Synthetic Minimal Medium Research Articles

Naturally-occurring, osmo-remedial variants of Escherichia coli.

Two clones of Escherichia coli O27:K1:H31 and O2:H7, isolated from patients with urinary tract infection or bacteraemia, failed to grow in a synthetic minimal medium (MM) of low osmolality. They were considered to be osmo-remedial because they grew well when sufficient amounts of NaCl, mannitol or sucrose were added to raise the osmolality of the medium to > 300 mOsm/kg. The defect could also be corrected by nicotinamide or its precursors quinolinic and aspartic acids. Each clone had a unique DNA restriction enzyme profile, fimbriae and antibiotic susceptibility patterns. The osmo-remedial variants were unstable and underwent phenotypic modulation to form mixtures with osmo-tolerant forms when grown in MM. They tended to form satellites of small colonies around large colonies of osmo-tolerant cells on MM agar plates. The penicillin method of Davis was used to separate the two forms. Nicotinamide induced the expression of ompF when the osmo-remedial strains were grown under conditions of low osmolality. It is possible that the variants are defective in the synthesis of membrane-derived oligosaccharides or outer-membrane proteins, but this has yet to be determined.

Effect of nutrient deficiency on accumulation and relative molecular weight of poly-?-hydroxybutyric acid by methylotrophic bacterium, Pseudomonas 135

Pseudomonas 135, a facultative methylotroph, was cultivated on methanol as a sole carbon and energy source for the accumulation of poly-β-hydroxybutyric acid (PHB). The cells grew fairly well on minimal synthetic medium containing 0.5% (v/v) of methanol at pH 7.0 and 30° C. The maximum specific growth rate was determined to be 0.26–0.28 h−1 with a growth yield of 0.38 in the optimized growth medium. For stimulation of PHB accumulation in the cells, deficiency of nutrients such as NHinf4sup+, Mg2+ and POinf4sup3−was crucial even though cell growth was significantly suppressed. The PHB content of a 40-h culture was determined to be 37% of the total cell mass in NHinf4sup+-limited medium, 42.5% on Mg2+-deficient medium, and 34.5% on POinf4sup3−-deficient medium. The maximum content of PHB in the cells could reach 55% in NHinf4sup+-limited fed-batch culture. The average relative molecular eight determined by gel permeation chromatography was 3.7 × 105 in NHinf4sup+-limited culture, 2.5 × 105 in Mg2+-deficientmedium, and 3.1 × 105 in POinf4sup3−-deficient medium. Polydispersity determined in each culture was relatively high (about 10–11). The solid PHB had a melting temperature of 173° C.

Growth of Cryptococcus neoformans in a thiamine-free medium.

The growth of Cryptococcus neoformans in a minimal liquid synthetic medium with or without thiamine (10 micrograms/ml) was investigated. In these media the presence or absence of thiamine had no effect on the development of C. neoformans. To check these results, we performed a series of experiments on a solid form of the minimal synthetic medium. In this study a series of six serial transfers were carried out to starve the cells of nutrients that may have been carried over from their growth on rich media. In each of the transfers on the solid synthetic medium, C. neoformans showed a similar and scarce growth. This finding indicates that C. neoformans could be autotrophic in respect to thiamine.

A new minimal synthetic medium for germ-tube production in Candida albicans

A new minimal synthetic medium, with low amount of glucose, without aminoacids, vitamins and neutral pH, which induces germ-tubes production in Candida albicans, is reported in this work. The results indicate a perfect agreement between the germ-tube test performed with the standard method in human or animal serum and this test performed in minimal synthetic medium. In this medium the germ-tube test for the presumptive identification of Candida albicans can be performed with the same formality, time and reproducibility as those in human or animal serum. This constitutes an interesting finding because it is easy to prepare, to store and is highly reproducible.

Optimal concentration of ammonium ion in a minimal synthetic medium for the growth of Candida albicans

Candida albicans strain B 311-10 with and without starvation was cultivated in the minimal synthetic medium of Shepherd et al., modified without biotin, amino acids, low glucose concentration and with decreasing amounts of (NH4)2SO4, to determine the optimal growth requirement for this strain. All the experiments were carried out under sterile conditions at 25 degrees C in a thermostat with initial O.D.s (675 nm) of 0.500 and 0.100. Cell growth was generally monitored everyday for six days with a spectrophotometer by determining the absorbance of the cultures at 675 nm. All the experiments were repeated three times and a statistical analysis of the data with a probability of 99% and 1% of error was performed to confirm the validity of the results. Best growth was obtained with starved cells at an initial O.D. of 0.100 and with a 0.1 g/L concentration of (NH4)2SO4. At this concentration, the growth of C. albicans B 311-10 was best between the first and the fourth day with the maximum at the third day. With (NH4)2SO4 concentrations of 0.05 and 0.5 g/L, cell growth was the same.

