Optimization of a synthetic nutrient medium for culturing Bacillus thuringiensis

The requirement of Bacillus thuringiensis subsp. galleriae 69/6 for amino acids and vitamins was studied. The composition of a synthetic nutrient medium was optimized. Alanine and nicotinic acid were found to be necessary for growth while other amino acids (aspartic and glutamic acids, histidine, threonine) were not indispensable although they increased the population density (yield). A deficiency as well as an excess of individual amino acids (threonine and glutamic acid) inhibited growth and decreased the yield of biomass. Elevated concentrations of aspartic and glutamic acids inhibited the formation of spores and crystals. As was demonstrated using the method of mathematical planning of an experiment, the synthetic medium contained optimal concentrations of nicotinic and amino acids and was suitable for the growth of B. thuringiensis strains as well as for the formation of spores and crystals by them.     

Growth, D-glucose utilization and malolactic fermentation by Leuconostoc œnos strains in 18 media deficient in one amino acid

Six Leuconostoc œnos strains were used to study the effect of the deficiency of one amino acid on growth, heterofermentative pathway and malolactic fermentation. All strains had an absolute requirement for four amino acids (isoleucine, glutamic acid, tryptophan and arginine) and needed six other amino acids (valine, methionine, cysteine, leucine, aspartic acid and histidine) for optimum growth. Each deficiency in one amino acid had a particular effect on D-glucose utilization. Overproduction and underproduction of D-lactic acid were observed. The rate of L-malic acid consumption in media deficient in one amino acid was lower than in the complete medium with all amino acids.
Although some deficiencies (glycine, phenylalanine, proline or tyrosine) had no influence on the growth, they noticeably limited the malolactic fermentation.     

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.     

Amino acid transport in a polyaromatic amino acid auxotroph of Saccharomyces cerevisiae

The initiation of growth of a polyaromatic auxotrophic mutant of Saccharomyces cerevisiae was inhibited by several amino acids, whereas growth of the parent prototroph was unaffected. A comparative investigation of amino acid transport in the two strains employing (14)C-labeled amino acids revealed that the transport of amino acids in S. cerevisiae was mediated by a general transport system responsible for the uptake of all neutral as well as basic amino acids. Both auxotrophic and prototrophic strains exhibited stereospecificity for l-amino acids and a K(m) ranging from 1.5 x 10(-5) to 5.0 x 10(-5) M. Optimal transport activity occurred at pH 5.7. Cycloheximide had no effect on amino acid uptake, indicating that protein synthesis was not a direct requirement for amino acid transport. Regulation of amino acid transport was subject to the concentration of amino acids in the free amino acid pool. Amino acid inhibition of the uptake of the aromatic amino acids by the aromatic auxotroph did not correlate directly with the effect of amino acids on the initiation of growth of the auxotroph but provides a partial explanation of this effect.     

Exogenous Valine Reduces Conversion of Leucine to 3-Methyl-1-Butanol in Saccharomyces cerevisiae

Mutant strains of the yeast Saccharomyces cerevisiae that require branched-chain amino acids must be supplemented with large concentrations (up to 10 mM) of these amino acids to satisfy their nutritional requirement. The utilization of one branched-chain amino acid, leucine, was examined in several leul strains of yeast grown aerobically in a glucose-ammonium salts minimal medium containing a limiting concentration (0.2 mM) of leucine. In this medium, the leucine requirement of the auxotrophic strains could be reduced by valine, another branched-chain amino acid. Increasing the valine concentration increased the cell yields of cultures and also reduced the levels of 3-methyl-1-butanol detected in the medium by gas chromatography. The concentration of 3-methyl-1-butanol was reduced from 122.0 to 48.9 μM when 5.0 mM valine was supplemented to limiting-leucine cultures. The amino acids isoleucine, threonine, norleucine, norvaline, α-amino-butyrate, alanine, and glycine also spared the leucine requirement of leucine auxotrophs, most likely because they resembled leucine and competed for its uptake. We propose that leucine analogs restrict the entry and degradation of leucine and thus reduce its conversion to 3-methyl-1-butanol, a major component of fusel oil.     

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.     

