The Influence of Inorganic Ions on the Heat Stability of a Thermophilic Bacteriophage

A thermophilic bacteriophage was isolated from soil. Heat inactivation of this phage, suspended in tryptone starch broth at 65°C and 70°C, was found to be a monomolecular reaction. The phage was more heat stable in tryptone broth than in tris buffer. When the tris buffer was supplemented with calcium or magnesium ions, the survival percentage increased from 0.0 to 18.0 after two hours of heating at 65°C. The addition of sodium or potassium ions to the tris buffer had no significant effect. Equimolar solutions of calcium and magnesium chloride had the same effect on the heat stability of the phage. Maximum stability was attained in 2.5 mM solutions of these salts, and a further increase in the concentration up to 10.0 mM did not increase the percentage of surviving phages.     

Effect of hydrogen ion buffers on photosynthetic oxygen evolution in the blue-green alga, Agmenellum quadruplicatum.

The photosynthetic oxygen evolution capacity of Agmenelium quadruplication suspended in four hydrogen ion buffers (pH 7.4, 0.05 M) and its synthetic marine growth medium was measured with an oxygen electrode. High rates of oxygen evolution were obtained in the growth medium and N-tris(hydroxymethyl)-methylglycine (Tricine) buffer. Compared to oxygen evolution in the growth medium, rates in phosphate buffer and N-tris(hydroxymethyl)-2-aminoethanesulphonic acid (TES) buffer were sometimes reduced by up to 30% and rates in tris (hydroxymethyl) amino-methane (Tris) were consistently reduced by 50%. An incubation-rinsing procedure caused inhibition of oxygen evolution in TES, phosphate, and Tris by 50 to 100%. Oxygen evolution could be restored to cells rinsed in TES or phosphate by resuspension in growth medium or in buffer plus magnesium and calcium ions. Bezoquinone-supported oxygen evolution was not affected by rinsing with any buffer tested except Tris. Ferricyanide was photoreduced at a low rate by cells rinsed in Tes but at a high rate in TES plus magnesium and calcium ions. We interpreted our results to mean that, in Agmenellum quadruplicatum, inhibition of photosynthetic oxygen evolution by Tris occurs at the level of photosystem 2 while the effects of TES and phosphate are on electron-transport occurring after the rate-limiting reaction.     

Buffer-induced rat liver mitochondrial aggregation during differential centrifugation

Use of buffers in homogenization media can result in loss of considerable particulate enzyme activity even with low-speed centrifugation. Addition of tris chloride buffer to 0.25 M sucrose homogenization media resulted in precipitation of 80 to 95% of the activity of two mitochondrial marker enzymes (3-hydroxy-3-methylglutaryl CoA lyase and citrate synthase) with the nuclear fraction during differential centrifugation. Lactate dehydrogenase, a cytoplasmic marker, was not precipitated under the same conditions, indicating that the precipitated enzymes were not associated with intact cells. Photomicrographs showed that tris chloride buffers resulted in mitochondrial aggregation. Isolated mitochondria resuspended in tris chloride or potassium phosphate buffer also aggregated, which resulted in a marked decrease in assayable mitochondrial enzyme activity.     

Role of calcium in the localization of maternal poly(A)+RNA and tubulin mRNA in Xenopus oocytes

Summary Poly(A)⁺RNA and tubulin mRNA are localized in the periphery of Xenopus oocytes and become delocalized during meiotic maturation. Delocalization of this RNA can be triggered by incubation in agents which reduce entry of calcium ions into the cell (e.g. lanthanum chloride and verapamil). Although these agents ordinarily promote meiotic maturation, addition of theophylline to the medium will inhibit maturation but not delocalization. Manipulations which prevent calcium entry without inducing meiotic maturation (e.g. calcium-free buffer) are also shown to trigger disruption of the RNA localization. In addition, manipulations which reduce chloride efflux from the cell (e.g. increasing the external chloride ion concentration with choline chloride) result in disruption of the localization of poly (A)⁺ RNA and tubulin mRNA without inducing meiotic maturation. The calcium-dependent chloride efflux present in Xenopus oocytes disappears after the oocyte has been stimulated to proceed through meiotic maturation. We show that reduction of the influx of calcium ions or efflux of chloride ions induces the delocalization of poly (A)⁺RNA and tubulin mRNA without inducing meiotic maturation. We suggest, therefore, that reducing the transmembrane movement of these ions is likely to be the natural trigger for the delocalization of poly(A)⁺RNA and tubulin mRNA.     

