Nutrition of Acetobacter rancens S3 and F11 Isolated from Tanks for Vinegar Production

The strains S3 and F11 which were isolated respectively from static and submerged tanks for vinegar production were identified as Acetobacter rancens. Neither strain grew in an ammonium defined medium containing ethanol, glucose, glycerol or organic acids as the sole carbon source. When casamino acids were added, they grew luxuriantly with lactate, ethanol or glycerol as the carbon source and less well with acetate or glucose. They grew, forming much acetic acid, in defined ethanol medium when alanine was supplied in place of casamino acids, but strain S3 showed a longer lag time than strain Fl1. This lag time could be shortened by addition of aspartate and glutamate. These amino acids could be replaced by succinate, fumarate, malate, lactate, pyruvate or propionate but not by glucose. Both strains required lactate or pyruvate in defined glucose medium but many other organic acids, which were effective in defined ethanol medium, were ineffective or slightly effective in glucose medium.     

Characterization of gluconeogenesis in hepatocytes isolated from the American eel,Anguilla rostrata LeSueur,

Summary Isolated hepatocyte preparations from fed immature American eels,Anguilla rostrata Le Sueur, were used to study gluconeogenic, lipogenic, glycogenic and oxidative rates of radioactively labelled lactate, glycerol, alanine and aspartate. Eel hepatocytes maintain membrane integrity and energy charge during a 2 h incubation period and are considered a viable preparation for studying fish liver metabolism.Incubating eel hepatocytes with 10 mM substrates, the following results were obtained: glycerol, alanine and lactate, in that order, were effective gluconeogenic substrates; these three substrates reduced glucose release from glycogen stores, while aspartate had no such effect; lactate, alanine and aspartate led to high rates of glycerol production, with subsequent incorporation into lipid; incorporation into glycogen was low from all substrates; and, alanine oxidation was seven times higher than that observed with other substrates.When eel hepatocytes were incubated with low or physiological substrate concentrations gluconeogenic rates from lactate were twice those from alanine; rates from aspartate were very low. Glucagon stimulated lactate gluconeogenesis, but not amino acid gluconeogenesis, and had no significant effect on glycogenolysis. Cortisol increased gluconeogenic rates from 1 mM lactate.Thus, in the presence of adequate substrate, eel liver gluconeogenesis is preferentially stimulated relative to glycogenolysis to produce plasma glucose. These data support three important roles for gluconeogenesis: the recycling of muscle lactate, the synthesis of glucose from dietary amino acids to supplement glucose levels, and the production of glycerol for lipogenesis.This work was supported from operating grants to TWM from the National Research Council of Canada (A6944)     

Role of sodium in the growth of a ruminal selenomonad

The ruminal selenomonad strain H18 grew rapidly (mu = 0.50 h-1) in a defined medium containing glucose, ammonia, purified amino acids, and sodium (95 mM); little if any ammonia was utilized as a nitrogen source. When the sodium salts were replaced by potassium salts (0.13 mM sodium), there was a small reduction in growth rate (mu = 0.34 h-1), and under these conditions greater than 95% of the cell nitrogen was derived from ammonia. No growth was observed when the medium lacked sodium (less than 0.35 mM) and amino acids were the only nitrogen source. At least six amino acid transport systems (aspartate, glutamine, lysine, phenylalanine, serine, and valine) were sodium dependent, and these systems could be driven by an electrical potential (delta psi) or a chemical gradient of sodium. H18 utilized lactate as an energy source for growth, but only when sodium and aspartate were added to the medium. Malate or fumarate was able to replace aspartate, and when these acids were added, sodium was no longer required. Glucose-grown cells accumulated large amounts of polysaccharide (64% of dry weight), and when the exogenous glucose was depleted, this material was converted to acetate and propionate as long as sodium was present. When the cells were incubated in buffers lacking sodium, succinate accumulated and exogenous succinate could not be decarboxylated. Because sodium had little effect on the transmembrane pH gradient at pH 6.7 to 4.5, it did not appear that sodium was required for intracellular pH regulation.     

Role of sodium in the growth of a ruminal selenomonad.

