Selection of Antagonistic Fluorescent Pseudomonas spp. and their Root Colonization and Persistence following Treatment of Seed Potatoes

One hundred and twelve fluorescent Pseudomonas isolates from potato periderm were screened for antagonistic activity in vitro against 5 pathogenic and saprophytic fungi, 5 Gram-positive bacteria including Streptomyces scabies and 4 Gram-negative bacteria including Erwinia car . var. carotovora and Erwinia car . var. atroseptica . Fifty-seven percent of the isolates showed wide spectrum inhibitory activity, mostly due to the production of fluorescent siderophores. Only 4 % of the isolates were very strong antagonists. By adding iron (Fe³+) to the test medium, the antagonistic activity drastically diminished but revealed some strong antagonists of which the antagonism was not affected. Apparently growth-inhibiting substances other than siderophores were involved. Antibiotic-resistant mutants of the antagonists were used to establish that the bacteria on the seed tubers migrated to the roots under different conditions. Drying of the treated potatoes gradually diminished the number of viable cells, but subsequent planting in moist soil led to substantial increases in the first three days. Initially high numbers of the antibiotic-resistant antagonists on the roots tended to drop quickly in time, probably due to loss of resistance to the antibiotics in the selective medium.     

The evaluation of novel chromogenic substrates for the detection of lipolytic activity in clinical isolates of Staphylococcus aureus and MRSA from two European study groups

Pinellia ternata , a traditional Chinese herb that has been used in China for over 1000 years, is susceptible to a soft rot disease, which may cause major loss of yield. The use of bacteria as potential antagonists against Pectobacterium carotovorum SXR1, the causal agent of the disease on P. ternata , was evaluated. Altogether, 1107 candidate bacteria were isolated from the rhizosphere and surface-sterilized plants of P. ternata . In Petri dish tests, 55 isolates inhibited the growth of strain SXR1, and 21 of these reduced the disease development on P. ternata slices by over 50%. Four selected antagonists significantly reduced the disease incidence on tissue culture seedlings, and also prevented the disease on the transplants. Agonist P-Y2-2 yielded a good prevention level of 81.9%. The four antagonists rapidly colonized the tissue culture seedlings and transplants, whereas greater populations of the antagonists (10⁷–10⁹ CFU g⁻¹ fresh tissues) were observed in the seedlings and in the preinoculated transplants than in those inoculated during transplanting. The use of pathogen-free tissue culture seedlings pre-inoculated with antagonist may provide a strategy for production of P. ternata plantlets resistant to soft rot disease. This is the first report on the efficacy of biocontrol agents against pathogens on P. ternata .     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5·2 with HCl and incubated at 30°C, inocula containing < 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give > 10⁸ bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10–20 weeks. Citric acid concentrations of 10–50 mmol/l at pH 5·2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10⁶. The citric acid also reduced the concentration of free Ca²⁺ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5·2 could be prevented by the addition of Ca²⁺ or Mg²⁺ and greatly reduced by Fe²⁺ and Mn²⁺. The addition of Ca²⁺, but not of the remaining divalent metal ions, restored the concentration of free Ca²⁺ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca²⁺. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5·2. The effect of citric acid and Ca²⁺ at pH 5·2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

DETECTION AND ENUMERATION OF H2S+ BACTERIA: APPLICATION TO SHEWANELLA PUTREFACIENS (CIP)

H₂S⁺ bacteria responsible for the degradation of sulfur-containing amino acids of fish muscle are currently little used to evaluate the microbiological pal quality of fish. Shewanella putrefaciens greatly predominates in this flora, and was therefore used to define a suitable culture method and medium. Inoculations by the Spiral surface method at 25C, with an incubation of 72h, gave the best counts on a medium containing two sources of sulfur (organic and inorganic) for H₂S⁺ bacteria. The culture medium and the NaCl concentration were determinant in the evaluation of this flora. At present there is no standard medium which meets these requirements.     