Determination of the sensitivity of bacteria to barium ions,a taxonomic marker of the genus <Emphasis Type="Italic">Pseudomonas</Emphasis>

Comparative efficacy of the determination of the sensitivity of bacterial cells to barium ions was evaluated on a synthetic nutrient medium, FMH agar, Mueller-Hinton agar, and AGV agar. The synthetic nutrient medium developed for this study contained L-proline and L-glutamine as the sole nitrogen and carbon source, which promoted growth of all Pseudomonas strains and ensured the minimal level of barium binding. The sensitivity of 80 strains belonging to 11 Pseudomonas species, including the type strains, as well as of 80 strains of 22 other bacterial species, was studied. The sensitivity of bacteria to barium ions was determined by using serial dilutions of barium chloride in the nutrient medium. The highest level of analytical sensitivity of pseudomonads to barium ions was determined on the synthetic nutrient medium: the minimal inhibitory concentration (MIC) values of barium chloride ranged from 0.5 to 6 g/L, the MIC₉₀ value was 2 g/L. At the same time, 86.1% of all strains of fluorescent Pseudomonas species produced fluorescein on the control BaCl₂-free synthetic nutrient medium. For representatives of other genera grown on all the studied nutrient media, the MIC values of barium chloride ranged from 20 to 50 g/L. The proposed method for determination of the sensitivity of bacteria to barium ions using the synthetic nutrient medium with 6 g/L of barium chloride as a criterion for the classification of barium-sensitive strains to the genus Pseudomonas is suitable for standardization.     

Inhibition of bacterial growth by metal salts. The accumulation of ribonucleic acid during inhibition of Escherichia coli by cobalt chloride

During inhibition of the growth of Escherichia coli by cobalt chloride protein synthesis was decreased more than the synthesis of RNA. Three species of particle accumulated during the incubation. These had sedimentation coefficients of about 44s, 33s and 23s in tris buffer containing 10 mm-magnesium acetate and 100 mm-potassium chloride, but their sedimentation properties were susceptible to changes in buffer composition. The particles contained RNA but were more readily degraded by ribonuclease than were the ribosomes. RNA isolated from the particles differed slightly in sedimentation properties from the major species of ribosomal RNA. The particles are likely to be closely related to ribosome precursors that have been detected in other circumstances. Changes in the polyribosome fraction during inhibition by cobalt chloride, nickel chloride and chloramphenicol provided further evidence that inhibition by Co(2+) involves specific effects on the protein-synthesizing machinery.     

The effect of osmotic `shock'' on the swelling pattern and respiratory control of rat-liver mitochondria

1. Rat-liver mitochondria suspended in 0.25m-sucrose were exposed for a few seconds to strongly hypo-osmotic conditions, and then the osmolarity of the medium was raised again to 0.25 with the aid of tris chloride (osmotic ;shock'). 2. Mitochondria after hypo-osmotic pretreatment lost their capacity for slow energy-dependent swelling in iso-osmotic tris buffer and showed no respiratory control. 3. Swelling could be induced in the ;shocked' mitochondria by ATP but not by addition of respiratory substrates. 4. It was shown that cytochrome c is lost from ;shocked' mitochondria when they come into contact with the tris buffer present in the assay medium, and that the changes observed in the pattern of swelling, as well as in respiratory control, are directly connected with this loss of cytochrome c. 5. The results of the investigation are discussed with regard to the role of cytochrome c in swelling and respiratory control.     

The production of extracellular thermostable neutral proteinase and α-amylase byBacillus stearothermophilus

Summary Extracellular thermostable neutral proteinase was produced byBacillus stearothermophilus strains NCIB 8924 and NRRL B-3880 growing at 55°C. The formation and stabilization of this proteinase was found to be dependent on the concentration of free calcium ions. Therefore, procedures that removed free calcium ions from the medium, such as the use of phosphate buffer, resulted in a lower production of proteinase. The calcium-deficient proteinase was denaturated or adsorbed by calcium phosphate compounds. During the sterilization procedure of the culture medium, the CaCO₃ precipitation, caused by the removal of CO₂, influenced the amount of proteinase produced in a phosphate buffered medium made with tap water. An improved medium without phosphate buffer was used for 10 and 300 l batch cultivations and the calcium requirement for proteinase formation by the two strains was determined.     

