A defined minimal medium for axenic strains of Dictyostelium discoideum.
A defined, minimal medium for the cellular slime mold Dictyostelium discoideum is reported.
Amino Acids↗
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The Pxp Complex Detoxifies 5-Oxoproline and Promotes the Growth of Clostridioides difficile.
Clostridioides difficile is an anaerobic enteric pathogen that disseminates in the environment as a dormant spore. For C. difficile and other sporulating bacteria, the initiation of sporulation is a regulated process that prevents spore formation under favorable growth conditions. In Bacillus subtilis, one such mechanism for preventing sporulation is the prokaryotic 5-oxoprolinase, PxpB (KipI), which impedes the activation of the main sporulation kinase. In addition, PxpB functions as part of a complex that detoxifies the intermediate metabolite, 5-oxoproline (OP), a harmful by-product of glutamic acid and its derivatives. In this study, we investigate the orthologous Pxp proteins in C. difficile to determine their roles in the regulation of sporulation and metabolism. Through deletion of the pxpAGBC operon, we show that, unlike in B. subtilis, the Pxp (Kip) proteins have no significant impact on sporulation. However, we found that the pxp operon encodes a functional oxoprolinase that facilitates detoxification of OP. Furthermore, our data demonstrate that PxpAGBC not only detoxifies OP but also allows OP to be used as a nutrient source that supports the growth of C. difficile, thereby facilitating the conversion of a toxic by-product of metabolism into an energy source.
Clostridioides difficile↗
Phenylalanine stimulation of gramicidin S formation.
Bacillus brevis produces the antibiotic gramicidin S, a cyclic peptide comprised of two pentapeptide units of the sequence d-Phe-l-Pro-l-Val-l-Orn-l-Leu. Synthesis of this antibiotic is carried out by two enzymes, GS synthetases I and II. By supplementing a defined minimal medium [glycerol, NH(4) (+), tris(hydroxymethyl)aminomethane, salts] with each of the constituent amino acids (in the l form), we found that only l-phenylalanine had a marked stimulatory effect on gramicidin S production. This effect was not caused by an increase in growth, by an induction of GS synthetases I or II, or by a stabilization of these enzymes. l-Phenylalanine apparently stimulates production via its role as a precursor of the d-phenylalanine moiety of the gramicidin S molecule.
Amino Acids↗
Novel iron uptake system specified by ColV plasmids: an important component in the virulence of invasive strains of Escherichia coli.
The enhanced virulence of invasive strains of Escherichia coli carrying ColV plasmids was shown to be due to a novel plasmid-mediated iron uptake system. Possession of a ColV plasmid conferred strong selective advantage on the host bacterial strain in experimental infections unless excess iron was administered in the inoculum. Moreover, supplementation of defined minimal medium with transferrin to complex available iron caused marked limitation of the growth of plasmid-free strains but had no effect on strains carrying a ColV plasmid. The activity of an efficient iron uptake process was clearly shown by experiments with a mutant of E. coli deficient in enterochelin biosynthesis. Although the mutant was dependent on the presence of citrate in the growth medium to facilitate iron transport, colicinogenic derivatives did not require added citrate for growth. Radioactive iron was shown to be taken up rapidly by nongrowing cells of the plasmid-carrying strain. Furthermore, it was observed that repression of the synthesis of specific outer membrane proteins normally induced by conditions of iron deficit was maintained after a shift of the colicinogenic strains from a rich medium to a medium low in iron. The ColV plasmid-mediated iron uptake system was independent of the active iron transport mechanisms known in E. coli, but like them it required tonB activity as a source of energy.
Animals↗
Repression of heat-stable enterotoxin synthesis in enterotoxigenic Escherichia coli.
Five different carbon sources were examined for their ability to control synthesis of heat-stable enterotoxin (ST) by enterotoxigenic (ENT+) Escherichia coli grown in either a defined medium containing four amino acids or a minimal salts medium. No ST activity was observed when D-glucose, D-gluconate, and L-arabinose were added separately to the defined medium, whereas glycerol and pyruvate decreased toxin levels. Similar results were obtained using a minimal salts medium, except with pyruvate, which did not support growth. Inhibition of ST synthesis by D-glucose was overcome by the addition of 3 X 10(-3) M cyclic adenosine 3',5'-monophosphate. Glucose repression of beta-galactosidase synthesis under conditions optimal for inhibition of ST synthesis was also reversed by exogenous cyclic adenosine 3',5'-monophosphate in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside. The data suggest that control mechanisms for the synthesis of plasmid gene products of bacterial pathogens are similar to those exerted on the host chromosome.
Cyclic AMP↗
Enzyme regulation in neuroblastoma cells in a salts/glucose medium: induction of ornithine decarboxylase by asparagine and glutamine.
