Sensitization of Escherichia coli to nisin by maltol and ethyl maltol

F. SCHVED, M.D. PIERSON AND B.J. JUVEN. 1996. When used separately, 20 mmol 1^-1 maltol or 1600 AU ml^-1 nisin resulted in a 0–0.6 log₁₀ reduction in viable counts of Escherichia coli in a buffer system. However, when added in combination they yielded a 1.8–5. 5–log-cycle reduction in viable counts of E. coli at pH 5.0 and 6.8 respectively. It is postulated that maltol (and ethyl maltol) destabilizes the cell outer membrane by chelation of Mg²⁺ and/or Ca²⁺, thus permeabilizing the E. coli cell to nisin.     

Effect of some factors on determination of viable microbial cells by peroxyoxalate chemiluminescence method

The effects of physical and chemical factors on the production of H₂O₂ from Escherichia coli cells were studied. When 20 mmol 1^-1 Tris-HCl buffer was used for this purpose the electron transport system (ETS) showed the highest activity at pH 7.6-8.2. KCN promoted the production of H₂O₂ from E. coli cells, and the optimum concentration was changed in different reaction times and pH values. Glucose, 5 mg ml^-1, increased the ETS activity about twofold. The other substrates and surfactants did not increase the chemiluminescence intensity. NaNO₂ and Na₂SO₄ in inorganic salts significantly reduced the ETS activity above 70%. In addition, the optimum temperature for the production of H₂O₂ was 30°C in this study. When glucose (5 mg ml^-1) and KCN (0.2 mmol 1^-1) were added to the reaction buffer containing 0.5 mmol 1^-1 menadione, the detectable minimum cell densities (averages of triplicate assay) of E. coli, Enterobacter cloacae and Serratia marcescens were 5 times 10³ cells ml^-1, 10⁴ cells ml^-1 and 10⁴ cells ml^-1 respectively.     

Nutrient reserves of Salicornia europaea seeds

Seeds of Salicornia europaea L. were analyzed for their nutrient reserves. The content of potassium and sodium was 216 and 39 mmol (kg dry seeds)^-1, respectively. Calcium and magnesium accounted for 30 and 138 mmol (kg dry seeds)^-1, respectively. Whereas most of the alkali metals were water soluble, the alkaline earth metals were mostly acid soluble. The acid-soluble calcium plus magnesium corresponded well with the acid-soluble phosphate. Chloride was accumulated to a level equivalent to that of sodium. Carbonate was present at a concentration of 9 mmol (kg dry seeds)^-1. Carbohydrates accounted for 93 g (kg dry seeds)^-1, nearly half of which was derived from sucrose. Fructose and glucose were present only in traces. Total nitrogen was determined to be 55 g (kg dry seeds)^-1, 16% of which was diethylether soluble. The remaining nitrogen was separated into 39 g (kg dry seeds)^-1 ethanol-insoluble and 8 g (kg dry seeds)^-1 ethanol-soluble nitrogen. About 10% of the ethanol-soluble nitrogen were derived from amino acids. Total lipid content was about 280 g (kg dry seeds)^-1. The alcoholic component of the storage lipids was glycerol and the glycerides were calculated from gas chromatography to be 66% of the total lipids. About 90% of the fatty acids consisted of unsaturated acids, linoleic and oleic acid, the majority (77%) of which was linoleic acid.     

Reduction of tetrazolium salts by sulfate-reducing bacteria

Abstract The reduction of tetrazolium salts by the sulfate-reducing bacteria, Desulfovibrio desulfuricans and Desulfotomaculum orientis , was examined. D. desulfuricans and D. orientis reduced triphenyltetrazolium chloride (TTC) and 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyltetrazolium chloride (INT) forming intracellular formazan deposits. The reduction rate of INT was higher than that of TTC. INT reduction was not inhibited by the addition of sulfate or molybdate, and sulfate uptake was inhibited by the addition of both INT and molybdate. The ratio of intracellular formazan forming cells to acridine orange direct counts in both strains decreased with culture age and starvation time.     

