Influence of agar and activated charcoal on uptake of gibberellin and plant morphogenesis In vitro

Summary Agar and activated charcoal (AC) are commonly used in tissue culture. However, their deeper actions and functions are largely unknown. This experiment investigated the effect of agar and AC, singly and jointly, on gibberellin (GA) uptake by corn shoots. Corn seeds were germinated on Murashige and Skoog medium (MS). Shoot excised from 1-wk-old seedlings were cultured on liquid (0.0 g l⁻¹ agar) or solid (8 g l⁻¹ agar) MS containing 3 μM indole-3-acetic acid, 13.3 μM N⁶-benzyladenine, and 6000 CPM ml⁻¹ [³H]GA₄ as tracer. Both liquid and solid media had two treatments, one without AC and one supplemented with 5 g l⁻¹AC. Uptake of [³H]GA₄ and morphogenesis of corn shoots were recorded after 2 wk of culture. Corn explants cultured in AC-free media acquired high levels of [³H]GA₄, while explants from AC-containing media showed only traces of [³H]GA₄. Explants cultured in AC-free liquid medium contained about twice the amount of [³H]GA₄ as those from AC-free solid medium. Addition of agar reduced shoot length, while addition of AC increased both shool and root length. It is concluded that: (1) agar reduced the uptake of GA₄; and (2) GA₄ was irreversibly adsorbed by AC, and thus became unavailable to corn explants.     

Bioreactor for continuous synthesis of compactin by Penicillium cyclopium

Compactin was synthesized by Penicillium cyclopium in submerged as well as in bioreactor systems and assayed spectrophotometrically with a detection limit of 0.5 μg ml⁻¹ solvent. Synthesis in submerged culture was affected by aeration, glucose level, pH, and type and molarity of buffer. Citrate or succinate (pH 4.0, 0.10 M) in malt glucose peptone broth (MGPB) stimulated cell specialization, sporulation, enhanced compactin permeation from mycelia and its production (60.05 μg ml⁻¹ after 12 days). Fungal spores, immobilized onto-into loofah sponge, in a bioreactor, using MGPB-citrate as feed stock, resulted in productivity of 23.04 mg compactin (L⁻¹ h⁻¹) during 50 days operation at 0.45 h⁻¹ dilution rate. Compactin synthesis in the bioreactor was also affected by culture age, substrate, incubation and dilution rates. Scanning electron micrographs of the loofah sponge, prior to, during and post-spores immobilization showed that loofah channels served well for fungal support in the bioreactor. Received 6 October 1997/ Accepted in revised form 2 September 1998     

Phytohormone production by three strains of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and possible physiological and technological implications

The aim of this work was to evaluate phytohormone biosynthesis, siderophores production, and phosphate solubilization in three strains (E109, USDA110, and SEMIA5080) of Bradyrhizobium japonicum, most commonly used for inoculation of soybean and nonlegumes in USA, Canada, and South America. Siderophore production and phosphate solubilization were evaluated in selective culture conditions, which had negative results. Indole-3-acetic acid (IAA), gibberellic acid (GA₃), and abscisic acid (ABA) production were analyzed by gas chromatography–mass spectrometry (GC-MS). Ethylene and zeatin biosynthesis were determined by GS–flame ionization detection and high-performance liquid chromatography (HPLC-UV), respectively. IAA, zeatin, and GA₃ were found in all three strains; however, their levels were significantly higher (p < 0.01) in SEMIA5080 (3.8 μg ml⁻¹), USDA110 (2.5 μg ml⁻¹), and E109 (0.87 μg ml⁻¹), respectively. ABA biosynthesis was detected only in USDA110 (0.019 μg ml⁻¹). Ethylene was found in all three strains, with highest production rate (18.1 ng ml⁻¹ h⁻¹) in E109 cultured in yeast extract mannitol medium plus l-methionine. This is the first report of IAA, GA₃, zeatin, ethylene, and ABA production by B. japonicum in pure cultures, using quantitative physicochemical methodology. The three strains have differential capability to produce the five major phytohormones and this fact may have an important technological implication for inoculant formulation.     

