Induction of pigmentation in nonproliferating cells of Serratia marcescens by addition of single amino acids

Addition of casein hydrolysate to suspensions of washed, nonpigmented, nonproliferating Serratia marcescens incubating at 27 C induced biosynthesis of prodigiosin. Four amino acids of casein hydrolysate, dl-aspartic acid, l-glutamic acid, l-proline, and l-alanine caused formation of pigment when added individually. dl-Ornithine also was effective. Optimal concentrations for maximal pigmentation were 5 to 10 mg/ml; at these high concentrations, d-serine also induced biosynthesis of some prodigiosin. dl-Alanine and -ornithine were as effective as the l-iosomers, but l-glutamic acid and l-proline gave better responses than their racemic mixtures. Kinetics of prodigiosin biosynthesis after addition of dl-alanine (20 mg/ml) were similar to those of cells suspended in 0.2% casein hydrolysate. The other amino acids were less effective. Addition of 5 mg of dl-alanine or casein hydrolysate per ml to minimal medium increased by 30% the amount of prodigiosin formed by growing cells after incubation for 7 days at 27 C. Cultures grown for 7 days at 27 C in 0.2% casein hydrolsate formed more prodigiosin than did suspensions of nonproliferating cells containing individual amino acids or casein hydrolysate. However, more pigment was produced by cells suspended in l-alanine (5 mg/ml) or l-proline (10 mg/ml) than when suspended in 0.4% natural or synthetic casein hydrolysate. Filtrates from suspensions of nonproliferating cells forming pigment in l-proline induced more rapid formation of prodigiosin, but filtrates from suspensions in dl-alanine did not. The data supported the hypothesis that pyrrole groups of prodigiosin may be synthesized from 5-carbon amino acids such as proline, ornithine, aspartic, and glutamic acids, but the role of alanine is unknown.     

Production of branched-chain volatile fatty acids by certain anaerobic bacteria.

Net production of isobutyric acid, isovaleric acid, and 2-methylbutyric acid by cultures of Bacteroides ruminicola and Megasphaera elsdenii on media that contained Trypticase or casein hydrolysate continued (up to 5 days) after growth had ceased. Only trace quantities of these acids were produced in a medium that contained a mixture of amino acids that did not include the branched-chain amino acids. M. elsdenii produced increased quantities of the branched-chain fatty acids in a medium that contained Trypticase when glucose was reduced or eliminated from the culture medium. However, B. ruminicola produced increased quantities of branched-chain fatty acids and of phenylacetic acid from Trypticase when glucose was supplied at 3 mg/ml rather than at 1 mg/ml. Single strains of Streptococcus bovis, Selenomonas ruminantium, Bacteroides amylophilus, and Butyrivibrio fibrisolvens did not produce branched-chain fatty acids.     

Production of branched-chain volatile fatty acids by certain anaerobic bacteria

Net production of isobutyric acid, isovaleric acid, and 2-methylbutyric acid by cultures of Bacteroides ruminicola and Megasphaera elsdenii on media that contained Trypticase or casein hydrolysate continued (up to 5 days) after growth had ceased. Only trace quantities of these acids were produced in a medium that contained a mixture of amino acids that did not include the branched-chain amino acids. M. elsdenii produced increased quantities of the branched-chain fatty acids in a medium that contained Trypticase when glucose was reduced or eliminated from the culture medium. However, B. ruminicola produced increased quantities of branched-chain fatty acids and of phenylacetic acid from Trypticase when glucose was supplied at 3 mg/ml rather than at 1 mg/ml. Single strains of Streptococcus bovis, Selenomonas ruminantium, Bacteroides amylophilus, and Butyrivibrio fibrisolvens did not produce branched-chain fatty acids.     

Repair and Enumeration of Injured Coliforms in Frozen Foods

Two strains of Escherichia coli manifested death and repairable injury after being frozen in water or sterile foods at -20 C. The injured survivors were inhibited from forming colonies on violet red bile agar (VRBA) or deoxycholate lactose agar; this inhibition was greater when enumeration was done by the pour plate method instead of the surface or surface-overlay method. Injured cells repaired rapidly in Trypticase soy broth (TSB), and the repair was about maximum after 1 h at 25 C. When the injured cells were added to different foods and incubated at 25 C, repair also occurred; however, recovery was better and more uniform when the samples were mixed with TSB and incubated 1 h at 25 C. Cell multiplication was not evident until after 90 to 120 min at 25 C. The enumeration of coliforms from commercially frozen foods was increased when the thawed samples were mixed with TSB and the cells were allowed to repair 1 h at 25 C. In some samples, the repair permitted at least a 20-fold increase in the coliform count. The associated flora in the commercially frozen foods gave no evidence of impairing the repair of coliforms, nor did they start multiplication prior to 90 min after being incubated in TSB at 25 C. Generally, the plating gave more reproducible recovery of coliforms than did the most probable number method. Also, a higher number of coliforms were obtained by the surface-overlay method of plating using VRBA.     