The ruminal selenomonad strain H18 grew rapidly (mu = 0.50 h-1) in a defined medium containing glucose, ammonia, purified amino acids, and sodium (95 mM); little if any ammonia was utilized as a nitrogen source. When the sodium salts were replaced by potassium salts (0.13 mM sodium), there was a small reduction in growth rate (mu = 0.34 h-1), and under these conditions greater than 95% of the cell nitrogen was derived from ammonia. No growth was observed when the medium lacked sodium (less than 0.35 mM) and amino acids were the only nitrogen source. At least six amino acid transport systems (aspartate, glutamine, lysine, phenylalanine, serine, and valine) were sodium dependent, and these systems could be driven by an electrical potential (delta psi) or a chemical gradient of sodium. H18 utilized lactate as an energy source for growth, but only when sodium and aspartate were added to the medium. Malate or fumarate was able to replace aspartate, and when these acids were added, sodium was no longer required. Glucose-grown cells accumulated large amounts of polysaccharide (64% of dry weight), and when the exogenous glucose was depleted, this material was converted to acetate and propionate as long as sodium was present. When the cells were incubated in buffers lacking sodium, succinate accumulated and exogenous succinate could not be decarboxylated. Because sodium had little effect on the transmembrane pH gradient at pH 6.7 to 4.5, it did not appear that sodium was required for intracellular pH regulation.     

Development of improved defined media for Clostridium botulinum serotypes A, B, and E

The minimal nutritional growth requirements were determined for strains Okra B and Iwanai E, which are representatives of groups I and II, respectively, of Clostridium botulinum. These type B and E strains differed considerably in their nutrient requirements. The organic growth factors required in high concentrations by the Okra B strain (group I) were arginine and phenylalanine. Low concentrations (less than or equal to 0.1 g/liter) of eight amino acids (methionine, leucine, valine, isoleucine, glycine, histidine, tryptophan, and tyrosine) and of five vitamins (pyridoxamine, p-aminobenzoic acid, biotin, nicotinic acid, and thiamine) were also essential for biosynthesis. The 10 required amino acids could be replaced by intact protein of known composition by virtue of the bacterium's ability to synthesize proteases. Glucose or other carbohydrates were not essential for Okra B, although they did stimulate growth. Quantitatively, the most essential nutrients for Okra B were arginine and phenylalanine. In contrast, the nonproteolytic strain, Iwanai E (group II), did not require either arginine or phenylalanine. It required glucose or another carbohydrate energy source for growth and did not utilize arginine or intact protein as a substitute source of energy. Iwanai E utilized ammonia as a nitrogen source, although growth was stimulated significantly by organic nitrogenous nutrients, especially glutamate and asparagine. Iwanai E also required biosynthesis levels of seven amino acids (histidine, isoleucine, leucine, tryptophan, tyrosine, valine, and serine), adenine, and six vitamins (biotin, thiamine, pyridoxamine, folic acid, choline, and nicotinamide). Calcium pantothenate also stimulated growth. On the basis of the nutritional requirements, chemically defined minimal media have been constructed for C. botulinum serotypes A, B, E, and F (proteolytic).(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of improved defined media for Clostridium botulinum serotypes A, B, and E.

The minimal nutritional growth requirements were determined for strains Okra B and Iwanai E, which are representatives of groups I and II, respectively, of Clostridium botulinum. These type B and E strains differed considerably in their nutrient requirements. The organic growth factors required in high concentrations by the Okra B strain (group I) were arginine and phenylalanine. Low concentrations (less than or equal to 0.1 g/liter) of eight amino acids (methionine, leucine, valine, isoleucine, glycine, histidine, tryptophan, and tyrosine) and of five vitamins (pyridoxamine, p-aminobenzoic acid, biotin, nicotinic acid, and thiamine) were also essential for biosynthesis. The 10 required amino acids could be replaced by intact protein of known composition by virtue of the bacterium's ability to synthesize proteases. Glucose or other carbohydrates were not essential for Okra B, although they did stimulate growth. Quantitatively, the most essential nutrients for Okra B were arginine and phenylalanine. In contrast, the nonproteolytic strain, Iwanai E (group II), did not require either arginine or phenylalanine. It required glucose or another carbohydrate energy source for growth and did not utilize arginine or intact protein as a substitute source of energy. Iwanai E utilized ammonia as a nitrogen source, although growth was stimulated significantly by organic nitrogenous nutrients, especially glutamate and asparagine. Iwanai E also required biosynthesis levels of seven amino acids (histidine, isoleucine, leucine, tryptophan, tyrosine, valine, and serine), adenine, and six vitamins (biotin, thiamine, pyridoxamine, folic acid, choline, and nicotinamide). Calcium pantothenate also stimulated growth. On the basis of the nutritional requirements, chemically defined minimal media have been constructed for C. botulinum serotypes A, B, E, and F (proteolytic).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Cellulose degradation by a new isolate from sewage sludge, a member of the Bacteroidaceae family.