Inhibition of galectin-3 mediated cellular interactions by pectic polysaccharides from dietary sources

Pectic polysaccharides from dietary sources such as Decalepis hamiltonii—swallow root (SRPP), Hemidesmus indicus (HPP), Nigella sativa—black cumin (BCPP), Andrographis serpyllifolia—(APP), Zingiber officinale—ginger (GRPP) and, citrus pectin (CPP) were examined for galectin inhibitory activity. Inhibition of (a) galectin-3 of MDA-MB-231 cells induced hemagglutination of red blood cells; (b) galectin-3 mediated interaction between normal/metastatic human buccal cells (NBC)/(MBC) and; (c) invasion of MDA-MB-231 and MBC in the invasive chamber was assessed. Results indicated that SRPP inhibited hemagglutination at Minimum Inhibitory Concentration (MIC) of 1.86 μg ml⁻¹ equivalent of carbohydrate as apposed to those of BCPP (130 μg ml⁻¹), APP (40 μg ml⁻¹), HPP (40 μg ml⁻¹) and CPP (25 μg ml⁻¹). GRPP even at concentration >1–6 mg ml⁻¹ did not inhibit agglutination. Also SRPP showed ∼15 and 2 fold potent anti hemagglutination activity relative to that of galectin-3 specific sugars—galactose (MIC-27.1 μg ml⁻¹) and lactose (MIC-4.16 μg ml⁻¹) respectively. Further, SRPP at 10 μg ml⁻¹ inhibited agglutination of NBC by galectin-3 of MDA-MB-231 cells. Modified swallow root pectic polysaccharide (MSRPP) of 50 kDa retained anti hemagglutination activity (MIC of 1.03 μg ml⁻¹) and inhibited MDA-MB-231 and MBC invasion by 73 and 50% with an IC₅₀ of 136 and 200 μg ml⁻¹ respectively. Both SRPP and MSRPP induced apoptosis up to 80% at 100 μg ml⁻¹ concentration by activating ∼2 and 8 folds of Caspase-3 activity. Sugar composition analysis and its correlation with the galectin inhibitory property indicated that pectic polysaccharides with higher arabinose and galactose content—arabinogalactan inhibited hemagglutination significantly.     

Long-term biological hydrogen production by agar immobilized Rhodobacter capsulatus in a sequential batch photobioreactor

In this study, agar immobilization technique was employed for biological hydrogen production using Rhodobacter capsulatus DSM 1710 (wild type) and YO3 (hup-mutant) strains in sequential batch process. Different agar and glutamate concentrations were tested with defined nutrient medium. Agar concentration 4% (w/v) and 4 mM glutamate were selected for bacterial immobilization in terms of rate and longevity of hydrogen production. Acetate concentration was increased from 40 to 60—100 and 60 mM gave best results with both bacterial strains immobilized in 4% (w/v) agar. Cell concentration was increased from 2.5 to 5 mg dcw mL⁻¹ agar and it was found that increasing cell concentration of wild-type strain caused decrease in yield and productivity while these parameters improved by increasing cell concentration of mutant strain. Also, the hydrogen production time has extended from 17 days up to 60 days according to the process conditions and parameters. Hydrogen production by immobilized photosynthetic bacteria is a convenient technology for hydrogen production as it enables to produce hydrogen with high organic acid concentrations comparing to suspended cultures. Besides, immobilization increases the stability of the system and allowed sequential batch operation for long-term application.

