Showing papers on "Growth medium published in 2000"

Anaerobic oxidation of alkanes by newly isolated denitrifying bacteria.

Abstract: The capacity of denitrifying bacteria for anaerobic utilization of saturated hydrocarbons (alkanes) was investigated with n-alkanes of various chain lengths and with crude oil in enrichment cultures containing nitrate as electron acceptor. Three distinct types of denitrifying bacteria were isolated in pure culture. A strain (HxN1) with oval-shaped, nonmotile cells originated from a denitrifying enrichment culture with crude oil and was isolated with n-hexane (C6H14). Another strain (OcN1) with slender, rod-shaped, motile cells was isolated from an enrichment culture with n-octane (C8H18). A third strain (HdN1) with oval, somewhat pleomorphic, partly motile cells originated from an enrichment culture with aliphatic mineral oil and was isolated with n-hexadecane (C16H34). Cells of hexane-utilizing strain HxN1 grew homogeneously in the growth medium and did not adhere to the alkane phase, in contrast to the two other strains. Quantification of substrate consumption and cell growth revealed the capacity for complete oxidation of alkanes under strictly anoxic conditions, with nitrate being reduced to dinitrogen.

Distribution and Exudation of Allelochemicals in Wheat Triticum aestivum

Abstract: Wheat allelopathy has potential for weed suppression. Allelochemicals were identified in wheat seedlings, and they were exuded from seedlings into agar growth medium. p-Hydroxybenzoic, trans-p-coumaric, cis-p-coumaric, syringic, vanillic, trans-ferulic, and cis-ferulic acids and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) were identified in both the shoots and roots of 17-day-old wheat seedlings and their associated agar growth medium. Wheat accessions with previously identified allelopathic activity tended to contain higher levels of allelochemicals than poorly allelopathic ones. The allelopathic compounds present in the shoots generally also were identified in the roots and in the agar medium. Allelochemicals were distributed differentially in wheat, with roots normally containing higher levels of allelochemicals than the shoots. When the eight allelochemicals were grouped into benzoic acid and cinnamic acid derivatives, DIMBOA, total coumaric, and total ferulic acids, the amount of each group of allelochemicals was correlated between the roots and the shoots. Most of the allelochemicals identified in the shoots and roots could be exuded by the living roots of wheat seedling into the agar growth medium. However, the amounts of allelochemicals in the agar growth medium were not proportional to those in the roots. Results suggest that wheat plants may retain allelochemicals once synthesized. The presence of allelochemicals in the agar growth medium demonstrated that wheat seedlings were able to synthesize and to exude phytotoxic compounds through their root system that could inhibit the root growth of annual ryegrass.

Contribution of NADH oxidase to aerobic metabolism of Streptococcus pyogenes.

Abstract: An understanding of how the heme-deficient gram-positive bacterium Streptococcus pyogenes establishes infections in O(2)-rich environments requires careful analysis of the gene products important in aerobic metabolism. NADH oxidase (NOXase) is a unique flavoprotein of S. pyogenes and other lactic acid bacteria which directly catalyzes the four-electron reduction of O(2) to H(2)O. To elucidate a putative role for this enzyme in aerobic metabolism, NOXase-deficient mutants were constructed by insertional inactivation of the gene that encodes NOXase. Characterization of the resulting mutants revealed that growth in rich medium under low-O(2) conditions was indistinguishable from that of the wild type. However, the mutants were unable to grow under high-O(2) conditions and demonstrated enhanced sensitivity to the superoxide-generating agent paraquat. Mutants cultured in liquid medium under conditions of carbohydrate limitation and high O(2) tension were characterized by an extended lag phase, a reduction in growth, and a greater accumulation of H(2)O(2) in the growth medium compared to the wild-type strain. All of these mutant phenotypes could be overcome by the addition of glucose. Either the addition of catalase to the culture medium of the mutants or the introduction of a heterologous NADH peroxidase into the mutants eliminated the accumulation of H(2)O(2) and rescued the growth defect of the mutants under high-O(2) conditions in carbohydrate-limited liquid medium. Taken together, these data show that NOXase is important for aerobic metabolism and essential in environments high in O(2) with carbohydrate limitation.

Genetic analysis reveals that FLO11 upregulation and cell polarization independently regulate invasive growth in Saccharomyces cerevisiae.

