Showing papers on "Growth medium published in 2006"

High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium.

Abstract: During the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium. Here, recombinant production and export of B. megaterium ATCC14945 penicillin G amidase (PGA) which is used in the reverse synthesis of β-lactam antibiotics were systematically improved. For this purpose, the PGA leader peptide was replaced by the B. megaterium LipA counterpart. A production strain deficient in the extracellular protease NprM and in xylose utilization to prevent gene inducer deprivation was constructed and employed. A buffered mineral medium containing calcium ions and defined amino acid supplements for optimal PGA production was developed in microscale cultivations and scaled up to a 2 Liter bioreactor. Productivities of up to 40 mg PGA per L growth medium were reached. The combination of genetic and medium optimization led to an overall 7-fold improvement of PGA production and export in B. megaterium. The exclusion of certain amino acids from the minimal medium led for the first time to higher volumetric PGA activities than obtained for complex medium cultivations.

Improved in vitro rooting and hyperhydricity in regenerating tissues of Thapsia garganica L.

Abstract: Stock cultures of Thapsia garganica grown on Murashige and Skoog agar medium (1962) (MS) (0.8% agar [w/v]; pH 5.8) with 0.5 mg l−1 NAA and 1.5 mg l−1 BA were best rooted by subjecting to half strength MS liquid medium with IBA (10 mg l−1) for 3 days prior to transfer to medium without plant growth regulators. A rooting frequency of 60% was noted with seven roots per rooted plant. Rooting was apparent after 10 days. The present study also aimed at reducing the occurrence of hyperhydric plants. The inclusion of 2% polyethylene glycol (w/v) in the growth medium or ventilation of cultures prior to acclimatization resulted in the production of plants true to type, closely resembling wild plants. Those plantlets that had been rooted and exposed to a better vented environment were able to acclimatize readily. Tissue culture propagation is therefore beneficial to the conservation of the medicinally important species, Thapsia garganica.

Rapid identification of target genes for 3-methyl-1-butanol production in Saccharomyces cerevisiae

Abstract: Extracellular conditions determine the taste of fermented foods by affecting metabolite formation by the micro-organisms involved. To identify targets for improvement of metabolite formation in food fermentation processes, automated high-throughput screening and cDNA microarray approaches were applied. Saccharomyces cerevisiae was cultivated in 96-well microtiter plates, and the effects of salt concentration and pH on the growth and synthesis of the fusel alcohol-flavoured substance, 3-methyl-1-butanol, was evaluated. Optimal fermentation conditions for 3-methyl-1-butanol concentration were found at pH 3.0 and 0% NaCl. To identify genes encoding enzymes with major influence on product formation, a genome-wide gene expression analysis was carried out with S. cerevisiae cells grown at pH 3.0 (optimal for 3-methyl-1-butanol formation) and pH 5.0 (yeast cultivated under standard conditions). A subset of 747 genes was significantly induced or repressed when the pH was changed from pH 5.0 to 3.0. Expression of seven genes related to the 3-methyl-1-butanol pathway, i.e. LAT1, PDX1, THI3, ALD4, ILV3, ILV5 and LEU4, strongly changed in response to this switch in pH of the growth medium. In addition, genes involved in NAD metabolism, i.e. BNA2, BNA3, BNA4 and BNA6, or those involved in the TCA cycle and glutamate metabolism, i.e. MEU1, CIT1, CIT2, KDG1 and KDG2, displayed significant changes in expression. The results indicate that this is a rapid and valuable approach for identification of interesting target genes for improvement of yeast strains used in industrial processes.

Spent growth medium of Pantoea agglomerans primes wheat suspension cells for augmented accumulation of hydrogen peroxide and enhanced peroxidase activity upon elicitation