Glutathione-coated Cadmium-Sulfide Crystallites in Candida glabrata

Cadmium-sulfide crystallites form in the yeast Candida glabrata cultured in the presence of cadmium salts. The particles function to sequester and detoxify intracellular cadmium ions. The crystallites are peptide-coated, but the coating peptide varies with the nutrient conditions of the growth medium. When cultured in rich nutrient broth the yeast forms intracellular CdS particles coated with a mixture of glutathione and the gamma-glutamylcysteine dipeptide. In contrast, cultures in synthetic minimal medium yield particles coated with polymerized gamma EC peptides of general structure (gamma-Glu-Cys)n-Gly. Glutathione/gamma-glutamylcysteine particles exhibit properties analogous to quantum, semiconductor-type crystallites. The optical properties are dependent on particle size, and irradiation results in photoluminescence and photoreduction not observed in bulk CdS mineral. Aerobic irradiation leads to particle decomposition presumably via oxidation of the sulfide ions within the crystallite.

Characterization of the biocontrol activity of Debaryomyces hansenii in the control of Penicillium digitatum on grapefruit

Interactions between Debaryomyces hansenii and Penicillium digitatum were studied in culture and on fruit to better characterize the observed biological control of green mold on grapefruit by the yeast. The antagonist did not produce antibiotic substances in culture and was ineffective in protecting against the disease when killed by heat or chemicals. Incidence of green mold was dependent upon the concentration of both the pathogen spores and the antagonist yeast cells. Control of green mold was most effective at 109 cfu/mL of D. hansenii. The role of available nutrients in the biological control activity of D. hansenii was assessed. Significant inhibition of spore germination and hyphal growth of P. digitatum in culture was achieved by the addition of the yeast cells to a minimal synthetic growth medium. Inhibition of P. digitatum by the antagonist in culture and on the fruit peel could be overcome by the addition of exogenous nutrients. Our results indicate that competition for nutrients may play a role in the biocontrol of P. digitatum by D. hansenii on grapefruit.Key words: biological control, Penicillium digitatum, Debaryomyces hansenii, grapefruit.

Importance of some factors on the dimorphism of Candida albicans.

The passage between the yeast and mycelial forms of Candida albicans B 311-10 was studied by using the minimal synthetic medium of Shepherd et al. modified without biotin and with low glucose concentrations. It was observed that biotin, aminoacids and particularly pH are not important factors in the dimorphism of C. albicans. The only factor of notable importance in the passage of yeast form to mycelial form in C. albicans was glucose concentration.

Growth of Candida albicans in a minimal synthetic medium without biotin.

Growth of Candida albicans strain B 311-10 was observed in a minimal synthetic biotin-free medium, using different glucose concentrations, during the first 30 hours of its development at 28 degrees C. The yeast's growth was observed spectrophotometrically at 675 nm reading its optical density every hour. The minimal medium of Shepherd et al., with glucose (15 g/L) and biotin was modified: this vitamin was eliminated and the concentration of glucose was gradually lowered to 0.5 g/L. At 5 g/L of glucose and without biotin very good growth was obtained. Based on our results during the first 30 hours of growth, biotin has no influence on the yeast's growth. This medium would be useful for the study of the physiology of C. albicans during the first period of its development.

Selective medium for isolation of Clostridium butyricum from human feces.

A selective medium, Clostridium butyricum isolation medium (BIM), is described for the isolation of C. butyricum from human feces. The BIM is a synthetic minimal medium and contains trimethoprim (16 micrograms/ml), D-cycloserine (10 micrograms/ml), and polymyxin B sulfate (20 micrograms/ml) as selective inhibitory agents. Qualitative tests indicated that C. butyricum and other butyric acid-producing clostridia grew on BIM, Clostridium sphenoides and Bacillus cereus produced small colonies, and other clostridia and other obligate anaerobic or facultatively anerobic bacteria were inhibited. Quantitative recovery of C. butyricum from cultures or seeded fecal samples was comparable with BIM and with complex medium, but the quantitative recovery of the other butyric acid-producing clostridia tested (C. beijerinckii, C. acetobutylicum) was lower with BIM than with complex medium. The BIM should aid the rapid isolation of C. butyricum from fecal samples and should be useful for bacteriological investigation of neonatal necrotizing enterocolitis.

