Effects of sub-minimal inhibitory concentrations of EDTA on growth of Escherichia coli and the release of lipopolysaccharide

Abstract Release of lipopolysaccharide from E. coli was studied in the presence of sub-minimal inhibitory concentrations of ethylenediaminetetraacetic acid (EDTA). In untreated cells no release was detected with 50 mM Mg²⁺ in the medium, but a steady release of over 50% of the synthesized lipopolysaccharide was observed with 0.1 mM Mg²⁺. EDTA at MIC/8 led to a 2- to 3-fold higher release, presumably by an adjustment of the concentration of unchelated Mg²⁺ to a value still sustaining normal growth but giving rise to a highly unstable outer membrane. No structural difference was observed between cell-bound and released lipopolysaccharide.     

Effects of Divalent Metal Cations in the Growth Medium upon Sensitivity of Batch-grown Pseudomonas aeruginosa to EDTA or Polymyxin B

The sensitivity of batch grown Pseudomonas aeruginosa to ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(2-aminoethyl ether)-N, N-tetraacetic acid (EGTA) and to polymyxin depended upon the nature and concentration of divalent metal cations (Ca²⁺, Mg²⁺, Ba²⁺, Mn²⁺, Sr²⁺, Zn²⁺, Fe²⁺, Al³⁺) present in the simple salts-glucose growth medium. Sensitivity to the antibacterial agents was measured in terms of per cent decrease in optical density which was proportional to total count. The rate of decrease in optical density and the per cent decrease after a given time were proportional to the cation content of the growth medium. The lytic action of the agents correlated to some extent with the stability constants of the cation for the two chelating agents.
The data support the theory that EDTA and polymyxin both act initially at a common site of action, namely the Mg²⁺ and Ca²⁺ cross bridges that stabilize components of the outer membrane.     

Characterization of acetylcholinesterase from Arthrobacter ilicis associated with the marine sponge (Spirastrella sp.)

The bacterium Arthrobacter ilicis isolated from the marine sponge Spirastrella sp. produces extracellular serine type acetylcholinesterase. The maximum enzyme activity was found at 45 °C and pH 8·0. The activation and deactivation energies, calculated from an Arrhenius plot, were 13·68 and 36·96 kcal mol⁻¹, respectively. The enzyme was not affected by the addition of the major cations of sea water, such as Ca²⁺ and Mg²⁺ at 25 mmol l⁻¹, and was strongly inhibited by EDTA and different organophosphorus and carbamate compounds at 5 mmol l^−1.     

MAGNESIUM REQUIREMENT IN FERTILIZATION PROCESS OF SEA URCHINS

The Mg²⁺ requirement in fertilization was investigated in sea urchins. It was found that when sea urchin eggs were inseminated in sea water free of Mg²⁺, little fertilization took place. Even when spermatozoa pre-treated with dissolved egg-jelly to induce the acrosome reaction, which needs Ca²⁺, were used, the fertilization rate remained quite low in the absence of Mg²⁺. In Strongylo-centrotus intermedius , the lowest concentration of Mg²⁺ required for 50% fertilization was 0.05 mM in the presence of 10 mM Ca²⁺, whereas that of calcium was 3 mM in the presence of 49 mM Mg²⁺. These critical concentrations increased when the concentration of the other ion decreased. Removal of Mg²⁺ or Ca²⁺ or both from the suspending medium had little adverse effect on sperm motility. The elevation of the fertilization membrane was also induced by butyric acid independent of the presence or absence of Mg²⁺ and/or Ca²⁺. These results indicate that Mg²⁺ are required at least in some process(es) between acrosome reaction and fertilization membrane elevation, such as sperm penetration or membrane fusion.     

Uptake of Magnesium by Chromaffin Granules In Vitro: Role of the Proton Electrochemical Gradient

Abstract: The chromaffin granule membrane in vitro is impermeable to protons as well as to Mg²⁺; however, when granules are incubated in the presence of the proton ionophore carbonyl cyanide p -trifluoromethoxy-phenylhydrazone or an inhibitor of the granule membrane Mg²⁺-dependent ATPase, the metal ion is accumulated inside the granules. This accumulation is dependent upon the granule transmembrane potential. The simultaneous presence of the ATPase inhibitor and the proton ionophore markedly increases metal ion incorporation. Mg²⁺ incorporation is also promoted by nigericin in the presence of potassium or sodium ions, indicating that Mg²⁺ accumulation is also dependent upon the transmembrane pH gradient. Concomitant with the Mg²⁺ accumulation, there is a significant loss of endogenous catecholamines. It is concluded that Mg²⁺ accumulation is determined by the electrochemical gradient maintained across the membrane. Once the metal ion has accumulated into the granules it displaces catecholamines from their storage sites.     