Abstract: Under inducing conditions, haploid Saccharomyces cerevisiae perform a dimorphic transition from yeast-form growth on the agar surface to invasive growth, where chains of cells dig into the solid growth medium. Previous work on signaling cascades that promote agar invasion has demonstrated upregulation of FLO11, a cell-surface flocculin involved in cell-cell adhesion. We find that increasing FLO11 transcription is sufficient to induce both invasive and filamentous growth. A genetic screen for repressors of FLO11 isolated mutant strains that dig into agar (dia) and identified mutations in 35 different genes: ELM1, HSL1, HSL7, BUD3, BUD4, BUD10, AXL1, SIR2, SIR4, BEM2, PGI1, GND1, YDJ1, ARO7, GRR1, CDC53, HSC82, ZUO1, ADH1, CSE2, GCR1, IRA1, MSN5, SRB8, SSN3, SSN8, BPL1, GTR1, MED1, SKN7, TAF25, DIA1, DIA2, DIA3, and DIA4. Indeed, agar invasion in 20 dia mutants requires upregulation of the endogenous FLO11 promoter. However, 13 mutants promote agar invasion even with FLO11 clamped at a constitutive low-expression level. These FLO11 promoter-independent dia mutants establish distinct invasive growth pathways due to polarized bud site selection and/or cell elongation. Epistasis with the STE MAP kinase cascade and cytokinesis/budding checkpoint shows these pathways are targets of DIA genes that repress agar invasion by FLO11 promoter-dependent and -independent mechanisms, respectively.

Understanding the Growth Phenotype of the Yeast gcr1 Mutant in Terms of Global Genomic Expression Patterns

Abstract: The phenotype of an organism is the manifestation of its expressed genome. The gcr1 mutant of yeast grows at near wild-type rates on nonfermentable carbon sources but exhibits a severe growth defect when grown in the presence of glucose, even when nonfermentable carbon sources are available. Using DNA microarrays, the genomic expression patterns of wild-type and gcr1 mutant yeast growing on various media, with and without glucose, were compared. A total of 53 open reading frames (ORFs) were identified as GCR1 dependent based on the criterion that their expression was reduced twofold or greater in mutant versus wild-type cultures grown in permissive medium consisting of YP supplemented with glycerol and lactate. The GCR1-dependent genes, so defined, fell into three classes: (i) glycolytic enzyme genes, (ii) ORFs carried by Ty elements, and (iii) genes not previously known to be GCR1 dependent. In wild-type cultures, GCR1-dependent genes accounted for 27% of the total hybridization signal, whereas in mutant cultures, they accounted for 6% of the total. Glucose addition to the growth medium resulted in a reprogramming of gene expression in both wild-type and mutant yeasts. In both strains, glycolytic enzyme gene expression was induced by the addition of glucose, although the expression of these genes was still impaired in the mutant compared to the wild type. By contrast, glucose resulted in a strong induction of Ty-borne genes in the mutant background but did not greatly affect their already high expression in the wild-type background. Both strains responded to glucose by repressing the expression of genes involved in respiration and the metabolism of alternative carbon sources. Thus, the severe growth inhibition observed in gcr1 mutants in the presence of glucose is the result of normal signal transduction pathways and glucose repression mechanisms operating without sufficient glycolytic enzyme gene expression to support growth via glycolysis alone.

The regulator of the yeast proline utilization pathway is differentially phosphorylated in response to the quality of the nitrogen source.