Studies on the effect of mercury and organomercurial on the growth and nitrogen fixation by mercury-resistant Azotobacter strains

Five nitrogen-fixing Azotobacter strains isolated from agricultural farms in West Bengal, India, were resistant to mercuric ion and organomercurials. Resistance of Hg-resistant bacteria to mercury compounds is mediated by the activities of mercuric reductase and organomercurial lyase in the presence of NADPH and GSH as cofactors. These bacteria showed an extended lag phase in the presence of 10–50 μmol 1^-1 HgCl₂. Nitrogen-fixing ability of these isolates was slightly inhibited when the mercuryresistant bacterial cells were preincubated with 10 μmol 1^-1 HgCl₂. Acetylene reduction by these bacteria was significantly inhibited (91-97%) by 50 μmol 1^-1 HgCl₂. However, when GSH and NADPH were added to the acetylene reduction assay mixture containing 50 μmol 1^-1 HgCl₂, only 42–50% inhibition of nitrogenase activity was observed. NADPH and GSH might have a role in suppressing the inhibition of N₂-fixation in the presence of Hg compounds either by assisting Hg-detoxifying enzymes to lower Hg concentration in the assay mixture or by formation of adduct comprising Hg and GSH which is unable to inhibit nitrogen fixation.     

Inhibition of Salmonella typhimurium by the products of tartrate metabolism by a Veillonella species

The inhibition of the growth of Salmonella typhimurium by a Veillonella species grown on media supplemented with tartrate was examined. Growth of Salmonella typhimurium was not inhibited by the concentrations of products metabolized by Veillonella cultures on media supplemented with 0 or 50 mmol 1^-1 of tartrate, but was inhibited on media supplemented with 100 or 150 mmol 1^-1 of tartrate. Inhibition of Salm. typhimurium was correlated with the increased production of acetate and propionate from tartrate by the Veillonella species.     

Joint action of l-leucine and sodium phosphate buffer solutions as feeding stimulants for protein-deprived females of the house fly Musca domestica

ABSTRACT. Potentiation in joint action was demonstrated between solutions of L-leucine and sodium phosphate buffer (pH 6.3) as feeding stimulants for protein-deprived females of the house fly, Musca domestica L. Both components alone elicited feeding. In two-choice feeding tests, mixtures consisting of equi-stimulating concentrations of the two components were taken in greater quantities than either component alone at twice the concentration in the mixture.
The presence of 1×10^-1 M phosphate buffer markedly lowered the threshold for detection of L-leucine. The presence of phosphate buffer strengthened the preferences shown by flies given choices of concentrations of L-leucine differing by a factor of 2 and enabled them to display preferences at lower concentrations.
The presence of 1×10^-3 M L-leucine increased, somewhat, the ability of flies to detect low concentrations of phosphate buffer. Its presence had relatively little effect on the strength of preference shown between two-fold differences in concentration of phosphate buffer when the higher concentration was 6.3×10^-3 M or less, but markedly strengthened the preferences when the higher concentration was 2.5×10^-2M or greater. Leucine increased the optimal concentration of phosphate buffer by a factor of more than 2 and converted 2×10^-1 M phosphate buffer from a mild feeding deterrent to a powerful feeding stimulant.     

THE IRREVERSIBLE INHIBITION OF BRAIN L-GLUTAMATE-I-DECARBOXYLASE BY (2RS,3E)-2-METHYL-3,4-DIDEHYDROGLUTAMIC ACID

Abstract— Incubation of chick embryo brain l -glutamate-1-dccarboxylase (GAD, EC 4.1.1.15) with (2RS,3E)-2-methyl-3,4-didehydroglutamic acid (MDG), a substrate analog of l -glutamic acid, results in a time-dependent irreversible inhibition of the enzymic activity. In the presence of 2.0 ± 10^-3 m inhibitor the half-life for inactivation is 11.6min. The inhibitor is a substrate for GAD and requires turnover prior to inactivating the enzyme and is therefore another example of the k _cat class of inactivator. The measured K _l is 6.6 ± 10^-4 m and the k _cat for its turnover is 1.01 ± 10^-3 s-¹ at 37°C (pH 7.2). The inhibitor has no effect on the apoenzymc or the holoenzyme treated with 1.0 ± 10^-3 m hydrazinc. Both l -and d -glutamate, but not mercaptoethanol, reduce the rate of enzymie inactivation by the inhibitor. The exceedingly high specificity implicit in the design of this inhibitor should render it useful in studies designed to uncover the physiological role of GABA.     

Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria

A study was undertaken to measure aerobic respiration by indigenous bacteria in a sand and gravel aquifer on western Cape Cod, MA using tetrazolium salts and by direct oxygen consumption using gas chromatography (GC). In groundwater and aquifer slurries, the rate of aerobic respiration calculated from the direct GC assay was more than 600 times greater than that using the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT). To explain this discrepancy, the toxicity of INT and two additional tetrazolium salts, sodium 3'-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to bacterial isolates from the aquifer was investigated. Each of the three tetrazolium salts was observed to be toxic to some of the groundwater isolates at concentrations normally used in electron transport system (ETS) and viability assays. For example, incubation of cells with XTT (3 mM) caused the density of four of the five groundwater strains tested to decline by more than four orders of magnitude. A reasonable percentage (>57%) of cells killed by CTC and INT contained visible formazan crystals (the insoluble, reduced form of the salts) after 4 h of incubation. Thus, many of the cells reduced enough CTC or INT prior to dying to be considered viable by microscopic evaluation. However, one bacterium (Pseudomonas fluorescens) that remained viable and culturable in the presence of INT and CTC, did not incorporate formazan crystals into more than a few percent of cells, even after 24 h of incubation. This strain would be considered nonviable based on traditional tetrazolium salt reduction assays. The data show that tetrazolium salt assays are likely to dramatically underestimate total ETS activity in groundwater and, although they may provide a reasonable overall estimate of viable cell numbers in a community of groundwater bacteria, some specific strains may be falsely considered nonviable by this assay due to poor uptake or reduction of the salts.     

Flight responses of tsetse flies (Glossina) to octenol and acetone vapour in a wind-tunnel

Abstract. Female Glossina morsitans morsitans Westwood were video-recorded in a wind-tunnel as they entered, in crosswind flight, a broad plume of either octenol or acetone (two components of ox odour). Both odours produced upwind turning responses (in-flight anemotaxis) to a range of concentrations, with thresholds at around 10^-8mg1^-l for octenol and 10^-6mg1^-1 for acetone. Kinetic responses were unaffected by octenol at low concentrations, but flight speed was significantly reduced and sinuosity (^om^-1) and angular velocity (^os^-1) significantly increased by concentrations at or above those in ox breath; for acetone, these effects were apparent but inconsistently related to concentration. It is concluded that octenol and acetone vapour are used by tsetse flies to locate hosts by upwind anemotaxis, probably combined with kinetic responses. The behavioural basis for the 'repellency' of high octenol concentrations in the field is discussed in the context of the virtual loss of upwind anemotaxis to octenol at the highest concentration tested in the tunnel (30 × ox breath).     

A Simple Cranial Window Technique for Optical Monitoring of Cerebrocortical Microcirculation and NAD/NADH Redox State. Effect of Mitochondrial Electron Transport Inhibitors and Anoxic Anoxia

Abstract: Fluorescence of NADH and vascular volume of the brain cortex of chloralose-anesthetized cats were measured by surface fluororeflectometry. A cranial window and superfusion technique was elaborated for the topical inhibition of mitochondrial electron transport in the brain cortex by amytal (inhibits at site I) and cyanide (inhibits at site III). The changes in NAD/NADH redox state and CVV evoked by these electron transport inhibitors were compared with those elicited by anoxic anoxia. Amytal (10^-3-10^-1 M ) and cyanide (10^-5-10^-2 M ) resulted in a concentration-dependent and reversible increase in cortical NAD reduction and vascular volume, but the cerebrocortical vessels were almost completely dilatated long before maximum NAD reduction was reached. Cyanide at 10^-2 M increased cortical NAD reduction and vascular volume as much as anoxic anoxia. Amytal at 10^-1 M induced approximately half of the NAD reduction evoked by 10^-2 M cyanide or anoxic anoxia, but resulted in only slightly less vasodilatation than that following cyanide and anoxic anoxia. Since amytal inhibits mitochondrial electron transport at site I—and cyanide and anoxia at site III—but induces a comparable degree of vasodilatation, it is concluded that cytochrome oxidase cannot be the single molecular oxygen sensor in the brain cortex.     