     

Efficient preservation in a silicon oxide matrix of Escherichia coli, producer of recombinant proteins

The aim of this work was to study the use of silicon oxide matrices for the immobilization and preservation of recombinant-protein-producing bacteria. We immobilized Escherichia coli BL21 transformants containing different expression plasmids. One contained DNA coding for a T-cell receptor β chain, which was expressed as inclusion bodies in the cytoplasm. The other two encoded bacterial superantigens Staphylococcal Enterotoxin G and Streptococcal Superantigen, which were expressed as soluble proteins in the periplasm. The properties of immobilization and storage stability in inorganic matrices prepared from two precursors, silicon dioxide and tetraethoxysilane, were studied. Immobilized E. coli was stored in sealed tubes at 4 and 20°C and the number of viable cells and level of recombinant protein production were analyzed weekly. Different tests showed that the biochemical characteristics of immobilized E. coli remained intact. At both temperatures selected, we found that the number of bacteria in silicon dioxide-derived matrix was of the same order of magnitude (10⁹ cfu ml⁻¹) as before immobilization, for 2 months. After 2 weeks, cells immobilized in an alkoxide-derived matrix decreased to 10⁴ cfu ml⁻¹ at 4°C, and no viable cells were detected at 20°C. We found that immobilized bacteria could be used as a starter to produce recombinant proteins with yields comparable to those obtained from glycerol stocks: 15 mg l⁻¹ for superantigens and 2 mg l⁻¹ for T-cell receptor β chain. These results contribute to the development of methods for microbial cell preservation under field conditions. Martín F. Desimone and Mauricio C. De Marzi contributed equally to this work     

Production of thermostable and neutral glucoamylase using immobilized <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae was immobilized in alginate, κ-carrageenan, agarose, agar, polyacrylamide and loofah (Luffa cylindrica) sponge (as such or coated with alginate/starch/Emerson YpSs agar), and used for the production of glucoamylase in submerged fermentation. The mycelium developed from alginate-immobilized sporangiospores secreted higher glucoamylase titres (22.7 U ml⁻¹) than those immobilized in other gel matrices and the freely growing mycelial pellets (18.5 U ml⁻¹). Loofah network provided a good support for mycelial growth, but the enzyme production was lower than that attained with alginate beads. Glucoamylase production increased with inoculum density and the optimum levels were achieved when 40 calcium alginate beads (∼5 × 10⁶ immobilized spores) were used to inoculate 50 ml production medium. The alginate bead inoculum displayed high storage stability at 4°C and produced comparable enzyme titres up to 120 days. The glucoamylase production by hyphae emerged from the immobilized sporangiospores was almost stable over eight batches of repeated fermentation. Scanning electron micrographs of alginate beads, after batch fermentation, revealed extensive mycelial growth inside and around the beads.     

Novel Antifouling and Antimicrobial Compound from a Marine-Derived Fungus Ampelomyces sp.

In this study, using a bioassay-guided isolation and purification procedure, we obtained 3-chloro-2,5-dihydroxybenzyl alcohol from a marine-derived Ampelomyces species that effectively inhibited larval settlement of the tubeworm Hydroides elegans and of cyprids of the barnacle Balanus amphitrite. The inhibitive effect on larval settlement was nontoxic and the EC₅₀ of 3-chloro-2,5-dihydroxybenzyl alcohol ranged from 3.19 μg ml⁻¹ to 3.81 μg ml⁻¹ while the LC₅₀ was 266.68 μg ml⁻¹ for B. amphitrite cyprids; EC₅₀ ranged from 0.67 μg ml⁻¹ to 0.78 μg ml⁻¹, and LC₅₀ was 2.64 μg ml⁻¹ for competent larvae of H. elegans, indicating that inhibitive effect of this compound was nontoxic. At a concentration of 50 μg per disc, this compound showed strong inhibitive effects on the growth of 13 out of 15 marine bacterial species tested in disc diffusion bioassay. Overall, the high inhibitory activities against bacteria and larval settlement as well as the non- or low-toxic nature of this compound to the barnacle and polychaete larvae suggest this compound could be a potent antifoulant and/or antibiotic.     