Abstract: Induced disease resistance in plants is based on multiple mechanisms, including cell “priming”, i.e. an enhancement of the capacity to mobilize cellular defense responses upon pathogen attack. Potent inducers of priming are, for example, salicylic acid, synthetic compounds such as a benzothiadiazole, and certain rhizosphere bacteria. While priming is well characterized for a number of dicot plants, only few cases of priming are documented in monocots. Here, we report that the spent growth medium of the Gram negative bacterium Pantoea agglomerans is capable of priming wheat cells (Triticum aestivum L. cv Prelude-Sr5) for elicitor-induced defense responses. Pre-incubation of suspension-cultured wheat cells with growth medium of P. agglomerans led to a strong enhancement of an oxidative burst that has been induced by chitin or chitosan and to an increase in extracellular peroxidase activity. Moreover, exopolysaccharides (EPS) were isolated from the spent growth medium and demonstrated to be sufficient for the induction of H2O2 priming. The EPS-induced priming was shown to be time- and concentration-dependent. We conclude that EPS are the or one of several priming-active component(s) in the spent growth medium of P. agglomerans. The present work is the first report of priming in a monocot plant by a specific component of bacterial origin. A comparison with known chemical inducers of resistance revealed that a benzothiadiazole was able to enhance the oxidative burst similar to the spent growth medium or the EPS of P. agglomerans, while salicylic acid was not.

Phosphate Metabolism in the Cyanobacterium Anabaena doliolum Under Salt Stress

Abstract: In the present study, we have investigated the effects of NaCl concentrations on the growth and phosphate metabolism of an Anabaena doliolum strain isolated from a paddy field, in order to determine the possible effects of salinization. Growth rate, chlorophyll content, and protein content decreased with increasing salt concentration in the growth medium, while carbohydrate concentration increased. Phosphate content and phosphate uptake rate decreased. There was an increase in total alkaline phosphatase activity, with an approximately 7-fold increase in extracellular activity compensating for an approximately 3-fold decrease in cell-bound activity. NaCl effects on protein and chlorophyll concentrations were greater in P-deficient medium, while presence or absence of P in the medium had little effect on cellular carbohydrate concentrations. It is concluded that growth in high salt likely leads to reduced phosphate uptake in A. doliolum.

Dynamics of Arabidopsis thaliana soluble proteome in response to different nutrient culture conditions.

Abstract: In an effort to determine the best extraction procedure compatible with the high-reproducible 2-DE, different methods of soluble protein extraction from Arabidopsis cell culture suspensions grown in Gamborg B5 medium were tested. A reference 2-DE map was established for this soluble extract revealing 1184 spots. The most abundant protein spots were excised, trypsin-digested, and mass spectra obtained via MALDI-TOF and/or LC coupled to ESI-MS. Three hundred and thirty one proteins were identified and their functions were defined based on sequence comparisons and classified in different protein families. In order to analyze the impact of culture medium on the Arabidopsis proteome, we performed the 2-DE map from Arabidopsis cell suspensions cultured in another growth medium Murashige and Skoog (M-S) and 327 major spots were identified. Using PDQuest imaging analysis, significant increases in the amount of several housekeeping enzymes, stress/defense proteins, and heat shock proteins were found in M-S medium. Modified expression of certain proteins and detection of new isoforms involved in nitrate assimilation, nitrogen, and sulfur metabolism were also observed in the M-S medium. This study provides the first 2-DE maps of the soluble proteome of Arabidopsis cell suspensions. The comparative analysis of the Arabidopsis proteome in respect to different nutrient supplies shows that the culture medium may significantly influence the expression pattern of major soluble proteins in Arabidopsis cells. This work also constitutes an important step for further proteomic analysis concerning cell responses to abiotic or biotic stresses.

Modification of a virulence-associated phenotype after growth of Listeria monocytogenes on food.