Preparation of 3-Methyl-3-(substituted phenyl)-pyruvic Acid

The relationships between dietary levels of the essential amino acids and hepatic polysome profiles of rats were investigated with special attention to the amino acid requirement pattern for the maximum rat growth as determined by other investigators. The basal diet contained a 7% essential amino acid mixture and a 3% non-essential amino acid mixture, with appropriate amounts of other nutrients. Rats were fed test diet for 5 hours and then the polysome profile was determined. The amounts of essential amino acids needed for maximum aggregation of polysome were low for methionine-cystine, leucine and tryptophan as compared with requirements for maximum growth. But in other essential amino acids, the amounts were in almost the same range as those reported for maximum growth by others. The differences between the amino acid requirement patterns for maximum aggregation of hepatic ribosomes and for maximum growth of rats might be due to a difference in amino acid requirements of the liver and whole body. Therefore, the hepatic polysome profile might be used to measure the effect of amino acid supplementation on dietary proteins. The requirement pattern of essential amino acids in other organs may be studied by polysome profile determination.     

Biological role of the general control of amino acid biosynthesis in Saccharomyces cerevisiae.

The biological role of the "general control of amino acid biosynthesis" has been investigated by analyzing growth and enzyme levels in wild-type, bradytrophic, and nonderepressing mutant strains of Saccharomyces cerevisiae. Amino acid limitation was achieved by using either bradytrophic mutations or external amino acid imbalance. In the wild-type strain noncoordinate derepression of enzymes subject to the general control has been found. Derepressing factors were in the order of 2 to 4 in bradytrophic mutant strains grown under limiting conditions and only in the order of 1.5 to 2 under the influence of external amino acid imbalance. Nonderepressing mutations led to slower growth rates under conditions of amino acid limitation, and no derepression of enzymes under the general control was observed. The amino acid pools were found to be very similar in the wild type and in nonderepressing mutant strains under all conditions tested. Our results indicate that the general control affects all branched amino acid biosynthetic pathways, namely, those of the aromatic amino acids and the aspartate family, the pathways for the basic amino acids lysine, histidine, and arginine, and also the pathways of serine and valine biosyntheses.     

氨基酸发酵生产的研究进展

由于氨基酸在食品、饲料、医药、农业和日用化工等方面有极其广泛的用途,尤其随着抗癌药物制剂、氨基酸输液制剂及甜味二肽生产的飞速发展,对原料氨基酸的需求量日益增长。传统的发酵工业越来越不能满足需求,势必被以基因工程为基础的新兴发酵工业所代替。通过对发酵法生产氨基酸的历史进行回顾,及对未来前景作出展望,指出了运用ＤＮＡ 重组、定向突变等手段,对代谢途径及关键酶进行了深入系统的研究的必要性。     

Amino acids in root exudates of cotton

Summary Cotton-field soils from a wilt-free zone (KPT) and from a wilt-sick zone (PLD), and root exudates of diploid and amphidiploid cotton strains grown in KPT and PLD soils were analyzed for 13 free amino acids employing microbiological assay techniques. Test organisms used wereLactobacillus arabinosus 17/5 andLeuconostoc mesenteroides P. 60.Arginine, proline, lysine, serine, glycine, cystine, and tyrosine occurred in both the soils; amounts of these were higher in PLD soil than in KPT soil.Seven to eleven amino acids were found in the root exudates of cotton. Aspartic acid, threonine (in one), phenylalanine and leucine were found in root exudates but not in the control soils. Diploid strains were found to exude greater amounts of amino acids than the amphidiploid strains. Comparing the effect of the two soils tested, it was found that amino acids in exudates of plants grown in PLD soil were higher except in the case of one diploid strain.In the inoculated series, amino acids in the exudates were markedly lower than the respective healthy controls in the diploid strains. In the exudates of amphidiploid strains also, amino acid contents were reduced but to a lesser extent.Significance of occurrence of cystine in the exudates of some strains is indicated.Part of Doctoral thesis, University of Madras.     

Nutritional requirements for growth of Helicobacter pylori.

A chemically defined medium consisting of a buffered mineral base supplemented with amino acids, a purine, and thiamine supported growth of 23 clinical isolates and the type strain of Helicobacter pylori. The growth of four strains was inhibited by the presence of certain amino acids. All but one strain required alanine for growth. The amino acids leucine, valine, phenylalanine, methionine, arginine, and histidine were generally required. Isoleucine either was required or stimulated growth. Strains could be differentiated into groups on the basis of a requirement for one or more of the amino acids cysteine, serine, and proline. Only one strain however, demonstrated a requirement for all three of these amino acids.     

