The regulation of carbohydrate metabolism in Klebsiella aerogenes NCTC 418 organisms,growing in chemostat culture

Klebsiella aerogenes NCTC 418 was grown in chemostat cultures (D=0.17 hr^-1; pH 6.8; 35° C) that were, successively, carbon-, sulphate-, ammonia-, and phosphate-limited, and which contained as the sole carbon-substrate first glucose, then glycerol, mannitol and lactate. Quantitative analyses of carbon-substrate used and products formed allowed carbon balances to be constructed and direct comparisons to be made of the effciency of substrate utilization. With all sixteen cultures, carbon recoveries of better than 90% were obtained.Optimum utilization of the carbon substrate was invariably found with the carbon-limited cultures, the sole products being organisms and carbon dioxide. But the extent to which excess substrate was over-utilized varied markedly with both the nature of the growth-limitation and the identity of the carbon-substrate. In general, sulphate-, ammonia-, and phosphate-limited cultures utilized glycerol more efficiently than mannitol, mannitol better than lactate, and glucose least efficiently. Glucose-containing cultures also synthesized some extracellular polysaccharide.When the carbon source was in excess, a range of acidic compounds generally were excreted. Sulphate-limited cultures, growing on glucose, excreted much pyruvate and acetate, whereas similarly-limited cultures growing on glycerol, mannitol or lactate produced only acetate. Ammonialimited cultures invariably excreted 2-oxoglutarate and acetate, whereas phosphate-limited cultures produced gluconic acid, 2-ketogluconic acid and acetate, when growing on glucose, but only acetate when growing on mannitol or lactate.From the rates of substrate and oxygen consumption, and the rates of cell synthesis, yield values for both substrate and oxygen were calculated. These showed different trends, but were similar in being highest under carbon-limitation and substantially lower under all other limitations.The physiological significance of these findings, and the probable nature of the regulatory mechanisms underlying overflow metabolism are discussed.     

Growth yields and energy generation by Campylobacter sputorum subspecies bubulus during growth in continuous culture with different hydrogen acceptors

Campylobacter sputorum subspecies bubulus was grown in continuous culture with excess of l-lactate or formate, and growth-limiting amounts of oxygen, fumarate, nitrate or nitrite. l-Lactate was oxidized to acetate, fumarate was reduced to succinate, and nitrate and nitrite were reduced to ammonia. The Y _lactate values (g dry weight bacteria/g mol lactate) for the respective hydrogen acceptors were much higher than the Y _formate values. Steady state cultures on formate and nitrite could only be obtained at a low dilution rate and low nitrite concentrations in the growth medium. In H⁺/2e measurements with lactate-grown cells proton ejections were observed with lactate or pyruvate as a hydrogen donor, and oxygen or hydrogen peroxide as a hydrogen acceptor. Proton ejection was also observed with pyruvate and nitrate. Proton ejection did not occur with lactate and nitrate, neither with lactate or pyruvate and fumarate or nitrite. With formate as a hydrogen donor acidification occurred with all hydrogen acceptors mentioned. It has been concluded that during growth on lactate and fumarate or nitrite substrate level phosphorylation at acetate formation is the sole ATP-generating system. Growth on formate and fumarate or nitrite is explained by a proton gradient generated as a result of oxidation of formate at the periplasmic side of the cytoplasmic membrane. With oxygen and nitrate additional ATP is formed by electron transport-linked phosphorylation. The low molar growth yields with formate are explained by the observation that formate-grown cells had a great permeability to protons.Abbreviations H⁺/2e value number of protons ejected per electron pair transported in the respiratory system - P/2e value mol of ATP formed per electron pair transported in the respiratory system - CCCP carbonyl cyanide m-chlorophenyl-hydrazone     

Interstitial lactate,lactate/pyruvate and glucose in rat muscle before,during and in the recovery from global hypoxia

Background

Hypoxia results in an imbalance between oxygen supply and oxygen consumption. This study utilized microdialysis to monitor changes in the energy-related metabolites lactate, pyruvate and glucose in rat muscle before, during and after 30 minutes of transient global hypoxia. Hypoxia was induced in anaesthetised rats by reducing inspired oxygen to 6% O₂ in nitrogen.

