Showing papers on "Growth medium published in 2003"

Polyamines protect Escherichia coli cells from the toxic effect of oxygen

Abstract: Wild-type Escherichia coli cells grow normally in 95% O2/5% CO2. In contrast, cells that cannot make polyamines because of mutations in the biosynthetic pathway are rapidly killed by incubation in 95% O2/5% CO2. Addition of polyamines prevents the toxic effect of oxygen, permitting cell survival and optimal growth. Oxygen toxicity can also be prevented if the growth medium contains an amino acid mixture or if the polyamine-deficient cells contain a manganese-superoxide dismutase (Mn-SOD) plasmid. Partial protection is afforded by the addition of 0.4 M sucrose or 0.4 M sorbitol to the growth medium. We also report that concentrations of H2O2 that are nontoxic to wild-type cells or to mutant cells pretreated with polyamines kill polyamine-deficient cells. These results show that polyamines are important in protecting cells from the toxic effects of oxygen.

The Gap1 general amino acid permease acts as an amino acid sensor for activation of protein kinase A targets in the yeast Saccharomyces cerevisiae.

Abstract: Addition of a nitrogen source to yeast (Saccharomyces cerevisiae) cells starved for nitrogen on a glucose-containing medium triggers activation of protein kinase A (PKA) targets through a pathway that requires for sustained activation both a fermentable carbon source and a complete growth medium (fermentable growth medium induced or FGM pathway). Trehalase is activated, trehalose and glycogen content as well as heat resistance drop rapidly, STRE-controlled genes are repressed, and ribosomal protein genes are induced. We show that the rapid effect of amino acids on these targets specifically requires the general amino acid permease Gap1. In the gap1Delta strain, transport of high concentrations of l-citrulline occurs at a high rate but without activation of trehalase. Metabolism of the amino acids is not required. Point mutants in Gap1 with reduced or deficient transport also showed reduced or deficient signalling. However, two mutations, S391A and S397A, were identified with a differential effect on transport and signalling for l-glutamate and l-citrulline. Specific truncations of the C-terminus of Gap1 (e.g. last 14 or 26 amino acids) did not reduce transport activity but caused the same phenotype as in strains with constitutively high PKA activity also during growth with ammonium as sole nitrogen source. The overactive PKA phenotype was abolished by mutations in the Tpk1 or Tpk2 catalytic subunits. We conclude that Gap1 acts as an amino acid sensor for rapid activation of the FGM signalling pathway which controls the PKA targets, that transport through Gap1 is connected to signalling and that specific truncations of the C-terminus result in permanently activating Gap1 alleles.

Employment of stressful conditions during culture production to enhance subsequent cold- and acid-tolerance of bifidobacteria

Abstract: Aims: This study examined whether exposure of early stationary phase Bifidobacterium longum and B. lactis cells to various combinations of reduced temperature, reduced pH and starvation would enhance the cells’ subsequent cold- and/or acid-tolerance. Methods and Results: Survival of B. longum in growth medium at 6°C significantly (P < 0·05) increased as a result of starving cells for 30 or 60 min without any simultaneous decrease in temperature or pH. Acid-tolerance of B. lactis (at pH 3·5 in synthetic gastric fluid) increased significantly when the growth medium pH was decreased from 6·0 to 5·2 and cells experienced 30 or 60 min of starvation. Enhanced B. lactis acid-tolerance persisted through 8–11 weeks of −80°C storage in the pH 5·2 growth medium. Upon addition to milk during yogurt manufacture, these cells initially had enhanced acid-tolerance relative to untreated cells but untreated cells became equally acid-tolerant during the first 2·5 h of yogurt manufacture. Conclusions: The cold- and acid-tolerance of bifidobacteria vary widely, but may be significantly increased by application of sub-lethal stress to early stationary phase cells during culture production. Significance and Impact of the Study: The enhancement of B. lactis acid-tolerance observed in this study may be of potential importance in the production of effective ready-to-consume probiotic dietary supplements.

De novo synthesis of bacterial glycogen: Agrobacterium tumefaciens glycogen synthase is involved in glucan initiation and elongation

Abstract: Evidence is presented indicating that initiation of glycogen synthesis in Agrobacterium tumefaciens does not require the presence of α(1,4)-linked glucans. Crude cell extracts incubated with ADP-glucose (Glc) were able to form α(1,4)-linked glucans despite the fact that cells used for extract preparation displayed a genotype that prevented synthesis of Glc-containing sugar nucleotides and thus preformation of α(1,4)-linked glucans and that the defined growth medium used contained glycerol as carbon source. A. tumefaciens glycogen synthase (GS) purified to homogeneity from the above-mentioned cells was able to build its own primer by transferring Glc residues from ADP-Glc to an amino acid(s) in the same protein. Primed GS then became the substrate for further GS-catalyzed glucan elongation. It was concluded that, contrary to what happens in mammalian and yeast cells in which two different proteins are required for linear α(1,4)-linked glucan formation (glycogenin for initiation and GS for further elongation), in A. tumefaciens and probably in all other bacteria, the same protein is involved in both glycogen initiation and elongation.