Abstract: Saccharomyces cerevisiae cells can sense the quality of the nitrogen source in their environment, enabling them to utilize preferred nitrogen-containing compounds over nonpreferred ones or to express pathways for the utilization of alternative nitrogen sources when the preferred ones have been consumed. Although very little is known about the sensing mechanism itself, work over the last decade has led to the discovery of a set of regulatory proteins, the GATA factors, whose role is to regulate, in both positive and negative directions, the expression of pathways of nitrogen assimilation in yeast. These proteins, Gln3p (26), Nil1p/Gat1p (10, 44), Dal80p/Uga43p (12, 13), and Nil2p/Gzf3p/Deh1p (11, 34, 42), are involved in a complex set of regulatory loops, competition for GATA binding sites, and possibly even some autoregulation. Recently, the coactivator Ada1p, isolated as Gan1p, was identified as a link between the GATA binding proteins and the basal transcriptional machinery (41). Global nitrogen repressor Ure2p is believed to interact with Gln3p to obtain appropriate expression of a variety of nitrogen assimilatory pathways (3; reviewed by Magasanik [23]). In their natural habitat, S. cerevisiae cells are found on grapes and in grape must, a nitrogen-poor environment where the most abundant nitrogen source is proline (2). Although proline is the least-preferred nitrogen source for many laboratory yeast strains and although its utilization results in the slowest growth rates, yeast cells have evolved a regulatory circuit that enables them to use the proline in the environment when preferred nitrogen sources are no longer available. The flux of proline into yeast cells is controlled by the activities of the general amino acid permease Gap1p and the proline-specific permease, Put4p (21). These permeases are regulated by nitrogen repression and do not respond to proline induction (17, 21, 43). The enzymes of the proline utilization pathway are induced by the presence of proline (6), and their levels reflect internal proline levels. The PUT1 and PUT2 genes encoding the enzymes of the pathway are regulated by Put3p, a member of the Zn(II)2Cys6 binuclear cluster protein family (4, 6, 7, 15, 24, 25, 40, 45, 49) and a close relative of Gal4p, the activator of the galactose utilization pathway. In vivo, Put3p binds the promoters of PUT1 and PUT2 in the presence or absence of proline and without regard to the quality of the nitrogen sources present in the growth medium (1) but activates transcription to a maximum level when proline is the sole source of nitrogen. PUT1 and PUT2 are repressed by Ure2p and in some, but not all, strain backgrounds are regulated by some of the GATA factors (9, 14, 50). This report presents the results of studies on wild-type and regulation-defective mutant Put3 proteins in cells grown in media containing different nitrogen sources. We show that Put3p is differentially phosphorylated as a function of the quality of the nitrogen source and that the slowest-migrating species of Put3p are correlated with elevated transcriptional activity. Analysis of the Put3p phosphoforms of activator-defective and activator-constitutive mutants leads to the suggestion that altered phosphorylation status may be one of two signals (proline induction being the other) that is required for maximum transcriptional activity by Put3p.

Biosynthesis and secretion of recombinant human growth hormone in Pichia pastoris

Abstract: Mature human growth hormone (hGH) cDNA was cloned by homologous recombination into the yeast Pichia pastoris genome. The hGH gene expression was placed under the control of the methanol-inducible alcohol oxidase 1 (AOX1) gene promoter and the Saccharomyces cerevisiae α-factor signal sequence to direct the secretion of recombinant human growth hormone (rhGH) into the growth medium. O2-limited induction of recombinant yeast strains in shake tubes with 3 ml of culture medium produced up to 11 mg rhGH l−1, while high cell density cultures using a 2-l bioreactor produced about 49 mg rhGH l−1 achieving 40% of total protein of the culture medium supernatant.

pH-dependent protein profiles of Helicobacter pylori analyzed by two-dimensional gels.

Abstract: Background.Helicobacter pylori survives transient exposure to extreme acid prior to adherence and growth on the gastric epithelium at neutral pH. Materials and Methods. The effect of pH stress on protein profiles of H. pylori was observed using two-dimensional gel electrophoresis (2-D gels). H. pylori 26695 was grown microaerobically in tryptone-yeast extract broth, 3% fetal bovine serum. Growth in acid alkalinized the medium, whereas growth in base caused acidification. For 2-D gel analysis of protein profiles, cultures were grown in media buffered at pH 5.7 and at pH 7.5. Results. Under all pH conditions, the most abundant proteins observed were the urease structural subunit UreB and the chaperonin GroEL. Growth in acid significantly increased the abundance of UreB. Thus, urease expression is not completely constitutive, as reported previously, but shows regulation by pH. Another protein observed only at low pH was identified as mammalian apolipoprotein A-I, possibly taken up by H. pylori from bovine serum in the growth medium. This finding, if confirmed, suggests that uptake of high-density lipoprotein from the human host may facilitate acquisition of cholesterol, required for formation of the unique cholesteryl glucosides in the membrane of H. pylori. In growth above pH 7, three stress proteins were induced: GroES (HspA), GroEL (HspB), and the antioxidant AhpC homolog TsaA. In addition, N-terminal sequence analysis identified five additional proteins that had not previously been reported on 2-D gels of H. pylori (FMN, SodB, TrxB, TsaA, and Tsr). Conclusions. In summary, our 2-D gel study reveals expression of several proteins dependent on growth pH.