Abstract: Aim: To assess the effect of different foods, which have been implicated or not in cases of listeriosis, on the in vitro virulence-associated phenotype level of different Listeria monocytogenes strains. Methods and Results: The virulence-associated phenotype level of L. monocytogenes was studied with the in vitro cell test based on a plaque-forming assay with a human adenocarcinoma cell line (HT-29) monolayer. Three strains of L. monocytogenes were grown in preparations (homogenate, 1-μm filtrate or 0·2-μm filtrate) of different food extracts [‘rillettes’ (potted minced pork), milk, raw salmon and cold-smoked salmon] or in a control medium, brain heart infusion (BHI). The bacterial suspensions grown in food extracts or in BHI at 37°C were diluted with their growth medium (food extract or BHI) or with minimum essential medium before seeding on confluent HT-29 cell monolayers. Filtration of food extracts had no significant effect on the plaque numbers formed by the bacteria. A significant decrease in the plaque numbers was noted for the three strains when they grew in the rillettes extracts, compared with the other food extracts and BHI. The levels of in vitro virulence-associated phenotype of the strains after growth in the rillettes extract were similar to or lower than that of the hypovirulent internal reference strain L. monocytogenes 442. After growth in milk and cold-smoked salmon, the impact on virulence-associated phenotype depended on the strain. In contrast, plaque-forming assay indicated increased virulence-associated phenotype when the strains were switched from a nutrient-rich medium (food extract or BHI) to a minimum essential medium. Conclusions: In vitro virulence-associated phenotype level of the studied strains grown in BHI or cold-smoked salmon was the same as the control virulent strain EGD. In contrast, the nutrients present in rillettes may therefore substantially reduce the number of plaques but not the growth of L. monocytogenes. The utilization of minimum essential medium as diluent attenuates changes the effect of the food extract on virulence-associated phenotype in vitro. Significance and Impact of the Study: In the experimental design of this study, we showed that the nature of the food could affect the in vitro virulence-associated phenotype level of L. monocytogenes.

Selenium enrichment and anti-oxidant status in baker's yeast, saccharomyces cerevisiae at different sodium selenite concentrations.

Abstract: The use of selenized yeast as enriched selenium supplements in human nutrition has become a topic of increasing interest over the last decade. The present study was designed with the aim to achieve a balance between selenium (Se) incorporation and optimal growth of yeast cells along with effect of Se enrichment on antioxidant defense status of yeast cells. Since oxidative stress has been known to play a role in the life span of all types of cells, so in the present studies anti-oxidant defense status was evaluated in the Se- enriched baker’s yeast cell culture model. Upon Se supplementation as sodium selenite at various concentrations in the growth medium, a continuous increase in glutathione peroxidase (GSH-Px) activity and Se content was observed. In case of reduced glutathione (GSH) decreasing trend were observed with increasing Se concentrations. An increasing trend in total glutathione as well as glutathione-s-transferase activity was observed at increasing Se concentrations. Thus, Se supplementation significantly enhanced GSH-Px levels along with alterations in other anti-oxidant enzymes, suggesting the role of Se in the enzyme defense system of yeast against oxidative damage. Further, as Se exerts growth inhibitory effect on cells, the growth inhibition study was carried out and decrease in biomass was observed with increasing concentrations of Se. Due to nutritional benefits, Se-enriched yeast may be considered a safe source of Se supplementation. (Nutr Hosp. 2006;21:704-708)

Growth of an Escherichia coli mutant deficient in respiration

Abstract: An Escherichia coli mutant deficient in genes for heme biosynthesis grew in medium of initial pH 8 containing 1% tryptone and glucose under aerobic growth conditions, and its doubling time was approximately 60 min at 37°C. The growth rate was not increased under O2-limiting conditions. When the mutant was grown in medium of initial pH 6, growth stopped at the middle of the exponential growth phase. This could be overcome and the growth yield increased by the addition of 20 mM lysine to the growth medium. Lysine did not prevent the decrease in the medium pH as growth proceeded, making it unlikely that lysine decarboxylation stimulates growth by the alkalinization of the medium. These results indicate that respiration is not obligatory for growth under aerobic conditions, but growth without respiration at low pH requires a large amount of lysine.

Glycerophosphate as a phosphorus source in a defined medium for Pichia pastoris fermentation

Abstract: Pichia pastoris has emerged as a commercially important yeast for the production of a vast majority of recombinant therapeutic proteins and vaccines The organism can be grown to very high cell densities using a defined basal salts media (BSM) However, BSM contains bi-cation or tri-cation phosphate, which precipitates out of the medium at pH above 55, although the optimal fermentation pH of most recombinant protein fermentation varies between 55 and 70 In this article, the application of glycerophosphates was investigated as a substitute phosphate source in an effort to eliminate precipitation The solubility of BSM containing sodium or potassium glycerophosphates was examined before and after autoclaving at various pHs Sodium glycerophosphate was found stable at autoclave temperature but formed complexes with coexisting magnesium and calcium ions that were insoluble above pH 70 Medium where sodium glycerophosphate was autoclaved separately and then added to the growth medium did not produce any precipitate up to pH 105 The performance of P pastoris fermentations expressing α-galactosidase and ovine interferon-τ using a glycerolphosphate-based medium was found to be comparable to a conventional BSM The results from this work demonstrate that sodium glycerophosphate can be assimilated by the P pastoris strains and can be employed as a reliable phosphorus source for both cell growth and recombinant protein production

Ecto-glycanases and metabolic stability of the capsule in Cryptococcus neoformans.