L-Asparagine is necessary and sufficient for the maximal induction of ornithine decarboxylase (ODC) (L-ornithine carboxy-lyase, EC 4.1.1.17) activity in confluent N18 mouse neuroblastoma cells in a salts/glucose medium; L-asparagine also induces maximal ODC activity when added to a tissue culture medium. L-Glutamine is about one-half as effective as asparagine. Cholera toxin and agents that are known to raise intracellular cyclic AMP concentrations have no effect on the induction of ODC activity unless suboptimal concentrations of asparagine are present in the salts/glucose medium. Whereas actinomycin D does not inhibit induction of ODC activity by asparagine, it inhibits the induction of ODC activity in association with cyclic AMP. In the salts/glucose medium, the rate of loss of ODC activity following the inhibition of protein synthesis by cycloheximide or puromycin depends upon the presence or absence of asparagine; loss is rapid only in the absence of asparagine and does not appear to be related to the inhibition of protein synthesis. These results are discussed in the context that the overlay of the growth medium tends to mask the minimal requirements for enzyme induction, because the composition of the medium defines: (a) the requirements for the induction of ODC activity; (b) the effect, or lack of effect, of cyclic AMP (and of inducers of intracellular cyclic AMP) on the induction of ODC activity; (c) the effect, or lack of effect, of actinomycin D on the induction of ODC activity; and (d) the action of puromycin and of cycloheximide on the rate of loss of ODC activity. It will be interesting to determine whether these results are uniquely applicable to ODC, whether many of the reactions attributed to cyclic AMP in the literature may be mediated by asparagine and glutamine, and whether actinomycin D, cycloheximide, and puromycin can be relied upon to differentiate between transcriptional and post-transcriptional control.
Asparagine↗
Methenamine and its salts as urinary tract antiseptics: variables affecting the antibacterial activity of formaldehyde, mandelic acid, and hippuric acid in vitro.
The activities of formaldehyde and of mandelic and hippuric acids, alone and in combination, have been tested against some 300 strains of bacteria typical of those causing urinary tract infections. In a chemically defined medium, which resembles urine in many respects, formaldehyde had a mean minimal inhibitory concentration of 13 mug per ml. Activity was several fold lower in media (nutrient agar and tryptic soy agar) that contained significant amounts of protein. The activity of formaldehyde is virtually unaffected by pH in the range of 5 to 8. Mandelic and hippuric acids (2 mg per ml) have limited antimicrobial activity at acid pH values only. The combination of formaldehyde with mandelic acid (2 mg per ml) was additive, most markedly at pH 5; the formaldehyde-hippuric acid combination, however, did not appear to be additive. Our findings suggest that, at pH values between 5 and 6, an antibacterial concentration of formaldehyde will be generated from methenamine within approximately 1 hr after being excreted into the urine.
Anti-Infective Agents, Urinary↗
Genetic regulation of galactokinase in Tetrahymena by cyclic AMP glucose, and epinephrine.
We have found evidence that transcription of the galactokinase (ATP:D-galactose 1-phosphotransferase; EC 2.7.1.6) gene is inhibited, in the animal-like protozoan Tetrahymena, by dibutyryl adenosine 3':5'-cyclic monophosphate, glucose, and epinephrine. The specific activities of galactokinase in Tetrahymena cells grown in defined media with galactose or glycerol as the principal carbon source are equivalent; the specific activity in glucose minimal medium is [unk] the value. Thus, while there seems to be no specific induction of the enzyme by the substrate, galactose, there is a strong "repression" by glucose. This repression by glucose is mimicked, in glycerol-grown cells, by the addition of millimolar amounts of dibutyryl adenosine 3':5'-cyclic monophosphate or phosphodiesterase inhibitors such as caffeine and theophylline. When glucose-grown cells are washed and resuspended in carbohydrate-free medium, the galactokinase specific activity increases by as much as 10-fold within 12 hr. This increase is blocked by dibutyryl adenosine 3':5'-cyclic monophosphate and by epinephrine (synthesized by Tetrahymena, and previously shown to activate a membrane-bound adenylate cyclase in extracts of this organism), as well as by inhibitors of mRNA synthesis, maturation, and translation. Our results suggest that glucose and epinephrine can regulate transcription of the galactokinase gene by modulation of cyclic nucleotide levels. The observation that the nonmetabolized sugars 2-deoxyglucose, 2-deoxygalactose, and alpha-methylglucoside are as effective as glucose suggests that the sugar itself, or an immediate metabolite such as the 1-phosphate derivative, may be the effector.
Animals↗
Chemical and biological properties of B16 murine melanoma cells grown in defined medium containing bovine serum albumin.