The effect of monovalent cations on calcium efflux in yeasts

The properties of the calcium efflux system in the yeast Saccharomyces cerevisiae were investigated. After growing the cells overnight in medium containing ⁴⁵Ca, the cells were transferred to medium containing glucose, Hepes buffer (pH 5.2) and monovalent cations. The presence of potassium or sodium in the medium induced efflux of calcium from the cells. The magnitude of the efflux was dependent on the concentration of these cations in the medium. The time course of calcium efflux was analyzed, and two types of exchangeable calcium pools, which turned over at different rates, were detected: ‘Fast turnover’ and ‘slow turnover’. Increase in the concentration of monovalent cations in the medium caused an increase in the fraction of cellular calcium which turned over at a fast rate, and activation of calcium efflux from the ‘slow turnover’ calcium pool. The specific changes in the parameters of calcium efflux induced by monovalent cations were different from those reported previously to be induced by divalent cations. Both processes, i.e. activation of calcium efflux by monovalent and by divalent cations, were found to be additive, indicating that they operate via different mechanisms. Experiments using the respiratory inhibitor Antimycin A, showed that stimulation of calcium efflux by monovalent cations is energy dependent. Lanthanum ions which are known to inhibit calcium influx into yeast cells, inhibitted the activation of calcium efflux by both divalent and monovalent cations. Determination of the cationic composition of the cells indicated that the stimulation of calcium efflux was accompanied by influx of potassium or sodium into the cells.     

Enhancement and stabilization of the production of glucoamylase by immobilized cells of Aureobasidium pullulans in a fluidized-bed reactor

Summary Glucoamylase production by Aureobasidium pollulans A-124 was compared in free-living cells, cells immobilized in calcium alginate gel beads aerated on a rotary shaker (agitation rate 150 rpm), and immobilized cells aerated in an air bubble column reactor. Fermentation conditions in the bioreactor were established for bead concentration, substrate (starch) concentration, calcium chloride addition to the fermentation medium, and rate of aeration. Production of glucoamylase was optimized at approximately 1.5 units of enzyme activity/ml medium in the bioreactor under the following conditions: aeration rate, 2.0 vol air per working volume of the bioreactor (280 ml) per minute; gel bead concentration, 30% of the working volume; substrate (starch) concentration, at 0.3% (w/v); addition of calcium chloride to the medium at a final concentration of 0.01 M. Productivity levels were stabilized through the equivalent of ten batches of medium with the original inoculum of immobilized beads. Offprint requests to: M. Petruccioli     

The influence of a specific microelemental environment in alginate gel beads on the course of propionic acid fermentation

In this work the exchange of calcium, cobalt, iron, magnesium, zinc and manganese ions between alginate gel beads and casein medium was investigated. The high release of calcium ions from alginate to the medium and the biosorption of some metal ions were observed. The pure alginate gel adsorbed all the metal ions examined, from a fermentative medium. Gel with immobilized cells of two strains of Propionibacterium freudenreichii subsp. shermanii showed an active ability to adsorb only cobalt, iron and zinc ions. In this way, a special microelemental environment was created in the alginate gel. This resulted in an increase of propionic acid production and a decrease of vitamin B₁₂ biosynthesis. Received: 30 April 1997 / Received revision: 2 July 1997 / Accepted: 4 July 1997     

On the Nature of the Heat Resistance of Thermophilic Bacteria

The importance of inorganic ions for the heat stability of thermophilic bacteria was investigated. Cells of Bacillus stearothermophilus, strain 1503, were incubated at elevated temperatures in various media and the number of surviving organisms was determined at suitable intervals. The bacteria rapidly died at temperatures ordinarily employed for their cultivation if the surrounding medium lacked calcium ions. Besides calcium ions, potassium and phosphate ions and glucose, or some other energy source, seemed to be required for the heat stability of the cells. A chemically defined stabilizing medium with these components was developed for the above-mentioned strain. When any component of this medium was excluded, the heat resistance of this organism was lost. This medium had a stabilizing effect also on the cells of three other strains of B. stearothermophilus. These requirements suggest that the heat stability of thermophilic bacteria is attributable to an active transport of calcium ions from the environment into the cells.     

Method for the lysis of Gram-positive, asporogenous bacteria with lysozyme.

A method developed for the lysis of oral streptococci that employed the action of lysozyme suspended in dilute tris(hydroxymethyl)aminomethane-hydrochloride buffer containing polyethylene glycol has been adapted for use with lactobacilli, actinomycetes, propionibacteria, and pediococci. Most of the cellular deoxyribonucleic acid was liberated from many strains of bacteria usually thought to be lysozyme resistant. The major observations were as follows: (i) supplementation of the growth medium with L-threonine, L-lysine, or both frequently produced cells that were more susceptible to lysis by lysozyme; (ii) glucose-containing media produced cells that were more easily lysed than those from cultures grown on other substrates; (iii) polyethylene glycol not only served as an osmotic stabilizer, it also enhanced the extent of lysis; and (iv) dilute tris(hydroxymethyl)aminomethane buffer was superior to the buffer systems most commonly employed in published muramidase-based lysis techniques. Stationary-phase cells of Lactobacillus casei and Streptococcus mutans were more easily lysed than those isolated from log-phase cultures. The method as detailed in this report should be generally applicable for the lysis of gram-positive, asporogenous bacteria.     