Investigation of the Cytotoxicity of Antimicrobial Peptide P40 on Eukaryotic Cells

The in vitro cytotoxicity of the antimicrobial peptide P40 was investigated. The food grade bacteriocin nisin was also analyzed for comparison. VERO cells were treated with different concentrations (0.02–2.5 μg ml⁻¹) of nisin and P40, and cell viability and plasma membrane integrity were checked by MTT, neutral red uptake (NRU), and lactate dehydrogenase (LDH) assays. In MTT and NRU assays the EC₅₀ to the purified peptide P40 were 0.30 and 0.51 μg ml⁻¹, while values found to nisin were 0.35 and 0.79 μg ml⁻¹, respectively. In the LDH assay, the EC₅₀ was 0.57 and 0.62 μg ml⁻¹ for P40 and nisin, respectively. The peptide P40 revealed higher hemolytical activity (19%) when compared to nisin (4.9%) at the highest concentration tested (2.5 μg ml⁻¹). Relatively few studies about the cytotoxicity of antimicrobial peptides are available. The determination of the cytotoxicity of antimicrobial peptides is an essential step to warrant their safe use.     

Major substrates for microbial sulfate reduction in the sediments of Ise Bay, Japan

To clarify the anaerobic microbial interactions in the process of carbon mineralization in marine eutrophic environments, the microbial sulfate reduction and methane production rates were examined in coastal marine sediments of Ise Bay, Japan, in autumn 1990. Sulfate reduction rates (51–210 nmol ml⁻¹ day⁻¹ at 24°C) were much higher than the methane production ones (<1.78 nmol ml⁻¹ day⁻¹) in the surface sediments (top 2 cm) at the six stations surveyed (water depth: 10.7–23.3 m). Substrates for sulfate-reducing bacteria (SRB) were estimated after the addition of a specific inhibitor for SRB (20 mmol l⁻¹ molybdate) into the sediment slurry, from the substrate accumulation rates. In the presence of the inhibitor, sulfate reduction was completely stopped and volatile fatty acids (mainly acetate) were accumulated, although hydrogen was not. Methane production occurred markedly accompanied by consumption of the accumulated acetate from the third day after the addition of molybdate. The maximum rate of methane production was 1.2–1.9 μmol ml⁻¹ day⁻¹, which was similar to those in highly polluted freshwater sediments such as the Tama River, Tokyo, Japan. These results show that acetate is a common major substrate for sulfate reduction and methane production, and SRB competitively inhibit potential acetoclastic methanogenesis in coastal sediments. Methanogens may potentially inhabit the sediments at low levels of population density and activity.     

Long-term production of soluble human Fas ligand through immobilization of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> in a fibrous bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.     

Growth promotion of wheat seedlings by <Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis> 1P (MTCC 8707) a cold tolerant bacterial strain from the Uttarakhand Himalayas

Exiguobacterium acetylicum strain 1P (MTCC 8707) is a gram-positive, rod-shaped, yellow pigmented bacterium isolated from soil on nutrient agar plates at 4°C. The identity of the bacterium was arrived on the basis of the biochemical characterization, BIOLOG sugar utilization pattern and sequencing of the 16S rRNA gene. It grew at temperatures ranging from 4 to 42°C, with temperature optima at 30°C. It expressed multiple plant growth promotion attributes such as phosphate solubilization, indole acetic acid (IAA), siderophore and hydrogen cyanide (HCN) production, differentially at suboptimal growth temperatures (15 and 4°C). At 15°C it solubilized phosphate (21.1 μg of P ml⁻¹ day⁻¹), and produced IAA (14.9 μg ml⁻¹ day⁻¹) in tryptophan amended media. Qualitative detection of siderophore production and HCN were possible at 15°C. At 4°C it retained all the plant growth promotion attributes. Seed bacterization with the isolate, positively influenced the growth and nutrient uptake parameters of wheat seedlings in glass house studies at suboptimal cold growing temperatures.     

