Growth inhibition of Escherichia coli K-12 by L-valine: a consequence of a regulatory pattern.

Summary We studied the production of the ilvG gene product, the valine resistant acetolactate synthase isoenzyme II, in an ilvO ⁺ G ⁺ ilvB ilvHI derivative of Escherichia coli K-12. This strain contains mutations in the structural genes for the valine sensitive acetolactate synthase isoenzymes I and III. We find that the ilvG gene is not expressed in this strain when grown with either isoleucine and valine or with isoleucine, leucine and valine, or when limited for either isoleucine or valine. Since we previously found that the ilvG gene is expressed in an ilvO603 containing strain (Favre et al., 1976), we presume that the mechanism by which E. coli K-12 regulates the ilv gene cluster is responsible for the lack of ilvG expression in the ilvO ⁺ strain. The valine sensitivity of E. coli K-12 is a consequence of this regulatory pattern.     

Sulfonylurea-resistant mutants and natural tolerance of cyanobacteria

The herbicide sulfometuron methyl (SM) inhibited the growth of the cyanobacterium Synechococcus sp. PCC7942, but not of Synechocystis sp. PCC6714. The inhibitory effect was alleviated by the simultaneous addition of valine, leucine and isoleucine. SM resistant mutants were isolated from Synechococcus 7942, two types of which were further analysed. In these mutants, SM3/20 and SM2/32, the activity of acetolactate synthase (ALS) — a key enzyme in the biosynthesis of branched-chain amino acids —appeared 2600- and 300-fold, respectively, more resistant to SM than that of their wild type. Strain SM2/32 also exhibited a low level of ALS activity. Although the growth of the latter mutant was extremely inhibited by valine, the sensitivity of its ALS activity to feed-back inhibition by the amino acid was unaltered. At high concentrations valine inhibited growth of the wild type strains and of the mutant SM3/20. Isoleucine alleviated the valine-induced growth inhibition. Unlike that of Synechococcus 7942, the ALS activity of Synechocystis was found to tolerate high concentrations (100-fold) of the herbicide. The study confirms that the SM mutations are correlated with a cyanobacterial ilv gene.Abbreviations ALS acetolactate synthase; ile, isoleucine - leu leucine - NTG N-methyl-N-nitro-N-nitrosoguanidine - SM sulfometuron methyl - SM^r sulfometuron methyl resistant - val valine     

Regulation of leucine transport by intracellular pH in Bacillus pasteurii

The kinetics, specificity and mechanism of leucine uptake were studied in the alkaliphilic bacterium Bacillus pasteurii DSM 33 (ATCC 11859). Leucine was accumulated up to 200-fold by a sodium-dependent secondary transport system for branched-chain amino acids. Apparent K_t values of 9.6 μM for leucine, 8.9 μM for isoleucine, 9.3 μM for valine, and 0.71 mM for sodium were determined, and maximum uptake activity was observed at an external pH of 8.5 and at 35°C. The effect of several ionophores indicated that transport was energized by the membrane potential and a sodium gradient; each gradient alone was sufficient to drive the uptake of leucine. The activity of the leucine transport system was regulated by the intracellular pH and was inhibited at an internal pH below 7.0. Received: 26 September 1995 / Accepted: 10 December 1995     

Age-related changes of muscle and plasma amino acids in healthy children

The aim of the study was to explore if changes in muscle and plasma amino acid concentrations developed during growth and differed from levels seen in adults. The gradient and concentrations of free amino acids in muscle and plasma were investigated in relation to age in metabolic healthy children. Plasma and specimens from the abdominal muscle were obtained during elective surgery. The children were grouped into three groups (group 1: < 1 year, n = 8; group 2: 1–4 years, n = 13 and group 3: 5–15 years, n = 15). A reference group of healthy adults (21–38 years, n = 22) was included in their comparisons and reflected specific differences between children and adults. In muscle the concentrations of 8 out of 19 amino acids analysed increased with age, namely taurine, aspartate, threonine, alanine, valine, isoleucine, leucine, histidine, as well as the total sums of branched chain amino acids (BCAA), basic amino acids (BAA) and total sum of amino acids (P < 0.05). In plasma the concentrations of threonine, glutamine, valine, cysteine, methionine, leucine, lysine, tryptophane, arginine, BCAA, BAA and the essential amino acids correlated with age (P < 0.05). These results indicate that there is an age dependency of the amino acid pattern in skeletal muscle and plasma during growth.     