Liquid Nitrogen Preservation of Saccharomyces carlsbergensis and its Use in a Rapid Biological Assay of Vitamin B6 (Pyridoxine)

Growth medium as well as freezing menstruum greatly influenced the recovery of Saccharomyces carlsbergensis when it was quickly frozen in liquid nitrogen at - 196 C and quickly thawed at 40 C. Nearly 90% recovery in viability was obtained when S. carlsbergensis was grown in Trypticase Soy Broth and frozen in vitamin B(6) basal assay medium. The growth phase of S. carlsbergensis also influenced recovery after freezing. When S. carlsbergensis was grown in Trypticase Soy Broth and frozen in the broth at the logarithmic-growth phase, only 7% viability was retained; the recovery rate increased to 81% when the culture was frozen in the maximal stationary phase. To have the least possible lag period of growth after thawing, a technique called growth-phase conditioning was introduced. After 1 hr of growth-phase conditioning, S. carlsbergensis was clearly out of lag phase, and budding was observed. A vitamin B(6) microbiological assay with a 6-hr incubation period and with the use of liquid nitrogen-frozen S. carlsbergensis is described.     

Physiology of growth and sporulation in Bacillus cereus. I. Effect of glutamic and other amino acids

Buono, F. (Syracuse University, Syracuse, N.Y.), R. Testa, and D. G. Lundgren. Physiology of growth and sporulation in Bacillus cereus. I. Effect of glutamic and other amino acids. J. Bacteriol. 91:2291-2299. 1966.-Growth and sporulation were studied in Bacillus cereus by use of an active culture technique and a synthetic medium. A high level of glutamic acid (70 mm) was required for optimal growth and glucose oxidation followed by sporulation even though relatively little glutamic acid was consumed (14 mm). Optimal growth occurred with a combination of 14 mm glutamic acid and 56 mm (NH(4))(2)SO(4), aspartic acid, or alanine. Ornithine or arginine at 70 mm could replace glutamic acid in the synthetic medium without affecting the normal growth cycle. Glutamic acid was not replaced by any other amino acid, by (NH(4))(2)SO(4), or by a combination of either alpha-ketoglutarate or pyruvate plus (NH(4))(2)SO(4). Enzyme assays of cell-free extracts prepared from cells harvested at different times were used to study the metabolism of glutamic acid. Glutamic-oxaloacetic and glutamic-pyruvate transaminases were completely activated (or derepressed) during early stages of sporulation (period of 6 to 8 hr). Alanine dehydrogenase responded in a similar manner, but the levels of this enzyme were much higher throughout the culture cycle. Neither glutamic dehydrogenase nor alpha-ketoglutarate dehydrogenase was detected. Sporulation in a replacement salts medium was studied with cells harvested at different times from the synthetic medium. Cultures 2 to 6 hr old were unable to sporulate in the replacement salts medium unless glutamic acid (7.0 mm) was present. By the 6th hr, cells were in the early stages of sporulation, showing spore septa development. Cultures 8 hr old sporulated in the replacement salts medium. Other metabolic intermediates able to replace glutamic acid in the replacement salts medium were alanine, aspartic acid, and glutamine at equimolar concentrations. Also, ammonium ions in combination with pyruvic, oxaloacetic, alpha-ketoglutaric, or fumaric acid replaced glutamic acid. The likely role of these metabolites is discussed.     

THE NUTRITION AND SOME BIOCHEMICAL STUDIES OF VIBRIO CYCLOSITES AND VIBRIO NEOCISTES

SUMMARY: Vibrio cyclosites and V. neocistes grew in a defined medium consisting of a mineral basis, nicotinamide or nicotinic acid and the amino acids methionine, histidine, aspartic acid, phenylalanine and iso leucine. Washed suspensions failed to oxidize any of these substances in the Warburg apparatus. It was not possible with these organisms to demonstrate transamination between the five essential amino acids and α-ketoglutaric acid or pyruvic acid.     