Production of lipase by high cell density fed-batch culture of Candida cylindracea

Candida cylindracea NRRL Y-17506 was grown to produce extracellular lipase from oleic acid as a carbon source. Through flask cultures, it was found that the optimum initial oleic acid concentration for cell growth was 20 g l⁻¹. However, high initial concentrations of oleic acid up to 50 g l⁻¹ were not inhibitory. The highest extracellular lipase activity obtained in flask culture was 3.0 U ml⁻¹ after 48 h with 5 g l⁻¹ of initial oleic acid concentration. Fed-batch cultures (intermittent and stepwise feeding) were carried out to improve cell concentration and lipase activity. For the intermittent feeding fed-batch culture, the final cell concentration was 52 g l⁻¹ and the extracellular lipase activity was 6.3 U ml⁻¹ at 138.5 h. Stepwise feeding fed-batch cultures were carried out to simulate an exponential feeding and to investigate the effects of specific growth rate (0.02, 0.04 and 0.08 h⁻¹) on cell growth and lipase production. The highest final cell concentration obtained was 90 g l⁻¹ when the set point of specific growth rate (μ_set) was 0.02 h⁻¹. High specific growth rate (0.04 and 0.08 h⁻¹) decreased extracellular lipase production in the later part of fed-batch cultures due to build-up of the oleic acid oversupplied. The highest extracellular lipase activity was 23.7 U ml⁻¹ when μ_set was 0.02 h⁻¹, while the highest lipase productivity was 0.31 U ml⁻¹ h⁻¹ at μ_set of 0.08 h⁻¹.     

Monitoring of <Emphasis Type="Italic">E. coli</Emphasis> immobilization on modified gold electrode: A new bacteria-based glucose sensor

Electrochemical impedance spectroscopy (EIS) technique has proved to be an effective method for monitoring the immobilization of various bioactive species such as enzymes, DNA, whole cells, and so forth. In this work we describe the development of an electrochemical whole cell based biosensor. Biotinylated fluorescent E. coli are immobilized onto a cysteamine, Sulfo-NHS-LC-biotin, and avidin modified gold electrodes. Immobilized bacteria are clearly observed using confocal microscopy. Electrochemical measurements are based on the charge-transfer kinetics of [Fe (CN)₆]^3−/4− redox couple. The experimental impedance data were modelised with a computer. SAM assembly and the subsequent immobilization of bacteria on the gold bare electrodes greatly increased the electrontransfer resistance (R _et ) and reduced the constant phase element (CPE). It’s interesting to note, the hard immobilization of bacteria on the surface of electrode and do not remove during measurements. The effect of glucose addition was studied in the range of 10⁻⁷ μM to 10 μM. The relation between the evolution of R _et and D-glucose concentration was found to be linear for values ranging from 10⁻⁵ μM to 10⁻¹ μM and reached saturation for higher concentrations. Such biosensor could be applied to a more fundamental study of cell metabolism and drugs effect.     

Endophytic fungi with anti-microbial,anti-cancer and anti-malarial activities isolated from Thai medicinal plants

A total of 81 Thai medicinal plant species collected from forests in four geographical regions of Thailand were examined for the presence of endophytic fungi with biological activity. Of 582 pure isolates obtained, 360 morphologically distinct fungi were selected for cultivation on malt Czapek broth and yeast extract sucrose broth, from which extracts were tested for biological activity. Extracts of 92 isolates could inhibit Mycobacterium tuberculosis (MIC 0.0625–200 μg ml⁻¹) when tested by the microplate Alamar blue assay, while extracts of six inhibited Plasmodium falciparum (IC₅₀ of 1.2–9.1 μg ml⁻¹) as determined by the [³H]hypoxanthine incorporation method. Strong anti-viral activity against Herpes simplex virus type 1 was observed in 40 isolates (IC₅₀ of 0.28–50 μg ml⁻¹). The sulphorhodamine B assay for activity against cancer cell lines revealed that 60 were active against human oral epidermoid carcinoma cells (EC₅₀ 0.42–20 μg ml⁻¹) and 48 against breast cancer cells (EC₅₀ 0.18–20 μg ml⁻¹). Bioactivity profile was affected by the type of culture medium. Given the high incidence of bioactive extracts and the fact that most of the isolated fungi could not be identified due to lack of spore formation, the results suggested that Thai medicinal plants can provide a wide variety of endophytes that might be a potential source of novel bioactive compounds. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of a cold-tolerant plant growth-promoting bacterium Pantoea dispersa 1A isolated from a sub-alpine soil in the North Western Indian Himalayas