Rational development of a simple synthetic medium for the sustained growth of Lactococcus lactis

The growth of two strains of Lactococcus lactis subsp. lactis from vegetable (NCDO 2118) and dairy origin (IL 1403) were compared on various culture media. Both strains grew more rapidly on a complex organic medium than on a defined synthetic medium. The best growth was obtained under nitrogen gas phase. The single omission technique was applied to each component of a non-optimized synthetic medium in order to determine the true nutritional requirements. Requirements for macro-elements, oligo-elements, bases and vitamins were identical for the two strains. As expected, the dairy strain (IL 1403) was seen to be auxotrophic for some amino acids, whereas the vegetable strain (NCDO 2118) was seen to be prototrophic for all amino acids when using the single omission technique. Growth was then characterized on progressively simplified media and the composition of the absolute minimal media for the growth of both strains was defined. Sustained growth of the vegetable strain was only possible in minimal media supplemented with six amino acids (Glu, Met, Ile, Leu, Val, Ser), indicating that the definition of prototrophy/auxotrophy is partly dependent upon the medium composition. The dairy strain showed a requirement for Arg, His and Thr in addition to the six amino acids necessary for growth of the vegetable strain. The removal of ammonium salt from the medium did not affect the growth, illustrating that the amino acids may satisfy the totality of the nitrogen requirement for biomass synthesis.     

Minicells of Bacillus subtilis a unique system for transport studies

Ultrasound-purified minicells produced by Bacillus subtilis mutant div IV-Bl have been studied for their ability to transport and incorporate into macromolecules a variety of amino acids, uracil and thymine. Minicells transport all 12 amino acids examined, but are unable to incorporate them into macromolecules. No significant differences were found in the initial uptake rates of glutamic acid, aspartic acid, and alanine by minicells and parental cells. The uptake of methionine and proline by minicells was shown to be inhibited by metabolic poisons, indicating an energy-metabolism requirement for transport in this system. The proline pool in minicells was found to be readily exchangeable with exogenous proline. In contrast metabolically poisoned minicells only slowly lose their pool proline, indicating an energy requirement for pool maintenance. Packed-cell experiments reveal that minicells accumulate proline against a concentration gradient.In addition to amino acids, minicells are able to transport uracil but cannot incorporate uracil into acid-precipitable material (RNA). Neither thymine transport nor its incorporation into macromolecules can be demonstrated in minicells.Minicells appear to be a new system, therefore, in which transport may be studied in the absence of macromolecular biosynthesis.     

Antibacterial Effects of Glycyrrhetinic Acid and Its Derivatives on Staphylococcus aureus

Staphylococcus aureus is a major pathogen in humans and causes serious problems due to antibiotic resistance. We investigated the antimicrobial effect of glycyrrhetinic acid (GRA) and its derivatives against 50 clinical S. aureus strains, including 18 methicillin-resistant strains. The minimum inhibitory concentrations (MICs) of GRA, dipotassium glycyrrhizate, disodium succinoyl glycyrrhetinate (GR-SU), stearyl glycyrrhetinate and glycyrrhetinyl stearate were evaluated against various S. aureus strains. Additionally, we investigated the bactericidal effects of GRA and GR-SU against two specific S. aureus strains. DNA microarray analysis was also performed to clarify the mechanism underlying the antibacterial activity of GR-SU. We detected the antimicrobial activities of five agents against S. aureus strains. GRA and GR-SU showed strong antibacterial activities compared to the other three agents tested. At a higher concentration (above 2x MIC), GRA and GR-SU showed bactericidal activity, whereas at a concentration of 1x MIC, they showed a bacteriostatic effect. Additionally, GRA and GR-SU exhibited a synergistic effect with gentamicin. The expression of a large number of genes (including transporters) and metabolic factors (carbohydrates and amino acids) was altered by the addition of GR-SU, suggesting that the inhibition of these metabolic processes may influence the degree of the requirement for carbohydrates or amino acids. In fact, the requirement for carbohydrates or amino acids was increased in the presence of either GRA or GR-SU. GRA and GR-SU exhibited strong antibacterial activity against several S. aureus strains, including MRSA. This activity may be partly due to the inhibition of several pathways involved in carbohydrate and amino acid metabolism.     

Different mechanisms of energy coupling for transport of various amino acids in cells of Mycobacterium phlei.