Rhizosecretion of a monoclonal antibody protein complex from transgenic tobacco roots.

Abstract: The secretion of a functional, full-length monoclonal antibody complex from transgenic Nicotiana tabacum roots has been demonstrated. Initially, seeds were germinated on nitrocellulose membranes and antibody secretion detected from the developing roots. Plants were then established in hydroponic culture and secretion into the growth medium measured over 25 days. Western blotting indicated that full-length antibody was present in the medium along with other fragments. Secreted antibody was shown to be functional by binding to antigen in ELISA studies. In contrast, no antibody could be detected from transgenic Nicotiana in which the same antibody was expressed as a membrane protein in the plasmalemma. These results indicate that antibody accumulation in the growth medium is genuinely caused by rhizosecretion and not cell damage. Addition of gelatin to plant growth medium markedly increased levels of antibody accumulation. The mean antibody yield per plant was calculated to be 11.7 μg per gram root dry weight per day. Rhizosecretion may be a viable alternative to agricultural production or cell culture for the generation of monoclonal antibodies in transgenic plants. It may also give rise to novel applications for antibodies expressed in plants such as removal or neutralisation of environmental pollutants and attenuation of pathogens which infect the plant via the rhizosphere.

The Saccharomyces cerevisiae alcohol acetyl transferase gene ATF1 is a target of the cAMP/PKA and FGM nutrient-signalling pathways.

Abstract: The ATF1-encoded Saccharomyces cerevisiae yeast alcohol acetyl transferase I is responsible for the formation of several different volatile acetate esters during fermentations. A number of these volatile esters, e.g. ethyl acetate and isoamyl acetate, are amongst the most important aroma compounds in fermented beverages such as beer and wine. Manipulation of the expression levels of ATF1 in brewing yeast strains has a significant effect on the ester profile of beer. Northern blot analysis of ATF1 and its closely related homologue, Lg-ATF1, showed that these genes were rapidly induced by the addition of glucose to anaerobically grown carbon-starved cells. This induction was abolished in a protein kinase A (PKA)-attenuated strain, while a PKA-overactive strain showed stronger ATF1 expression, indicating that the Ras/cAMP/PKA signalling pathway is involved in this glucose induction. Furthermore, nitrogen was needed in the growth medium in order to maintain ATF1 expression. Long-term activation of ATF1 could also be obtained by the addition of the non-metabolisable amino acid homologue β- L -alanine, showing that the effect of the nitrogen source did not depend on its metabolism. In addition to nutrient regulation, ATF1 and Lg-ATF1 expression levels were also affected by heat and ethanol stress. These findings help in the understanding of the effect of medium composition on volatile ester synthesis in industrial fermentations. In addition, the complex regulation provides new insights into the physiological role of Atf1p in yeast.

Effect of bioaccumulation of cadmium on biomass productivity, essential trace elements, chlorophyll biosynthesis, and macromolecules of wheat seedlings

Abstract: Soil contamination with heavy metals has become a worldwide problem, leading to losses in agricultural yield and hazardous human health effects as they enter the food chain. The present investigation was undertaken to examine the influence of cadmium (Cd2+) on the wheat (Triticum aestivum L.) plant. Cd2+ accumulation and distribution in 3-wk-old seedlings grown in nutrient medium containing varying concentrations of Cd2+ (control, 0.25, 0.50, 1.0, 2.5, and 5.0 mg/L) was monitored. The effect of varying Cd2+ concentrations up to 21 d on biomass productivity, plant growth, photosynthetic pigments, protein, amino acids, starch, soluble sugars, and essential nutrients uptake was studied in detail to explore the level up to which the plant can withstand the stress of heavy metal. Plants treated with 0.5, 1.0, 2.5, and 5.0 mg/L Cd2+ showed symptoms of heavy-metal toxicity as observed by various morphological parameters which were recorded with the growth of plants. The root, shoot-leaf length and the root, shoot-leaf biomass progressively decreased with increasing Cd2+ concentration in the nutrient medium. Cd2+ uptake and accumulation was found to be maximum during the initial growth period. Cd2+ also interfered with the nutrients uptake, especially calcium (Ca2+), magnesium (Mg2+), potassium (K+), iron (Fe2+), zinc (Zn2+), and manganese (Mn2+) from the growth medium. Growth reduction and altered levels of major biochemical constituents such as chlorophyll, protein, free amino acids, starch, and soluble sugars that play a major role in plant metabolism were observed in response to varying concentrations of Cd2+ in the nutrient medium. In the present study, the effects of Cd2+ on growth, biomass productivity, mineral nutrients, chlorophyll biosynthesis, protein, free amino acid, starch, and soluble sugars in wheat plants was estimated to establish an overall picture of the Cd2+ toxicity at structural and functional levels.

