Inhibition of Salmonella typhimurium by the products of tartrate metabolism by a Veillonella species

The inhibition of the growth of Salmonella typhimurium by a Veillonella species grown on media supplemented with tartrate was examined. Growth of Salmonella typhimurium was not inhibited by the concentrations of products metabolized by Veillonella cultures on media supplemented with 0 or 50 mmol 1^-1 of tartrate, but was inhibited on media supplemented with 100 or 150 mmol 1^-1 of tartrate. Inhibition of Salm. typhimurium was correlated with the increased production of acetate and propionate from tartrate by the Veillonella species.     

Antibacterial activity of the metabolic by-products of a Veillonella species and Bacteroides fragilis

A Veillonella species and Bacteroides fragilis were isolated from the cecal contents of adult chickens. When growth on an agar medium supplemented with 0.4% glucose and adjusted to pH 6.5, mixed cultures containing Veillonella and B. fragilis inhibited the growth of Salmonella typhimurium; Salmonella enteritidis, Escherichia coli 0157:H7 and Pseudomonas aeruginosa. Decreasing the glucose concentration of the agar decreased the inhibitory activity of the mixed culture. Mixed cultures grown on agar media supplemented with 0.5% glucose and adjusted to pH 6.5, 7.0 or 7.5 also inhibited the growth of S. typhimurium, S. enteritidis, E. coli 0157:H7 and P. aeruginosa. However, increasing the pH of the agar decreased the inhibitory activity of the mixed culture. Pure cultures of Veillonella or B. fragilis did not inhibit the growth of S. typhimurium, S. enteritidis, E. coli 0157:H7 or P. aeruginosa on any of the agar supplemented with different concentrations of glucose or on any of the agar adjusted to different pH levels. The inhibitory activity of the mixed culture was correlated with the concentration of volatile fatty acids that were formed as B. fragilis metabolized glucose to produce succinate and acetate and as the succinate produced by B. fragilis was decarboxylated by Veillonella to produce propionate.     

Chemically defined media for growing anaerobic bacteria of the genus Veillonella

A chemically defined medium for Veillonella parvula and V. alcalescens is described. Some nutritional aspects of the two strains used were examined: the optimum concentration of reducing agents, the requirement for amino acids, diamines, vitamins and other growth factors, and the conditions needed for well balanced nutrition.No specific requirements for single amino acids were observed. A combination of l-cysteine, dl-aspartic acid, l-glutamic acid, l-serine and l-tyrosine, promoted growth. In V. alcalescens, serine could substitute both arginine and tryptophan (or histidine). No growth was obtained with ammonium salts as the sole N source.Decarboxylation of l-ornithine, l-lysine and l-arginine was not demonstrated in the Veillonella parvula strain, which required putrescine or cadaverine for growth. Spermine, spermidine, l-lysine, l-ornithine and l-arginine, could not substitute putrescine in Veillonella parvula. Veillonella alcalescens, which does not require putrescine in the medium, was able to decarboxylate l-ornithine while forming putrescine.     

Metabolism of Aspartate by Propionibacterium freudenreichii subsp. shermanii: Effect on Lactate Fermentation

More than 90% of the aspartate in a defined medium was metabolized after lactate exhaustion such that 3 mol of aspartate and 1 mol of propionate were converted to 3 mol of succinate, 3 mol of ammonia, 1 mol of acetate, and 1 mol of CO₂. This pathway was also evident when propionate and aspartate were the substrates in complex medium in the absence of lactate. In complex medium with lactate present, about 70% of the aspartate was metabolized to succinate and ammonia during lactate fermentation, and as a consequence of aspartate metabolism, more lactate was fermented to acetate and CO₂ than was fermented to propionate. The conversion of aspartate to fumarate and ammonia by the enzyme aspartase and subsequent reduction of fumarate to succinate occurred in the five strains of Propionibacterium freudenreichii subsp. shermanii studied. The ability to metabolize aspartate in the presence of lactate appeared to be related to aspartase activity. The specific activity of aspartase increased during and after lactate utilization, and the levels of this enzyme were lower in cells grown in defined medium than levels in those cells grown in complex medium. Under the conditions used, no other amino acids were readily metabolized in the presence of lactate. The possibility that aspartate metabolism by propionibacteria in Swiss cheese has an influence on CO₂ production is discussed.     