Results

Basal values for lactate, the lactate/pyruvate ratio and glucose were 0.72 ± 0.04 mmol/l, 10.03 ± 1.16 and 3.55 ± 0.19 mmol/l (n = 10), respectively. Significant increases in lactate and the lactate/pyruvate ratio were found in the muscle after the induction of hypoxia. Maximum values of 2.26 ± 0.37 mmol/l for lactate were reached during early reperfusion, while the lactate/pyruvate ratio reached maximum values of 35.84 ± 7.81 at the end of hypoxia. Following recovery to ventilation with air, extracellular lactate levels and the lactate/pyruvate ratio returned to control levels within 30-40 minutes. Extracellular glucose levels showed no significant difference between hypoxia and control experiments.

Conclusions

In our study, the complete post-hypoxic recovery of metabolite levels suggests that metabolic enzymes of the skeletal muscle and their related cellular components may be able to tolerate severe hypoxic periods without prolonged damage. The consumption of glucose in the muscle in relation to its delivery seems to be unaffected.

     

Isolation and characterization of Dehalospirillum multivorans gen. nov., sp. nov., a tetrachloroethene-utilizing,strictly anaerobic bacterium

A strictly anaerobic bacterium dechlorinating tetrachloroethene (perchloroethylene, PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE) was isolated from activated sludge with pyruvate plus PCE as energy substrates. The organism, called Dehalospirillum multivorans, is a gram-negative spirillum that does not form spores. The G+C content of the DNA was 41.5 mol%. According to 16S rRNA gene sequence analysis, D. multivorans represents a new genus and a new species belonging to the epsilon subdivision of Proteobacteria. Quinones, cytochromes b and c, and corrinoids were extracted from the cells. D. multivorans grew in defined medium with PCE and H₂ as sole energy sources and acetate as carbon source; the growth yield under these conditions was 1.4g of cell protein per mol chloride released. Alternatively to PCE, fumarate and nitrate could serve as electron acceptors; sulfate could not replace fumarate, nitrate, or PCE in this respect. In addition to H₂, the organism utilized a variety of electron donors for dechlorination (pyruvate, lactate, ethanol, formate, glycerol). Upon growth on pyruvate plus PCE, the main fermentation products formed were acetatc, lactate, DCE, and H₂. At optimal pH (7.3–7.6) and temperature (30°C), and in the presence of pyruvate (20mM) and PCE (160M), a dechlorination rate of about 50 nmol min^-1 (mg cell protein)^-1 and a doubling time of about 2.5h were obtained with growing cultures. The ability to reduce PCE to DCE appears to be constitutive under the experimental conditions applied since cultures growing in the absence of PCE for several generations immediately started dechlorination when transferred to a medium containing PCE. The organism may be useful for bioremediation of environments polluted with tetrachloroethene.Abbreviations PCE Perchloroethylene, tetrachloroethene - TCE Trichloroethene - DCE cis-1,2-Dichloroethene - CHC Chlorinated hydrocarbon     

Heterotrophic growth of Thiobacillus A2 on sugars and organic acids

Thiobacillus A2 grew on a number of organic acids, pentoses, hexoses and -linked disaccharides, but not on -linked disaccharides or galactosides. Growth was slow on glucose, although fast-growing strains were selectively isolated. Additive growth rates occurred on glucose and galactose; growth on glucose with fructose, pyruvate or gluconate was biphasic rather than diauxic; fructose was used preferentially over glucose; slow growth on glucose was accelerated by some disaccharides; growth on acetate, fumarate or succinate with glucose gave diauxic growth with preferential use of the acid and repression of glucose incorporation. Acetate and succinate tended to be used preferentially even with cultures grown on them in mixture with fructose or sucrose.     