Laboratory assessment of the allelopathic effects of fine leaf fescues.

Abstract: Laboratory screening studies were conducted to evaluate the allelopathic potential of fine leaf fescues. Of the seven accessions selected from prior field evaluations for weed-suppressive ability, all inhibited root growth of large crabgrass and curly cress in laboratory assays. Grown in agar as a growth medium and in the presence of living fescue seedlings for 14 or 21 days, test species were sensitive depending on the fescue cultivars. Growth inhibition increased when fescue was grown for increasing periods of time in agar. Seedling fescues produced significant quantities of bioactive root exudates, which were released into the agar medium. Bioactive root exudates were extracted from living fescue roots by using methylene chloride. Shoot tissue was extracted in water and the aqueous extract was partitioned against hexane, ethyl acetate, and methylene chloride. Extracts were tested for inhibitory activity on seedling growth as measured by inhibition of curly cress germination and radicle elongation. Root exudates were more toxic (70% inhibition) than shoot extracts (up 40% inhibition), when formulated at 0.25 mg/ml concentration. Light microscopy and transmission electron microscopy were utilized in an attempt to identify the cellular location of production of secondary products contained in bioactive root exudates. Ultrastructural analysis indicated that the exudate is produced in actively dividing tips of fibrous root cells. The mode of release of these exudates into the environment remains unknown.

Antifungal activity of lactic acid bacteria

Abstract: Enrichment culture techniques produced more than 1200 isolates of lactic acid bacteria (LAB) that were screened for antifungal activity against the indicator mould Aspergillus fumigatus. Approximately 10% of the LAB were active, but only 4% had medium or strong activity in an agar plate assay. The majority of isolates with strong antifungal activity were Lactobacillus coryniformis strains, but Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified. Some of the isolates lost activity during storage but most maintained their fungal inhibitory effect. Large variations in sensitivity were observed between different moulds and yeasts. Antifungal cyclic dipeptides and phenyllactic acid were detected in culture filtrates from several of the LAB isolates. Lactobacillus coryniformis subsp. coryniformis strain Si3 produced an antifungal compound that lost activity when treated with proteinases. The antifungal peptide(s) was heat stable, with a size of approx. 3kDa and had maximum activity at pH 3.0 to 4.5. Addition of ethanol to the growth medium of strain Si3 prevented a decline in observed antifungal activity during the stationary phase. Glycerol addition to agar plates with L. coryniformis strains, overlaid with soft agar suspensions of yeast cells or fungal spores, strongly enhanced the antifungal effect. This was particularly true with spoilage moulds and yeasts, e.g. Penicillium roqueforti and Pichia anomala, not normally affected by the antifungal metabolites of L. coryniformis. Chemical and genetic data suggested that reuterin (3-hydroxypropionaldehyde) was the cause of this effect. The glycerol/diol dehydratase operon of L. coryniformis was partially elucidated and found to be similar to that Lactobacillus collinoides. Bioassay-guided isolation of new metabolites from LAB revealed that Lactobacillus plantarum MiLAB 14 produces hydroxylated fatty acids with strong antifungal effects. 3-Hydroxydecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid and 3-hydroxy-5-cis-dodecenoic acid were characterized from the supernatant of MiLAB 14. The hydroxy fatty acids had total inhibitory effects in the range 10 to >100 µg ml-1 against several moulds and yeasts.

Effects of Addition of Sucrose and Salt, and of Starvation upon Thermotolerance and Survival During Storage of Freeze-dried Lactobacillus delbrueckii ssp bulgaricus

Abstract: Increased survival of freeze-dried cells of Lactobacillus bulgaricus was observed when the drying medium was supplemented with sucrose; however, the magnitude of such protection was dependent on the growth medium used. Supplementing the growth medium with NaCl markedly increased survival of dried cells, and only a small effect was exerted by the composition of the drying medium or prior to starvation of cells. The D57 values of Lactobacillus bulgaricus cells grown in MRS were about half of those of cells grown in MRS supplemented with sucrose, with sucrose plus NaCl, or with NaCl.