Nitrogen Metabolism of Lemna minor. I. Growth, Nitrogen Sources and Amino Acid Inhibition

Lemna minor grown in sterile culture on a minerals-sucrose medium can utilize as nitrogen source, in order of increasing growth rate: ammonia, nitrate, a mixture of glutamic and aspartic acids plus arginine, or a balanced mixture of amino acids (hydrolyzed casein). Maximum growth is found with nitrate plus hydrolyzed casein.Many synthetic mixtures of amino acids are unable to support growth. Many single amino acids are inhibitory, and when added (at 2 mm or less) to cultures, growing in the presence of nitrate, cause a decrease in growth rate or even death of the plants (e.g. with alanine, valine, methionine or leucine). Some of these inhibitory effects are also found when the amino acid is added to cultures growing on ammonia or hydrolyzed casein. Arginine was the only amino acid of those tested which gave a marked stimulation of growth when added to cultures growing with inorganic nitrogen.The rapid rate of growth, sterile nature of tissue, decreased biological variation of samples containing many plants and ability to utilize different culture media make this an attractive organism for studies on higher plant metabolism.     

Effect of Dietary Addition of Amino Acids and Proteins on the Activities of Some Urea Cycle Enzymes in Rat Liver

The activities of ornithine transcarbamylase, arginine synthetase and arginase in the liver of rats receiving basal diets containing 25% casein supplemented respectively with arginine, aspartic acid, glutamic acid, glycine, a mixture of arginine, aspartic acid and glutamic acid, egg albumin, casein, wheat gluten and gelatin have been determined.

These urea cycle enzymes in rats receiving diets supplemented with the various nitrogen sources were generally increased, but the increments were due to the increase of the ingested amount of nitrogen, and not the specific effect of the individual amino acids or proteins. The excretion of urinary urea in general was increased proportionally with the elevations of these enzyme activities, independent of the nature of the dietary nitrogen.     

Plant regeneration from root callus protoplasts of sour cherry (Prunus cerasus L.)

Summary Callus protoplasts of sour cherry clone CAB4D entered sustained division in Murashige and Skoog's (1962) medium with 1-naphthaleneacetic acid, 6-beneylaminopurine and zeatin. Further to callusing, organogenesis was induced from the protoplastderived callus, in a basal regeneration medium with these same growth regulators at 0.01 mg/l, 2,0 mg/l and 0.05 mg/l, respectively. The regeneration pathway, from such callus, could be altered by adding different organic compounds to this medium. Casein hydrolysate, added alone, promoted rhizogenesis, with shoot buds regenerated from the protoplast-derived roots, while in a basal regeneration medium with casein hydrolysate and a group B vitamin mixture direct caulogenesis occurred.Abbreviations BAP 6-benzylaminopurine - BR basal regeneration medium - CEH casein enzymatic hydrolysate - FPE final plating efficiency - fwt fresh weight - IPE initial plating efficiency - MES 2-N-morpholinoethane sulfonic acid - MPE intermediate plating efficiency - MS Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid     

THE EFFECTS OF AMINO ACIDS UPON THE DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA

Young excised floral buds of Aquilegia were grown on a chemically defined medium containing various concentrations of single amino acids or mixtures of amino acids. γ-Amino butyric acid significantly promoted floral development through the initiation and differentiation of carpels. These floral organs were generally absent on the basal medium. Alanine, glutamic acid, and aspartic acid had no effect upon floral development. All other amino acids were either ineffective at lower concentrations and inhibitory at higher concentrations or were inhibitory at all concentrations. Casein hydrolysate and a mixture of amino acids found in coconut milk were ineffective. The addition of both γ-amino butyric acid and alanine to the basal medium promoted development approaching that achieved on the coconut-milk medium. However, further growth factors appear to be required before development on coconut-milk medium is equalled or exceeded.     

Multivalent Induction of Biodegradative Threonine Deaminase

To determine the inducer(s) of the biodegradative threonine deaminase in Escherichia coli, the effects of various amino acids on the synthesis of this enzyme were investigated. The complex medium used hitherto for the enzyme induction can be completely replaced by a synthetic medium composed of 18 natural amino acids. In this synthetic medium, the omission of each of the seven amino acids threonine, serine, aspartic acid, methionine, valine, leucine, and arginine resulted in the greatest loss of enzyme formation. These seven amino acids did not significantly influence the uptake of other amino acids into the cells. Furthermore, they did not stimulate the conversion of inactive enzyme into an active form, since they did not affect the enzyme level in cells in which protein synthesis was inhibited by chloramphenicol. Threonine, serine, aspartic acid, and methionine failed to stimulate enzyme production in cells in which messenger ribonucleic acid synthesis was arrested by rifampin, whereas valine, leucine, and arginine stimulated enzyme synthesis under the same conditions. Therefore, the first four amino acids appear to act as inducers of the biodegradative threonine deaminase in E. coli and the last three amino acids appear to be amplifiers of enzyme production. The term "multivalent induction" has been proposed for this type of induction, i.e., enzyme induction only by the simultaneous presence of several amino acids.     