Quantitative aspects of glucose metabolism by Escherichia coli B/r,grown in the presence of pyrroloquinoline quinone

Escherichia coli B/r was grown in chemostat cultures under various limitations with glucose as carbon source. Since E. coli only synthesized the glucose dehydrogenase (GDH) apo-enzyme and not the appropriate cofactor, pyrroloquinoline quinone (PQQ), no gluconate production could be observed. However, when cell-saturating amounts of PQQ (nmol to mol range) were pulsed into steady state glucose-excess cultures of E. coli, the organisms responded with an instantaneous formation of gluconate and an increased oxygen consumption rate. This showed that reconstitution of GDH in situ was possible.Hence, in order to examine the influence on glucose metabolism of an active GDH, E. coli was grown aerobically in chemostat cultures under various limitations in the presence of PQQ. It was found that the presence of PQQ indeed had a sizable effect: at pH 5.5 under phosphate- or sulphate- limited conditions more than 60% of the glucose consumed was converted to gluconate, which resulted in steady state gluconate concentrations up to 80 mmol/l. The specific rate of gluconate production (0.3–7.6 mmol·h^-1·(g dry wt cells)^-1) was dependent on the growth rate and the nature of the limitation. The production rate of other overflow metabolites such as acetate, pyruvate, and 2-oxoglutarate, was only slightly altered in the presence of PQQ. The fact that the cells were now able to use an active GDH apparently did not affect apo-enzyme synthesis.Abbreviations HEPES N-2-hydroxy-ethylpiperazine-N-2-ethane sulphonic acid - MES 2-morpholinoethane sulphonic acid - PQQ pyrroloquinoline quinone (systematic name: 2,7,9-tricarboxy-1H-pyrrolo-(2,3-f)-quinoline-4,5-dione) - WB Wurster's Blue (systematic name: 1,4-bis-(dimethylamino)-benzene perchlorate     

Kinetics of conversion ofNeisseria gonorrhoeae to resistance to complement by cytidine 5′-monophospho-N-acetyl neuraminic acid

Freshly isolated gonococci upon subculture are readily lysed by normal human serum although a few strains remain inherently resistant to the complement activity. The sensitive gonococci can be converted to serum resistance by incubation with a host derived factor referred to as cytidine 5-monophospho-N-acetylneuraminic acid (CMP-NANA). These gonococci resist complement mediated killing due to their sialylation of an epitope structure on a component of lipo-oligosaccharide (LOS). In the present study, the kinetics of conversion to serum resistance by the action of sialyltransferase (STase) inNeisseria gonorrhoeae was followed with very low concentrations of CMP-NANA. This conversion could not be perceived at 2×10^–3 nmol.ml^–1 but was fully attainable from 8×10^–3 to 2×10^–2 nmol.ml^–1 CMP-NANA. When pretreated up to 100 min in presence of the very low concentration of 2×10^–3 nmol.ml^–1, a potentiating effect on the conversion of gonococci by 2×10^–2 nmol.ml^–1 was observed in relation to the time of preincubation. This action was abolished after exposure to a subinhibitory concentration of chloramphenicol (0.5 µg.ml^–1). The gonococci recovered their ability to convert to serum resistance following adequate washing. The potential for increase in STase activity should be of interest for understanding the conversion from a serum sensitive to a serum resistance state.     

Control of pyruvate kinase activity during glycolysis and gluconeogenesis in Propionibacterium shermanii

The concentrations of glycolytic intermediates and ATP and the activities of certain glycolytic and gluconeogenic enzymes were determined in Propionibacterium shermanii cultures grown on a fully defined medium with glucose, glycerol or lactate as energy source. On all three energy sources, enzyme activities were similar and pyruvate kinase was considerably more active than the gluconeogenic enzyme pyruvate, orthophosphate dikinase, indicating the need for regulation of pyruvate kinase activity. The intracellular concentration of glucose 6-phosphate, a specific activator of pyruvate kinase in this organism, changed markedly according to both the nature and the concentration of the growth substrate: the concentration (7-10 mM) during growth with excess glucose or glycerol was higher than that (1-2 mM) during growth with lactate or at growth-limiting concentrations of glycerol or glucose. Other glycolytic intermediates, apart from pyruvate, were present at concentrations below 2 mM. Glucose 6-phosphate overcame inhibition of pyruvate kinase activity by ATP and inorganic phosphate. With 1 mM-ATP and more than 10 mM inorganic phosphate, a change in glucose 6-phosphate concentration from 1-2 mM was sufficient to switch pyruvate kinase from a strongly inhibited to a fully active state. The results provide a plausible mechanism for the regulation of glycolysis and gluconeogenesis in P. shermanii.     