Whole cells of Mycobacterium phlei were shown to actively accumulate proline, leucine, lysine, tryptophan, histidine, glutamine, and glutamic acid to different steady state levels. The transport of proline, in contrast to that of other amino acids, was found to be insensitive to various respiratory inhibitors, e.g. cyanide, arsenate, azide, and sulfhydryl reagents. However, oxygen was an obligatory requirement for the uptake of proline, as well as for the other amino acids. The results indicate that the energy requirements for proline uptake are different from those of other amino acids. In contrast to the system from Escherichia coli, the mode of energy transduction for the uptake of proline, glutamine, and glutamic acid is different even though these amino acids are shock resistant in the M. phlei system.     

General control of amino acid biosynthesis in mutants of Candida spec. EH 15/D

The general control of amino acid biosynthesis was investigated in Candida spec. EH 15/D, using single and double mutant auxotrophic strains and prototrophic revertants starved for their required amino acids. These experiments show that starvation for lysine, histidine, arginine, leucine, threonine, proline, serine, methionine, homoserine, asparagine, glutamic acid or aspartic acid can result in derepression of enzymes. A correlation was found between the degree of derepression, growth of strains, and concentration of required amino acids. The amino acids pool pattern of mutants and revertants is different from that in the wild type strain.     

Proline-dependent wild strains and proline-dependent mutants of the plague microbe]

It is found that the growth of Yersinia pestis wild strains, isolated from Citellus musicus Menetrié in the Central Caucasus, depends on the presence of proline in the medium. Proline can not be substituted by glutamic acid, other amino acids or vitamins. 28 proline-requiring mutants were selected from Y. pestis marmot strain 20b. Three groups of proline-requiring Y. pestis mutants are established, similar to those of Escherichia coli. The requirement of proline does not affect the virulency, pigment formation and calcium dependence.     

Sequence variability of the meningococcal lactoferrin-binding protein LbpB

The lactoferrin receptor of Neisseria meningitidis consists of two proteins, LbpA and LbpB. LbpB is considered a promising vaccine candidate, and therefore its sequence variability was studied. LbpB from five different strains exhibited 70-80% mutual identity at the amino acid level. Most sequence variability was found in two stretches with a high content of negatively charged amino acids. These stretches were sequenced from six additional strains. One of the stretches is of variable length and is missing in some of the strains. The other stretch is present in all strains, but varies considerably in its exact amino acid sequence. The high degree of variability is disadvantageous for vaccine development, but may be useful for epidemiological studies.     

Selection and characterization of L-ethionine resistant mutants of Trichosporon cutaneum

Trichosporon cutaneum R57 and its L-ethionine resistant mutant NZ94 strain were investigated. The amino acid analyses of cell content of both strains were carried out. The pool of free methionine in the mutant strain is enhanced 16.5 times. The total amount of sulphur-containing amino acids in the mutant cells was significantly increased from 36.8 in the wild strain to 113.4 mg/g protein in the mutant strain. In the process of mutant strain cultivation there was found a high excretion of free methionine (259 microg/ml) in the medium. It was shown that the amino acid content of both wild and mutant strains would be helpful for formulating of new improved animal nutritional diets.     

Tempe fermentation: some aspects of formation of gamma-linolenic acid, proteases and vitamins

During a tempe fermentation the concentrations of linoleic, and alpha-linolenic acids (ALA) decreased while the concentration of oleic acid increased. During fatty acid synthesis Rhizopus sp. produced only gamma-linolenic acid (GLA) instead of ALA. The amount of GLA in tempe were influenced by varying external parameters.The proteolytic capacity of 36 strains of the genus Rhizopus isolated from Indonesian tempe or tempe inocula was examined. There was a distinct increase in the amount of free amino acids during tempe fermentation. Fermentations with mixed populations of bacteria and Rhizopus yielded a lower level of free amino acids, but an increase in total amount of amino acids. In comparison to intracellular, and extracellular proteases the proteases of the cell wall fraction are most responsible for proteolytic capacity of the different Rhizopus strains.Two isolated strains of Citrobacter freundii were found to be the best vitamin B(12) producers during the soaking of soybeans. In the solid substrate fermentation the Rhizopus molds formed vitamin B(6), riboflavin, and nicotinic acid. The addition of bacteria to the solid substrate fermentation resulted in a strong increase of active vitamin B(12) in tempe. In the presence of the Rhizopus mold, the vitamin B(12) formation by C. freundii was three times higher than that of a fermentation without the mold.