Beijerinckia derxii releases plant growth regulators and amino acids in synthetic media independent of nitrogenase activity.

Abstract: D . S . T H U L E R , E . I . S . F L O H , W . H A N D R O A N D H . R . B A R B O S A . 2003. Aims: This study aims at evaluating the ability of Beijerinckia derxii, a free-living nitrogen (N)-fixing bacterium frequently isolated from tropical soils, to release certain plant growth regulators [indoleacetic acid (IAA), ethylene, polyamines] and amino acids into the growth medium. Methods and Results: The production of those substances was compared using both cultures in which nitrogenase was active (N-free medium) and cultures in which nitrogenase was repressed (combined-N cultures). Those cultures were grown under agitation and in absence of agitation. Total IAA production was higher in agitated, N-free cultures but specific production was greater in combined-N cultures under agitation. Putrescine and spermidine were detected under all conditions tested. Ethylene was produced in both N-free and combined-N cultures. A greatest diversity of amino acids was released in N-free cultures. Conclusions: There was no inhibition of the production of the analysed substances under conditions where nitrogenase was inactive. Significance and Impact of the Study: Beijerinckia derxii is potentially a producer of plant-active substances; its presence in the natural environment suggests that this bacterium may contribute to the development of other living organisms.

Influence of Culture Conditions on the Fatty Acid Profiles of Laboratory-Adapted and Freshly Isolated Strains of Helicobacter pylori

Abstract: Cellular fatty acids of Helicobacter pylori have taxonomic, physiological, and pathogenic implications. However, little is known about the fatty acid composition under various culture conditions. H. pylori is usually grown on blood-supplemented complex media, and the fatty acids in the blood may affect the fatty acids in the cells. In addition, frequently subcultivated laboratory-adapted strains may have properties different from those of fresh clinical isolates, which are culturable only for a limited number of passages. Therefore, the cellular fatty acid profiles of laboratory-adapted strains (LAS) and freshly isolated strains (FIS) were compared after growth on agar that was fatty acid free and growth on blood agar that contained fatty acids. LAS ATCC 43504, 51932, and 700392 and the FIS IMMi 88, 89, and 92, each with <10 subcultures, were cultured in parallel on a fatty acid-free agar (ISAF) and on 5% sheep blood agar (SBA), which contained oleic acid (18:1 9c), hexadecanoic acid (16:0), and octadecanoic acid (18:0). ISAF-grown cultures showed no 18:1 9c and no appreciable differences between the profiles of FIS and LAS. After culture on SBA, the strains showed 18:1 9c and increased 16:0 and 18:0 content combined with decreased tetradecanoic acid (14:0) content compared to ISAF-grown cells. The changes in the fatty acid profiles were much more pronounced in FIS than in LAS. LAS are obviously characterized by a lower uptake of the fatty acids from the growth medium than FIS. Furthermore, it could be shown that this LAS behavior is most likely a primary strain attribute that is favored under laboratory conditions. The pronounced uptake of fatty acids by strains with FIS behavior may be associated with the expression of virulence properties.

Toxicity of parathion-methyl to cells, akinetes and heterocysts of the cyanobacterium Cylindrospermum, sp. and the probit analysis of toxicity

Abstract: The toxicity of a commercial formulation of the insecticide parathion-methyl to the N 2 -fixing filamentous cyanobacterium (blue-green alga) Cylindrospermum sp. was studied. A concentration of parathion-methyl of 0.5 ppm caused growth increase in liquid growth media. The minimum inhibitory concentration of parathion-methyl for both types (N 2 -fixing and nitrate supplemented) of liquid and solid media was 1.0 ppm. LC 50 values were: 4.4 ppm (liquid, N 2 -fixing), 5.5 ppm (liquid, nitrate supplemented), 3.3 ppm (agar, N 2 -fixing) and 4.0 ppm (agar, nitrate supplemented). LC 100 values for N 2 -fixing liquid and both types of agar media were 10.0 ppm, while for the liquid nitrate supplemented medium the LC 100 was 12.0 ppm. Both akinete (spore) formation and germination were inhibited below the highest permissive concentration of 8.0 ppm, with the insecticide incorporated in the agar media. In soil, the LC 50 and LC 100 values for parathion-methyl were 13.6 and 30 ppm, respectively. Both the dehydrogenase activity of heterocysts (monitored by 2,3,5-triphenyl tetrazolium chloride reduction) and the nitrogen concentration of cultures (estimated by the micro-Kjeldahl method) were affected by the insecticide, but the latter (N 2 -fixation) was more sensitive. The Kruskal-Wallis H test on the numbers of vegetative cells in the filaments revealed that the insecticide significantly affected the division of vegetative cells. The cyanobacterium could detoxify the growth medium containing high levels (30 and 40 ppm) of the insecticide in short-term exposures at the expense of cell viability.