Improving culture performance and antibody production in CHO cell culture processes by reducing the Warburg effect

Lactate is one of the key waste metabolites of mammalian cell culture. High lactate levels are caused by high aerobic glycolysis, also known as the Warburg effect, and are usually associated with adverse culture performance. Therefore, reducing lactate accumulation has been an ongoing challenge in the cell culture development to improve growth, productivity, and process robustness. The pyruvate dehydrogenase complex (PDC) plays a crucial role for the fate of pyruvate, as it converts pyruvate to acetyl coenzyme A (acetyl‐CoA). The PDC activity can be indirectly increased by inhibiting the PDC inhibitor, pyruvate dehydrogenase kinase, using dichloroacetate (DCA), resulting in less pyruvate being available for lactate formation. Here, Chinese hamster ovary cells were cultivated either with 5 mM DCA or without DCA in various batch and fed‐batch bioreactor processes. In all cultures, DCA increased peak viable cell density (VCD), culture length and final antibody titer. The strongest effect was observed in a fed batch with media and glucose feeding in which peak VCD was increased by more than 50%, culture length was extended by more than 3 days, and the final antibody titer increased by more than twofold. In cultures with DCA, lactate production and glucose consumption during exponential growth were on average reduced by approximately 40% and 35%, respectively. Metabolic flux analysis showed reduced glycolytic fluxes, whereas fluxes in the tricarboxylic acid (TCA) cycle were not affected, suggesting that cultures with DCA use glucose more efficiently. In a proteomics analysis, only few proteins were identified as being differentially expressed, indicating that DCA acts on a posttranslational level. Antibody quality in terms of aggregation, charge variant, and glycosylation pattern was unaffected. Subsequent bioreactor experiments with sodium lactate and sodium chloride feeding indicated that lower osmolality, rather than lower lactate concentration itself, improved culture performance in DCA cultures. In conclusion, the addition of DCA to the cell culture improved culture performance and increased antibody titers without any disadvantages for cell‐specific productivity or antibody quality.     

Studies on the mode of action of the antibacterial agent diethyltin dichloride onEscherichia coli

The mode of action of diethyltin dichloride as a representative of the antibacterial dialkyl-substituted tin compounds was studied forEscherichia coli. The oxygen uptake by resting cells ofEicoli with glucose, lactate, pyruvate or glutamate as substrates is strongly inhibited by diethyltin dichloride at a concentration of 0.08mm which is still below the minimum inhibitory concentration for growth on a rich medium. Inhibition of oxygen uptake with glucose or lactate as substrates is accompanied by an increase in the amount of pyruvic acid accumulating. A slight rise in the concentration of -ketoglutaric acid results from the inhibition of succinate and malate oxidation by diethyltin dichloride, while inhibition of -keto acid production is obtained with glutamate as a substrate. Anaerobically, with glucose as a substrate, ethanol formation as well as the production of carbon dioxide and hydrogen are inhibited by diethyltin dichloride. In many respects the effects of diethyltin dichloride on metabolism resemble those of arsenite.The inhibition of pyruvate metabolism probably results in a lack of acetyl-CoA, and this is suggested to be a possible cause of growth inhibition.     

Significance of phosphoglucose isomerase for the shift between heterolactic and mannitol fermentation of fructose by Oenococcus oeni

The bacterium Oenococcus oeni employs the heterolactic fermentation pathway (products lactate, ethanol, CO₂) during growth on fructose as a substrate, and the mannitol pathway when using fructose as an electron acceptor. In this study, [U-¹³C]glucose, [U-¹³C]fructose, HPLC, NMR spectroscopy, and enzyme analysis were applied to elucidate the use of both pathways by the hexoses. In the presence of glucose or pyruvate, fructose was metabolized either by the mannitol or the phosphoketolase pathways, respectively. Phosphoglucose isomerase, which is required for channeling fructose into the phosphoketolase pathways, was inhibited by a mixed-type inhibition composed of competitive (K _i=180 M) and uncompetitive (K_i=350 M) inhibition by 6-phosphogluconate. Erythrose 4-phosphate inhibited phosphoglucose isomerase competitively (K _i=1.3 M) with a low contribution of uncompetitive inhibition (K_i=13 M). The cellular 6-phosphogluconate content during growth on fructose plus pyruvate (<75 M) was significantly lower than during growth on fructose alone or fructose plus glucose (550 and 480 M). We conclude that competitive inhibition of phosphoglucose isomerase by 6-phosphogluconate (and possibly erythrose 4-phosphate) is responsible for exclusion of fructose from the phosphoketolase pathway during growth on fructose plus glucose, but not during growth on fructose plus pyruvate.     