一株人消化道中韦荣氏球菌的分离、鉴定及其代谢特性研究

【目的】分离与鉴定人肠道乳酸利用菌,研究其乳酸利用的代谢特性。【方法】利用Hungate滚管技术从人粪便中分离厌氧细菌,通过形态、生化和16S r RNA基因鉴定;通过体外发酵技术研究乳酸代谢,并且与乳酸产生菌共培养,研究二者之间交互饲喂。【结果】验证了乳酸可作为代谢中间产物被人肠道混合菌群利用;分离到一株乳酸利用菌,在24 h内消耗乳酸超过50 mmol/L,经鉴定为韦荣氏球菌,并将其形成乙酸和丙酸。当和肠球菌共培养时,可以有效地减缓乳酸的积累。【结论】本株乳酸利用菌可以作为潜在的益生菌,和乳酸菌一起调节人肠道的乳酸动态平衡。     

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.     

Growth yield increase and ATP formation linked to succinate decarboxylation in Veillonella parvula

Veillonella parvula strain 259 (=DSM 2007) was able to grow on a mineral salts medium supplemented with (per litre) 1 g yeast extract, 1 g Tween-80, and 3 mg putrescine. 2 HCl, with 6 mM thioglycolate as reductant and lactate as growth substrate. Succinate did not serve as a growth substrate, but when added in conjunction with lactate, it was decarboxylated to propionate and resulted in a measurable increase in growth yield, corresponding to the formation of 2.4 g cell dry mass per mol succinate. A growth yield increase linked to succinate metabolism occurred only while lactate was also being metabolised. Experiments with cell suspensions showed that succinate decarboxylating activity was constitutive. Addition of succinate produced clear increases in cellular ATP levels in ATP-depleted washed cells.     

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.     

Chemotaxis toward amino acids in Escherichia coli

Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.     

Amino acid consumption by the parasitic, amoeboid protists Entamoeba histolytica and E. invadens

Abstract Amino acid consumption by Entamoeba histolytica and E. invadens has been measured in order to assess the possible roles of amino acids as energy substrates. Mixtures of amino acids enhanced the growth of the parasites in complex medium and their survival in simple medium. The consumption of several amino acids by the parasites suspended in simple media was greater when glucose was absent, suggesting that they may act as alternative energy sources. Under these conditions, asparagine was consumed extremely rapidly by E. histolytica in particular, and arginine, leucine and threonine were used greatly by both species. There was also a marked consumption of aspartate, but this occurred even when glucose was present. These five amino acids and phenylalanine were the ones consumed in greatest amounts during growth of E. histolytica in complex medium. Under the same growth conditions, E. invadens also used asparagine, arginine, leucine and threonine and in addition there was a large consumption of serine and especially glutamate. In contrast, the aspartate concentration in the complex medium increased and there was also a net increase in the concentration of some other amino acids. Alanine was produced by both species when the parasites were incubated in simple medium with glucose, and in greater amounts during growth in complex media, suggesting that it is an end product of energy metabolism. The findings provide support for the suggestion that energy generation through amino acid catabolism may be a characteristic feature of anaerobic parasitic protists.     

Metabolic footprinting of the anaerobic bacterium Fusobacterium varium using 1H NMR spectroscopy

Metabolic footprinting of the anaerobic bacterium Fusobacterium varium demonstrated the accumulation of six carboxylic acids as metabolic end-products and revealed specific growth requirements and utilization capabilities towards amino acids. Guided by (1)H NMR determinations of residual amino acids in spent medium, a modified chemically defined minimal medium (CDMM*) was developed by minimizing the amino acid composition while satisfying nutritional requirements to support abundant growth of F. varium. Quantitative determinations of carboxylate salts and residual substrates were readily performed by (1)H NMR analysis of lyophilized residues from CDMM* cultures without interference from initial medium components. Only small concentrations of alanine, arginine, glycine, isoleucine, leucine, methionine, proline and valine were required to support growth of F. varium, whereas larger quantities of aspartate, asparagine, cysteine, glutamine, glutamate, histidine, lysine, serine and threonine were utilized, most likely as energy sources. Both bacterial growth and the distribution of carboxylate end-products depended on the composition of the chemically defined medium. In cultures provided with glucose as the primary energy source, the accumulation of butyrate and lactate correlated with growth, consistent with the regeneration of reduced coenzyme formed by the oxidative steps of glucose catabolism.     

Nutritional requirements of some non-pathogenic Neisseria grown in simple synthetic media.