Abstract: We have identified a number of ecto-glycanases (glycosylhydrolases) associated with the capsule and/or the cell wall of Cryptococcus neoformans. The enzyme activities detected included α -mannosidase, α -, and β -glucosidase, α -, and β -galactosidase, β -xylosidase, β -glucuronidase, and endo-β -1,3-glucanase. Small portions of the enzymes were also secreted into the growth medium. Cell-wall associated endo-β -1,3-glucanases exhibited highest activity in the acidic range between pH 2.5 and 5.0. The products of laminarin hydrolysis by the enzymes located on the cell surface were glucose and β -1,3-linked glucooligosaccharides. The same products were released from isolated cell walls incubated in the buffer. Endo-β -1,3-glucanase activity extracted from the cell surfaces by mild sonication consisted of six isoforms separable by isoelectric focusing. In spite of the presence of the whole array of glycanase activities on the cell surfaces, capsular polysaccharides released from C. neoformans cells into the growth medium were practically metabolically stable. From the defined polysaccharides tested, only laminarin (β -1,3-glucan) and to some extent also mixed-linkage β -1,3/β -1,4-glucan and/or 4-O-methyl-D-glucurono-D-xylan were able to support the yeast growth. The activities of majority of identified ecto-glycanases were low when the yeast was grown on glucose but were considerably elevated when the cells were grown on glycerol indicating that their synthesis is regulated by catabolite repression. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Effect of L-serine on the biosynthesis of aromatic amino acids in Escherichia coli.

Abstract: It is well known that some amino acids inhibit bacterial growth. L-Serine is known to inhibit the growth of Escherichia coli by inhibition of homoserine dehydrogenase (EC 1.1.1.3). It has been reported that this L-serine inhibition may be prevented by the addition of L-isoleucine or L-threonine to the medium. In our study, however, recovery of the growth inhibition of Escherichia coli by L-serine occurred in the presence of several amino acids, especially L-phenylalanine. In an attempt to further elucidate this inhibition mechanism, different intermediates of aromatic amino acid biosynthesis were added to the growth medium. Recovery from the inhibition did not occur in the presence of prephenate but did occur when phenylpyruvate was added to the medium. The specific activity of prephenate dehydratase decreased in cells grown in the presence of L-serine. However. L-serine did not inhibit in vitro prephenate dehydratase activity, and the expression of pheA, which encodes the prephenate dehydratase, was not depressed by L-serine. We suggest that L-serine acts via another inhibition mechanism. Although this inhibition mechanism has not been fully elucidated, our results suggest that the addition of L-serine to the growth medium inhibits prephenate dehydratase synthesis and thus affects L-phenylalanine biosynthesis.

The growth of Saccharomyces cerevisiae yeast in cadmium enriched media

Abstract: The effect of Cd ions on the growth of Saccharomyces cerevisiae yeasts grown in a natural and a synthetic medium was studied. Two strains, B1 i G1, were used. Both strains displayed high sensitivity to increased cadmium salt contents, particularly those grown in a synthetic medium. High concentrations of cadmium (> 50 μM) practically stopped the growth of the studied strains. The negative effect of cadmium ions was partly altered by adding zinc or calcium salts to the synthetic YM medium.

SYNTHESIS OF SIDEROPHORES BY SOIL BACTERIA OF THE GENUS Pseudomonas UNDER VARIOUS CULTURE CONDITIONS

Abstract: The ability of six strains belonging to the genus Pseudomonas isolated from the rhizosphere of wheat to produce pyoverdin was examined. The studied strains demonstrated a varied level of production of the si derophore, depending on the culture conditions. The highest level of pyoverdin was dete rmined after 72 hours of growth at 20- 25 °C in iron-free medium supplemented with succinate. The synthesis of pyoverdin by all the strains studied was strongly repressed by the a ddition of iron ions (III) to the growth medium. Calcium, cadmium and magnesium ions stimulated the synthesis of the siderophore examined, whereas zinc and lead ions partially decreased its level. Enrichment of the growth medium in cobalt ions completely inhibited the synthesis of siderophores as well as growth of the bacteria.