De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene

Role of ethylene in de novo shoot morphogenesis from explants and plant growth of mustard ( Brassica juncea cv. India Mustard) in vitro was investigated, by culturing explants or plants in the presence of the ethylene inhibitors aminoethoxyvinylglycine (AVG) and AgNO₃. The presence of 20 μ M AgNO₃ or 5 μ M AVG in culture medium containing 5 μ M naphthaleneacetic acid and 10 μ M benzyladenine were equally effective in promoting shoot regeneration from leaf disc and petiole explants. However, AgNO₃ greatly enhanced ethylene production which reached a maximum after 14 days, whereas ethylene levels in the presence of AVG remained low during 3 weeks of culture. The promotive effect of AVG on shoot regeneration was overcome by exogenous application of 25 μ M 2-chloroethylphosphonic acid (CEPA), but AgNO_3-induced regeneration was less affected by CEPA. For whole plant culture, AVG did not affect plant growth, although it decreased ethylene production by 80% and both endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC by 70–80%. In contrast, AgNO₃ stimulated all 3 parameters of ethylene synthesis. Both AgNO₃ and CEPA were inhibitory to plant growth, with more severe inhibition occuring in AgNO₃. Leaf discs derived from plants grown with AVG or AgNO₃ were highly regenerative on shoot regeneration medium without ethylene inhibitor, but the presence of AgNO₃ in the medium was inhibitory to regeneration of those derived from plants grown with AgNO₃.     

Platelet activation by a relapsing fever spirochaete results in enhanced bacterium–platelet interaction via integrin αIIbβ3 activation

Borrelia hermsii , a spirochaete responsible for relapsing fever in humans, grows to high density in the bloodstream and causes thrombocytopenia. We show here that B. hermsii binds to human platelets. Extended culture in bacteriological medium resulted in both diminished infectivity in vivo and diminished platelet binding in vitro . Platelet binding was promoted by the platelet integrin α_IIbβ₃: the bacterium bound to purified integrin α_IIbβ₃, and bacterial binding to platelets was diminished by α_IIbβ₃ antagonists or by a genetic defect in this integrin. Integrin α_IIbβ₃ undergoes a conformational change upon platelet activation, and bacteria bound more efficiently to activated rather than resting platelets. Nevertheless, B. hermsii bound at detectable levels to preparations of resting platelets. The bacterium did not recognize a point mutant of α_IIbβ₃ that cannot acquire an active conformation. Rather, B. hermsii was capable of triggering platelet and integrin α_IIbβ₃ activation, as indicated by the expression of the platelet activation marker P-selectin and integrin α_IIbβ₃ in its active conformation. The degree of platelet activation varied depending upon bacterial strain and growth conditions. Prostacyclin I₂, an inhibitor of platelet activation, diminished bacterial attachment, indicating that activation enhanced bacterial binding. Thus, B. hermsii signals the host cell to activate a critical receptor for the bacterium, thereby promoting high-level bacterial attachment.     

Random mutagenesis of Legionella pneumophila with mini-Tn10

Abstract The degradation of sheets of poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (BIOPOL^®) by aerobic sewage sludge was analyzed. Degradation of the polymer was highly dependent on the pH of the culture medium and was maximal between pH 7 and pH 8.5. Below pH 6 and above pH 9 the degradation rate was very low. Agitation of the culture fluid had relatively little influence on the rates of degradation. 1.2×10⁵ aerobic polymer-degrading bacteria per ml sewage sludge were identified by halo formation on solid poly(3-hydroxybutyrate) (PHB)-containing media. The number of PHB-degrading bacteria in other ecosystems amounted to 3.8×10³ per ml sludge of a fresh-water lake, 9.2×10⁵ per g garden-soil, 1.3×10⁶ per g field-soil and 4.3×10⁶ per g compost.     

