Aspartame as a source of essential phenylalanine for the growth of oral anaerobes

Abstract Phenylalanine and aspartic acid requirements were determined for 13 species of oral bacteria using the chemically defined medium OMIZ-W1. None of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Eikenella corrodens, Selenomonas sputigene, Treponema pectinovorum, T. socranskii , or Wolinella recta required either of these amino acid constituents of aspartame ( l -aspartyl- l -phenylalanine methylester). Phenylalanine was essential for the growth of Capnocytophaga gingivalis, Eubacterium timidum, Fusobacterium nucleatum, Porphyromonas gingivalis, T. denticola , and T. vincentii , while aspartic acid was not required. With the exception of E. timidum , all phenylalanine-dependent strains could grow when the free amino acid was replaced by aspartame at concentrations at least 10-fold lower than those used for aspartame as an artificial sweetener.     

Amino acid requirements and peptidase activities of Streptococcus salivarius subsp. thermophilus

The aim of this work was to investigate the relationship between amino acid requirements and peptidase enzyme systems in three Streptococcus salivarius subsp. thermophilus strains. A synthetic medium without nitrogen components and a milk (RD milk) without its non-protein nitrogen fraction were prepared with different mixtures of amino acids. The strains showed different amino acid requirements. Some amino acids proved to be essential, some were required, while others did not affect growth. In the synthetic medium, only leucine and glutamic acid were essential for growth. In RD milk, the amino acid requirements were found to be lower, with only the absence of glutamic acid causing complete inhibition of growth. Relationships between aminopeptidase activities of the strains and their amino acid requirements were observed. Strains with higher amino acid requirements were also found to express a wider range of peptidases.     

A Synthetic Medium for Comparative Nutritional Studies of Lactobacilli

The composition of a synthetic medium supporting the growth of lactobacilli is given (Table 1). The medium, containing glucose, amino acids, vitamins, mineral salts, purines and pyrimidines, allows the study of nutritional requirements of different strains of lactobacilli under identical environmental conditions. It was found that all the strains tested needed L-glutamic acid, L-valine and L-leucine, and a group of them also required L-arginine, L-tyrosine and L-tryptophan. Some strains required vitamins, e.g. L. bulgaricus (pantothenic acid), L. fermenti (pantothenic acid and niacin). These results are compared with those found by others employing different media.     

Relationship between Na+-dependent cytoplasmic pH homeostasis and Na+-dependent flagellar rotation and amino acid transport in alkalophilic Bacillus

The cytoplasmic pH homeostasis of alkalophilic Bacillus strains required the presence of Na+ in the medium, and Li+ was found to be equivalently substitutable for Na+. Flagellar rotation and amino acid transport of these bacteria also required Na+ but Li+ was not substitutable for Na+. Na+ concentration of about 1 mM was enough for the cytoplasmic pH homeostasis, while more than 10 mM Na+ was required for the full activities of flagellar rotation and amino acid transport. The addition of 150 mM ethanolamine to the cells at pH 9.6 disrupted the pH homeostasis and increased the cytoplasmic pH close to the external pH. Under this condition, however, flagellar rotation and amino acid transport were not so much affected. Thus, it is clear that flagellar rotation and amino acid transport themselves require the presence of Na+ in the medium, independent of the Na+-dependent cytoplasmic pH homeostasis.     

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.     

Partial suppression of the effect of gene re1A in Fusr-mutant Escherichia coli K-12 cells]

Mutants of Escherichia coli K-12 re1A+-strain CP 78 resistant to fusidic acid (Fusr) were isolated and forms sensitive to high concentration of leucine (500 g/ml) were selected. When shifted down from nutrient broth to minimal medium M9 with supplemented glucose and required amino acids, these leucine-sensitive mutants continued RNA synthesis and demonstrated the prolonged lag-phase in contrast to the parent strain CP 78. Both properties are known to be characteristic of the Rel- strains. At the same time withdrawal of the required amino acids results in cessation of RNA synthesis in Fusr mutants, in the parent Rel+ strain. Thus, leucine-sensitive Fusr mutants show Rel- phenotype only upon amino acid starvation caused by shift down from nutrient broth to minimal medium.     

Development of a minimal chemically-defined medium for the exponential growth of Streptococcus thermophilus

AIMS: The present work aimed to define a minimal chemically-defined medium which could sustain the growth of most (if not all) strains of Streptococcus thermophilus. METHODS AND RESULTS: A minimal medium containing 20 components, including one carbohydrate source, six amino acids, two metallic ions, six vitamins and urea allowed for growth of 13 out of 15 Strep. thermophilus strains. Growth of the two last strains required the presence of additional amino acids, the number of which depended on the strain. Growth rates of the strains in the minimal medium ranged from 0.38 to 0.64 h(-1), and final populations were about 10(8) cfu ml(-1). CONCLUSIONS: Streptococcus thermophilus appears much less demanding than other lactic acid bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The definition of such a growth medium will be very useful for metabolic flux studies as well as peptide transport studies.     