Effect of organic N source on bacterial growth, lipo-chitooligosaccharide production, and early soybean nodulation by Bradyrhizobium japonicum.

Abstract: Production of Bradyrhizobium japonicum inoculants is problematic because high inoculation rates are necessary but expensive, while production of rhizobial Nod factors (lipo-chitooligosaccharides (LCOs)), key signal molecules in the establishment of legume-rhizobia symbioses, may be inhibited at high culture cell densities. We conducted experiments to determine the effects of growth medium N source on B. japonicum growth, LCO production, and early nodulation of soybean. We found that 1.57 mmol ammonium nitrate x L(-1) resulted in less rhizobial growth and rhizobial capacity to produce LCOs (on a per cell basis) than did 0.4 g yeast extract x L(-1), which contained the same amount of N as the ammonium nitrate. Increasing yeast extract to 0.8 g x L(-1) increased rhizobial growth and LCO production on a volume basis (per litre of culture) and did not affect cell capacity to produce LCOs; however, at 1.4 g yeast extract x L(-1) per cell, production was reduced. A mixture of 0.8 g yeast extract x L(-1) and 1.6 g casein hydrolysate x L(-1) resulted in the greatest bacterial growth and LCO production on a volume basis but reduced LCO production per cell. Changes in organic N level and source increased production of some of the measured LCOs more than others. LCO production was positively correlated with cell density when expressed on a volume basis; however, it was negatively correlated on a per cell basis. We conclude that although quorum sensing affected Nod factor production, increased levels of organic N, and specific compositions of organic N, increased LCO production on a volume basis. Greenhouse inoculation experiments showed that the medium did not modify nodule number and N fixation in soybean, suggesting that it could have utility in inoculant production.

Effect of monocrotophos and butachlor on N-fixing cyanobacteria and associated biochemical activities

Abstract: Both the pesticides caused stimulation of growth and nitrogen fixation at lower concentrations and partial/complete inhibition at higher concentrations. Anabaena sp. was found to be the most susceptible and Westiellopsis prolifica as the most tolerant strain to the pesticides. The glutamine synthetase activity and release of free amino acids in the culture medium of cyanobacteria were also reduced in presence of pesticides. It is suggested that the NH4+ that remained underutilized due to reduced activity of glutamine synthetase might be released in the growth medium, which might also be available to the paddy crop under the conditions of the treatment.

Expression of 5-aminolevulinic acid synthase in recombinant Escherichia coli

Abstract: The Rhodobacter sphaeroides hemA gene codes for 5-aminolevulinic acid synthase (EC.2.3.1.3), catalysing the pyridoxal phosphate-dependent condensation of succinyl coenzyme A and glycine to give 5-aminolevulinic acid. The gene was transformed into Escherichia coli K12 using the pALA vector systems. Effects of host strains, vector plasmids, growth substrates and precursors on the expression and activity of 5-aminolevulinic acid synthase were studied. The E. coli host strain had an enormous effect on 5-aminolevulinic acid synthase activity and production of 5-aminolevulinic acid, with E. coli DH1 being best suited. RT-PCR of hemA mRNA indicated that the transcription of the hemA gene in the recombinant strain appeared to be higher than that of the wild-type strain. 5-Aminolevulinic acid synthase activity was maximal when hemA had the same transcription direction as the lac promoter. Distance between lac promoter and hemA affected the expression of 5-aminolevulinic acid synthase on different growth substrates. 5-Aminolevulinic acid synthase activities were also dependent on the carbon sources and precursors. l-Malate gave the highest activity of 5-aminolevulinic acid synthase, while lactose as a carbon source resulted in a repression of 5-aminolevulinic acid synthase. Production of 5-aminolevulinic acid by recombinants were limited by the availability of glycine, and repeated addition of glycine (20 mM) to the growth medium increased production of 5-aminolevulinic acid to 33.8 mM.

