Synthetic Growth Medium Research Articles

Trinucleotide insertions, deletions, and point mutations in glucose transporters confer K+ uptake in Saccharomyces cerevisiae.

Deletion of TRK1 and TRK2 abolishes high-affinity K+ uptake in Saccharomyces cerevisiae, resulting in the inability to grow on typical synthetic growth medium unless it is supplemented with very high concentrations of potassium. Selection for spontaneous suppressors that restored growth of trk1delta trk2delta cells on K+-limiting medium led to the isolation of cells with unusual gain-of-function mutations in the glucose transporter genes HXT1 and HXT3 and the glucose/galactose transporter gene GAL2. 86Rb uptake assays demonstrated that the suppressor mutations conferred increased uptake of the ion. In addition to K+, the mutant hexose transporters also conferred permeation of other cations, including Na+. Because the selection strategy required such gain of function, mutations that disrupted transporter maturation or localization to the plasma membrane were avoided. Thus, the importance of specific sites in glucose transport could be independently assessed by testing for the ability of the mutant transporter to restore glucose-dependent growth to cells containing null alleles of all of the known functional glucose transporter genes. Twelve sites, most of which are conserved among eukaryotic hexose transporters, were revealed to be essential for glucose transport. Four of these have previously been shown to be essential for glucose transport by animal or plant transporters. Eight represented sites not previously known to be crucial for glucose uptake. Each suppressor mutant harbored a single mutation that altered an amino acid(s) within or immediately adjacent to a putative transmembrane domain of the transporter. Seven of 38 independent suppressor mutations consisted of in-frame insertions or deletions. The nature of the insertions and deletions revealed a striking DNA template dependency: each insertion generated a trinucleotide repeat, and each deletion involved the removal of a repeated nucleotide sequence.

Premating Isolation Is Determined by Larval Rearing Substrates in CactophilicDrosophila mojavensis. III. Epicuticular Hydrocarbon Variation Is Determined by Use of Different Host Plants inDrosophila mojavensis andDrosophila arizonae

Adult epicuticular hydrocarbon profiles of male and femaleDrosophila mojavensis have been implicated as determinants of mate choice leading to premating isolation between geographically isolated populations. Hydrocarbon profiles of a Baja California and a mainland Sonora population ofDrosophila mojavensis, ayellow body mutant strain ofD. mojavensis, and a population ofD. arizonae were compared among flies that had been reared on two cactus substrates and a synthetic laboratory growth medium in order to assess the degree to which natural rearing substrates influence adult hydrocarbon composition. Twenty epicuticular hydrocarbon components, ranging from C29 to C41, were recovered by gas chromatography that represented major classes of alkanes, alkenes, and alkadienes. We found differences in relative amounts of epicuticular hydrocarbons among Baja and mainlandD. mojavensis, and theyellow body mutants. There were few differences betweenD. mojavensis andD. arizonae. The effects of rearing substrates were remarkable: most of the differences were due to the effects of lab food vs. cactus, but there were significant rearing substrate effects due to differences in the two cacti used. Eleven hydrocarbon components differed in abundance between males and females or showed significant sex × rearing substrate interactions from ANOVA. The effects of rearing substrates on epicuticular hydrocarbon composition inD. mojavensis are concordant with changes in the intensity of premating isolation between populations, implicating host ecology as a major determinant in patterns of mate choice in this species.

Studies on Heavy Metal Biosorption by Phanerochaete chrysosporium ME-446

In this study, Basidiomycetes strains Phanerochaete chrysosporium ME-446 and Pleurotus sajor-cqju were grown in synthetic complete medium and petri plates containing Cu, Co, Cd, Zn and Ag as metal salts, Through preliminary plate tests Phanerochaete chrysosporium ME-446 was found to be more resistant to toxic heavy metals as compared to Pleurotus sajor-caju. Specific growth rate and maximum dry mycelium mass were inhibited by increasing metal concentrations. Metal biosorption was also observed during the course of growth in synthetic media, and 80-95% of metals were found to be absorbed by the end of active growth.