Growth requirements of san francisco sour dough yeasts and bakers' yeast

The growth requirements of several yeasts isolated from San Francisco sour dough mother sponges were compared with those of bakers' yeast. The sour dough yeasts studied were one strain of Saccharomyces uvarum, one strain of S. inusitatus, and four strains of S. exiguus. S. inusitatus was the only yeast found to have an amino acid requirement, namely, methionine. All of the yeasts had an absolute requirement for pantothenic acid and a partial requirement for biotin. Inositol was stimulatory to all except bakers' yeast. All strains of S. exiguus required niacin and thiamine. Interestingly, S. inusitatus, the only yeast that required methionine, also needed folic acid. For optimal growth of S. exiguus in a molasses medium, supplementation with thiamine was required.     

Growth factor requirements of two strains ofSphaerotilus discophorus

The relatively complex growth-factor requirements of two strains ofSphaerotilus discophorus, strains 31 and 32, have been elucidated. In addition to thiamin and biotin which are required by other strains ofS. discophorus, the two strains must be supplied also with adenine or guanine and with cyanocobalamin for growth in a glucose — Casamino Acids — mineral salts medium. The cyanocobalamin can be replaced by methionine but only if relatively large amounts of this amino acid, 100 µg or more per ml, are added to the medium. The methionine requirement of the two strains is approximately 3 times greater than that of otherS. discophorus strains.     

The effect of 2-oxoglutarate and biotin in the release of amino acids by Citrobacter intermedius C3

Excretion of amino acids by Citrobacter intermedius C3 was assayed in a mineral medium with glucose as carbon source. Glutamic acid is the main amino acid excreted in liquid medium and it is also detected at the colonial level in solid medium. Mutants with different behaviour with respect to the excretion of amino acids are studied. The presence of 2-oxoglutarate in the medium induced excretion in all strains. On the other hand when biotin was added to the culture media amino acid excretion was partially reduced.     

Influence of Incubation Atmosphere on Growth and Amino Acid Requirements of Streptococcus mutans

The growth response of Streptococcus mutans representing antigenic type a or d in a chemically defined medium was influenced by the oxygen concentration of the growth atmosphere. Under controlled aerobic (1.5% O(2)) conditions these cultures attained a greater density than when the atmosphere contained 0.006% O(2) or less. The growth of S. mutans strains representing antigenic types b or c in the defined medium was independent of the oxygen concentration of the growth environment. Under the conditions used in this study, none of the strains tested could utilize ammonium ion as a sole source of nitrogen for growth. The requirement for certain amino acids and inhibition by other amino acids varied with antigenic type and relative oxygen concentration of the growth environment. Under conditions where the atmospheric oxygen was reduced to 0.0006% O(2) or less, the amino acid requirements of the cultures became either more numerous or more stringent. S. mutans strains of type c generally required the least number of amino acids, whereas cultures of type d had more numerous requirements. Nearly every culture tested under the anaerobic atmosphere was inhibited by one of the branched-chain amino acids, leucine, valine, or isoleucine. Methionine and lysine were also found to be inhibitory, particularly toward the type c strains.     

Growth Requirements of San Francisco Sour Dough Yeasts and Bakers' Yeast

The growth requirements of several yeasts isolated from San Francisco sour dough mother sponges were compared with those of bakers' yeast. The sour dough yeasts studied were one strain of Saccharomyces uvarum, one strain of S. inusitatus, and four strains of S. exiguus. S. inusitatus was the only yeast found to have an amino acid requirement, namely, methionine. All of the yeasts had an absolute requirement for pantothenic acid and a partial requirement for biotin. Inositol was stimulatory to all except bakers' yeast. All strains of S. exiguus required niacin and thiamine. Interestingly, S. inusitatus, the only yeast that required methionine, also needed folic acid. For optimal growth of S. exiguus in a molasses medium, supplementation with thiamine was required.     

Utilization of Ammonia Nitrogen by Intestinal Bacteria Isolated from Pigs

In a medium containing ammonia, proteose peptone, and cysteine as nitrogen sources, 17 of 24 Bacteroidaceae strains, 3 of Selenomonas strains, 1 of 7 curved rods, 3 of 7 Spirochaetaceae strains, 8 of 20 Eubacterium strains, 8 of 13 Peptococcaceae strains, 3 of 4 Clostridium strains, 19 of 20 Enterobacteriaceae strains, and 1 of 8 Streptococcus strains utilized ammonia nitrogen preferentially to proteose peptone nitrogen. To determine the ability of intestinal microbes to synthesize amino acids from ammonia, ammonia utilization by Bacteroides ruminicola strain 9 was studied in defined media containing ammonia and other nitrogen sources. In another medium containing ammonia, proteose peptone, and cysteine as nitrogen sources, ammonia was preferentially utilized even when the proteose peptone nitrogen content was eight times greater than that of ammonia nitrogen. In a medium containing ammonia, an amino acid, and cysteine, the lowest uptake of ammonia nitrogen was observed when the medium contained aspartic acid, glutamic acid, threonine, or alanine; but ammonia was utilized more effectively than any of the amino acids. Incorporation of ¹⁵N from [¹⁵N]ammonia into bacterial amino acids was studied. ¹⁵N was incorporated into every amino acid of B. ruminicola strain 9, and the highest uptake was observed in aspartic acid and alanine.     