Analysis of trehalose-6-phosphate control over carbon allocation and growth in plants

Abstract: Trehalose is the non-reducing alpha-alpha-1, 1-linked glucose disaccharide. The biosynthesic precursor of trehalose, trehalose-6-phosphate (T6P), is essential for plant development, growth, carbon utilization and alters photosynthetic capacity but its mode of action is not underestood. This thesis describes a genetic approach to dissecting the pleiotropic effects of T6P in the model plant Arabidopsis thaliana. Trehalose supplied to the growth medium of Arabidopsis seedlings causes T6P accumulation that inhibits growth and allocation of carbon to the root and shoot apices. 19 trehalose resistant (trr) mutant that are resistant to 100 mM trehalose are identified from the Leclere and Bartle mutant collection. These mutants contain a T-DNA expressing randomly cloned cDNAs. The trr phenotype segregated as a dominant trait in 13 of the 19 trr mutants with over-expression of PS-I, GR-RBP2, TRR14 and repression of LHCB1 causing trehalose resistance. Mutatant analysis confirms that T6P control over starch and growth uses two separate pathways . TRR14 suppresses both the massive accumulation of starch in the cotyledons and the growth inhiition due to T6P accumulation on 100 mM trehalose. A model of interactions surrounding T6P is thus presented with TRR14 affecting immediate targets of T6P that are in common to both starch and growth control and with GR-RBP2 and LHCB1 affecting remote targets of T6P in pathway controlling growth.

Viable Hybrids between Adherent Cells: Generation, Yield Improvement, and Analysis

Abstract: Publisher Summary This chapter describes how somatic cell hybridization has not only shown that tissue-specific genes are regulated by transacting factors, but also has provided strong evidence for the existence of tumor suppressor gene frequency. A spontaneous fusion of cells in culture occurs at a very low frequency. The best known of such methods is the application of hypoxanthine + aminopterin + thymidine (HAT) selection for the fusion of cells deficient in hypoxanthine guanosine phosphoryl transferase with cells deficient in thymidine kinase, but different combinations of selectable markers can be used. Inoculate the mixture of parental cells to be fused into several 50-ram tissue culture dishes containing complete growth medium. The total number of cells has to be adjusted to occupy the entire dish surface, such that the fusion will be done on cells that are in close contact. Resuspend thoroughly the cell pellet in a complete growth medium and count the cell number in a hemacytometer.

Method of indefinite culture of hepatitis C virus

Abstract: The invention discloses a method of maintaining hepatitis C virus (HCV) growing indefinitely in cell culture. The method includes providing a culture of cells susceptible to infection by HCV; introducing to the cell culture an inoculum containing an infective dose of HCV; contacting the inoculated cell culture with a growth medium containing an excess of uridine and cytidine; and changing spent growth medium with fresh growth medium containing the excess of uridine and cytidine on a predetermined schedule.

Spent growth medium of Pantoea aggtomerans primes wheat suspension cells for augmented accumulation of hydrogen peroxide and enhanced peroxidase activity upon elicitation

Abstract: Induced disease resistance in plants is based on multiple mechanisms, including cell "priming", i.e. an enhancement of the capacity to mobilize cellular defense responses upon pathogen attack. Potent inducers of priming are, for example, salicylic acid, synthetic com pounds such as a benzothiadiazole, and certain rhizo sphere bacteria. While priming is well characterized for a number of dicot plants, only few cases of priming are documented in monocots. Here, we report that the spent growth medium of the Gram negative bacterium Pan toea agglomerans is capable of priming wheat cells (Triti cum aestivum L. cv Prelude-Sr5) for elicitor-induced defense responses. Pre-incubation of suspension-cultured wheat cells with growth medium of P. agglomerans led to a strong enhancement of an oxidative burst that has been induced by chitin or chitosan and to an increase in extra cellular peroxidase activity. Moreover, exopolysaccha rides (EPS) were isolated from the spent growth medium and demonstrated to be sufficient for the induction of H202 priming. The EPS-induced priming was shown to be time- and concentration-dependent. We conclude that EPS are the or one of several priming-active compo nents) in the spent growth medium of P. agglomerans. The present work is the first report of priming in a monocot plant by a specific component of bacterial ori gin. A comparison with known chemical inducers of resistance revealed that a benzothiadiazole was able to enhance the oxidative burst similar to the spent growth medium or the EPS of P. agglomerans, while salicylic acid was not.