Effect of amino acids on growth ofSphaerotilus discophorus

The effect of casein hydrolysate, of mixtures of amino acids and of individual amino acids on the growth of 4 strains ofSphaerotilus discophorus was determined. Growth was virtually completely inhibited by 1.0% Bacto Casamino Acids, 0.54% simulated casein hydrolysate and 0.2% of a uniform mixture of 18 amino acids. The latter were prepared withl amino acids except thatdl-serine,dl-valine anddl-threonine were present in the uniform amino acid mixture.Experiments designed to test the toxicity of the 18 individual amino acids at 0.018 – 0.36% concentration indicated that arginine, glutamic acid, leucine, lysine and proline were non-toxic. However, aspartic acid and methionine were moderately toxic; growth was greatly repressed at a concentration of 0.36%. The remaining 11 amino acids which included alanine, cystine, glycine, tyrosine, histidine, isoleucine, phenylalanine, serine, threonine, tryptophane and valine were the most toxic of the group. They prevented growth partially or completely, at a concentration of 0.18% or 0.36%.dl-Serine anddl-valine were especially toxic and prevented growth at a concentration of 0.018%. The toxicity of the individuall-amino acids can account for the toxicity of Casamino Acids and simulated casein hydrolysate. l-Methionine or cyanocobalamin (vitamin B₁₂) is required for the growth ofS. discophorus. Alsod- anddl-methionine can replace cyanocobalamin although they completely repress growth when used at the relatively high concentration of 200 µg per ml of medium.     

Ultra-high expression of a thermally responsive recombinant fusion protein in E. coli

Elastin-like polypeptides (ELPs) are recombinant peptide-based biopolymers that contain repetitive sequences enriched in glycine, valine, proline, and alanine. Because of the unusually large fraction of these amino acids in ELPs as compared to other cellular proteins, we hypothesized that intracellular pools of these amino acids can be selectively depleted and limit protein yields during expression. In this study, we examined how culture conditions and individual medium components affect protein yields by monitoring cell growth and protein expression kinetics of E. coli expressing an ELP tagged with a green fluorescent protein (GFP). By determining the underlying principles of superior fusion protein yields generated by the hyperexpression protocol, we further improved protein yields through the addition of glycerol and certain amino acids such as proline and alanine and found that amino acid concentrations and the type of basal medium used strongly influenced this beneficial effect. Surprisingly, amino acids other than those that are abundant in ELPs, for example, asparagine, aspartic acid, glutamine, and glutamic acid, also enhanced protein yields even in a nutrient-rich medium. Compared to commonly used Luria-Bertani medium, the protein yield was improved by 36-fold to the remarkable level of 1.6 g/L in shaker flask cultures with a modified medium and optimized culture conditions, which also led to a 8-fold reduction in the cost of the fusion protein. To our knowledge, this is the highest yield of an ELP-fusion protein purified from E. coli cultured in shaker flasks. This study also suggests a useful strategy to improve the yields of other ELP fusion proteins and repetitive polypeptides.     

MAIZE ENDOSPERM TISSUE GROWN IN VITRO III. DEVELOPMENT OF A SYNTHETIC MEDIUM

Straus , Jacob . (U. Oregon, Eugene.) Maize endosperm tissue grown in vitro. III. Development of a synthetic medium. Amer. Jour. Bot. 47(8) : 641–647. Illus. 1960.—The development of a synthetic medium for the growth of endosperm tissue cultures derived from the maize variety, ‘Black Mexican Sweet,‘ is described. Previously, these tissues required yeast extract, casein hydrolyzate, or tomato juice in the medium in order to grow. The growth-supporting activity of these complexes could be attributed to their organic nitrogen content. The effect of juice extracted from fresh tomatoes is enhanced by autoclaving under acid conditions. Presumably this treatment increases the free amino acid content of the tomato juice. One-dimensional paper chromatograms of tomato juice autoclaved under acid conditions indicated the presence of a large amount of free amino acids. Addition of 1.5 × 10^–2 M asparagine to the basal mineral-sugar-vitamin medium (White's medium plus Nitsch's trace-element solution) resulted in better growth than that supported by yeast extract, tomato juice, or casein hydrolyzate. Arginine was ineffective. Glutamine, glutamic acid and aspartic acid (all at 1.5 × 10^–2 M) supported appreciable growth of the tissue but none of them were nearly so good as asparagine in this respect. Thus, a medium containing minerals, sugar, vitamins, and asparagine is capable of supporting excellent growth of maize endosperm tissue cultures.     