Branched chain amino acids as source of specific branched chain volatile fatty acids during the fermentation process of fish sauce

Summary.  The source of the formation of branched chain volatile fatty acids (VFA) in fish sauce was investigated. Certain branched VFA were derived from the degradation of specific amino acids as iso-butyric acid from valine and iso-valeric acid from leucine. Short and long straight chain VFA were significantly higher in the linoleic acid added sample than in the control but did not significantly bring changes to the branched chain VFA. It is suggested that straight chain VFA developed from fish fats. Alanine and isoleucine did not have a clear influence on the production of volatile fatty acids. Received November 23, 2001 Accepted June 20, 2002 Published online December 18, 2002 RID="*" ID="*" Part of this paper was presented in the 7th International Congress on Amino Acids and Proteins in Vienna, Austria from August 6–10, 2001. Authors' address: Norlita G. Sanceda, Ph.D., Institute of Environmental Science for Human Life, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112, Japan, Fax: + 81-3-5978-5805, E-mail: lita@cc.ocha.ac.jp     

Determination of amino acid tissue concentrations by microdialysis: method evaluation and relation to plasma values

Summary. Microdialysis is an in vivo technique to monitor tissue concentrations of low molecular weight substances by means of a continuously perfused artificial capillary with a semipermeable membrane placed into the region of interest. The suitability of microdialysis to determine tissue concentrations of amino acids was evaluated in vitro by placing the catheter into Ringer buffer or into a plasma protein (50 g/l) solution containing 32 different amino acids (150 μmol/l each). All amino acids tested crossed freely the microdialysis membrane with recoveries close to 100%. Microdialysis fluid was sampled from subcutaneous tissue of five newborns and amino acid content analysed. Total and non protein bound amino acids were determined in the patients plasma by acid precipitation or ultrafiltration, respectively. Mean subcutaneous tissue concentrations were lower as compared to plasma for taurine, serine, alanine, aspartate, glutamate and ornithine and higher for valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and arginine, indicating net uptake or release of amino acids from subcutaneous tissue. Thus, microdialysis offers a convenient and minimal invasive way to study tissue amino acid composition and appears to be a promising analytical tool for the study of amino acid metabolism in vivo. Received August 7, 2000 Accepted January 7, 2001     

Expression of a valine-resistant acetolactate synthase activity mediated by the ilv O and ilv G genes of Escherichia coli K-12

Summary A strain carrying the ilv0603 mutation has been isolated in E. coli K-12 and its characteristics were found to be very similar to those previously reported by Ramakrishnan and Adelberg (1965a) for other ilv0 mutants.The strain carrying the ilv0603 mutation is resistant to valine inhibition (Val^r) and we show that this resistance depends on the expression of a newly recognized gene, ilvG, which is located at min 75, between ilvE and ilvD on the E. coli K-12 map. The ilvG gene causes the expression of a Val^r acetolactate synthase, which is detectable only when the ilv0603 mutation is also present in cis on the same chromosome. Under these conditions the Val^r acetolactate synthase activity is eluted, on a hydroxylapatite column, at an ionic strength slightly lower than that required for elution of the remaining acetolactate synthase activity (sensitive to valine inhibition). The Val^r peak is missing in a strain carrying an ilvG (amber) mutation.     

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and γ-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra × maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and γ-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.     

Degradation of proteins and amino acids by Caloramator proteoclasticus in pure culture and in coculture with Methanobacterium thermoformicicum Z245