Simple Colorimetric Estimation of Cyclopiazonic Acid in Contaminated Food and Feeds

A method has been developed for quantitative determination of cyclopiazonic acid, a mycotoxin produced by a common food contaminant, Penicillium cyclopium. The organism was grown successively in synthetic minimal medium, rice, corn, and wheat for 15 days. The toxin was extracted with chloroform followed by separation by thin layer chromatography. A colorimetric assay procedure has been successfully developed for the analysis of cyclopiazonic acid present in infected rice, corn, and wheat. The sensitivity of the method was tested by using recovery experiments.

Pattern of protein degradation during germination of Streptomyces antibioticus spores.

The pattern of protein degradation during germination of Streptomyces antibioticus spores was studied by the pulse and chase technique. Two different protein fractions were found. First, a fraction of the proteins synthesized during the darkening process (20-30%) was quickly degraded in the 30 min following the labelling period. This rapid protein degradation was partially inhibited by protease inhibitors: p-chloromercuribenzoic acid, phenylmethylsulphonylfluoride, and o-phenanthroline. Second, the remaining 70-80% and the entire protein population formed during spore swelling and germ tube emergence were degraded with a lower and constant rate (3.3-6.0% /h). A stable mRNA fraction of the dormant spores was translated upon incubation of the spores in a minimal synthetic medium (MSM) or in distilled water. However, the degradation of these proteins did not occur unless the spores were then incubated in the MSM. A strong correlation between the degradation pattern of these proteins and that of those quickly degraded at the beginning of germination was observed. Protease activity in cell-free extracts of dormant spores was detected. Inhibition studies suggest the presence of serine, thiol, and metalloproteases. The protease activity, using casein as substrate, remained constant during the darkening process and started to increase progressively from the beginning of spore swelling.

Bound and spontaneously released toxic products of NAG vibrios cultivated in minimal synthetic media

Properties of toxic substances released spontaneously and by ultrasonic disintegration from NAG vibrio cells cultivated in minimal synthetic medium were compared.The released material was separated on the AMICON ultramembranes, dialysed and fractionated on the column of Sephadex G-100. This procedure revealed three fractions released by ultrasonic disintegration. The culture released spontaneously only one fraction separated by the above metioned procedure. The molecular weight estimated by the elution from calibrated G-100 column of the fractions released by the ultrasound was a follows: first fraction (H1) over 100,000, second fraction (H2) 25,000–30,000 and third one (H3) below 10,000. The molecular weight of the spontaneously released fraction (F1) was over 100,000. The biological activity of the fractions was tested by the rabbit skin test and by the rabbit ligated ileal loop test. The fractions H1, H2 and F1 were positive in both while the fraction H3 was negative. Fraction F1 was inactivated at 56 °C while at 100 °C its activity was restored. In agar gel diffusion test, the presence of endotoxin in fraction F1 was proved by means of specific antiserum. Results show that the toxic substances of enterotoxin nature are bound also to cell structures. They reveal the biological activity qualitatively and quantitatively different of the spontaneously released substances. Besides the endotoxin also other cell components can contribute to these effects.

Thymineless death in recombination deficient mutants of Escherichia coli K12.

When grown in thymine-free minimal synthetic medium, thyA recA mutants of Escherichia coli K12 were comparatively more resistant and thyA recBC mutants more sensitive to thymineless death (TLD) than their respective parent strains. No excessive numbers of single-strand breaks were observed in the DNA of the recBC mutant strain starved for thymine, hence the hypersensitivity for TLD in this strain was not caused by this type of DNA damage. Although experiments were performed with the same recBC mutant strain as used by earlier workers, results presented here contradict earlier findings that recBC mutants are no more sensitive and recA mutants are no more resistant to TLD.

Nutritional Requirements of Microbacterium thermosphactum

Microbacterium thermosphactum requires cysteine, alpha-lipoate, nicotinate, pantothenate, p-aminobenzoate, biotin, and thiamin for aerobic growth in glucose-mineral salts medium. Glucose cannot be replaced by Casamino Acids or acids of the tricarboxylic acid cycle as sole carbon and energy sources. The organism can also grow anaerobically in the minimal synthetic medium.