Liquid Nitrogen Freezing in Microbiological Assay Systems: I. Preservation of Lactobacillus leichmannii for Direct Use in the Vitamin B12 Assay

Suspensions of Lactobacillus leichmannii were stored in liquid nitrogen and were used as direct inocula in vitamin B₁₂ assays. Complete recovery of viable cells was obtained when the suspensions in basal B₁₂ medium were rapidly frozen by direct immersion into liquid nitrogen and rapidly thawed by agitating the suspensions in a water bath at 40 C. Greater than 90% destruction occurred when the suspensions were in saline. However, both suspensions were usable in the B₁₂ assay system. Assay results on a number of test materials indicated good correlation between freshly prepared suspensions and frozen suspensions in basal medium stored 3 months. Suspensions in saline stored for 1 year in liquid nitrogen showed no detectable difference from the first day after freezing. Suspensions frozen slowly at the rate of 1 degree per min from 4 to -40 C and subsequently immersed in liquid nitrogen had a longer lag period of growth and were not usable in the 18-hr assay incubation system. A major advantage of a stored inoculum for direct use in a microbiological assay is the reduced day-to-day variation in the inoculum.     

The penicillin G acylase production by B. megaterium is amino acid consumption dependent

Aiming at to enhance the production of penicillin G acylase (PGA) by Bacillus megaterium, we have performed flasks experiments using different medium composition. Using 51 g/L of casein hydrolyzed with Alcalase and 2.7 g/L of phenylacetic acid (PhAc), the following carbon substrates were tested, individually and combined: glucose, glycerol, and lactose (present in cheese whey). Glycerol and glucose showed to be effective nutrients for the microorganism growth but delayed the PGA production. Cheese whey always increased enzyme production and cell mass. However, lactose (present in cheese whey) was not a significant carbon source for B. megaterium. PhAc, amino acids, and small peptides present in the hydrolyzed casein were the actual carbon sources for enzyme production. Replacement of hydrolyzed casein by free amino acids, 10.0 g/L, led to a significant increase in enzyme production (app. 150%), with a preferential consumption of alanine, aspartic acid, glycine, serine, arginine, threonine, lysine, and glutamic acid. A decrease of the enzyme production was observed when 20.0 g/L of amino acids were used. Using the single omission technique, it was shown that none of the 18 tested amino acids was essential for enzyme production. The use of a medium containing eight of the preferentially consumed amino acids lead to similar enzyme production level obtained when using 18 amino acids. PhAc, up to 2.7 g/L, did not inhibit enzyme production, even if added at the beginning of the cultivation.     

Physiological and nutritional features of Corynebacterium pyogenes

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

Growth of Suspension Cultures of Sugarcane Cells in Chemically Defined Media

A chemically defined medium is desirable for nutritional studies and is frequently necessary for biochemical investigations. Several defined media are available for use with tissue and cell cultures from dicotyledonous plants. A fully defined medium has now been developed for cell suspension cultures from sugarcane. Prior to this, the only medium successfully used for cell cultures of monocotyledonous plants was a modification of Straus' synthetic medium (used to grow cell suspensions of corn). Cell suspension cultures from sugarcane stalk parenchyma, originally established in complex media containing coconut milk or yeast extract, can be grown in this synthetic medium, which consists of inorganic salts, vitamins, sucrose, 2.4-dicliloropheuoxyacetic acid, and a mixture of 13 amino acids. The most important of the amino acids are arginine aspartic acid, and glutamic acid. This simplified medium wilt aid in the investigation of the unusual and important role of arginine in sugar-cane growth and metabolism.     

Requirement for GLY-60 of Escherichia coli adenylyl cyclase for ATP binding and catalytic activity.

The region of Escherichia coli adenylyl cyclase spanned by glycine-55 to threonine-65 was tested for its importance for enzyme activity. Site-directed mutagenesis was used to replace glycine-55 and glycine-60 as well as lysine-59, leucine-63 and threonine-65 with other amino acids. While substitution of glycine-55 with aspartic acid produced no significant change in kinetic parameters, the change of glycine-60 to aspartic acid or asparagine eliminated binding to 8-azido-ATP and decreased the Vmax (two orders of magnitude) and Km (factor of four-five). Smaller effects on kinetic parameters were observed with substitutions of lysine-59, leucine-63 or threonine-65.