Transport ofl-lactate by cultured rat brain astrocytes

Several reports indicate that lactate can serve as an energy substrate for the brain. The rate of oxidation of this substrate by cultured rat brain astrocytes was 3-fold higher than the rate with glucose, suggesting that lactate can serve as an energy source for these cells. Since transport into the astrocytes may play an important role in regulating nutrient use by individuals types of brain cells, we investigated the uptake ofl-[U-¹⁴C]lactate by primary cultures of rat brain astrocytes. Measurement of the net uptake suggested two carrier-mediated mechanisms and an Eadie-Hofstee type plot of the data supported this conclusion revealing 2 Km values of 0.49 and 11.38 mM and Vmax values of 16.55 and 173.84 nmol/min/mg protein, respectively. The rate of uptake was temperature dependent and was 3-fold higher at pH 6.2 than at 7.4, but was 50% less at pH 8.2. Although the lactate uptake carrier systems in astrocytes appeared to be labile when incubated in phosphate buffered saline for 20 minutes, the uptake process exhibited an accelerative exchange mechanism. In addition, lactate uptake was altered by several metabolic inhibitors and effectors. Potassium cyanide and -cyano-4-hydroxycinnamate inhibited lactate uptake, but mersalyl had little or no effect. Phenylpyruvate, -ketoisocaproate, and 3-hydroxybutyrate at 5 and 10 mM greatly attenuated the rate of lactate uptake. These results suggest that the availability of lactate as an energy source is regulated in part by a biphasic transport system in primary astrocytes.This data was presented in part at the meeting of the Federation of American Societies for Experimental Biology in May 1989.     

L-lactate production from xylose employingLactococcus lactis IO-1

Summary The growth, substrate utilisation and L-lactate production ofLactococcus lactis IO-1 were examined on xylose, and glucose and xylose media. The yield of lactate on xylose was 0.47 g lactate/g xylose at an initial xylose concentration of 51.2 g/l and the _max was 0.72 h^–1. Xylose cultures were more susceptible to lactate inhibition than were glucose cultures but showed similar kinetic behaviour. The organism was capable of complete sugar utilisation when grown on a mixture of 20 g/l xylose and 20 g/l glucose and synthesised 0.66 g lactate/g sugar.     

Regulation of T-cell functions by L-lactate

Lactate is a product of glycolytically active macrophages. After stimulation with concanavalin A accessory cell-depleted splenic T-cell populations were found to produce only minute amounts of T-cell growth factor (TCGF); but substantial amounts of TCGF were produced if the cultures were supplemented either with splenic adherent cells or with lactate but not with interleukin-1 (IL-1). IL-1 was capable, however, of supporting TCGF production by the thymoma subline EL4-6.1. TCGF production in cultures of accessory cell-depleted splenic T-cell populations was demonstrable with 10(-3) M L-lactate, and optimal responses (plateau level) were obtained with 4-6 X 10(-2) M L-lactate. Cultures of macrophages were found to accumulate up to 5 X 10(-2) M lactate. Our experiments indicate, therefore, that lactate serves as a regulatory signal by which macrophage-like accessory cells enhance helper-T-cell functions. Lactate is apparently not the only mediator of accessory cell function since plateau levels of TCGF production were markedly lower with lactate than with splenic accessory cells; but L-lactate was found also to determine the magnitude of T-cell-mediated immune responses in vivo and in cultures of unfractionated lymphocyte populations. The production of interferon in accessory cell-depleted and concanavalin A-treated T-cell cultures, however, was not significantly affected by lactate. Concanavalin A-stimulated splenic T-cell populations were found to consume glucose rapidly and to release lactate into the supernatant. This indicates that the cells contain more lactate and pyruvate than they can utilize by their respiratory metabolism. The administration of external lactate or pyruvate was found to inhibit the utilization of glucose by the mitogenically stimulated T cells.     