Kinetic and metabolic effects of nitrogen, magnesium and sulphur restriction in Xanthomonas campestris batch cultures

By reducing the concentration of nitrogen (from 5.0 to 2.5 mmol 1^-1), batch cultures of Xanthomonas campestris induced the enzyme UDP-glucose dehydrogenase and stimulated the Entner-Doudoroff pathway enzyme glucose-6-P dehydrogenase. The surplus energy generation was directed to xanthan biosynthesis resulting in a 10% polysaccharide increase. The nitrogen restriction led to a higher consumption of nitrogen (93%) whereas glucose consumption did not surpass 75% utilization. Low concentrations of both magnesium and sulphur exerted a negative effect on xanthan formation. Both restrictions reduced the phosphomannose isomerase enzyme activity by 10-fold turning the mannose transference presumably into the rate-limiting step for xanthan biosynthesis. Conversely, the rate of synthesis of glucuronic acid residues did not affect the rate of xanthan biosynthesis. Polysaccharide synthesis in magnesium and sulphur cultures was negatively affected in comparison with cell formation as the cell volumetric production rate increased from 0.037 to 0.091 g 1^-1 h^-1 and the xanthan volumetric production rate dropped from 0.133 g 1^-1 h^-1 to the minimum obtained at 0.083 g 1^-1 h^-1. The efficiency of the carbon substrate conversion was also greatly changed.     

β-Amylase from Clostridium thermocellum SS8 - a thermophilic, anaerobic, cellulolytic bacterium

The extracellular amylase produced by Clostridium thermocellum strain SS8 on starch was characterized as a β-amylase based on blue value reduction test and the production of maltose from starch. The enzyme had a temperature and pH optima of 60°C and 6.0, respectively. Of the metal ions tested, Ca^{2 +} and Mg^{2 +} had little effect on enzyme activity, but their presence increased its thermal stability. Ca^{2 +} displayed a higher stabilizing effect and at 10 mmol 1^-1 Ca^{2 +}, the enzyme retained 86% activity even after exposure at 70°C for 30 min. The amylase was induced on starch or maltose but was repressed strongly by glucose.     

Characterization of L-asparaginase from Bacillus sp. isolated from an intertidal marine alga (Sargassum sp.)

B.R. MOHAPATRA, R.K. SANI AND U.C. BANERJEE. 1995. The bacterial flora associated with an intertidal marine alga ( Sargassum sp.) were screened for the presence of extracellular L-asparaginase; one out of five Bacillus strains was found positive. The maximum L-asparaginase activity was found at 37°C and pH 8.0. The optimum NaCl concentration for enzyme activity was found to be 2% (w/v). The enzyme activity was not affected by the addition of different metal ions (Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺and Ni²⁺) at 10 mmol 1^-1, but was strongly inhibited by EDTA.     

The effects of two synthetic putrescine analogues on germination and appressorium formation by uredospores of Uromyces viciae-fabae on artificial membranes

M. REITZ, D.R. WALTERS, B. MOERSCHBACHER AND D.J. ROBINS. 1995. An examination was made of the effects of two synthetic putrescine analogues, ( E )-1,4-diaminobut-2-ene (E-BED) and ( E )-( N, N, N, N -tetraethyl)-1,4-diaminobut-2-ene (E-TED), on germination and appressorium formation by uredospores of the rust fungus Uromyces viciaefabae on artificial membranes. E-BED reduced germination by just 11% at 0.1 mmol 1^-1and by 24% at 1 mmol 1^-1, while appressorium formation was reduced by 37% at 0.05 mmol 1^-1and was completely prevented at 1 mmol 1^-1E-BED. The E-BED derivative E-TED reduced uredospore germination by 45% at 0.05 mmol 1^-1, while no appressoria were formed when uredospores were exposed to 0.05 mmol 1^-1E-TED. These results support previous suggestions that E-BED and E-TED exert their main effect on fungal development on the leaf surface.     

Effect of 2-chloroethylphosphonic acid on capsule formation and alkaloid content in Papaver somniferum

Application of different concentrations of ethephon (2-chloroethylphosphonic acid) to Papaver somniferum L. at the times of stem elongation, bud, and capsule formation produced different effects. Ethephon (10^-2 M ) retarded growth of the plant and inhibited capsule formation during stem elongation, significantly reduced capsule size during the flowering period, but did not alter capsule development during capsule formation. When applied during the period of stem elongation, ethephon (10^-3 M and 10^-4 M ) reduced capsule size; alkaloid accumulation was reduced by ethephon at a concentration of 10^-3 M , but slightly increased by 10^-4 M . Ethephon (10^-3 M and 10^-4 M ) did not alter capsule development or alkaloid content significantly when applied during bud formation, but stimulated capsule size and alkaloid content when applied during capsule formation. Pretreating the plants with Ag⁺ (silver nitrate) did not reverse the ethephon effect. The results suggest that capsule maturation and alkaloid accumulation in P. somniferum are modified by ethylene, which is produced as a result of exogenous ethephon treatment.     