Regulation of cellular Mg 2+ by Saccharomyces cerevisiae

Regulation of cellular Mg 2+ by S. cerevisiae was investigated. The minimal concentration of Mg 2+ that results in optimal growth of S. cerevisiae is about 30 μM and a half-maximum growth rate is attained at about 5 μM Mg 2+. Since the plasma membrane has an electrical potential greater than 100 mV, passive equilibration of Mg 2+ across the plasma membrane would provide sufficient cytosolic Mg 2+ (0.1–1 mM). The total cellular Mg 2+ of cells grown in synthetic medium containing 1 mM Mg 2+ is about 400 nmol/mg protein, most of which is bound to polyphosphate, nucleic acids, and ATP. Total cellular Mg 2+ decreases to about 80 nmol/mg protein as the Mg 2+ in synthetic growth medium is reduced to 0.02 mM, but remains relatively constant in growth medium containing 1 to 100 mM Mg 2+. Cells shifted into Mg 2+-free medium continue to grow by utilizing the vacuolar Mg 2+ stores. Mg 2+-starved cells replenish vacuolar Mg 2+ stores with a halftime of 30 min. following the addition of 1 mM Mg 2+ to the growth medium. The data indicate that cytosolic Mg 2+ is maintained by the regulation of Mg 2+ fluxes across both the vacuolar and plasma membranes.

X-ray photoelectron spectroscopy analysis of the surface composition of Azospirillum brasilense in relation to growth conditions

X-ray photoelectron spectroscopy (XPS) has been used to determine the surface chemical composition of the Gramnegative bacterium Azospirillum brasilense as a function of growth conditions. As opposed to growth in complex medium, growth in synthetic medium led to low reproducibility of XPS results, and did not show a significant variation of the cell surface composition as a function of culture time; moreover, the molecular composition obtained by modelling the XPS data was found to be generally poorer in proteins, and richer in polysaccharides and in hydrocarbon-like compounds. The direct correlation between the phosphate concentration and the concentration of carbon bound only to carbon and hydrogen, C (C,H), indicated that hydrocarbon-like compounds detected at the surface of cells grown in synthetic medium are essentially phospholipids. The poor reproducibility of the XPS data obtained for cells grown in synthetic medium and the random observation of phospholipids at the outermost cell surface are not due to cell disruption or to migration of intracellular components during sample preparation; exposure of phospholipids at the cell surface reflects either actual variations of the composition of the native surface induced by nutrient limitation or reorganization of cell surface polymers upon freeze drying.

Production of ligninolytic activities when treating paper pulp effluents by Trametes versicolor

In this work, the ability of Trametes versicolor to decolorize the effluents from the alkaline cooking of cereal straw produced at a paper and paper pulp making plant is studied. Enzymatic activities related to the metabolism of lignin during fungal treatment have also been evaluated. The results that have been attained show the necessity of a carbon source for the progress of the decolorization process, which can reach color elimination values higher than 70%. The study of the enzymatic activities shows the production (starting from the first hours of treatment) of laccase activity much higher than that obtained under the same conditions in synthetic growth media. There is also a clear relationship between the effluent concentration in the medium and the laccase activity. In the decolorization media, manganese-dependent peroxidase activity has also been detected, when SO 4Mn is added to those media. No LiP activity was detected in all the conditions assayed.

Use of a genetic algorithm in the development of a synthetic growth medium for Arthrobacter simplex with high hydrocortisone Δ1‐dehydrogenase activity

AbstractThe production process for Arthrobacter simplex high specific hydrocortisone Δ1‐dehydrogenase activity and biomass yield (full experimental plan: 1·5 × 1014 experiments). The specific hydrocortisone Δ1‐dehydrogenase activity was improved by a vector of 4·5 to 1800 U g−1. With the optimized medium composition in a stirred tank reactor for batch production of A. simplex, a specific hydrocortisone Δ1‐dehydrogenase activity of 2000–3000 U g−1 dry biomass was achieved depending on harvest time (previously reported data: 18–37 U g−1).