Effect of sugars on the association between cowpea vicilin (7S storage proteins) and fungal cells.

Abstract: Vicilins (7S storage proteins) found in various legume seeds have been previously shown to interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. In the present work vicilins from cowpea (Vigna unguiculata) seeds were added to the growth medium of Saccharomyces cerevisiae cells and Fusarium oxysporum conidia. Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin were used to identify differences in the binding of the vicilins to the surface of cells of S. cerevisiae and F. oxysporum treated with this protein. After the growth period, the material in suspension (yeast cells) was centrifuged and the final pellet was also treated with different sugar (glucose, sucrose, glucosamine, N-acetyl-glucosamine) concentrations and 0.1 M HCl for extraction of vicilins associated to chitinous structures present in yeast cells. Our results showed that vicilin sub-units were present in the different sugar extracts of yeast cells pretreated with the vicilins and these proteins were eluted by 0.5 M solutions of sugars in the following order of efficiency of elution: N-acetyl-glucosamine, sucrose/glucose and glucosamine.

Production of a-amylase by thermotolerant Bacillus subtilis in the presence of some carbon, nitrogen containing compounds and surfactants

Abstract: The effect of some carbon, nitrogen sources and surfactants on the α-amylase production of Bacillus subtilis isolated from hot-spring water was investigated. Galactose, glucose, xylose, sucrose, fructose or corn starch were used as a carbon sources for production of α-amylase. Methionine, glycine, aspartic acid, lysine, (NH 4 ) 2 SO 4 , NH 4 Cl, (NH 4 ) 2 S 2 O 8 and NH 4 NO 3 were used as a organic and inorganic nitrogen sources, respectively. The high- er α-amylase production was determined in corn starch, sucrose, fructose and galactose com- pared to other sugars and with lysine and methionine than compared with other organic nitrogen sources. Nitrate and aspartic acid are clearly not as good a nitrogen source for α- amylase production. When Triton X100, sodium dodecyl sulphate (SDS) and Tween 40 were employed in growth medium, although bacterial growth was found high, enzyme production was not detectable. Soluble starch and yeast extract were used in order to evaluate the influ- ence of the medium composition on the α-amylase production instead of sole carbon and nitrogen sources. Maximum enzyme production was found in 3.5 % and 1.5% soluble starch and yeast extract growth media, respectively.

The glutamine synthetase of Prevotella bryantii B14 is a family III enzyme (GlnN) and glutamine supports growth of mutants lacking glutamate dehydrogenase activity

Abstract: Prevotella spp. are believed to play a central role in ruminal nitrogen metabolism, but little is understood about the genetics and biochemistry of nitrogen assimilation and regulation in these bacteria. The gene encoding a family III glutamine synthetase (GSIII, glnN ) in Prevotella bryantii B 1 4 was cloned by Escherichia coli mutant complementation, and enzyme assays as well as Northern blot analysis showed that maximal enzyme activity and glnN transcription occurred in cells grown under nitrogen-limiting conditions. Addition of methionine sulfoximine (MSX), a GS inhibitor, terminated bacterial growth when ammonium was provided as the sole nitrogen source, but the inhibitory effect could be overcome by the inclusion of either L -glutamine or trypticase in the growth medium. A P. bryantii mutant lacking glutamate dehydrogenase (GdhA) activity was isolated by ethylmethylsulfonate mutagenesis. Growth studies with different nitrogen sources showed that the mutant strain was still capable of growth with ammonium as the sole nitrogen source, albeit at a decreased growth rate. The mutant strain could also grow with L -glutamine as a nitrogen source in the presence of MSX. These data suggest that GlnN provides an effective route of ammonium assimilation for P. bryantii, in addition to that afforded by the glutamate dehydrogenase pathway.