On the Nature of the Heat Stabilizing Effect of Inorganic Ions on Escherichia coli

The growth lag of Escherichia coli at 45°C was reduced by the addition of sodium, potassium, magnesium and calcium ions to the growth medium. A method to quantitatively determine the lag-reducing effect of these ions was developed. The results obtained showed that equivalent amounts of the ions produced the same reduction of the growth lag. According to the results of plasmolysis experiments cells of E. coli suspended in peptone broth were permeable to all four ions. The course of plasmolysis and subsequent deplasmolysis was registered as changes in the cells' ability to scatter light. The heat stability of catalase from E. coli was increased by addition of the four ions. This was observed in experiments with intact cells and with a crude cell-free preparation of catalase. The results of our experiments are most easily explained by assuming a stabilizing effect of the ions tested on the intracellular bacterial proteins.     

Activity of Stereoisomers of Serricornin,Sex Pheromone of the Cigarette Beetle ( Lasioderma serricorne F.)

Formation of quinoprotein methanol dehydrogrnase (MDH) in Methylobacillus glycogenes, an obligate methylotroph, was strictly controlled by calcium (Ca) in the culture medium. Both the growth of the organism and the total enzyme activity of MDH were also repressed at less than 1 ppm of Ca, although specific activity of MDH showed a similar level. Ca in MDH was replaced with other metals during cultivation of M. glycogenes. When strontium (Sr) chloride was fed instead of CaCl₂, Ca in MDH was completely replaced by Sr and showed a specific activity over ten times higher than Ca-containing MDH, although there was no appreciable increase in the MDH content in cells cultured in Sr medium. Methanol oxidase activity was also elevated in the cells that were cultured in the medium with Sr.     

Effect of calcium and anaerobiosis on the thermostability of Bacillus stearothermophilus.

Bacillus stearothermophilus NCA 2184 lost viability and subsequently released cytoplasmic components when suspended in 0.1 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.2) and incubated at 60 degrees C. Cell lysis was prevented by the addition of 10 mM CaCl2 to the Tris-buffer suspension. Cells which were incubated under anaerobic conditions for 20 min in the growth medium before they were collected were stable in the Tris-buffer suspension without added calcium. Anaerobic incubation effected an increase in membrane cardiolipin which appeared to be related to the increase in the thermostability of the cells.     

Energy-dispersive X-ray analysis of the extracellular cadmium sulfide crystallites of Klebsiella aerogenes

Klebsiella aerogenes forms electron-dense partieles on the cell surface in response to the presence of cadmium ions in the growth medium. These particles ranged from 20 to 200 nm in size, and quantitative energy dispersive X-ray analysis established that they comprise cadmium and sulfur in a 1:1 ratio. This observation leads to the conclusion that the particles are cadmium sulfide crystallites. A combination of atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, and acid-labile sulfide analysis revealed that the total intracellular and bound extracellular cadmium:sulfur ratio is also 1:1, which suggests that the bulk of the cadmium is fixed as extracellular cadmium sulfide. The tolerance of K. acrogenes to cadmium ions and the formation of the cadmium sulfide crystallites were dependent on the buffer composition of the growth medium. The addition of cadmium ions to phosphate-buffered media resulted in cadmium phosphate precipitates that remove the potentially toxic cadmium ions from the growth medium. Electrondense particles formed on the surfaces of bacteria grown under these conditions were a combination of cadmium sulfide and cadmium phosphates. The specific bacterial growth rate in the exponential phase of batch cultures was not affected by up to 2mM cadmium in Tricine-buffered medium, but formation of cadmium sulfide crystallites was maximal during the stationary phase of batch culture. Cadmium tolerance was much lower (10 to 150 M) in growth media buffered with Tris, Bistris propane, Bes, Tes, or Hepes. These results illustrate the importance of considering medium composition when comparing levels of bacterial cadmium tolerance.Abbreviations EDXA Energy dispersive X-ray analysis - AAS Atomic absorption spectroscopy - TEM Transmission electron microscopy - SEM Scanning electron microscopy - ICP-MS Inductively coupled plasma mass spectrometry - ALSA Acid-labile sulfide analysis     

The causes of the biological action of electrochemically activated solutions by changes in the growth of Escherichia coli cells