An iron-dependent mutant of Listeria monocytogenes of attenuated virulence

A bank of Tn 917-insertional mutants from the facultative intracellular pathogen Listeria monocytogenes was screened by an original method based on bacterial growth on synthetic medium under iron-limiting conditions. One mutant, whose in vitro growth in synthetic medium was specifically dependent upon the availability of iron in its environment, was isolated and characterized. The insertional event occurred in a non-coding region, upstream of a rrn operon and located within a 1100-kb NotI fragment of the physical map, where the virulence genes already identified in L. monocytogenes were also present. Protein analysis by SDS-PAGE revealed a pleiotropic effect of the insertional event on cell-associated proteins, suggesting a polar effect of the transposon on adjacent unknown gene(s). The virulence in the mouse of this mutant was strongly impaired, although it was capable in vitro of growing intracellularly and of spreading from cell to cell, as shown by the production of lytic plaques on cell culture.

An iron-dependent mutant of Listeria monocytogenes of attenuated virulence.

A bank of Tn917-insertional mutants from the facultative intracellular pathogen Listeria monocytogenes was screened by an original method based on bacterial growth on synthetic medium under iron-limiting conditions. One mutant, whose in vitro growth in synthetic medium was specifically dependent upon the availability of iron in its environment, was isolated and characterized. The insertional event occurred in a non-coding region, upstream of a rrn operon and located within a 1100-kb NotI fragment of the physical map, where the virulence genes already identified in L. monocytogenes were also present. Protein analysis by SDS-PAGE revealed a pleiotropic effect of the insertional event on cell-associated proteins, suggesting a polar effect of the transposon on adjacent unknown gene(s). The virulence in the mouse of this mutant was strongly impaired, although it was capable in vitro of growing intracellularly and of spreading from cell to cell, as shown by the production of lytic plaques on cell culture.

Pentose-phosphate pathway in Saccharomyces cerevisiae: analysis of deletion mutants for transketolase, transaldolase, and glucose 6-phosphate dehydrogenase

Deletion mutants for the yeast transketolase gene TKL1 were constructed by gene replacement. Transketolase activity was below the level of detection in mutant crude extracts. Transketolase protein could be detected as a single protein band of the expected size by Western-blot analysis in wild-type strains but not in the deletion mutant. Deletion of TKL1 led to a reduced but distinct growth in synthetic medium without an aromatic amino-acid supplement. We also isolated double and triple mutants for transketolase (tkl1), transaldolase (tal1), and glucose 6-phosphate dehydrogenase (zwf1) by crossing the different mutants. A tal1 tkl1 double mutant grew nearly like wild-type in rich medium. Only the tkl1 zwf1 double and the tal1 tkl1 zwf1 triple mutant grew more slowly than the wild-type in rich medium. This growth defect could be partly alleviated by the addition of xylulose but not ribose. The triple mutant still grew slowly on a synthetic mineral salts medium without a supplement of aromatic amino acids. This suggests the existence of an alternative but limited source of pentose phosphates and erythrose 4-phosphate in the tkl1 zwf1 double mutants. Hybridization with low stringency showed the existence of a sequence with homology to transketolase, possibly a second gene.

Studies on metal resistance system in Kluyveromyces marxianus.

Through preliminary plate tests, Kluyveromyces marxianus was found to be much more resistant to toxic heavy metals compared to a CUP1R strain of Saccharomyces cerevisiae. Specific growth rate and maximum dry weights affected by increasing metal concentrations were determined to obtain precise patterns of resistance. Metal biosorption was also monitored during the course of growth in synthetic media containing respective metals at 0.5 mM final concentration. Although Zn- and Co-binding was negligible, as much as 90% of silver, 60% of copper, and 65% of cadmium were found to be absorbed by the end of active growth. Analysis of the protein profiles of S. cerevisiae and K. marxianus on metal exposure suggested constitutive production of metallothionein in K. marxianus. Furthermore, a smaller protein synthesized by K. marxianus on induction by silver or cadmium accounts for the high resistance of the organism to these metals.