A mesophilic anaerobe, a member of the Bacteroidaceae family (NRC2248), isolated from a cellulose-enrichment culture, digested untreated Whatman cellulose powder and HCl-treated cotton battings while producing hydrogen, carbon dioxide, cellobiose, glucose, and acetic acid as the sole volatile acid. This organism also utilized cellobiose as carbon and energy source but did not utilize glucose. It grew well in synthetic medium containing ammonium salts as nitrogen source and having a pH value of 7.0-7.1 and an Eh value of -160mV or lower. The nutrient requirements of this organism were found to be similar to those of other anaerobes except for Na2S which inhibited cellulose degradation in concentrations above 0.75 mM. Best cellulose degradation occurred under an atmosphere of 80% N2-20% CO2. Use of H2 or 80% H2-20% CO2 as headspace gas inhibited growth. Although accumulation of acetic acid in about 15-16 mM concentrations inhibited the further formation of H2, CO2, and acetic acid in the broth, it did not stop the degradation of cellulose. The results indicate that this organism has the ability to grow in media containing up to 20 g/L of cellulose and to produce industrially important and easily separable end products from cellulose.     

Pathways of glucose catabolism in Mycobacterium smegmatis.

Glucose metabolism in Mycobacterium smegmatis was investigated by the radiorespirometric method and by assaying for key enzymes of the major energy-yielding pathways. Glucose is oxidized in this organism mainly through the Embden-Meyerhof-Parnas pathway, irrespective of the carbon source used for growth. The pentose phosphate pathway plays only a minor role and its extent depends on the carbon source used for growth. Enzymes of glycolytic and oxidative pathways were detected in cells grown on glucose, glycerol, or pyruvate but enzymes of the Entner-Duodroff pathway could be detected only in glucose-grown cells. Labeled acetate is utilized by cells cultured on glucose, glycerol, and pyruvate. In all cases more of C1 of acetate was converted to CO2 while incorporation into cellular constituents was maximum from C2 of acetate.     

Growth of Ferrobacillus ferrooxidans on Organic Matter

Following a brief adaptation period to glucose, Ferrobacillus ferrooxidans was grown on glucose, mannitol, several other sugars, and a few amino acids in the absence of an oxidizable iron source. Prolonged growth on an organic substrate free from iron rendered the organism obligately organotrophic. The growth rate of the bacterium was greater in heterotrophic culture; the doubling time was approximately 4.5 hr on glucose. The bacterium retained its acidophilic properties during adaptation and growth on glucose and would not grow in neutral or slightly alkaline media. Addition of p-aminobenzoic acid was necessary for abundant growth of the cells on glucose. Of the eight strains of Fe(++)-oxidizing bacteria studied, only two strains grew on glucose in a nondialyzed system. The results of manometric studies are discussed with regard to metabolic efficiency of organic matter in this organism.     

The mitochondrial dicarboxylate carrier is essential for the growth of Saccharomyces cerevisiae on ethanol or acetate as the sole carbon source

The dicarboxylate carrier (DIC) is an integral membrane protein that catalyses a dicarboxylate-phosphate exchange across the inner mitochondrial membrane. We generated a yeast mutant lacking the gene for the DIC. The deletion mutant failed to grow on acetate or ethanol as sole carbon source but was viable on glucose, galactose, pyruvate, lactate and glycerol. The growth on ethanol or acetate was largely restored by the addition of low concentrations of aspartate, glutamate, fumarate, citrate, oxoglutarate, oxaloacetate and glucose, but not of succinate, leucine and lysine. The expression of the DIC gene in wild-type yeast was repressed in media containing ethanol or acetate with or without glycerol. These results indicate that the primary function of DIC is to transport cytoplasmic dicarboxylates into the mitochondrial matrix rather than to direct carbon flux to gluconeogenesis by exporting malate from the mitochondria. The delta DIC mutant may serve as a convenient host for overexpression of DIC and for the demonstration of its correct targeting and assembly.     