Pantoea dispersa strain 1A is a Gram-negative rod-shaped, yellow-pigmented bacterium isolated on nutrient agar plates incubated at 4°C. The identity of the bacterium was confirmed by sequencing of the 16 S rRNA gene. It was capable of growing at temperatures ranging from 4 to 42°C, but maximum growth was observed at 30°C. It is endowed with multiple plant growth promotion attributes such as phosphate solubilization, IAA production, siderophore production and HCN production, which are expressed differentially at sub-optimal temperatures (15 and 4°C). It was able to solubilize phosphate (17.6 μg of P₂O₅ ml⁻¹ day⁻¹), and produce IAA (3.7 μg ml⁻¹ day⁻¹), at 15°C. Qualitative detection of siderophore production and HCN were also observed at 15°C. At 4°C it was found to express all the plant growth promotion attributes. This bacterial isolate was able to positively influence and promote the growth and nutrient uptake parameters of wheat (cv. VL.802) under glasshouse conditions. Hence in the context, of cold wheat-growing environments, it is proposed that Pantoea dispersa 1A (MTCC 8706), could be deployed as an inoculant to attain the desired results of bacterization.     

Plant-growth-promoting compounds produced by two agronomically important strains of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>, and implications for inoculant formulation

We evaluated phytohormone and polyamine biosynthesis, siderophore production, and phosphate solubilization in two strains (Cd and Az39) of Azospirillum brasilense used for inoculant formulation in Argentina during the last 20 years. Siderophore production and phosphate solubilization were evaluated in a chemically defined medium, with negative results. Indole 3-acetic acid (IAA), gibberellic acid (GA₃), and abscisic acid (ABA) production were analyzed by gas chromatography-mass spectrometry. Ethylene, polyamine, and zeatin (Z) biosynthesis were determined by gas chromatography-flame ionization detector and high performance liquid chromatography (HPLC-fluorescence and -UV), respectively. Phytohormones IAA, Z, GA₃, ABA, ethylene, and growth regulators putrescine, spermine, spermidine, and cadaverine (CAD) were found in culture supernatant of both strains. IAA, Z, and GA₃ were found in all two strains; however, their levels were significantly higher (p < 0.01) in Cd (10.8, 2.32, 0.66 μg ml⁻¹). ABA biosynthesis was significantly higher (p < 0.01) in Az39 (0.077 μg ml⁻¹). Ethylene and polyamine CAD were found in all two strains, with highest production in Cd cultured in NFb plus l-methionine (3.94 ng ml⁻¹ h⁻¹) and Az39 cultured in NFb plus l-lysine (36.55 ng ml⁻¹ h⁻¹). This is the first report on the evaluation of important bioactive molecules in strains of A. brasilense as potentially capable of direct plant growth promotion or agronomic yield increase. Az39 and Cd showed differential capability to produce the five major phytohormones and CAD in chemically defined medium. This fact has important technological implications for inoculant formulation as different concentrations of growth regulators are produced by different strains or culture conditions.     

Anomalies in the enumeration of starved bacteria on culture media containing nalidic acid and tetracycline

Culturable counts of antibiotic resistant, genetically engineeredPseudomonas fluorescens were determined on antibiotic-containing plate count agar during starvation in water. Prior to starvation, colony counts obtained on all media separated into two groups. The mean of the colony counts on plate count agar with or without tetracycline (4.9 × 10⁶ ml⁻¹) was significantly higher than the mean colony counts on plate count agar containing either nalidixic acid or nalidixic acid plus tetraclycline (2.5×10⁶ ml⁻¹). After 20 days of starvation the highest mean colony counts continued to be obtained on plate count agar (7.2 × 10⁶ ml⁻¹) with slightly, but significantly, lower counts obtained on plate count agar containing either nalidixic acid (5.6 × 10⁶ ml⁻¹) or tetraclycline (1.5×10⁶ ml⁻¹). A combination of nalidixic acid and tetracycline in plate count agar, however, dramatically reduced colony counts (8.3 × 10² ml⁻¹) after this starvation period. The addition of catalase to plate count agar containing nalidixic acid and tetracycline negated the effect caused by this combination of antibiotics. When colony counts obtained over the entire 20 day incubation were considered, the addition of MgSO₄ to plate count agar containing nalidixic acid and tetracycline resulted in a significant increase in colony counts. Other combinations of antibiotics, nalidixic acid+carbenicillin, nalidixic acid+kanamycin, streptomycin+tetracycline, streptomycin+carbenicillin, rifampicin+tetracycline, rifampicin+carbenicillin, and rifampicin+kanamycin, did not inhibit colony formation of starved cells. Antibiotic resistant strains ofP. putida andEscherichia coli also displayed sensitivity to the combination of nalidixic acid and tetracycline in plate count agar after starvation.     