Growth Responses of Coliform Bacteria to Recombinant Bovine Cytokines

Growth responses of 10 coliform isolates to recombinant bovine cytokines were measured in vitro. Six Escherichia coli and four Klebsiella pneumoniae isolates obtained from bovine MI were tested for growth responses to recombinant bovine interleukin-β, interleukin-2, and interferon-γ. Cytokines were tested at 104, 103, 102, and 10 U/ml of media. Media used were a synthetic tissue culture medium, a chemically defined synthetic bacterial growth medium, and UHT sterilized milk. Bacterial counts in the synthetic tissue culture medium and UHT milk increased slightly as concentration of interferon-γ in the media increased. Recombinant bovine interferon-γ increased bacterial populations during the log growth phase but did not affect the number of bacteria in stationary growth phase. Bacterial growth responses were not related to either interleukin-2 or interleukin-1β concentrations in any of the three media. Bacterial growth responses to cytokines were not related to differences in either serum susceptibility, growth of isolates in dry cow secretion, duration of IMI from which isolates were obtained, or bacterial species.

Effect of temperature and medium composition on mycelial growth of Streptomyces tendae in submerged culture.

To investigate the influence of culture conditions on growth of filamentous bacteria in submerged culture, S. tendae was cultivated at various temperatures and in various media. For this purpose, a temperature-controlled growth chamber was constructed, which allows the cultivation of filamentous bacteria and fungi oxygen saturated medium. To observe the development of mycelia emerging from spores, this growth chamber was mounted on a microscope stage and series of images were analyzed by an image processing system. Growth kinetics obtained in liquid culture were identical to those determined on solid media. Specific growth rate and apical extension rates of individual hyphae seemed to be higher than those observed on solid media, due to a better supply of medium in submerged culture. A two-fold increase in specific growth rate, mean apical extension rate and branching rate was observed when the temperature was increased from 27 degrees C to 37 degrees C, therefore, the length of the hyphal growth unit was not changed. Growth in synthetic media used here was considerably slower compared with complex medium. A decrease in glucose concentration from 40.0 g l-1 to 1.0 g l-1 resulted in an increase in branching rate and specific growth rate, while apical extension rate of individual hyphae was unchanged.

Regulation ofTermitomyces clypeatusprotoplast regeneration by Krebs cycle acids

Abstract Mycelial derived protoplasts of the mushroom, Termitomyces clypeatus , did not regenerate in synthetic growth medium but did regenerate if casein hydrolysate was added to the same medium. Aspartic acid, glutamic acid, histidine, of serine and alanine when added individually had a similar effect on regeneration to that of casein hydrolysate. Krebs cycle acids also supported protoplast regeneration in the presence of glucose. Glucose alone, in the regenerating medium supported osmotically stable germ tube formation from protoplasts while Krebs cycle acid alone could not even change the osmotic fragility of the protoplasts. Electron microscopy of germ tube preparations indicated the presence of an incomplete wall structure when regeneration took place in the presence of glucose alone.

Regulation of Termitomyces clypeatus protoplast regeneration by Krebs cycle acids

Mycelial derived protoplasts of the mushroom, Termitomyces clypeatus, did not regenerate in synthetic growth medium but did regenerate if casein hydrolysate was added to the same medium. Aspartic acid, glutamic acid, histidine, of serine and alanine when added individually had a similar effect on regeneration to that of casein hydrolysate. Krebs cycle acids also supported protoplast regeneration in the presence of glucose. Glucose alone, in the regenerating medium supported osmotically stable germ tube formation from protoplasts while Krebs cycle acid alone could not even change the osmotic fragility of the protoplasts. Electron microscopy of germ tube preparations indicated the presence of an incomplete wall structure when regeneration took place in the presence of glucose alone.

Biotoxicity of mercury as influenced by mercury(II) speciation

Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolated to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s; mercuric chloride, on the other hand, always produced lower IC50s. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1% glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)2(2+)], chloride (HgCl+, HgCl2(0), HgClOH0, and HgCl3-), phosphate (HgHPO4(0), ammonia (HgNH3(2+), glycine [Hg(GLY)+], alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl2(0), and HgClOH0 were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)2(2-) was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.