Serine synthesis by an isolated perfused rat kidney preparation.

The isolated perfused rat kidney was shown to synthesize serine from aspartate or glutamate, both of which are also precursors of glucose. The major products of aspartate metabolism were ammonia, serine, glutamate, glucose, glutamine and CO2. Perfusion of kidneys with aspartate in the presence of amino-oxyacetate resulted in a near-complete inhibition of aspartate metabolism, illustrating the essential role of aspartate aminotransferase in the metabolism of this substrate. Radioactivity from 14C-labelled aspartate and from 14C-labelled glycerol was incorporated into serine and glucose. Production of both glucose and serine from aspartate was suppressed in the presence of 3-mercaptopicolinic acid. These data provide evidence for the operation of the phosphorylated and/or non-phosphorylated pathway for serine production to the presence of 3-mercaptopicolinic acid. This is explained by simultaneous glycolysis. The rate of glucose production, but not that of serine, was greater in kidneys perfused with glutamate or with aspartate plus glycerol than the rates obtained by perfusion with aspartate alone. These data are taken to suggest that serine synthesis occurred at a near-maximal rate, and that the capacity of the kidney for serine synthesis from glucose precursors is lower than that for glucose synthesis.     

Transition of Chemolithotroph Ferrobacillus ferrooxidans to Obligate Organotrophy and Metabolic Capabilities of Glucose-Grown Cells

Transition of chemolithotrophic Ferrobacillus ferrooxidans to organotrophy occurred after 60 hr of incubation in an organic medium. Three distinct phases, based on metabolic activities of cells, were observed during the course of transition. Conversion of cellular nutrition to organotrophy resulted in a gradual loss of Fe(2+) oxidation and cessation of CO(2) fixation. These changes were concomitant with a rapid increase in uptake of glucose and phosphate during the latter part of transition period. The outcome of transition was governed by the pH of the medium, temperature of incubation, availability of oxygen, age of the chemolithotrophic cells, and the type of energy and carbon source available to the bacterium. Presence or absence of p-aminobenzoic acid and Fe(2+) ions did not influence transition of cells. A defined medium containing glucose, mineral salts, and p-aminobenzoic acid at pH 2.5 was found to be most suitable for transition and for culture of heterotrophic convertants. Maximum growth rate of the heterotrophic cells was attained with vigorous aeration at 35 C. The bacterium could be cultured on a variety of organic compounds, including complex organic media, provided they were used in low concentrations. Serological studies on autotrophic cells and the heterotrophic convertant have shown a definite antigenic relationship between the two cell types.     

Physiological and nutritional features of Corynebacterium pyogenes

Growth and acid metabolic products were similar when Corynebacterium pyogenes was grown aerobically or anaerobically in a serum-free medium (SFM). This indicated that C. pyogenes obtains energy for growth primarily by fermentative metabolism even under aerobic growth conditions. Growth yield was reduced by 90% in SFM minus glucose, 50% in SFM minus NaHCO3, 90% in SFM minus yeast extract, 100% in SFM minus Trypticase and yeast extract, and 30% in SFM minus haemin or Trypticase. Growth was not detectable when a known mixture of amino acids, vitamins, and nucleic acid bases were substituted for Trypticase and yeast extract in SFM; addition to the latter medium of a peptide source such as Trypticase or casitone supported good growth of the organism. When NaHCO3 was omitted from SFM and dissolved CO2 in the medium was rigorously excluded, growth was undetectable indicating that C. pyogenes has an obligate requirement for CO2 for growth. Succinate, formate and acetate were the major fermentation products in SFM, whereas in SFM minus HCO-3 or haemin, lactate was the major product and only small quantities of other acids accumulated.     

Comparison of Hup Trait and Intrinsic Antibiotic Resistance for Assessing Rhizobial Competitiveness Axenically and in Soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.     