To study the causes of the biological effect of electrochemically activated solutions, nutrient growth media M 9 were prepared using catholyte and anolyte solutions containing separate components of the nutrient medium, such as distilled water, phosphate buffer, phosphate buffer with chlorides (NaCl, NH4Cl), and chlorides. The biological activity of different nutrient media was assessed by a comparison with the stimulation or inhibition of the growth of Escherichia coli cells in the catholyte and anolyte of the complete nutrient medium M 9. It was shown that medium M 9 prepared on the catholytes of different initial solutions acquired the stimulating properties only if the initial solution contained salts containing chlorine. The stimulating effect of the initial solution was 18-24%. Electrochemical treatment of solutions containing no chlorides (distilled water, phosphate buffer) and subsequent addition of the components of nutrient medium to exposed solutions had neither a stimulating nor the inhibiting effect on cell growth. The cultivation of cells in a nutrient medium based on the catholyte of preliminarily treated hydrochloric acid showed that it is the presence of chlorine ions in solution during electrolysis that causes the stimulating effect of the nutrient medium based on the catholyte. The formation of oxidizers and the inhibitory effect of the anolyte described previously was also observed if the solution contained chlorine ions during electrolysis. Possible mechanisms of the biological effect of catholytes containing chlorides during electrolysis were discussed.     

Blood-Free Medium for the Rapid Growth of Pasteurella tularensis

A medium composed of (in g/100 ml) Tryptose broth with thiamine (Difco), 2.6; cysteine-HCl, 0.12; glucose, 1; FeSO₄, 7H₂O, 0.005; KCl, 0.02; histidine, 0.1; tris(hydroxymethyl)aminomethane (tris) buffer, 0.3; and agar, 1; will support rapid growth of the fully virulent SCHU-S4 strain of Pasteurella tularensis. Although the test organism grew rapidly on medium from which KCl and tris buffer were omitted, these two components increased the stability of the medium upon storage at 4 C. It was necessary to (i) control carefully the relative concentration of the ferrous iron and cysteine-HCl, (ii) incubate the prepared medium overnight prior to use, and (iii) incubate the inoculated plates in an atmosphere of high relative humidity. Rapid growth of the organism was obtained also from very small inocula in the liquid form of the medium. Biochemical studies designed to elucidate the mechanisms involved in the enhancement of growth of P. tularensis in this relatively simple blood-free medium were initiated.     

Vasoconstrictor hormones depolarize renal glomerular mesangial cells by activating chloride channels

Mesangial cells are smooth muscle-like cells of the renal glomerulus which contract and produce prostaglandins in response to vasopressin and angiotensin. These responses serve to regulate the glomerular capillary filtering surface area. We have used the membrane potential-sensitive fluorescent dye bis-oxonol and the intracellular fluorescent calcium-sensitive probe Indo-1 to study the changes in membrane potential (Em) and intracellular free calcium concentration ([Ca2+]i) in cultured rat mesangial cells in response to vasoconstrictor hormones. Basal [Ca2+]i was 227 +/- 4 nM, and stimulation by maximal concentrations of either vasopressin or angiotensin resulted in a transient 4-6-fold rise. Resting membrane potential was 45.8 +/- 0.9 mV and vasoconstrictor hormones caused a depolarization of 14-18 mV. The following extracellular ion substitutions indicated that chloride efflux was the predominant ion flux responsible for depolarization: 1) depolarization persisted when sodium in the medium was substituted with N-methylglucamine; 2) substitution of medium sodium chloride with sodium gluconate, which enhances the gradient for chloride efflux, augmented vasoconstrictor-stimulated depolarization; 3) suspension of cells in potassium chloride medium resulted in depolarization, following which, stimulation by either vasopressin or angiotensin resulted in hyperpolarization; and 4) this hyperpolarization did not occur when potassium gluconate medium was used to depolarize the cells. The calcium ionophore ionomycin also resulted in membrane depolarization. However, prevention of the rise in [Ca2+]i by prior exposure to ionomycin in calcium-free medium or by loading mesangial cells with the intracellular calcium buffer BAPTA did not abrogate the depolarization response to vasoconstrictor hormones. This indicates that a rise in intracellular calcium is not necessary for depolarization. In contrast, prior depolarization of the cells using varying concentrations of KCl in the external medium, which dissipated the electrochemical gradient for chloride efflux, resulted in a corresponding prolongation of the transient calcium response to vasopressin and angiotensin. These findings indicate that angiotensin and vasopressin depolarize mesangial cells by activating chloride channels and that this activation can occur by both calcium-dependent and -independent mechanisms. In addition, activation of chloride channels with resulting depolarization may serve to modulate the calcium signal.