Evidence for the presence of Pir-like proteins in Candida albicans

Abstract: Pir proteins are unique proteins with internal repeat sequences that are reported to be present in the cell wall of Saccharomyces cerevisiae. They are covalently attached to the cell wall and can be released by mild alkali treatment. In this study the biotinylated cell wall preparations from Candida albicans and S. cerevisiae were extracted by alkali and β-1,3 glucanase and analyzed in parallel. Among the four bands detected by streptavidin, two proteins were recognized by the antibody to the S. cerevisiae Pir protein Hsp150. The antibody also detected a high molecular mass protein secreted in the growth medium of C. albicans. Using S. cerevisiae HSP150/PIR2 gene as a probe, Southern and Northern hybridizations were performed with DNA and RNA of C. albicans. Hybridization with DNA digested with different restriction enzymes showed more than one hybridized fragment. An increased level of mRNA was found in heat shocked cells (37°C for 45 min compared to 25°C). Hybridization of ScHSP150 gene to mRNAs from cells grown in different media was also determined. Two transcripts of size approximately 3.5 kb and 2.0 kb were detected in mRNAs from cells grown in defined medium with glucose as carbon source or in the same medium supplemented with hemoglobin. The lower transcript of size 2.0 kb was absent in cells grown in medium with galactose as carbon source. A single band was also observed when cells were grown in rich medium. Together these results demonstrated the existence of β1,3 glucan linked proteins in C. albicans, which are related to Pir family proteins of S. cerevisiae.

Quantitative assessment of marine sponge cells in vitro: development of improved growth medium

Abstract: As sources of natural products with potential human therapeutic value, marine sponges are important subjects for cell culture studies. A critical component of any cell culture system is its growth medium. Proceeding from the hypotheses that the thawed, cryopreserved, primary cells would display detectable differential responses and that those responses could be comparatively quantified, this study has established that multiwell screening assays are useful tools for improving medium formulations in cell cultures of the marine sponge, Teichaxinella morchella. Fluorescent probe signals were correlated with known cell densities and viabilities in a 96-well format. Analysis of variance and post-test methods were applied to judge the significance of signal differences seen in a variety of medium formulations. Results from a series of experiments suggested that reducing glutamine and selenium concentrations in the standard medium would result in greater DNA, protein, and esterase activity signals. This was confirmed by the direct comparison of the standard and improved medium formulations. Significantly higher protein content and esterase activity were associated with the improved medium. DNA content was also higher, though not significantly. The result is a new medium formulation that may be more able to support cell growth and division, providing an improved cell culture system for marine sponge cell studies. The assays can be used in additional studies to further improve the in vitro conditions for marine sponge cell culture.

Regulation of pyc1 encoding pyruvate carboxylase isozyme I by nitrogen sources in Saccharomyces cerevisiae

Abstract: In Saccharomyces cerevisiae, the existence of PYC1 and PYC2 encoding cytosolic pyruvate carboxylase isoform I and II is rather puzzling, owing to the lack of potent differential gene regulation by the carbon sources We report several findings indicating that these two genes are differentially regulated by the nature of the nitrogen source In wild-type cells, the activity of pyruvate carboxylase, which is the sum of pyruvate carboxylase isoform I and II, was two- to fivefold lower in carbon medium containing aspartate, asparagine, glutamate or glutamine instead of ammonium as the nitrogen source, whereas it was 15- to threefold higher when the ammonium source was substituted by arginine, methionine, threonine or leucine These enzymatic changes were independent of the nature of the carbon source and closely correlated to the changes in β-galactosidase from PYC1-lacZ gene fusion and in PYC1 transcripts Transfer of exponentially growing cells of the pyc2 mutant from an aspartate or a glutamate medium to an ammonium medium caused a fivefold increase in PYC1 mRNA in less than 30 min, whereas in the inverse experiment, PYC1 transcripts returned within 30 min to the low levels found in aspartate/glutamate medium By contrast, these conditions affected neither the pyruvate carboxylase activity encoded by PYC2 nor PYC2 mRNA Considering that changes in PYC1 expression inversely correlated with changes in α-ketoglutarate concentration or in α-ketoglutarate/glutamate ratio following the nitrogen shift experiments, and taking into account the pivotal role of this metabolite in ammonium assimilation, it is suggested that changes in α-ketoglutarate or in the α-ketoglutarate/glutamate ratio might be implicated in triggering the nitrogen effects on PYC1 expression The physiological significance of the differential sensitivity of PYC1 and PYC2 genes with respect to the nitrogen source in the growth medium is also discussed

Threshold level of protein kinase A activity and polarized growth in Mucor rouxii.