Comparison of hup trait and intrinsic antibiotic resistance for assessing rhizobial competitiveness axenically and in soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.     

Lactate dehydrogenases in cyanobacteria

NAD-linked lactate dehydrogenases specific for the D- and L-lactate have been demonstrated in a number of strains of unicellular cyanobacteria. The D-lactate dehydrogenase of one strain (Synechococcus 6716) was partially purified and its properties were studied. The enzyme has a molecular weight of ca. 115000-120000, is highly specific, autooxidizable, and susceptible to inhibition by iodoacetamide, oxamate and ATP. The possible physiological functions of the enzyme in the metabolism of the organism were investigated. D-lactate carbon was incorporated in cell material during photosynthetic growth with CO2, but lactate was not used as sole source for carbon for photosynthetic or chemosynthetic development. D-lactate and pyruvate were oxidized aerobically in the dark by resting cell suspensions with the assimilation mainly of the C2 and the C3 carbon atoms. In the oxidation of lactate, acetate was excreted into the medium. No fermentation of glucose was found, but a small amount of D-lactate was detected as a product of endogenous dark metabolism of the cell. All enzymes required for the production of lactate from glucose and from glycogen were found in exponentially growing cells, but the activity of some key enzymes was low or undetectable in old cultures.     

Function of Growth Factors for Rumen Microorganisms I. Nutritional Characteristics of Selenomonas ruminantium

Nutritional characteristics of Selenomonas ruminantium var. lactilytica isolated from a sheep rumen were studied. The organism required for growth the addition of a clarified rumen fluid to a Trypticase-yeast extract medium with either lactate or glucose as an energy source. The requirement for rumen fluid was found to be satisfied by volatile fatty acids in glucose media and by biotin in lactate media. Straight-chain saturated fatty acids with C(3) to C(10) carbon skeleton had been found to be effective. Among them, n-valerate was most effective at the lowest concentration. An abnormal morphology was observed with n-valerate-deficient glucose media. n-Valerate was essential in glucose media, and it was stimulatory in lactate media. Fermentation products from glucose were lactate, propionate, and acetate, and fermentation products from lactate were propionate and acetate. When cells were grown in a glucose medium containing n-valerate-C(14), the label was present in cell fractions. Almost all of the activity was found in lipid materials.     

Nutrition of Myxococcus xanthus, a fruiting myxobacterium.

The minimal requirements for vegetative growth of Myxococcus xanthus have been sought. Isoleucine, leucine, and valine were required, and vitamin B12 was needed for the synthesis of methionine. Pyruvate was an excellent energy source and an efficient source of cellular carbon. Acetate, aspartate, glutamate, and most tricarboxylic acid cycle intermediates could also be utilized, but were less efficient sources of carbon and energy than was pyruvate. Many mono- and disaccharides were tested, but, in agreement with earlier results, none served as carbon-energy sources. A minimal medium (A1) has been devised that includes the essential amino acids and vitamin B12, with pyruvate and aspartate as carbon-energy sources. In this medium, M. xanthus could propagate indefinitely, and on it vegetative cells formed colonies with greater than 75% efficiency; hence, it is likely that no organic cofactors other than those present in A1 are required in more than trace amounts.     

Substrate utilization by rat stomach in vivo. Arteriovenous differences for glucose, lactate, ketone bodies, fatty acids and glycerol under control and acid-secreting conditions.

Arteriovenous differences for several potential metabolic substrates were measured across the fundic wall of the stomach of rats that had been starved overnight. There was an uptake of glucose and D-3-hydroxybutyrate, but no significant arteriovenous differences for acetoacetate, pyruvate, non-esterified fatty acids and glycerol were apparent. Lactate output represented a substantial fraction of glucose uptake when the arterial lactate concentration was within the resting physiological range, but when the arterial lactate concentration was above 1.3 mM, lactate was taken up by the stomach. Stimulation of acid secretion by pentagastrin did not affect the value of arteriovenous differences. Thus blood flow to the fundic mucosa and substrate metabolism may be similarly enhanced by pentagastrin. It is concluded that metabolism of glucose and D-3-hydroxybutyrate, and to a lesser extent of glutamine and branched-chain amino acids [Anderson & Hanson (1983) Biochem. J. 210, 451-455], could supply energy to power acid secretion.