Production of laccase by the phytopathogenic fungus Rhizoctonia solani

Abstract: This thesis is an investigation into the production of laccases by the phytopathogenic soil fungus Rhizoctonia solani. The fungus causes maceration of plant tissue by the production of a variety of plant cell wall degrading enzymes. Whilst most attention has focused on the role of pectinases in maceration, the laccases which degrade lignin are likely to be important in this process. The production of laccase by the AG-11 isolate VR20 in V8 medium reached a maximum after 6 days incubation. Laccase activity was unaffected by variation in temperatures over the range 4-15 degrees C, but as the temperature increased the activity increased to a maximum at 25 degrees C. This high level activity was maintained as the temperature was increased to 37 degrees C. The effects of pH on laccase activity was also determined. Activity was stable over the pH range 4.5-6. Outside this range the activity decreased significantly. The composition of the growth medium also had a significant effect on laccase production. Similar levels of activity were observed during growth in V8, apple pectin media, or in media containing ground up lupin hypocotyls as a carbon source. However, approx 20 fold higher levels were obtained after growth in Czapek-Dox medium. Different laccase activity band patterns were obtained by zymogram analysis of culture supernatants. The production of the other cell wall degrading enzymes pectinase,xylanase and cellulase in these media was assessed for comparison. Whilst all three were produced in V8 and apple pectin media, cellulase was not produced in lupin medium, and none of these were produced in Czapek-Dox medium. Attempts to increase laccase production by the addition of the reported laccase inducers CuSO4.5H2O, p-anisidine, ethanol, MnSO4.7H2O, resveratrol, and tannic acid to the growth medium showed mixed results. The only case where enhancement of synthesis was observed was with the addition of MnSO4 to Czapek-Dox medium. This compound did not enhance production in the other media tested. With the exception of p-anisidine, the other inducers had minimal effect in V8, apple pectin or lupin media. Para anisidine completely inhibited production in lupin medium. With the exception of MnSO4, all inducers inhibited laccase production in Czapek-Dox medium, with p-anisidine causing complete inhibition. The production of xylanase and cellulase was also inhibited by these inducers but in a growth medium dependent manner. Cellulase production in V8 medium was inhibited by ethanol, MnSO4, resveratrol, and tannic acid whilst only the latter two inhibited xylanase production and none of these inhibited pectinase production. In contrast, p-anisidine had a greater inhibitory effect on pectinase and xylanase production in V8 medium than on cellulase production. Para-anisidine also inhibited xylanase (and laccase) but not pectinase production in lupin medium but not in apple pectin medium. Resveratrol and tannic acid also inhibited xylanase production in lupin medium. The effects of the inhibitory compounds EDTA and SDS on laccase activity was determined. With SDS the % inhibition increased as the concentration of inhibitor decreased from 5% to 0.5%. With EDTA the opposite trend was observed. The effects of arginine on laccase activity was also tested. At concentrations of 0.5 to 5% arginine, laccase activity was completely inhibited. Laccase activity was purified from the culture supernatant by anion exchange chromatography, and by electroelution from a native-PAGE gel. The degree of purification by each method was greater than 50 fold. Electrophoresis of the purified protein on an SDS-PAGE gel followed by staining with Coomassie blue showed two protein bands of 66 and 38 KDa. Measurement of the absorption spectrum of the purified protein showed two absorbance maxima, at 240 and 340nm. Laccases produced by isolates from different anastomosis groups were analysed by staining gels for laccase activity. Variations in the band pattern were observed both between and within anastomosis groups. Analysis of single spore isolates from AG-8 and AG-11 showed segregation of band patterns. However the sample sizes were too small to make conclusions about the numbers of laccase enzymes produced by or the number of genes in the parent field isolates. The role of laccases in maceration of lupin radicle tissue was investigated. Microscopic staining showed the presence of lignin in radicle tissue, and when incubated in the fungal enzymes the tissue lost integrity, characteristic symptoms of maceration. Maceration wasreadily observed as discolouring when the radicle was incubated in a solution of fungal enzyme. The degree of maceration was quantified by measuring the length of the discoloured region. Enzymes from all of the isolates tested caused maceration of lupin radicle. The degree of maceration ranged from 80-100%. No maceration was observed when agrinine was included in the reaction mixture. Arginine does not inhibit the activity of pectinases, xylanases, or cellulases. In maceration assays with potato tuber tissue which does not contain lignin, the addition of arginine to the reaction did not inhibit maceration. The results show that laccases are required for maceration of lignified tissue, but not for non-lignified tissue. Laccase gene sequences were cloned from three isolates, SCR122 (AG-6), 11034 (AG-8), and VR20 (AG-11) using degenerate primers to conserved sequences to amplify the gene sequences. The amplicons were cloned and sequenced. A BLAST search of the NCBI database with the derived amino acid sequences confirmed that the sequences were from laccase genes.