Defined minimal media for the growth of prototrophic and auxotrophic strains of Bacillus stearothermophilus

Minimal chemically defined media for Bacillus stearothermophilus were developed at 60°C and quantitative requirements for each nutrient were determined. A prototrophic strain of B. stearothermophilus was grown in medium containing only glucose and mineral salts whereas auxotrophic strains in addition required biotin, thiamine, nicotinic acid and DL-methionine. Metabolic interaction between L-valine and L-leucine was observed with auxotrophic organisms. The presence of L-leucine in minimal medium necessitated the addition of L-valine. Growth took place in the absence of both amino acids.     

Chemically defined media for the cultivation of Naegleria: pathogenic and high temperature tolerant species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCC 30100, ATCC 30863, and ATCC 30896) and two strains of N. lovaniensis (ATCC 30467 and ATCC 30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCC 30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysin for ATCC 30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCC 30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCC 30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B12, nicotinamide, and Ca pantothenate) was required.     

Porphyrin and corrinoid mutants of Bacillus subtilis.

Porphyrin auxotrophs of Bacillus subtilis can be divided into two groups. Strains belonging to the first group (hemA, hemB, or hemC) are not able to synthesize or metabolize porphobilinogen. These strains require cysteine, cystine, and methionine, respectively. Traces of aminolevulinic acid, in a hemin-containing medium, can replace the cysteine requirement in a mutant lacking aminolevulinic acid synthetase. In bacteria belonging to the second group (hemE, hemF, or hemG), porphyrin biosynthesis is blocked at later steps, and the amino acids mentioned above are not required. It is of interest that both the activity of ribonucleotide reductase and the amount of vitamin B12 were significantly lower in the first group. The addition of vitamin B12 to the medium did not promote the growth of strains examined. We assume that porphobilinogen deaminase is essential for the synthesis of corrinoids.     

Design and characterization of auxotrophy-based amino acid biosensors

Efficient and inexpensive methods are required for the high-throughput quantification of amino acids in physiological fluids or microbial cell cultures. Here we develop an array of Escherichia coli biosensors to sensitively quantify eleven different amino acids. By using online databases, genes involved in amino acid biosynthesis were identified that - upon deletion - should render the corresponding mutant auxotrophic for one particular amino acid. This rational design strategy suggested genes involved in the biosynthesis of arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, and tyrosine as potential genetic targets. A detailed phenotypic characterization of the corresponding single-gene deletion mutants indeed confirmed that these strains could neither grow on a minimal medium lacking amino acids nor transform any other proteinogenic amino acid into the focal one. Site-specific integration of the egfp gene into the chromosome of each biosensor decreased the detection limit of the GFP-labeled cells by 30% relative to turbidometric measurements. Finally, using the biosensors to determine the amino acid concentration in the supernatants of two amino acid overproducing E. coli strains (i.e. ΔhisL and ΔtdcC) both turbidometrically and via GFP fluorescence emission and comparing the results to conventional HPLC measurements confirmed the utility of the developed biosensor system. Taken together, our study provides not only a genotypically and phenotypically well-characterized set of publicly available amino acid biosensors, but also demonstrates the feasibility of the rational design strategy used.     

Extracellular amino acids of aerobic spore-forming bacteria]

An ability to synthesize and accumulate in the growth medium for extracellular amino acids of 108 strains of 13 aerobic sporeforming bacteria species at deep cultivation on the simple synthetic glucose-mineral liquid nutrient medium optimized for the representatives of this genus. Has been studied the bacillus strains studied in the present conditions synthesize 19 amino acids and 2 amino carbons in different quantities and combinations. Amino acids being synthesized the most and least by these microorganisms have been determined in quantitative expression. Extracellular amino acid accumulation was proved to be a strain property not of genus. In general, the genus studied was evaluated as active one in amino acid production. The most active strains may present an interest as initials for further selective obtaining of producers of separate amino acids. The outlook for aerobic bacilli strains having marked amino-synthesizing properties and complex of other biological activities is being discussed for construction of medicinal and prophylactic of live microbial culture preparations.     

Nutrient requirements of Bacillus anthracis strains and the growth characteristics of strain STI-1 in batch cultivation on synthetic broths

A synthetic culture medium is proposed. This medium satisfies the nutritional requirements of most cultivated strains under study by a single amino acid: valine. The possibility of detecting strains with other nutritional requirements has been shown. The determination of LD50 in white mice has revealed no differences between the virulence of valine-dependent strains and that of strains with other nutritional requirements. During the batch cultivation of strain STI-1 in a liquid medium glucose shows the effect of substrate limitation and inhibition. The optimum concentration of glucose can be different, depending on the composition of the medium. The influence of different amino acids, the sources of carbon and energy, and nitrogen on the growth of the strain is evaluated.