Optimization of medium constituents for improved chitinase production by Paenibacillus sp. D1 using statistical approach

Aims:  Statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1.
Methods and Results:  Urea, K₂HPO₄, chitin and yeast extract were identified as significant components influencing chitinase production by Paenibacillus sp. D1 using Plackett–Burman method. Response surface methodology (central composite design) was applied for further optimization. The concentrations of medium components for improved chitinase production were as follows (g l⁻¹): urea, 0·33; K₂HPO₄, 1·17; MgSO₄, 0·3; yeast extract, 0·65 and chitin, 3·75. This statistical optimization approach led to the production of 93·2 ± 0·58 U ml⁻¹ of chitinase.
Conclusions:  The important factors controlling the production of chitinase by Paenibacillus sp. D1 were identified as urea, K₂HPO₄, chitin and yeast extract. Statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 2·56-fold increase in chitinase production.
Significance and Impact of the Study:  The present investigation provides a report on statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. Paenibacillus species are gram-positive, spore-forming bacteria with several PGPR and biocontrol potentials. However, only few reports concerning mycolytic enzyme production especially chitinases are available. Chitinase produced by Paenibacillus sp. D1 represents new source for biotechnological and agricultural use.     

DOMOIC ACID PRODUCTION IN NITZSCHIA SP. (BACILLARIOPHYCEAE) ISOLATED FROM A SHRIMP-CULTURE POND IN DO SON, VIETNAM

Domoic acid (DA), a neuroexcitatory amino acid, was detected in batch culture of the newly recognized species Nitzschia navis-varingica Lundholm et Moestrup . The production of DA by this diatom was confirmed by electrospray ionization mass spectrometry. The diatom was collected from a shrimp-culture pond in Do Son, Vietnam. The production of DA (1.7 pg·cell ^{− 1}) is within the levels reported for Pseudo-nitzschia multiseries (Hasle) Hasle. The DA production started during the late exponential growth phase and reached a maximum during the early stationary growth phase. Maximum DA levels in the axenic culture decreased to about half that of the nonaxenic culture (0.9 pg·cell ^{− 1} vs. 1.7 pg·cell ^{− 1}), suggesting that DA production by the new species is influenced by bacteria.     

5-Hydroxytryptamine Receptors

5-Hydroxytryptamine (5-HT) receptors consist of at least three distinct types of molecular structures (Table 1): guanine nucleotide binding protein (G protein)-coupled receptors, ligand-gated ion channels, and transporters. Prior to the introduction of molecular biological techniques, the classification of 5-HT receptors was based predominantly on the pharmacological properties of the receptors. For example, "5-HT₁" receptors were defined as membrane binding sites that displayed nanomolar affinity for [³H]5-HT (Peroutka and Snyder, 1979). Subsequently, Bradley et al. (1986) defined "5-HT_1-like" receptors by their susceptibility to antagonism by methiothepin and/or methysergide, resistance to antagonism by 5-HT₂ antagonists, and potent agonism by 5-carboxamidotryptamine (5-CT). Thus, these classification systems are dependent upon the availability of selective pharmacological agents.     

Effects of culture conditions on Pectinatus cerevisiiphilus and Pectinatus frisingensis metabolism: a physiological and statistical approach

The genus Pectinatus has been often reported in beer spoilage with off-flavours. The bacteria are strictly anaerobic, Gram-negative rods. Propionate and acetate are the main fermentation products from glucose in the two species belonging to the genus, P. cerevisiiphilus and P. frisingensis. Amino acids routinely present at a high level in beer were not growth substrates for both species, and a significant accumulation of succinate was observed with lactate as growth substrate. Both Pectinatus ssp. showed almost identical fermentation balances on glucose. Growth kinetics of both glucose-grown species were unchanged under a N₂, H₂ or 20% CO₂-containing atmosphere. Combinations of culture medium pH values from pH 3·9 to pH 7·2, of glucose levels between 5 and 55 mmol l^-1, and of lactate concentrations varied from 4 to 40 mmol l^-1 demonstrated that biomass and volatile fatty acids production were proportional to glucose concentration for both Pectinatus species. A significant increase of volatile fatty acid production was measured for both species at the lowest pH values with a lactate or a glucose concentration increase. The maximum biomass production was observed at pH 6·2 for P. cerevisiiphilus , and between pH 4·5 and pH 4·9 for P. frisingensis. Glucose and lactate or pH value were dependent with regard to propionate and acetate production in P. frisingensis. On the other hand, the variations of these three parameters were independent with regard to biomass production for both strains, and to volatile fatty acids production for P. cerevisiiphilus. Addition of ethanol to glucose-grown cultures completely inhibited growth at 1·3 mol l^-1 ethanol for P. cerevisiiphilus , and at 1·8 mol l^-1 for P. frisingensis.     