Thiaisoleucine resistant mutants in Saccharomyces carlsbergensis increase the content ofd-amyl alcohol in beer

Four presumably dominant thiaisoleucine resistant mutants of a brewing strain of Saccharomyces carlsbergensis were previously shown to produce mored-amyl alcohol than the parent strain when grown in synthetic minimal medium with aeration. In the present work an increased production ofd-amyl alcohol was also found in fermenting wort under brewing conditions resulting in a beer with an altered ratio betweend-amyl alcohol and isoamyl alcohol. The synthesis of 2,3-pentanedione plus its precursor, α-aceto-α-hydroxybutyrate, was generally higher in the mutants. The mutants showed a somewhat lower rate of increase in cell concentration during fermentation than the parent strain. With respect to other analytical data and taste, the three mutants isolated after moderate mutagenesis did not show deviations outside the range of the parent. One mutant obtained from a more heavity mutagenized culture deviated in several respects.

Development of Defined, Minimal, and Complete Media for the Growth of Hyphomicrobium neptunium

A complete synthetic medium containing 15 amino acids, a minimal synthetic medium (GAMS) containing 4 amino acids, and a supplemented minimal medium (GAMS + calcium pantothenate) have been developed for the cultivation of Hyphomicrobium neptunium ATCC 15444. Depending on the complexity of the synthetic media, generation times were approximately 2 to 3 times longer, and maximum cell densities were 0.3 to 0.9 log(10) lower than in ZoBell marine broth 2216. The fates of C-labeled amino acids in GAMS were monitored. Results suggested that H. neptunium was auxotrophic for methionine, utilized glutamic acid as a primary energy source, and readily anabolized and catabolized serine and aspartic acid. Individual amino acid concentrations above 125 mM induced prolonged lag periods, whereas only methionine was not growth limiting at a concentration as low as 2 mM.

Colorimetric Determination of Terreic Acid Produced by Aspergillus terreus

Abstract Two methods have been developed for determining terreic acid, which is a mycotoxin produced by Aspergillus terreus, a common food contaminant. The organism was grown independently in synthetic minimal medium, bread, and rice. After 15 days of growth, the toxin was extracted with ethyl acetate, cleaned up by thin layer chromatography, and determined colorimetrically, using Folin's reagent or 2,4- dinitrophenylhydrazine. The production of terreic acid was greater on bread than on rice or minimal medium. Recoveries of 200–400 ≧g terreic acid/100 g bread and rice ranged from 62 to 98%. The lower limit of detection was 200 ≧g/100 g.

Brewing performance of a yeast after prolonged growth on a synthetic medium

Two clones of brewers yeast, grown for more than 300 cell generations on a synthetic minimal medium were propagated on wort and used in pilot brewing experiments. Parallel brews with wort cultured yeast were carried out for comparison. Analytical data showed no differences between beer brewed with yeast grown on minimal medium and wort cultured yeast. One out of the two clones grown on minimal medium gave a beer with a somewhat inferior taste.

Growth cycles of filamentous Escherichia coli: II. Induced filaments

The effect of inhibition of cell division by gentian violet and thymine deficiency on the course of division of two E. coli strains, growing in batch cultures, was studied. These factors inhibited division and induced filament formation only in the pre-log phase and the exponential phase of growth. In the late logarithmic phase, the division was spontaneously derepressed even in the presence of inhibitor or thymine deficiency, and the filaments broke up into rods. Division of the filaments in the early log phase can be induced in the presence of the dye or thymine deficiency by transfering the filaments to media without any added carbon- or nitrogen source. In the thy − mutant, filament formation was suppressed during growth in a synthetic minimal medium with a low thymine concentration. Derepression of cell division under growth-limiting conditions is not caused by adaptation, selection of mutants or impermeability for the dye. The hypothesis is expressed that inhibition of the cell division, which can be reversed by growth-limiting conditions, acts indirectly on the division mechanism by way of unbalanced macromolecular synthesis. Die Einwirkung von Gentianaviolett und Thyminmangel auf die Hemmung der Zellteilung und den Verlauf derselben von Escherichia coli in Batch-Kulturen wurde studiert. Diese Faktoren hemmen die Zellteilung und induzieren Fadenbildung nur in der pre-logarithmischen und logarithmischen Phase des Wachstums; in der späten logarithmischen Phase wurde die Zellteilung spontan reaktiviert auch unter den Bedingungen eines Thyminmangels oder bei Anwesenheit eines Inhibitors. Die Filamente fragmentieren unter Bildung von Kurzstäbchen. Durch Übertragung der fadenförmigen Zellen aus der frühen logarithmischen Phase in ein Stickstoff- oder Kohlenstoff-freies Minimalmedium konnte Filamentteilung bei Anwesenheit eines Inhibitors oder bei Thyminmangel induziert werden. Die Neuerwerbung der Teilungsfähigkeit in der späten logarithmischen Phase wurde nicht durch Adaptation, Selektion von Mutanten oder Impermeabilität für Gentianaviolett verursacht.