Rhodopseudomonas cryptolactis, sp. nov., a new thermotolerant species of budding phototrophic purple bacteria

Rhodopseudomonas cryptolactis, sp. nov., was enriched from a hot spring environment using conditions that favor growth of anoxygenic phototrophs able to use N2 as the sole nitrogen source for growth. Phototrophic growth rate of the budding bacterium is optimal at ca. 40°C, and the maximum growth temperature is ca. 46°C. R. cryptolactis requires vitamin B12, nicotinic acid, and p-aminobenzoic acid for optimal growth in synthetic media, and has restricted capacities in respect to phototrophic carbon and nitrogen nutrition. Molecular nitrogen, NH4+, and urea can serve as nitrogen sources; pyruvate and lactate as carbon sources. Utilization of lactate, however, is dependent on the presence of bicarbonate, suggesting a linkage between lactate photometabolism and operation of the Calvin reductive pentose cycle. The photopigments of R. cryptolactis, including bacteriochlorophyll a, are housed in lamellar stacks parallel to and underlying the cell membrane. When grown at low light intensity, the cells apparently produce an additional species of light- harvesting Bchl that has a major in vivo absorbancy peak at 822 nm. The bacterium also grows as an aerobic chemoheterotrophs in darkness, and the presence of O2 causes severe repression of photopigment synthesis.

Rhodopseudomonas cryptolactis, sp. nov., a new thermotolerant species of budding phototrophic purple bacteria

Rhodopseudomonas cryptolactis, sp. nov., was enriched from a hot spring environment using conditions that favor growth of anoxygenic phototrophs able to use N 2 as the sole nitrogen source for growth. Phototrophic growth rate of the budding bacterium is optimal at ca. 40°C, and the maximum growth temperature is ca. 46°C. R. cryptolactis requires vitamin B 12, nicotinic acid, and p-aminobenzoic acid for optimal growth in synthetic media, and has restricted capacities in respect to phototrophic carbon and nitrogen nutrition. Molecular nitrogen, NH 4 +, and urea can serve as nitrogen sources; pyruvate and lactate as carbon sources. Utilization of lactate, however, is dependent on the presence of bicarbonate, suggesting a linkage between lactate photometabolism and operation of the Calvin reductive pentose cycle. The photopigments of R. cryptolactis, including bacteriochlorophyll a, are housed in lamellar stacks parallel to and underlying the cell membrane. When grown at low light intensity, the cells apparently produce an additional species of light- harvesting Bchl that has a major in vivo absorbancy peak at 822 nm. The bacterium also grows as an aerobic chemoheterotrophs in darkness, and the presence of O 2 causes severe repression of photopigment synthesis.

An in vivo replica method for the site-specific detection of Candida albicans on the denture surface in denture stomatitis patients: Correlation with clinical disease

A site-specific agar replica technique for detecting Candida albicans on the acrylic resin denture surface of denture stomatitis patients has been developed. The method is selective for C. albicans during a finite incubation period with a specific synthetic growth medium. C. albicans colonies can be geographically observed on the replica and their presence can be correlated with inflammatory lesions visible on the mucosa of the maxillary and mandibular residual ridges. In 12 denture stomatitis patients studied, a close clinical correlation of Newton type III patients was noted but this clinical correlation could not be observed in Newton type I and II patients. In general, the number of C. albicans colonies increased with the severity of the inflammation. The findings are discussed in light of lack of knowledge of the etiology of the stomatitis. The importance of the replica method is also discussed.

Requirement of chelating compounds for the growth of Corynebacterium glutamicum in synthetic media

The effects of various iron-complexing substances on the growth of Corynebacterium glutamicum in synthetic medium were investigated. The data obtained indicate that C. glutamicum has an absolute requirement for the presence of an iron-complexing compound as a growth factor for rapid and abundabnt growth in synthetic media. This requirement can be met by adding low concentrations (10−5 M) of certain dihydroxyphenols (catechol, protocatechuate) or relatively high concentratuions (0.1%) of citrate to the medium or by autoclaving a small amount of glucose together with other media components. The addition of catechol or protocatechuate to synthetic media has advantages over media preparation with citrate or autoclaved glucose. In the described synthetic broth supplemented with catechol or protocatechuate growth is largely independent of the iron content of the medium in a range between 0.037 and 0.37 mM.