Follicular fluid concentrations of glucose, lactate and pyruvate in buffalo and sheep, and their effects on cultured oocytes, granulosa and cumulus cells

The objective was to determine ovarian follicular fluid concentrations of glucose, lactate, and pyruvate in relation to follicle size in buffalo and sheep. The effect of varying concentrations of these substances on in vitro oocyte maturation, oocyte protein content, and granulosa and cumulus cell growth was also investigated. Follicular fluid was aspirated from various sizes of follicles (from ovaries without a dominant follicle) collected from adult, cycling nonpregnant buffalo (Bubalus bubalis) and sheep (Ovis aries) during the breeding season. Overall, mean (+/-S.E.M.) concentrations (mM) were glucose 2.42+/-0.31 and 1.40+/-0.22, lactate 7.56+/-2.61 and 10.42+/-1.64, and pyruvate 0.02+/-0.01 and 0.002+/-0.00, in buffalo and sheep, respectively. In both species, as follicles became larger, concentrations of glucose significantly increased, lactate significantly decreased, but pyruvate was not affected. Oocyte maturation was higher (P<0.05) in medium containing supra-physiological concentrations of either glucose (5 mM), or pyruvate (10 mM) alone, or physiological concentrations of glucose, lactate and pyruvate in combination, compared to supra-physiological concentrations of lactate (15 mM) alone, or sub- or supra-physiological concentrations of glucose, lactate and pyruvate in combination (both species). The protein content of oocytes was not significantly affected by the concentration of glucose, lactate, and pyruvate in the maturation medium. However, growth of granulosa and cumulus cells was higher (P<0.05) in medium containing supra-physiological concentrations of glucose (5 mM) alone, or pyruvate (10 mM) alone, or physiological, or supra-physiological concentrations of glucose, lactate and pyruvate in combination, compared to supra-physiological concentrations of lactate (15 mM) alone, or sub-physiological concentrations of glucose, lactate and pyruvate in combination (both species). In conclusion, concentrations of glucose, pyruvate and lactate in the medium had cell type-specific effects on oocyte maturation, and on growth of granulosa and cumulus cells. Furthermore, glucose and pyruvate were the principal energy sources for oocytes and follicular somatic cells in buffalo and sheep.     

Density-dependent changes in hexose transport, glycolytic enzyme levels, and glycolytic rates, in uninfected and murine sarcoma virus-transformed rat kidney cells

In normal rat kidney (NRK) cell cultures, increased cell density results in a decrease in the rates of hexose transport, glucose utilization, and lactate production and an increase in the level of hexokinase activity. A murine sarcoma virus (Kirsten)-transformed cell line (KNRK) showed little or no density-dependent variation in sugar uptake, glucose consumption, or lactate production. On the other hand, hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase activities were elevated in dense transformed cultures as compared to sparse or uninfected cultures. In another virus-transformed cell line (ts339/NRK) exhibiting temperature-dependent morphology, growth pattern, and transport of 2-deoxy- -glucose, the levels of glycolytic enzyme activity were related to cell density but not to the culture temperature. The lack of correlation between glycolytic enzyme activity and lactate production by either uninfected or murine sarcoma virus-transformed cultures supports the suggestion that enhanced growth and/or hexose transport capacity rather than elevated glycolytic enzyme activity are responsible for the increased rate of lactate production by virus-transformed NRK cells.     

The role of carbonate in the metabolism of glucose by Butyrivibrio fibrisolvens

The amount of Na2CO3 added to semi-synthetic medium determined the length of the lag phase, the growth rate and the dry weight of three strains of Butyrivibrio fibrisolvens (WV1, NOR37, B835). With increasing CO3(2-) concentration the molar growth yield of bacteria, from glucosewas increased and, of the fermentation products, formate increased more than the other acids. CO3(2-)-limited cultures of strain WV1 (Group 2 Butyrivibrio) and strain NOR37 (Troup 1 Butyrivibrio) incorporated 14CO3(2-) into lactate and formate. In NOR37, lactate and formate had equal specific activities; in WV1, the formate specific activity was twice that of lactate. Strain WV1 had an active pyruvate synthase and an energy-dependent exchange between CO3(2-) and formate was demonstrated. In strain WV1 butyrate was produced mainly from glucose.     

