the cell during oxidative metabolism. In vitro inactivation was observed in this study by incubating the purified enzyme in the presence of O2, Fe2+, and ascorbate or dihydroxyfumarate. It appears that O2 and the reducing agent formed H2O2 and that H2O2 reacted with Fe2+ to generate an activated species of oxygen which attacked the enzyme. The in vitro-oxidized enzyme, like the in vivo-inactivated enzyme, showed an increased Km for NAD (but not glycerol) and could no longer be activated by Mn2+ which increased the Vmax of the native enzyme but decreased its apparent affinity for NAD. Ethanol dehydrogenase and 1,3-propanediol oxidoreductase, two enzymes with anaerobic function, also lost activity when the cells were incubated aerobically with glucose. However, glucose 6-phosphate dehydrogenase (NADP-linked), isocitrate dehydrogenase, and malate dehydrogenase, expected to function both aerobically and anaerobically, were not inactivated. Thus, oxidative modification of proteins in vivo might provide a mechanism for regulating the activities of some anaerobic enzymes.     

A yeast mutant with glucose-resistant formation of mitochondrial enzymes

Summary Yeast mutants with glucose-insensitive formation of mitochondrial enzymes were isolated starting with a strain completely lacking alcohol dehydrogenase activity. The mutations could uniquely be attributed to a single nuclear gene, designated CCR80. They were largely dominant. Glucose-resistant enzyme formation was most prominent with regard to mitochondrial enzymes succinate dehydrogenase and NADH: cytochrome c oxidoreductase. The effect of CCR80 ^r mutations was rather small but significant on the gluconeogenetic enzymes isocitrate lyase, malate synthase and fructose-1,6-bisphosphatase and on invertase synthesis. The repressive effect of maltose in CCR80 ^r mutants was also reduced showing that glucose-resistance is not caused by a mere hexose uptake defect. This regulatory disorders were not accompanied by reduced levels of glycolytic enzymes or drastically altered levels of glycolytic intermediates.Aerobic fermentation of glucose was almost completely inhibited in the mutants; anaerobic glucose degradation was reduced but not completely abolished. Therefore, the mutants appear to be altered in the regulation of glycolysis. A largely glucose-resistant synthesis of respiratory enzymes is obviously a corollary of this alteration.     

Nitrate reduction in Aerobacter aerogenes

Summary Mutants of A. aerogenes blocked in aerobic and anaerobic nitrate assimilation and deficient in the reduction of nitrate and chlorate were found to give a positive methylred reaction and no gas formation from glucose. Resting cells, grown anaerobically in minimal medium with glucose, did not show gas production from formate. These results show that these mutants are also deficient in formate hydrogenylase. Revertants could readily be obtained by plating on minimal medium with nitrate as sole nitrogen source, indicating that in these mutants a pleiotropic point mutation is present, which affects both nitrate reductase and formate hydrogeny lase. It is suggested, that these mutants are deficient in the formation of an enzyme complex or particle on which these enzymes are present.     

A succinate transport mutant of Bradyrhizobium japonicum forms ineffective nodules on soybeans.

Biochemical evidence has shown that dicarboxylic acids actively support symbiotic nitrogen fixation by both fast- and slow-growing Rhizobium. Mutants defective in the active uptake of succinate have been previously described only in species of the fast-growing rhizobium. This article is a report on the isolation of mutants defective in dicarboxylate transport in a slow-growing species of rhizobium, Bradyrhizobium japonicum. One of these presumptive dicarboxylate transport mutants, GTS, was characterized further. Cultured GTS was unable to accumulate [14C]succinate above background levels but possessed normal rates of malate dehydrogenase, fumarase, and hydroxybutyrate dehydrogenase activities. When inoculated onto soybeans, GTS produced a Nod+, Fix- phenotype. The bacteroids isolated from these nodules failed to accumulate labelled succinate. Electron micrographs of nodules formed by inoculation with GTS appeared normal with the exceptions of more prominent peribacteroid spaces in the infected cells and the appearance of starch granules in the noninfected cells. The phenotypical and morphological changes observed for B. japonicum are similar to those previously reported for the fast-growing species.     

Mutants of Saccharomyces cerevisiae resistant to carbon catabolite repression.