Efflux of ions and ATP depletion induced by pediocin PA-1 are concomitant with cell death in Listeria monocytogenes Scott A

Domination of Carnobacterium divergens LV13 by a bacteriocin-producing (bac⁺) organism Carnobacterium piscicola LV17 was dependent on the level of inoculum of the producer strain and its bacteriocin production. When C. piscicola LV17 was grown in APT broth from an initial inoculum of α-10⁴ cfu ml^-1, bacteriocin was not produced (bac^-) although maximum population was reached. The culture remained bac^- during subsequent inoculation at 10²-10⁷ cfu ml^-1 unless it was first grown on solid medium or if heat-treated supernatant fluids from a bac⁺ culture of C. piscicola LV17, LV17A or LV17B were added to the culture prior to the stationary phase of growth. Use of purified carnobacteriocins from C. piscicola LV17A and LV17B confirmed their role in regulation of the bac⁺ phenotype. The need for induction might account in part for differences in bacteriocin production by cultures in liquid and on solid growth media.     

Nitrate reduction by Pseudomonas spp.: antagonism by fermentative bacteria

The reduction of nitrate by Pseudomonas denitrificans in a culture medium containing glycerol, yeast extract and 700 mg/1 NO₃–N, was antagonized by Aeromonas hydrophila, Escherichia coli and Enterobacter aerogenes. Nitrate reduction by Ps. denitrificans in mixed culture with a fermentative heterotroph was inhibited when 100–150 mg/1 NO₂–N had accumulated in the medium. The number of Ps. denitrificans declined concomitantly with the appearance of NO₂ in the culture medium, but there was only a slight increase in the numbers of fermentative hetero-trophs. The fermentative heterotrophs did not antagonize nitrate reduction by Ps. denitrificans , when the culture medium contained 140 mg/1 NO₃–N. When mixtures of equal parts of Ps. denitrificans and Esch. coli cultures were added to autoclaved river water relatively high concentrations of NO₂ were produced from the nitrate present in the water.     

Growth of thermophilic obligately autotrophic hydrogen-oxidizing bacteria on thiosulfate

Thermophilic obligately autotrophic H₂-oxidizing bacteria from Icelandic hot springs were tested for growth on thiosulfate. Ten strains were tested and all grew on thiosulfate but not on sulfite or sulfur. The product of thiosulfate oxidation was sulfate. The growth rate on thiosulfate was slower (μ=0.12 h^-1) than on H₂ (μ=0.34 h^-1). Washed cells which had been grown on thiosulfate could oxidize thiosulfate rapidly but H₂-grown cells oxidized thiosulfate much more slowly and with about a 3 h lag time. The bacteria would not grow on agar medium under H₂ but grew on agar medium containing thiosulfate.     

Galacto-oligosaccharide production using a recycling cell culture of Sterigmatomyces elviae CBS8119

Addition of small amounts of Fe²⁺, Zn²⁺, Cu²⁺ and thiamine-HCl to the culture medium was required for promoting the galacto-oligosaccharide (Gal-OS)-producing activity of Sterigmatomyces elviae CBS8119, when the concentration of yeast extract in the medium was lowered to 0·1 g l⁻¹. Galacto-oligosaccharide production using a recycling cell culture was performed in a medium containing 360 mg ml⁻¹ of lactose supplemented with optimal concentrations of Fe²⁺ (1·5 mg l⁻¹ of FeSO₄.7H₂O), Zn²⁺ (15 mg l⁻¹ of ZnSO₄.7H₂O), Cu²⁺ (0·5 mg l⁻¹ of CuSO₄.5H₂O) and thiamine-HCl (1 mg l⁻¹ ) . Galacto-oligosaccharide production was maintained at high levels during six cycles of production, with the amount of Gal-OS produced in each cycle being more than 216 mg ml⁻¹ (weight yield of more than 60%).     