Environmental and genetic factors affecting mutability to aminoglycoside antibiotics among Escherichia coli K12 strains

Abstract: Environmental and genetic factors affecting the in vitro spontaneous mutation frequencies to aminoglycoside resistance in Escherichia coli K12 were investigated. Spontaneous mutation frequencies to kanamycin resistance were at least 100 fold higher on modified Luria agar (L2) plates, when compared to results obtained in experiments carried out with Nutrient agar (NA) plates. In contrast to rifampincin, the increased mutability to kanamycin resistance could not be attributed to a mutator phenotype expressed by DNA repair defective strains. Kanamycin mutant selection windows and mutant preventive concentrations on L2 plates were at least fourfold higher than on NA plates, further demonstrating the role of growth medium composition on the mutability to aminoglycosides. Mutability to kanamycin resistance was increased following addition of sorbitol, suggesting that osmolarity is involved on the spontaneous mutability of E. coli K12 strains to aminoglycosides. The spontaneous mutation rates to kanamycin resistance on both L2 and NA plates were strictly associated with the selective antibiotic concentrations. Moreover, mutants selected at different antibiotic concentrations expressed heterogeneous resistance levels to kanamycin and most of them expressing multiple resistance to all tested aminoglycoside antibiotics (gentamicin, neomycin, amykacin and tobramycin). These results will contribute to a better understanding of the complex nature of aminoglycoside resistance and the emergence of spontaneous resistant mutants among E. coli K12 strains.

Changes in growth and polyglucose synthesis in response to fructose metabolism by Fusobacterium nucleatum grown in continuous culture.

Abstract: Fusobacterium nucleatum, grown in a chemically defined medium at micro(rel) = 0.5, produced greater cell yields and undetectable levels of intracellular polyglucose (IP) when fructose was substituted for glucose. The utilisation and metabolism of fructose by growing cells was studied and the effect of the energy-yielding amino acids, glutamate, serine, histidine and lysine on cell yield, IP synthesis and acidic end-products was investigated. When F. nucleatum was grown on elevated amino acid levels, IP was synthesised from fructose and amino acids were metabolised to lactate, acetate, butyrate and formate. Under these conditions, IP synthesis was associated with the cells being replete with amino acid-derived energy; an observation supported by the absence of IP when the levels of (energy yielding) amino acids were reduced. Compared with fructose, glucose was less efficiently removed from the growth medium and produced less biomass and markedly lower levels of IP during energy-limited growth.

Culture medium for human cell and culture method

Abstract: PROBLEM TO BE SOLVED: To provide a growth medium for a human cell by using human serum, and to provide a method for growing the cell. SOLUTION: This growth medium for the human cell contains the human serum and a growth factor. The method for growing the human cell comprises seeding the medium with the cell and growing the cell in the medium. The growth factor preferably comprises at least one kind selected from a group comprising a nerve cell growth factor, hepatic cell growth factor, epithelial cell growth factor, thrombopoietin, stem cell factor, and fibroblast growth factor. The human serum is preferably collected from an individual from which the human cell subjected to the growing is collected. A mesenchymal cell derived from human bone marrow, or another mesenchymal cell derived from human umbilical cord, is preferably grown in the method. Further, it is preferable that tissue cells containing the human cell are grown in the medium, after the medium is directly seeded therewith, without conducting any operation for separating the tissue cells. COPYRIGHT: (C)2003,JPO

High production system of the antibacterial peptide PLG-1.

Abstract: Propionibacterium thoenii P-127 produces and releases to the growth medium antibacterial agents that can be used as natural preservatives. The concentrations of these antibacterial agents in the growth medium are very low, and their activity can be detected only in concentrated medium, even in a bioreactor. A simple and efficient system to produce propionicin PLG-1 without the use of a bioreactor was investigated. Fermentation in screw-cap bottles without shaking produced antibacterial activity similar to that of fermentation in plates, but in a shorter time. Sodium lactate medium (NaLa) was found to be the most supportive for PLG-1 production compared to lactic acid bacteria media such as M-17 or beet molasses/corn. The initial concentration of the carbon source, sodium lactate, agar concentration, and the initial pH of the medium affected the synthesis of PLG-1. Additions of NaCl up to 1% showed no effect on the antibacterial agent production. The optimal conditions for production of the antibacterial agent were fermentation for 9 days in screw-cap bottles in modified NaLa medium (M-NaLa) containing 1% yeast extract, 1% tryptic soy broth, 0.9% lactic acid, and 0.6% agar, adjusted to pH of 9.

Influence of growth media on potential virulence factors of Pseudomonas aeruginosa.

Abstract: Potential virulence factors of three Pseudomonas aeruginosa strains after growth in three complex media (CM) and in one mineral medium (MM) were evaluated. Cell surface hydrophobicity demonstrated by adherence of bacteria to xylene as well as enzymatic activity (elastase, protease, lipase) of the strains grown in CM varied with composition of CM and with strain. All strains cultivated in CM showed higher hydrophobicity and higher elastase, protease and lipase (with the exception of one strain) activity in comparison with bacteria incubated in MM. Even no production of elastase was detected in the strains after growth in MM. Motility of bacteria was affected by culture media the least. In vitro composition of growth media influenced some potential virulence factors of P. aeruginosa.