Abstract: A model system to study the involvement of cAMP-mediated regulation of a cellular process such as hyphal morphogenesis was investigated. Impairment of polarized growth was observed when Mucor rouxii sporangiospores were grown in the presence of N(6)-cAMP analogues and of SQ 65,442, a cAMP phosphodiesterase inhibitor. Together with an effect on isodiametric growth, there was increased pigmentation, increased cell fragility and loss of cell adhesiveness. The total effect on morphology was attained even after adding the compounds shortly before germ-tube emergence; when added after this time growth continued in a non-polarized form and rounding of the germ tip was observed. The morphological effect was observed under all the nutritional and environmental conditions studied (aerobic conditions and defined medium with maltose or glucose, Casamino acids medium with glucose, or rich medium; anaerobic conditions with rich medium; and following a shift from anaerobiosis to aerobiosis). The time of germ-tube emergence, and the size of the cell at this time, was dependent on the growth medium. Protein kinase A (PKA) specific activity was followed during the germination process under three growth conditions. It was found that the total activity of PKA correlated with differentiation and not with growth, and that the total specific activity at the time of germination was the same, independent of the culture medium. The time of germ-tube emergence correlated with the time of attainment of a threshold level of PKA total specific activity. The concentration of dibutyryl-cAMP needed to promote isodiametric growth correlated with the total units of activity of PKA to be activated per cell. It was concluded that PKA is involved in the morphogenetic process of the fungus grown under all the nutritional and ambient conditions tested.

Bioaccumulation of metal ions by yeast cells of Saccharomyces cerevisiae

Abstract: The ability of two baker's yeast strains for bioaccumulation of chosen metals was investigated in the presence of other elements (ion pairs), during dynamic culturing in the brewer's wort and in the Rose medium. Yeast cells accumulated relatively small amounts of calcium, magnesium and manganese. Magnesium uptake was greatly affected by Co 2+ and Zn 2+ ions. Cobalt, zinc and copper accumulation was at the level between 0.1 to over 6 μmol per gram of dry weight of yeast, depending on the yeast strain, the kind of growth medium, and the presence of other metals. Increased level of Zn 2+ limits the accumulation of Co 2+ . Microscopic documentation indicates that Zn 2+ and Mn 2+ ions lower the destructive effect of increased levels of Co 2+ and Cu 2+ on yeast cell.

Biosynthesis and secretion of recombinant human growth hormone in Pichia pastoris

Abstract: Mature human growth hormone (hGH) cDNA was cloned by homologous recombination into the yeast Pichia pastoris genome. The hGH gene expression was placed under the control of the methanol-inducible alcohol oxidase 1( AOX1 )g ene promoter and theSaccharomyces cerevisiae-factor signal sequence to direct the secretion of recombinant human growth hormone (rhGH) into the growth medium. O2-limited induction of recombinant yeast strains in shake tubes with 3 ml of culture medium produced up to 11 mg rhGH l 1 , while high cell density cultures using a 2-l bioreactor produced about 49 mg rhGH l 1 achieving 40% of total protein of the culture

Formation of lipolytic enzymes by Brevibacterium linens

Abstract: When Brevibacterium linens ATCC 9172 was grown in shake flasks, it produced a cell-associated lipase with a specific activity of 152 to 188 U g−1 cells depending on the composition of the growth medium. There was no growth in media containing tributyrine as the sole carbon source. The cell-associated lipase had maximum activity at pH 8.0 and 37 °C and was strongly inhibited by 3,4-dichloroisocoumarin, an inhibitor specific for serine esterases. Cell-associated activity was released from the cells by treatment with lysozyme. The kinetics of lipase formation was closely related to the amount of biomass formed during growth.

Quantificação de bactérias totais e esporuladas no solo

Abstract: This work studied the effect of the temperature and incubation time and of the inoculum dilution, in two culture media, on the quantification of total and sporulating bacteria. The quantitative and qualitative growth of total and sporulating bacteria depended on the growth medium, temperature, dilution and incubation time, as well as on the interaction of these factors. Tryptic Soil Agar medium presented a greater number of colony forming units (CFU) for total bacteria. However, for Bacillus spp. there were higher counts on Thorton medium. Colonies grown on Tryptic Soy Agar medium were of larger size and had a more clearly defined shape. An incubation temperature of 30°C yielded more CFU than incubation at 25°C, especially during the first days of incubation. Bacterial growth was fit to a mathematical model and corresponded to a third degree equation. Depending on the growth culture medium, incubation temperature and dilution, periods of maximum growth were between 4.9 and 6.9 days for total bacteria and between 4.4 and 7.2 days for Bacillus spp. Although decimal inoculum dilutions were used, proportions between counts based on different conditions ranged from 6.3 to 10.0 times for total bacteria and from 2.0 to 7.0 times for sporulating bacteria.