Nitrogen regulation of erythromycin formation in Streptomyces erythreus

Abstract Erythromycin formation decreased in Streptomyces erythreus as a function of the ammonium concentration present in the medium. Total inhibition of synthesis was obtained with 100 mM NH₄Cl but medium pH and culture growth were not significantly affected. A similar effect was obtained with NH₄NO₃ or (NH₄)₂SO₄ indicating that ammonium ion probably repressed formation of antibiotic.     

On the screening of hydrogen peroxide-generating lactic acid bacteria

The published plate methods for the detection of hydrogen peroxide-producing lactic acid bacteria, which employ horseradish peroxidase and a chromogen, clearly fail to detect all the organisms that produce H₂O₂. Whilst keeping the same principle, the use of a novel growth medium and of the chromogen tetramethyl-benzidine allows the detection of H₂O₂ production by strains previously classified as non-producers.     

Taxonomic identification of Streptomyces exfoliatus K10 and characterization of its poly(3-hydroxybutyrate) depolymerase gene

Abstract Using fungi grown on synthetic agar medium, we evaluated and compared the concentration of various H₂O₂-producing enzymes. Our results showed that oxidase production in solid medium was better than that found in liquid medium and as high as that detected in wood samples. High yields of oxidases made it possible to compare different oxidases in the same culture extracts and under different conditions. Our results also indicated that H₂O₂ production is ubiquitous in the white rot fungi tested and that enzyme levels are influenced by the substrate composition.     

Studies on the Production of Extracellular Proteinases by a Non-pigmented Strain of Chromobacterium lividum Isolated from Abattoir Effluent

A highly proteolytic bacterium isolated from abattoir effluent was identified as a non-pigmented strain of Chromobacterium lividum. Ferrous or ferric ions at concentrations between 1·8 × 10^-5 and 9 × 10^-4 g ions/1, which is 2–3 orders of magnitude greater than that required for growth, were essential for extracellular proteinase production in aerated but not in static culture. Co²⁺, Ni²⁺, Mn²⁺, Cu²⁺ or Zn²⁺ ions could not replace iron. Four proteinases (I-IV) were produced in static culture, but only proteinase I was formed in significant quantities in aerated culture. With both forms of culture amino nitrogen was essential for proteinase production; glucose inhibited formation in aerated, but not static, cultures. Growth occurred over the range 1–33 °C, whereas proteinase production ceased at 27 °C, with maximum activity at 13 °C. Proteinase production appeared to be controlled by an interaction between iron, oxygen tension and glucose.     

Effects of Non-NMDA Receptor Modulators on [3H]Dopamine Release from Rat Mesencephalic Cells in Primary Culture

Abstract: The effects of AMPA and kainate on [³H]dopamine release from fetal (embryonic day 15) rat mesencephalic neurons in primary culture were enhanced markedly in a dose-dependent fashion by cyclothiazide, a recently described inhibitor of AMPA receptor desensitization. The EC₅₀ value for cyclothiazide was 2.2 ± 0.8 µ M . The release of [³H]dopamine induced by both AMPA (or kainic acid) and the combination of AMPA (or kainic acid) with cyclothiazide was antagonized by specific antagonists like 6-cyano-7-nitroquinoxaline-2,3-dione or the noncompetitive benzodiazepine GYKI 52466. Unlike cyclothiazide, the lectin concanavalin A did not stimulate [³H]dopamine release. These results established the involvement of AMPA-preferring receptors on [³H]dopamine release from rat mesencephalic neurons in primary culture and provided further evidence for the existence of regulatory allosteric sites on AMPA receptor subunits.