The addition of 1 percent (w/v) bovine serum albumin (BSA) to the F12 medium utilized for the growth of the B16 melanoma cells significantly stimulated the growth of this cell line. The synthesis of mucopolysaccharides and sialoglycopeptides in this medium is identical with that in Eagle's minimal essential medium with Earle's balanced salt solution supplemented with 2 mM L-glutamine, twice the recommended concentration of vitamins, nonessential amino acids, sodium pyruvate, and 10 percent (v/v) fetal calf serum. Cell volume and morphology did not change significantly, under the different growth conditions and tumorigenicity, as assayed by injection of cultured cells into syngeneic animals, was not decreased. Analysis of the BSA used indicated the presence of a sialoglycoprotein contaminant. This sialoglycoprotein contaminant was present in all lots examined and contains N-acetyl-and N-glycolylneuraminic acid, mannose, galactose, and glucosamine. The sialoglycoprotein can be removed by chromatography on acetate form anion-exchange resin at pH 4.3. F12 media containing the purified BSA plus selenite and the sodium salts of palmitic, oleic, and linoleic acids supported growth of the melanoma cells to the same extent as did the media containing unpurified BSA, indicating that the sialoglycoprotein has no role in sustaining the growth of the cells.
Cell Division↗
[A method for testing susceptibility of Haemophilus influenzae to co-trimoxazole (author's transl)].
Various methods for determining the activity of co-trimoxazole against haemophilus influenzae have been compared. The results were cortically influenced by antagonistic substances in the medium. The content of p-aminobenzoeacid and thymidine in the medium should be as low as possible. The antagonistic influences are compensated by lysed horseblood, which contains thymidine-phosphorylase. Haeminchloride and nicotinamide adenine dinucleotide (NAD) are better growth-factors than other poorly defined preparations (Supplement C, Fil-des-Enrichment, Iso-VitaleX). For the determination of the minimal inhibition concentration (MIC) we propose the microtiter technique. Likewise a modified agar diffusion test can be recommended for screening purposes. Of the 143 haemophilus influenzae strains tested against co-trimoxazole 134 (94%) were sensitive.
4-Aminobenzoic Acid↗
Defined conditions for synthesis of Bacillus cereus enterotoxin by fermenter-grown cultures.
A strain of Bacillus cereus produced high levels of enterotoxin when grown in a semidefined medium in a laboratory scale fermenter. The optimum conditions for enterotoxin synthesis by cultures grown in this medium, which contained Casamino Acids and yeast extract, were found to be: inoculation of vigorously gorwing culture at the 1% level, addition of glucose at a concentration of 1%, control of culture pH at 8.0, incubation at 32 degrees C, use of a moderate stirring rate, and addition of air at low flow rates to minimize foaming. The enterotoxin yield in fermenter-grown cultures was approximately 20 to 50 times higher than the yield obtained in shake flask cultures.
Bacillus cereus↗
Effect of different lots of Mueller-Hinton agar on the interpretation of the gentamicin susceptibility of Pseudomonas aeruginosa.
Population distributions and quality control data for strains of Pseudomonas aeruginosa tested for gentamicin susceptibility on six lots of Mueller-Hinton agar were analyzed. The lots of agar were used in three University of Washington hospitals from April 1975 through October 1977. The analyses indicated that the performance of members of the P. aeruginosa populations in each hospital closely followed the performance of the quality control strain, P. aeruginosa ATCC 27853, when tested on each lot of Mueller-Hinton medium. The variability of zone diameters with the P. aeruginosa populations and the quality control strain indicated that a fixed indeterminate range (13 to 16 mm) of gentamicin susceptibility was not applicable to these organisms as it was with the Enterobacteriaceae. Variability in gentamicin susceptibility results was demonstrated in both minimal inhibitory concentration and disk diffusion tests when eight selected P. aeruginosa strains and the quality control strain were tested on each lot of medium. This variation in susceptibility to gentamicin was not related to the total Ca(2+), Mg(2+), or Zn(2+) content of each lot of medium. The data demonstrated that a moving indeterminate range of gentamicin susceptibility, 3 to 6 mm below the mean zone diameter of the quality control strain, was a suitable criterion for strains tested on a single medium lot. These results illustrate the importance of defining stringent performance standards for media used in the susceptibility testing of P. aeruginosa with gentamicin and other aminoglycoside antibiotics.
Agar↗
Improved basal medium for Y-1 mouse adrenal cortex tumor cells in culture. I. Dependence of growth and steroid response on calcium ion concentration.
An improved basal medium is presented that required only minimal supplementation with dialyzed fetal bovine serum or bovine serum albumin and fetuin to be comparable to Ham's F-10, which requires 15% horse serum (HS) and 2.5% fetal bovine serum (FBS) for the growth and function of Y-1, mouse adrenal cortex tumor, cells. Cell monolayers maintained for up to 2 weeks without any protein supplementation have retained their steroid response to ACTH. The medium differs from Ham's F-10 in its buffer composition and higher calcium-ion concentration. This medium should be a useful adjunct to studies pertaining to steroid and lipid intermediary metabolism, the retention of a specialized physiological function in a chemically defined medium, and the mechanism of hormonal response.
Adrenal Gland Neoplasms↗