The influence of substrate concentration and growth phase on expression of Butyrivibrio sp. Mz5 endoxylanases

Abstract: Butyrivibria represent a significant proportion of bacterial isolates from different ruminants. The strain Butyrivibrio sp. Mz5 was originally isolated from the rumen of a black and white Frisian cow. It possesses very high xylanolytic activity, at least 1.65 times higher than any of the tested anaerobic bacteria, as shown earlier. The inducibility by substrate, the influence of substrate concentration and growth phase on expression of its endoxylanases were tested in the present work. Xylanase activity of Butyrivibrio sp. Mz5 was found to be inducible, the specific activity of cells grown on xylan being increased 34-fold in comparison with cells grown on soluble carbon sources. The 51 kDa- and 145 kDa-endoxylanases are constitutive. The highest xylanolytic activity was detected after 16 hours growth and at 0.5% xylan concentration in growth medium. The majority of the xylanolytic activity was cell bound. Lower concentrations of xylan promoted the release of xylanolytic enzymes into the medium. The 26.7 kDa endoxylanase showed resistence to lower pH. The data obtained will help to achieve maximal xylanolytic activity for the needs of enzyme purification.

Identification and characterization of an extracellular acid trehalase from the ectomycorrhizal fungus Amanita muscaria.

Abstract: The non-reducing disaccharide trehalose is a major storage compound in most fungi. For a better understanding of carbon metabolism in the ectomycorrhizal-forming basidiomycete Amanita muscaria, trehalase activity was analysed from mycelium growing in liquid culture or on agar plates, and from mycorrhizas. Trehalase activity was found in both the culture medium and in mycelial extracts. The excreted trehalase was purified to apparent homogeneity by anion-exchange chromatography, gel filtration and preparative gel electrophoresis. The identified enzyme belongs to the group of acid trehalases. The apparent molecular mass of the native enzyme was estimated to be 165 kDa by gel filtration and 135 kDa by SDS–PAGE. Isoelectrofocusing indicated an isoelectric point (pI) of approx. 3.7, which is typical for acid trehalases. The enzyme is highly specific for its substrate trehalose, with an apparent Km of 0.38 mM. Validamycin and sucrose act as competitive inhibitors with Ki values of 45 nM and 15 mM, respectively. The activity of acid trehalase excreted into the growth medium is independent of the carbon source (glucose or trehalose), revealing that the enzyme is not regulated by its substrate trehalose. Nevertheless, fungal hyphae grown in the absence of an external carbon source showed enhanced enzyme excretion. The biochemical characteristics of the trehalase activity measured in the mycelium are in the same range as those determined for the excreted enzyme. The enzyme is localized in the cell wall and its activity is strongly decreased in ectomycorrhizas.

GABA Increases the Rate of Nitrate Uptake and Utilization in Arabidopsis Roots

Abstract: GABA (4-aminobutyric acid) is a non-protein amino acid widely found in plant tissues that accumulates in response to various environmental stresses including heat, cold, drought, salinity, and anaerobic stress. Although there is limited information on its biological function in plants, some studies suggest that GABA acts as a carbon and/or nitrogen source in microorganisms, and it has been suggested that GABA metabolism under stress is related to excessive glutamate accumulation and/or cytoplasmic pH regulation. We found that exogenously applied GABA had an effect on root growth and development in Arabidopsis thaliana L. seedlings. This effect was modulated between inhibition of root elongation when seedlings were grown on full strength Murashige and Skoog salts (1/1X MS) to stimulation of root elongation when plants were grown on 1/8 strength MS salts (1/8X MS). When the concentration of single ions in MS salts was varied, the primary effect was found to be a direct interaction between GABA and the level of nitrate (NO3) in the growing medium. At NO3 concentrations below 40 mM, root growth was stimulated by the addition of GABA to the growth medium, whereas at concentrations above 40 mM NO3 addition of GABA to the growth medium inhibited root elongation. The uptake of NO; and tissue levels of nitrate were also investigated at high (40 mM NO3) and low (5 mM NO,) with and without GABA in the growing medium. In correlation with the growth results GABA promotes NO3 uptake at low NO3, while GABA inhibits NO3 uptake at high NO3. However, tissue levels of NO3 are increased by GABA at high NO3 while they are decreased by GABA at low NO3. These results can be interpreted to indicate that GABA produced during stress is capable of regulating NO3 uptake and metabolism during stress.