Fermentative degradation of glycolic acid by defined syntrophic cocultures

Three different defined cocultures of glycolatedegrading strictly anaerobic bacteria were isolated from enrichment cultures inoculated with freshwater sediment samples. Each culture contained a primary fermenting bacterium which used only glycolate as growth substrate. These cells were gram-positive, formed terminal oval spores, and did not contain cytochromes. Growth with glycolate was possible only in coculture with either a homoacetogenic bacterium or a hydrogen-utilizing methanogenic bacterium; the overall fermentation balance was either 4 glycolate 3 acetate + 2CO₂, or 4 glycolate 3 CH₄+5 CO₂. These transformations indicate that glycolate was converted by the primary fermenting bacterium entirely to CO₂ and reducing equivalents which were transferred to the partner organisms, probably through interspecies hydrogen transfer. The key enzymes of fermentative glycolate degradation were identified in cell-free extracts. An acetyl-CoA and ADP-dependent glyoxylate-converting enzyme activity, malic enzyme, pyruvate synthase, and methyl viologen-dependent hydrogenase were found at comparably high activities suggesting that these bacteria oxidize glycolate through a new pathway via malyl-CoA, and that ATP is synthesized by substrate-level phosphorylation, in a similar manner as found in a recently isolated glyoxylatefermenting anaerobe.     

Streptomycin-resistant, asporogenous mutant of Bacillus subtilis.

Summary Mutants of Saccharomyces cerevisiae lacking pyruvate kinase (EC 2.7.1.40) are described. These have less than 0.5% of the pyruvate kinase activity of the wild type. All the other glycolytic enymes are present in normal amounts in these mutants. The mutation is recessive and segregates in diploids as a single gene. Five alleles examined fail to complement one another. Tetrad analysis and mitotic recombination data place the mutation on the left arm of chromosome I distal to cys 1. The majority of single-step spontaneous revertants on glucose regain the enzyme activity fully and this activity appears, by a number of criteria, to be due to the same enzyme present in the wild type. Some of these revertants become nuclear petites. The mutants do neither grow on nor ferment sugars but do grow on ethyl alcohol or pyruvate. Glucose addition to cultures growing on alcohol arrests growth until glucose is exhausted. The steady state rate of glucose utilization is slower than in the wild type. This is associated with the accumulation of as much as 5 moles P-enolpyruvate per g wet weight of cells and proportional amounts of 2-P-glyceric and 3-P glyceric acids.The mutation is believed to involve some regulatory element in the synthesis of pyruvate kinase.     

Modifications by chronic intermittent hypoxia and drug treatment on skeletal muscle metabolism

The energy metabolism was evaluated in gastrocnemius muscle from 3-month-old rats subjected to either mild or severe 4-week intermittent normobaric hypoxia. Furthermore, 4-week treatment with CNS-acting drugs, namely, -adrenergic (-yohimbine), vasodilator (papaverine, pinacidil), or oxygen-increasing (almitrine) agents was performed. The muscular concentration of the following metabolites was evaluated: glycogen, glucose, glucose 6-phosphate, pyruvate, lactate, lactateto-pyruvate ratio; citrate, -ketoglutarate, succinate, malate; aspartate, glutamate, alanine; ammonia; ATP, ADP, AMP, creatine phosphate. Furthermore the Vmax of the following muscular enzymes was evaluated: hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase; citrate synthase, malate dehydrogenase; total NADH cytochrome c reductase; cytochrome oxidase. The adaptation to chronic intermittent normobaric mild or severe hypoxia induced alterations of the components in the anaerobic glycolytic pathway [as supported by the increased activity of lactate dehydrogenase and/or hexokinase, resulting in the decreased glycolytic substrate concentration consistent with the increased lactate production and lactate-to-pyruvate ratio] and in the mitochondrial mechanism [as supported by the decreased activity of malate dehydrogenase and/or citrate synthase resulting in the decreased concentration of some key components in the tricarboxylic acid cycle]. The effect of the concomitant pharmacological treatment suggests that the action of CNS-acting drugs could be also related to their direct influence on the muscular biochemical mechanisms linked to energy transduction.