Summary Mutants with defective carbon catabolite repression have been isolated in the yeast Saccharomyces cerevisiae using a selective procedure. This was based on the fact that invertase is a glucose repressible cell wall enzyme which slowly hydrolyses raffinose to yield fructose and that the inhibitory effects of 2-deoxyglucose can be counteracted by fructose. Repressed cells were plated on a raffinose-2-deoxyglucose medium and the resistant cells growing up into colonies were tested for glucose non-repressible invertase and maltase. The yield of regulatory mutants was very high. All were equally derepressed for invertase and maltase, no mutants were obtained with only non-repressible invertase synthesis which was the selected function. A total of 61 mutants isolated in different strains were allele tested and could be attributed to three genes. They were all recessive. Mutants in one gene had reduced hexokinase activities, the other class, located in a centromere linked gene, had elevated hexokinase levels and was inhibited by maltose. Mutants in a third gene were isolated on a 2-deoxyglucose galactose medium and had normal hexokinase levels. A partial derepression was observed for malate dehydrogenase in all mutants. Isocitrate lyase, however, was still fully repressible.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : I. Oxidative Activities with Soluble Substrates

This paper describes experiments conducted with membranous and soluble fractions obtained from Escherichia coli that had been grown on succinate, malate, or enriched glucose media. Oxidase and dehydrogenase activities were studied with the following substrates: nicotinamide adenine dinucleotide, reduced form (NADH), nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), succinate, malate, isocitrate, glutamate, pyruvate, and α-ketoglutarate. Respiration was virtually insensitive to poisons that are commonly used to inhibit mitochondrial systems, namely, rotenone, antimycin, and azide. Succinate dehydrogenase and NADH, NADPH, and succinate oxidases were primarily membrane-bound whereas malate, isocitrate, and NADH dehydrogenases were predominantly soluble. It was observed that E. coli malate dehydrogenase could be assayed with the dye 2,6-dichlorophenol indophenol, but that porcine malate dehydrogenase activity could not be assayed, even in the presence of E. coli extracts. The characteristics of E. coli NADH dehydrogenase were shown to be markedly different from those of a mammalian enzyme. The enzyme activities for oxidation of Krebs cycle intermediates (malate, succinate, isocitrate) did not appear to be under coordinate genetic control.     

Evidence for succinate production by reduction of fumarate during hypoxia in isolated adult rat heart cells

It has been demonstrated that perfusion of myocardium with glutamic acid or tricarboxylic acid cycle intermediates during hypoxia or ischemia, improves cardiac function, increases ATP levels, and stimulates succinate production. In this study isolated adult rat heart cells were used to investigate the mechanism of anaerobic succinate formation and examine beneficial effects attributed to ATP generated by this pathway. Myocytes incubated for 60 min under hypoxic conditions showed a slight loss of ATP from an initial value of 21 +/- 1 nmol/mg protein, a decline of CP from 42 to 17 nmol/mg protein and a fourfold increase in lactic acid production to 1.8 +/- 0.2 mumol/mg protein/h. These metabolite contents were not altered by the addition of malate and 2-oxoglutarate to the incubation medium nor were differences in cell viability observed; however, succinate release was substantially accelerated to 241 +/- 53 nmol/mg protein. Incubation of cells with [U-14C]malate or [2-U-14C]oxoglutarate indicates that succinate is formed directly from malate but not from 2-oxoglutarate. Moreover, anaerobic succinate formation was rotenone sensitive. We conclude that malate reduction to succinate occurs via the reverse action of succinate dehydrogenase in a coupled reaction where NADH is oxidized (and FAD reduced) and ADP is phosphorylated. Furthermore, by transaminating with aspartate to produce oxaloacetate, 2-oxoglutarate stimulates cytosolic malic dehydrogenase activity, whereby malate is formed and NADH is oxidized. In the form of malate, reducing equivalents and substrate are transported into the mitochondria where they are utilized for succinate synthesis.     

Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation.

Escherichia coli mutants lacking alcohol dehydrogenase (adh mutants) cannot synthesize the fermentation product ethanol and are unable to grow anaerobically on glucose and other hexoses. Similarly, phosphotransacetylase-negative mutants (pta mutants) neither excrete acetate nor grow anaerobically. However, when a strain carrying an adh deletion was selected for anaerobic growth on glucose, spontaneous pta mutants were isolated. Strains carrying both adh and pta mutations were observed by in vivo nuclear magnetic resonance and shown to produce lactic acid as the major fermentation product. Various combinations of adh pta double mutants regained the ability to grow anaerobically on hexoses, by what amounts to a homolactic fermentation. Unlike wild-type strains, such adh pta double mutants were unable to grow anaerobically on sorbitol or on glucuronic acid. The growth properties of strains carrying various mutations affecting the enzymes of fermentation are discussed in terms of redox balance.