Ten species of non-pathogenic Neisseria were grown in simple defined liquid media containing amino acids, biotin, nicotinic acid, calcium pantothenate, ferrous sulfate, magnesium sulfate, and potassium phosphate. Two of these Neisseria were induced to grow with glutamic acid as the carbon and nitrogen source. The remaining eight Neisseria grew in glutamic acid medium supplemented with from one to four additional amino acids, lactate, or lactate and glucose. A strain of N. flavescens grew in the absence of added growth factors whereas the remaining nine species of Neisseria required either biotin or nicotinic acid; pantothenate was required by two and was stimulatory for three of these species. Use of carbohydrates by the non-pathogenic Neisseria in synthetic medium was tested. Two strains failed to use any of the 14 carbohydrates tested; one strain used only glucose; the remaining seven strains used fructose, glucose, maltose, and sucrose to varying degrees.     

Alteration in the Amino Acid Content of Yeast During Growth Under Various Nutritional Conditions

Yeast cells grown under optimal and suboptimal concentrations of biotin were analyzed for the amino acid content of their soluble pool and cellular protein. Optimally grown yeast cells exhibited a maximum amino acid content after 18 hr of growth. Biotin-deficient cells were depleted of all amino acids at 26 and 43 hr, with alanine, arginine, aspartate, cysteine, glutamate, isoleucine, leucine, lysine, methionine, serine, threonine, and valine being present in less than half the concentration observed in biotin-optimal cells. At early time intervals, the amino acid pool of biotin-deficient yeast contained lower concentrations of all amino acids except alanine. After more prolonged incubation, several amino acids accumulated in the pool of biotin-deficient yeast, but citrulline and ornithine accumulated to appreciable levels. The addition of aspartate to the growth medium resulted in a decrease in the amino acid content of biotin-optimal cells but caused a marked increase in the concentration of amino acids in biotin-deficient cells. The pools of biotin-deficient yeast grown in the presence of aspartate displayed a marked reduction in every amino acid with the exception of aspartate itself. These data provide evidence that the amino acid content of yeast cells and their free amino acid pools are markedly affected by biotin deficiency as well as by supplementation with aspartate, indicating that aspartate plays a major role in the nitrogen economy of yeast under both normal as well as abnormal nutritional conditions.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.     

Study of amino acid utilization by isogenic strains of Escherichia coli in relation to the culture growth and biosynthesis of penicillin amidase

Dynamics of free amino acid utilization by isogenic strains of Escherichia coli differing in intensity of their growth and levels of penicillin acylase biosynthesis in media containing corn steep liquor or peptone was studied. It was shown that in both the media some amino acids such as serine, threonine, glutaminic and asparaginic acids were actively utilized by the strains mainly during the culture intensive growth while others such as glycine, alanine and tyrosine were actively utilized during the enzyme biosynthesis. Intensively utilized arginine and proline were probably used for the growth and biosynthesis. The other amino acids were not utilized completely from the media. The lowest levels of their utilization were observed when the strains were cultivated in the medium with peptone.     

The simultaneous biosynthesis and uptake of amino acids by Lactococcus lactis studied by (13)C-labeling experiments

Uniformly (13)C labeled glucose was fed to a lactic acid bacterium growing on a defined medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with (13)C disclosing a substantial de novo biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange flux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium.     

Reduced nitrogen influences somatic embryo quality and plant regeneration from suspension cultures of orchardgrass

Summary Production of somatic embryos in suspension cultures ofDactylis glomerata L. (orchardgrass) was stimulated by the addition of various compounds containing reduced nitrogen to Schenk and Hildebrandt (SH) basal medium. Equimolar concentrations of combinations of proline and either serine or threonine supported embryogenesis, whereas, these amino acids individually did not promote embryogenesis. SH medium supplemented with 6 to 25 mM ammonium ion or Murashige-Skoog basal medium also supported embryo production. Ammonium ion did not act synergistically with either proline or serine to enhance embryogenesis. Embryos produced in media containing amino acid combinations were formed singly, did not exhibit secondary embryogenesis, and had significantly higher conversion rates compared to those formed in either SH medium supplemented with ammonium ion only or in combination with amino acids.     