This study investigated the degradation of proteins and amino acids by Caloramator proteoclasticus, an anaerobic thermophilic (55 °C) fermentative bacterium isolated from an anaerobic bioreactor. Experiments were performed in the presence and absence of Methanobacterium thermoformicicum Z245, a methanogen that can use both hydrogen and formate for growth. Higher production rates and yields of the principal fermentation products from gelatin were observed in methanogenic coculture. The specific proteolytic activity in coculture tripled the value obtained in pure culture. C. proteoclasticus fermented glutamate to acetate, formate, hydrogen and alanine. In methanogenic coculture, a shift towards higher amounts of acetate and hydrogen with no alanine production was observed. Extracts of glutamate-grown cells possessed high activities of β-methylaspartase, a key enzyme of the mesaconate pathway leading to acetate. The presence of two enzymes (alanine-α-ketoglutarate aminotransferase and NADH-dependent alanine dehydrogenase) usually involved in the biosynthesis of alanine from pyruvate was also detected. The fermentation of amino acids known to be oxidatively deaminated (leucine and valine) was improved in the presence of both methanogenesis and glycine, a known electron acceptor in the Stickland reaction. Culture conditions seem to be very important in the way C. proteoclasticus disposes of reducing equivalents formed during the degradation of amino acids. Received: 29 March 1999 / Received revision: 2 July 1999 / Accepted: 1 August 1999     

The acetolactate synthase isoenzymes of Escherichia coli K-12.

Summary Strains of Escherichia coli K-12 possessing only one of the three genes coding for acetolactate synthetase activity present either in the wild type or in its ilv0603 derivative were prepared and analyzed. Extracts prepared from these strains show different values of acetolactate synthase specific activity and different sensitivity to valine inhibition. These strains show a unique pattern of growth inhibition by different substances.Temperature sensitive (ts) mutations in the ilvB and ilvG genes, have been isolated and characterized. Extracts of these strains were found to have an acetolactate synthase activity more heat labile than that of a strain containing the corresponding wild type allele. We conclude that ilvB and ilvG are the structural genes for two different forms of acetolactate synthase activity, most likely two isoenzymes. Moreover, since the strains containing a ts mutation show a temperature sensitive auxotrophy for isoleucine and valine, these two acetolactate synthases participate in isoleucine and valine biosynthesis. Similar evidence for a third acetolactate synthase, the product of the ilvHI genes, has been reported previously.We propose the following names for the acetolactate synthase isoenzymes: acetolactate synthase I (AHAS I), the product of the ilvB gene; acetolactate synthase II (AHAS II), the product of ilvG gene; and acetolactate synthase III (AHAS III), the product of the ilvHI genes.     

Improvement of a Chemically Defined Medium for the Sustained Growth of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>: Nutritional Requirements

The aims of this work were to improve a basal synthetic medium (BM) for the growth of Lactobacillus plantarum strains and to establish their amino-acid requirements. Amino-acid use was analyzed in the most nutritionally demanding bacterium. First, the improved BM (L. plantarum synthetic medium [LPSM]) was created by increasing some vitamins in the BM, especially p-aminobenzoic acid, vitamin B₁₂, and biotin; 5-fold phenylalanine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, and tryptophan; and 10-, 60-, and 75-fold valine, arginine, and tyrosine, respectively. With these additions, the N8 and N4 strains of L. plantarum grew rapidly to reach final cell densities similar to those obtained in Mann–Rogosa–Sharpe medium. When cysteine, leucine, valine, isoleucine, threonine, and glutamic acid were individually removed from this medium, bacterial growth significantly decreased or ceased, indicating that these amino acids are essential for growth. The N4 strain also required lysine and tryptophan in addition to the six amino acids necessary for growth. L. plantarum N4 mainly consumed essential amino acids, such as valine, lysine, cysteine, and threonine as well as the stimulatory amino acid, arginine. Thus, the BM was improved mainly on the basis of annulling limitations with respect to amino acids. With this, improved medium cell densities in the order of 10⁹ colony-forming units/mL have been achieved, indicating that LPSM medium could be used for conducting metabolic and genetic studies on L. plantarum. Their low levels in orange juice suggest that these amino acids may not satisfy the total nitrogen requirement for the development of L. plantarum in the natural environment.     

Amino acid degradation by the mesophilic sulfate-reducing bacterium Desulfobacterium vacuolatum

Desulfobacterium vacuolatum strain IbRM was able to grow using casamino acids as a source of carbon, energy and nitrogen. Growth was accompanied by utilization of several amino acids and sulfide production. Proline and glutamate were used preferentially and to the greatest extent. Glycine, serine and alanine were used more slowly and only after proline and glutamate were used. Isoleucine, valine, leucine and aspartate decrease was slowest and occurred in a linear fashion throughout the growth phase. Amino acids used from casamino acids, excluding aspartate, were also used as single carbon, energy and nitrogen sources. As a single amino acid, aspartate could only be used as a nitrogen source. Aspartate was not used as an electron acceptor. No growth occurred on any amino acid in the absence of sulfate. As single substrates, isoleucine, proline and glutamate were oxidized without formation of acetate and with molar yields of 13.1, 9.4 and 7.7 g mol^–1, respectively. Received: 24 June 1997 / Accepted: 10 September 1997     