Modification of an algal culture medium for sustained growth of a saxitoxin-producing isolate of Alexandrium minutum

Abstract: In order to study saxitoxin (STX) production bymicro-algae in the laboratory, a defined algal culture medium which supports optimum growth over a longtime-period is a requirement. In the development of such a medium, a number of modifications were made to a standard algal culture medium (GP) and growth of a STX-producing isolate of Alexandrium minutum in the different formulations was assessed by measuring maximum cell densities and mean generation times (MGT). All experiments were carried out under controlled conditions in an aerobic atmosphere with increased CO2. Whilst maximum cell densities in the different modifications were similar, the MGT was significantly shortened by the addition of Tris buffer and the trace metals strontium, selenium and molybdenum. Replacement of natural with artificial seawater and removal of soil extract did not adversely affect algal growth. Five of the six media formulations supported the growth of A. minutumover a 9-month period.

The cellular location of proteolytic enzymes of Bacillus intermedius

Abstract: The activities of proteinases in the culture fluid and cellular fractions ofBacillus intermedius 3–19 grown under various conditions were studied. Thiol-dependent serine proteinase was the prevalent enzyme in the total pool of extracellular proteinases (70%); its catalytically active form was also detected in the cell membrane and, during active enzyme production, in the cell wall. Another enzyme, glutamyl endopeptidase (10% of the total pool), was detected in the cell membrane; it was also found in the cell wall and cytoplasm during active enzyme secretion into the growth medium. The production of these enzymes was maximal on medium containing inorganic phosphate and gelatin and decreased 2-to 4-fold on medium with glucose and lactate. The level of activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of C0Cl2 (2 mM) into the medium caused an essential increase in extracellular glutamyl endopeptidase activity and promoted the release of the membrane-bound enzyme into the culture fluid. Proteolytic activity towards casein was also detected in the cytoplasm. The proteinases localized in the cytoplasm were shown to differ in their properties from those secreted.

Comparison of Effects of Medium Composition and Atmospheric Conditions on Detection of Bilophila wadsworthia β-Lactamase by Cefinase and Cefinase Plus Methods

Abstract: The influence of growth medium and incubation conditions on the detection of Bilophila wadsworthia β-lactamase was tested with Cefinase and Cefinase Plus disks. The tests involved aerobic and anaerobic incubation with conventional disk and quantitative tube assays. The production of β-lactamase was correlated with penicillin G, ampicillin, and ampicillin-sulbactam MICs and inhibition zones on penicillin (2-U) disks. The strains were grown on (i) brucella agar (brucella), (ii) brucella agar supplemented with 1% pyruvate (brucella-pyruvate), and (iii) brucella agar supplemented with 1% taurine (brucella-taurine). With the aerobic disk assay, 100, 100, and 7% of strains were positive after 30 min from growth on brucella-pyruvate, brucella, and brucella-taurine plates, respectively; of strains grown on brucella-taurine, 54% remained negative by the Cefinase assay, and 23% remained negative by the Cefinase Plus assay at 2 h. In quantitative assays, the strains became positive after 30 min from brucella-pyruvate plates and after 1 h from brucella plates. The intensities of the reactions were strongest with brucella-pyruvate plates under anaerobic test conditions. Anaerobic incubation enhanced β-lactamase detection of growth on brucella-taurine: at 3 h, 85% of strains were positive in comparison to 38% with aerobic incubation. All β-lactamase-negative strains were susceptible to penicillin G and ampicillin; all β-lactamase-positive strains were resistant to ampicillin and, with the exception of two strains, penicillin G. In conclusion, β-lactamase production correlated with susceptibility to penicillin G and ampicillin. Brucella agar supplemented with 1% pyruvate was the most reliable medium for testing B. wadsworthia β-lactamase, and anaerobic incubation expedited positive results. Brucella agar supplemented with taurine was unsuitable for B. wadsworthia β-lactamase testing. Cefinase and Cefinase Plus results were in agreement, but Cefinase Plus yielded faster reactions.