Amino acid-dependent growth of Campylobacter jejuni: key roles for aspartase (AspA) under microaerobic and oxygen-limited conditions and identification of AspB (Cj0762), essential for growth on glutamate

Amino acids are key carbon and energy sources for the asaccharolytic food-borne human pathogen Campylobacter jejuni . During microaerobic growth in amino acid rich complex media, aspartate, glutamate, proline and serine are the only amino acids significantly utilized by strain NCTC 11168. The catabolism of aspartate and glutamate was investigated. An aspartase ( aspA ) mutant (unable to utilize any amino acid except serine) and a Cj0762 c ( aspB ) mutant lacking aspartate:glutamate aminotransferase (unable to utilize glutamate), were severely growth impaired in complex media, and an aspA sdaA mutant (also lacking serine dehydratase) failed to grow in complex media unless supplemented with pyruvate and fumarate. Aspartase was shown by activity and proteomic analyses to be upregulated by oxygen limitation, and aspartate enhanced oxygen-limited growth of C. jejuni in an aspA -dependent manner. Stoichiometric aspartate uptake and succinate excretion involving the redundant DcuA and DcuB transporters indicated that in addition to a catabolic role, AspA can provide fumarate for respiration. Significantly, an aspA mutant of C. jejuni 81-176 was impaired in its ability to persist in the intestines of outbred chickens relative to the parent strain. Together, our data highlight the dual function of aspartase in C. jejuni and suggest a role during growth in the avian gut.     

Conversion of [U-14C]threonine into 14C-labelled amino acids in the brain of thiamin-deficient rats.

In confirmation of the findings of Gaitonde et al. (1974), a decrease in the brain concentration of threonine and serine, and an increase in glycine, were observed in rats maintained on a thiamin-deficient diet. Similar changes were found in the blood, and the concentration of several other amino acids in the blood decreased significantly. There was a correlation between the concentrations of threonine, serine, aspartate and asparagine in the brain and blood. In experiments in which [U-14C]threonine was injected into rats most of the radioactivity in the brain and blood of control rats was, as expected, in threonine in the acid soluble metabolites. In contrast, a considerable proportion of radioactivity was also found in other amino acids, namely glutamate, glutamine, aspartate, gamma-aminobutyrate and alanine, in the brain of thiamin-deficient rats. [U-14C]Threonine was also converted into 14C-labelled lactate and glucose, but the extent of this conversion was severalfold higher in thiamin-deficient than in control rats. This finding gave evidence of the stimulation in thiamin-deficient rats of the catabolism of [U-14C]threonine to [14C]lactate by the aminoacetone pathway catalysed by threonine dehydrogenase, and into succinate via propionate by the alpha-oxobutyrate pathway catalysed by threonine dehydratase (deaminase). The measurement of specific radioactivities of glutamate, aspartate and glutamine after injection of [U-14C]threonine, indicated a stimulation of the activities of threonine dehydrogenase and threonine dehydratase (deaminase) in the brain of thiamin-deficient rats. The specific radioactivities of glutamate, asparatate and glutamine int he brain were consistent with an alteration in the metabolism of threonine, mainly in the 'large' compartment of the brain of thiamin-deficient rats. The measurement of relative specific radioactivity of proteins after injection of [U-14C]threonine indicated a marked decrease in the synthesis of proteins, mainly in the liver of thiamin-deficient rats.     

Enhanced glycolysis after maturation of bovine oocytes in vitro is associated with increased developmental competence

The effect of maturation in vitro on metabolism of individual bovine oocytes was examined. Three maturation media were used: standard, consisting of tissue culture medium 199 supplemented with serum and pyruvate, and a chemically defined medium supplemented with either amino acids or lactate. Development to blastocyst was significantly higher (P < 0.05) after maturation in standard medium (47%) than in defined medium with lactate (17%) but was not different than maturation in defined medium with amino acids (29%). Glucose metabolism through the Krebs cycle was not different after maturation in standard or defined medium with amino acids or lactate (0.48, 0.43, 0.38 pmol/oocyte/3 hr, respectively) but was affected by the removal of unlabeled pyruvate from the metabolic measurement medium (0.16, 0.21, 0.27 pmol/oocyte/3 hr, respectively). When physiological concentrations of glucose (0.52 mM) and pyruvate (0.5 mM) were used, oxidation of pyruvate was not different after maturation in standard or defined medium with amino acids or lactate (1.38, 1.13, 1.13 pmol/ oocyte/3 hr, respectively); however, glycolysis was significantly increased (P < 0.05) in treatments that supported higher blastocyst development (standard medium, 1.77 pmol/oocyte/3 hr; defined medium with amino acids, 1.58 pmol/oocyte/3 hr; defined medium with lactate, 1.32 pmol/oocyte/3 hr). Metabolism of glucose through the Krebs cycle was low in all media. In contrast, oxidation of pyruvate readily occurred after maturation in vitro. Metabolism of glucose through the Embden-Meyerhof pathway is important during oocyte maturation in vitro, and higher glycolytic rates in in vitro matured oocytes may reflect increased developmental competence.