Neutrophils as a source of branched-chain,aromatic and positively charged free amino acids

Neutrophils release branched-chain (valine, isoleucine, leucine), aromatic (tyrosine, phenylalanine) and positively charged free amino acids (arginine, ornithine, lysine, hydroxylysine, histidine) when adhere and spread onto fibronectin. In the presence of agents that impair cell spreading or adhesion (cytochalasin D, fMLP, nonadhesive substrate), neutrophils release the same amino acids, except for a sharp decrease in hydroxylysine and an increase in phenylalanine, indicating their special connection with cell adhesion. Plasma of patients with diabetes is characterized by an increased content of branched-chain and aromatic amino acids and a reduced ratio of arginine/ornithine compared to healthy human plasma. Our data showed that the secretion of neutrophils, regardless of their adhesion state, can contribute to this shift in the amino acid content.

Abbreviations: BCAAs: branched-chain amino acids; Е2: 17β-estradiol; LPS: lipopolysaccharide from Salmonella enterica serovar Typhimurium; fMLP: N-formylmethionyl-leucyl-phenylalanine.     

Derepressed leucine transport activity in Escherichia coli

Transport activity and synthesis of binding protein for the amino acids leucine, isoleucine and valine in E. coli are coordinately controlled by the level of leucine in the growth medium. Spontaneous mutants (dlu) which can utilize D-leucine as a source of L-leucine show derepressed transport activity for the three-branched chain amino acids. The increased transport activity is a result of an increase in the binding protein for these amino acids. Azaleucine-resistant mutants have been isolated which have a defect in leucine transport but normal levels of the binding protein for leucine.     

Neuronal Metabolism of Branched-Chain Amino Acids: Flux Through the Aminotransferase Pathway in Synaptosomes

Abstract: The metabolism of branched-chain amino acids (BCAAs) was studied in cortical synaptosomes. With [¹⁵N]leucine (1 mM) as precursor, the cumulative appearance of ¹⁵N in [¹⁵N]glutamate and [¹⁵N]aspartate was 0.2 nmol/min/mg of protein without supplemental α-ketoglutarate and 0.3 nmol/min/mg of protein in the presence of α-ketoglutarate (0.5 mM). The BCAA aminotransferase reaction also proceeded in the “reverse” direction [α-ketoisocaproate (KIC) + glutamate → leucine + α-ketoglutarate]. This was documented by incubating synaptosomes with [¹⁵N]glutamate and measuring the formation of [¹⁵N]leucine. Without KIC in the medium, the rate of [¹⁵N]leucine production was 0.13 nmol/min/mg of protein. In the presence of 25 µM KIC the rate was 0.79 nmol/min/mg of protein and even greater (1.0 nmol/min/mg of protein) in the presence of 500 µM KIC. The reamination of KIC was two- to threefold faster with [2-¹⁵N]glutamine as precursor compared with [¹⁵N]glutamate. The ketoacid of valine, α-ketoisovalerate (KIV), was reaminated to [¹⁵N]valine at a rate comparable to that observed with respect to KIC. The BCAA transaminase mediated not only the bidirectional transfer of amino groups between leucine or valine and glutamate, but also the direct transfer of nitrogen between leucine and valine. This was ascertained in studies in which the incubation medium was supplemented with either [¹⁵N]leucine and KIV or [¹⁵N]valine and KIC (amino acids at 1 mM and ketoacids at 25 or 500 µM). The rate was faster in the direction of leucine formation at both the lower (6.1-fold) and higher (1.7-fold) KIC concentration. It is suggested that in synaptosomes the BCAA transaminase (a) functions predominantly in the direction of leucine formation and (b) maintains a constant ratio of BCAAs and ketoacids to one other.     