Antimicrobial activity of an endophytic <Emphasis Type="Italic">Xylaria</Emphasis> sp.YX-28 and identification of its antimicrobial compound 7-amino-4-methylcoumarin

An endophytic Xylaria sp., having broad antimicrobial activity, was isolated and characterized from Ginkgo biloba L. From the culture extracts of this fungus, a bioactive compound P3 was isolated by bioactivity-guided fractionation and identified as 7-amino-4-methylcoumarin by nuclear magnetic resonance, infrared, and mass spectrometry spectral data. The compound showed strong antibacterial and antifungal activities in vitro against Staphylococcus aureus [minimal inhibitory concentrations (MIC) 16 μg·ml⁻¹], Escherichia coli (MIC, 10 μg·ml⁻¹), Salmonella typhia (MIC, 20 μg·ml⁻¹), Salmonella typhimurium (MIC, 15 μg·ml⁻¹), Salmonella enteritidis (MIC, 8.5 μg·ml⁻¹), Aeromonas hydrophila (MIC, 4 μg·ml⁻¹), Yersinia sp. (MIC, 12.5 μg·ml⁻¹), Vibrio anguillarum (MIC, 25 μg·ml⁻¹), Shigella sp. (MIC, 6.3 μg·ml⁻¹), Vibrio parahaemolyticus (MIC, 12.5 μg·ml⁻¹), Candida albicans (MIC, 15 μg·ml⁻¹), Penicillium expansum (MIC, 40 μg·ml⁻¹), and Aspergillus niger (MIC, 25 μg·ml⁻¹). This is the first report of 7-amino-4-methylcoumarin in fungus and of the antimicrobial activity of this metabolite. The obtained results provide promising baseline information for the potential use of this unusual endophytic fungus and its components in the control of food spoilage and food-borne diseases.     

Studies on inositol-mediated expression of MAL gene encoding maltase and phospholipid biosynthesis in Schizosaccharomyces pombe

In this study, the effects of inositol addition on expression of the MAL gene encoding maltase and phosphatidylinositol (PI) biosynthesis in Schizosaccharomyces pombe (a naturally inositol-requiring strain) were examined. We found that specific maltase activity was at its maximum when the concentration of added inositol reached 6 μg ml⁻¹ in a synthetic medium containing 2.0% (w/v) glucose. When the concentration of added inositol was 1 μg ml⁻¹ in the medium, repression of MAL gene expression occurred at glucose concentration higher than 0.2% (w/v). However, when S. pombe was cultured in the synthetic medium containing 6 μg ml⁻¹, repression of maltase gene expression occurred only at initial glucose concentration above 1.0% (w/v). More mRNA encoding maltase was detected in the cells grown in the medium with 6 μg ml⁻¹ inositol than in those grown in the same medium with 1 μg ml⁻¹ inositol. These results demonstrate that higher inositol concentrations in the synthetic medium could derepress MAL gene expression in S. pombe. PI content of the yeast cells grown in the synthetic medium with 6 μg ml⁻¹ of inositol was higher than that of the yeast cells grown in the same medium with 1 μg ml⁻¹ of inositol. This means that PI may be involved in the derepression of MAL gene expression in S. pombe.