N2-fixing rice-field cyanobacterial mutant resistant against uracil herbicide

Abstract: The herbicide isocil (5-bromo-3-isopropyl-6-methyluracil), a superseded compound, has been found inhibitory to growth (in N-free or 5 mM NO 3 - medium) and heterocyst formation (in N-free medium) of the N 2 -fixing wetland rice paddy field cyanobacterium Nostoc muscorum at a dose of 400 - 800 μM for 15 min. Inhibition was reversed on exogenous supplementation of growth medium with 3.0 mM glucose and/or 5.0 μM NADPH 2 . A spontaneous 4 mM isocil-resistant mutant of this organism has been selected in N-free medium. This mutant shows better growth than the parental strain in both N-free and NO 3 - media, as well as higher heterocyst frequency and nitrogenase activity in N-free medium. The resistance factor (determined by dividing the LD 5 0 value of the mutant by that obtained for the parental strain) is nearly 100. The mutant has a simultaneous cross-resistance to 10 μM DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. The resistance of this mutant against herbicides, affecting the photosynthetic electron transport chain, in N. muscorum in N-free or NO 3 - medium makes this cyanobacterial strain valuable in herbicide-treated wetland rice paddy fields because of its unimpaired growth and efficient nitrogen fixation. Such strains could be potent candidates for use as natural biofertilizer in rice agriculture and nutrient cycling in rice-field ecosystems.

Synthetic growth medium for culture of tuberculinogenic strains of mycobacteria

Abstract: The synthetic growth medium for culture of tuberculinogenic strains of mycobacteria contains monobasic potassium phosphate, magnesium sulfate heptahydrate, ammonia ferric citrate, citric acid, glycerol, glycocol, zinc sulfate, nicotinic acid, and the distilled water. The modified composition provides for improving adaptive and growth properties of the medium allowing for the increased yield of bacterial mass and tuberculin.

Laboratory studies with purified iturin a, and with bacillus spp. grown in complex and defined growth media, to ascertain the identity and ability of compounds that inhibit fusarium graminearum

Abstract: Certain endospore-forming bacteria in the genus Bacillus are able to antagonize Fusarium graminearum in laboratory, greenhouse, and field-plot studies. We have worked with four strains of Bacillus spp. (likely related to Bacillus amyloliquefaciens and/or Bacillus subtilis) that have demonstrated ability to antagonize this wheat pathogen. Research has not yet entirely elucidated the mechanism of the antagonism, but it is probably due at least in part to bacterial antibiotics, such as cyclic lipopeptides in the iturin family. It is known from the literature that iturin production is enhanced in some growth media and suppressed in others. We have cultured these bacterial strains in potato dextrose broth (PDB), a complex medium containing glucose which may suppress iturin production to some degree. We have also cultured the Bacillus spp. in the defined broth medium of Besson et al. lacking glucose, containing mannitol, glutamic acid and inorganic salts. Bacterial cell numbers in this original formulation were lower than desirable for application of cells to wheat plants, so a modification of the original medium was used increasing the mannitol by 2.3 times, and increasing the glutamic acid by 2.1 times. After 10 days of growth in the modified broth medium with increased carbon and nitrogen sources, bacterial strain 1BA grew to over 10 times the optical density it achieved in the initial medium formulation. Plate count data also showed better growth of 1BA in the modified defined medium having elevated carbon and nitrogen, with plate counts of 10 9 CFU/ml or greater in the richer formulation, and plate counts that were orders of magnitude lower in the original growth medium formulation. Higher numbers of cells in the modified defined growth medium should allow better coverage of bacterial cells sprayed onto wheat surfaces when these bacteria are used in biocontrol trials. In addition, plate assays were done to see whether pure iturin would antagonize F. graminearum, and if the defined broth media in solidified form allowed the bacteria to antagonize the fungus. Purified iturin A was found to inhibit F. graminearum at a concentration of 40 µg/ml applied to a paper disk, challenging growth of the fungus on Potato Dextrose Agar. Analysis of extracts of broth cultures in defined media by absorption spectroscopy and HPLC indicated that iturin-like compounds were produced by all four Bacillus strains we have studied. Better understanding of the productioin of iturin and other compounds that might act in concert with iturin would allow better understanding and use of these and related bacteria as biocontrol agents to control FHB.