The biochemical metabolite screen in the Munich ENU Mouse Mutagenesis Project: determination of amino acids and acylcarnitines by tandem mass spectrometry

Background: Gene mutations often result in altered protein expression and, in turn, lead to changes in metabolite levels in one or more distinct biochemical pathways. Traditional analytical methods for metabolite determination are usually time consuming, expensive, and, thus, not suitable for high throughput analysis. However, recent developments in electrospray-tandem-mass-spectrometry allow comprehensive metabolite scanning from very small amounts of blood with high speed, cost effectiveness, and accuracy. Methods: A blood spot from a filter paper equivalent to 3 μl of blood was punched out and transferred to a 96-well microtiter plate. After addition of a set of 14 stable isotope-labeled internal standards, amino acids and acylcarnitines were extracted with methanol. The dried residue was derivatized with butanolic hydrochloric acid and subjected to MSMS analysis. Results: Acyl-carnitines were all determined by a precursor ion scan of 85 Da. Neutral loss scanning of 102 Da was suitable for the quantitation of threonine, serine, proline, histidine, alanine, aspartic acid, glutamic acid, methionine, tyrosine, phenylalanine, isoleucine/leucine and valine. Glycine was detected by a loss of a 56-Da fragment, whereas a 119-Da loss was suitable for the measurement of citrulline, ornithine, arginine, and lysine. Specific problems encountered: owing to their identical molecular weight, isoleucine and leucine could not be quantitated separately, and, owing to their instability, glutamine and asparagine were found to be decarboxylated to their respective acids. Determination was linear over the concentration range tested (20 to 1000 μmol/L), and intraassay and interassay coefficients of variation were in the range of 10–15%. Conclusion: ESI-MSMS proved to be a highly sensitive, linear, and sufficiently precise method for the quantitative determination of amino acids and acylcarnitines in mouse blood, allowing large-scale screening applications when speed and cost effectiveness are mandatory. Received: 16 December 1999 / Accepted: 17 December 1999     

Mechanism of the effect of levorin on transport of amino acids inCandida albicans

By use of incubation media of various ionic compositions and pH's, it was possible to reveal that inhibition of the transport of neutral amino acids having long hydrophobic chains (leucine, phenylalanine) inCandida albicans by the polyene antibiotic levorin was mainly due to redistribution—caused by the antibiotic—of monovalent cations in the cells and protoplasts of the yeast. In an acidic medium, levorin was also found to induce a decrease of proton release from the cells, accompanied by some inhibition of leucine transport. Transport of neutral amino acids having short hydrophobic chains (alanine, glycine, proline) was inhibited by this polyene antibiotic, irrespective of the ionic composition or pH of the medium.     

The distribution of acetohydroxyacid synthase in soil bacteria

Most bacteria possess the enzyme acetohydroxyacid synthase, which is used to produce branched-chain amino acids. Enteric bacteria contain several isozymes suited to different conditions, but the distribution of acetohydroxyacid synthase in soil bacteria is largely unknown. Growth experiments confirmed that Escherichia coli, Salmonella enterica serotype Typhimurium, and Enterobacter aerogenes contain isozymes of acetohydroxyacid synthase, allowing the bacteria to grow in the presence of valine (which causes feedback inhibition of AHAS I) or the sulfonylurea herbicide triasulfuron (which inhibits AHAS II) although a slight lag phase was observed in growth in the latter case. Several common soil isolates were inhibited by triasulfuron, but Pseudomonas fluorescens and Rhodococcus erythropolis were not inhibited by any combination of triasulfuron and valine. The extent of sulfonylurea-sensitive acetohydroxyacid synthase in soil was revealed when 21 out of 27 isolated bacteria in pure culture were inhibited by triasulfuron, the addition of isoleucine and/or valine reversing the effect in 19 cases. Primers were designed to target the genes encoding the large subunits (ilvB, ilvG and ilvI) of acetohydroxyacid synthase from available sequence data and a ∼355 bp fragment in Bacillus subtilis, Arthrobacter globiformis, E. coli and S. enterica was subsequently amplified. The primers were used to create a small clone library of sequences from an agricultural soil. Phylogenetic analysis revealed significant sequence variation, but all 19 amino acid sequences were most closely related to published large subunit acetohydroxyacid synthase amino acid sequences within several phyla including the Proteobacteria and Actinobacteria. The results suggested the majority of soil microorganisms contain only one functional acetohydroxyacid synthase enzyme sensitive to sulfonylurea herbicides.