2,4-Dichlorophenoxyacetic acid inhibition of potassium transport in barley seedlings

Growth, potassium uptake and translocation as well as transpiration rates were measured in intact low-salt barley seedlings ( Hordeum vulgare L. cv. Union) in the presence of different 2,4-D concentrations at pH 6.5. Growth was only affected at 10^-3 M .
Above 10^-7 M 2,4-D both uptake by the roots and transport to the shoots were inhibited. The inhibition at 10^-5 M remained constant for at least 24 h. Furthermore inhibition of uptake was measurable within 1 h. Excised roots and roots of intact plants showed the same uptake pattern.
It is suggested that the observed effects were caused by 2,4-D-induced changes in uptake and translocation systems in the roots. Pre-treatment with 10^-5 M 2,4-D had no effect upon subsequent potassium uptake. Transpiration was reduced within 1 h in 10^-4 or 10^-3 M 2,4-D, probably due to changes in water transport or root permeability.     

Citrate metabolism in 16 Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum strains

Citrate metabolism was studied in non-growing cells of Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum with respect to energetics, formation of degradation products and stoichiometry. The use of selective ionophores and uncoupler showed that citrate utilization was coupled to the proton motive force generated by ATP hydrolysis. Differences in citrate metabolism observed in 20 Leuconostoc strains were related to strains but not to the species or subspecies studied. Citrate metabolism was stimulated by glucose up to a concentration of 25 mmol 1^-1 and decreased at higher concentrations. The main degradation products resulting from the co-metabolism of citrate (10 mmol 1^-1) and glucose (2 mmol 1^-1) were acetate, lactate and pyruvate. Only four Leuconostoc strains produced low levels of acetoin and diacetyl. No strains produced ethanol or acetaldehyde. Citrate degradation ability was stable for at least 130 generations in 81% of the Leuconostoc strains.     

Isolation and properties of free and immobilized β-galactosidase from the psychrotrophic enterobacterium Buttiauxella agrestis (strain NC4)

A study of the β-galactosidase produced by the psychrotrophic bacterium Buttiauxella agrestis has been carried out. This micro-organism was isolated from raw milk and the enzyme isolated using standard methods. Molecular mass was estimated to be 515 kDa. The isoelectric point was close to 4·45. Optimum pH was 7·25. Maximal activity was observed at 50°C and activation energy was estimated to be 39·1 kJ mol^-1. Lactose enhanced thermal stability. Using α-nitrophenyl-β-D-galactopyranoside as the substrate, the K _m was 11 μmol 1^-1 and V _max was 85 U mg^-1 protein. β-Mercaptoethanol and ethanol were inhibitors; glycerol acted as a complex effector. The enzyme required divalent cations for activity while it was inhibited by EDTA. When the enzyme was immobilized in diethyl aminoethylcellulose the optimum pH of activity was 8. K _m was 47 μmol 1^-1 and V _max was 96 U mg^-1 protein.     

Sterilization of soybean cotyledons by boiling in lactic acid solution

F. RUÍZ-TERÁN AND J.D. OWENS. 1996. The effect of pH on the heat resistance of Bacillus stearothermophilus spores at 100°C in the presence of 0.11 mol 1^-1 lactic acid and 0.2 mol 1^-1 sodium phosphate buffer was examined. At pH values of 7.0 and 6.0 spores survived 60 min exposure unharmed but at pH 4.3 and 3.0 they died with decimal reduction times (DRTs) of 27 min and 2.8 min, respectively. Death rates were similar in the presence or absence of hydrated soybean cotyledons. In the presence of phosphate buffer and cotyledons at mean pH 3.6 the DRT was 118 min but in the presence, in addition, of lactic acid it was 11 min. It is suggested that the enhanced death rate was due to toxic effects of undissociated lactic acid. Rhizopus oligosporus NRRL 2710 grew well on cotyledons, having pH values from 7.0 to 3.7, prepared by boiling for 60 min in the presence of 0.11 mol 1^-1 lactic acid and 0.2 mol 1^-1 phosphate buffer.