Biochemical characterization of pectate lyases produced by fluorescent pseudomonads associated with spoilage of fresh fruits and vegetables

An improved method for purification of pectate lyases (PLI and PLII) from culture fluids of Pseudomonas fluorescens CY091 and Ps. viridiflava PJ-08-6 by using a phosphocellulose cation exchanger was described. Analysis of purified PLI and PLII by sodium dodecyl sulphate-polyacrylamide and isoelectric focusing gel electrophoresis revealed that both enzymes had been purified to near homogeneity. Optimal Ca²⁺ concentration required for PLI and PLII activity was determined to be 0·5 mmol l⁻¹. The Ca²⁺ requirement could not be replaced by other metal cations such as Mg²⁺, Cu²⁺, Zn²⁺, Fe³⁺ and Co²⁺. Optimal pH for activity was determined to be between 8·5 and 9·0. The K _m values for sodium polygalacturonate were 1·28 and 1·11 mg ml⁻¹ for PLI and PLII, respectively. Both PLI and PLII were stable at low temperatures (25°C or below) for at least 1 month. However, at 37°C, the activity decreased 50% in 36 h. Optimal temperatures for activity were estimated to be 46° and 52°C for PLI and PLII, respectively. Thermal stability of both enzymes at elevated temperatures (48°C or higher) increased when CaCl₂ or a positively charged molecule such as polylysine was present, but decreased when polygalacturonate or a negatively charged molecule such as heparin was present. PLI and PLII exhibit differential degrees of sensitivity to group-specific inhibitors, including iodoacetic acid and diethylpyrocarbonate. This result suggests that both sulphydryl and imidazole groups are important for the catalytic function of PLI and PLII.     

Mg2+-free buffer elevates transformation efficiency of Vibrio parahaemolyticus by electroporation

Aims:  The ability to transform Vibrio spp. is limited by the extracellular nuclease that their cells secrete. The reported transformation efficiency of this organism is 10²–10⁵ transformants per microgram DNA. We tried different buffers and conditions, aiming to elevate its transformation efficiency.
Methods and Results:  MgCl₂ and sucrose are often included in the washing and/or electroporation buffers to stabilize the cell membrane. However, Mg²⁺ is required for production and activity of the extracellular nuclease. A simple electroporation buffer lacking Mg²⁺ was found to increase transformation efficiency dramatically, to levels 50-fold more than the buffers containing Mg²⁺. To maintain the stability of the cell membranes, Mg²⁺ was replaced with high concentrations of sucrose, from 272 to 408 mmol l⁻¹. With the new buffers, the transformation efficiency of Vibrio parahaemolyticus was increased to 2·2 × 10⁶ transformants per microgram DNA.
Conclusions:  Mg²⁺ in the buffer adversely affected transformation of V. parahaemolyticus by electroporation. The cell membranes of vibrio can be stabilized by high concentration of sucrose when Mg²⁺ is absent.
Significance and Impact of the Study:  A greater transformation efficiency can facilitate the genetic analysis of an organism and its pathogenicity. Buffers lacking Mg²⁺ can be used for other nuclease-producing organisms.     

Magnesium Transport by Brain Mitochondria: Energy Requirement and Dependence on Ca2+ Fluxes

Abstract: The association of Mg²⁺ ions with mitochondria isolated from guinea pig cerebral cortex is investigated and resolved into two components, that bound to the surface of both the outer and the inner membranes and that transported into the mitochondrial matrix. When rotenone-treated mitochondria are preincubated in a Mg²⁺ -containing medium, Mg²⁺ binding can be measured and actual Mg²⁺ transport determined after the addition of succinate. Mg²⁺ uptake as well as retention within mitochondria is an energy-dependent process linked to substrate oxidation. EGTA completely prevents Mg²⁺ uptake, while the Ca²⁺ uniporter inhibitor Ruthenium Red, along with prevention of Mg²⁺ uptake, induces a slow efflux of accumulated Mg²⁺ ions. These findings suggest that both inward and outward Mg²⁺ movements follow Ca²⁺ fluxes across the mitochondrial membrane. Modulation of Mg²⁺ movements by mitochondria is therefore suggested to occur within nerve terminals.     

Characterization of the interaction of bovine plasmin with Streptococcus uberis

The binding of plasmin to Streptococcus uberis strain 0140 J was optimal in the pH range 5·0–5·5. Plasmin binding decreased exponentially with increasing NaCl concentration (0–0·8 mol l⁻¹), reaching a minimum at NaCl concentrations exceeding 0·55 mol l⁻¹. Neither K⁺, Mg²⁺ nor the metal chelator EDTA had any effect on the interaction. Plasmin binding was prevented, in a concentration-dependent manner, by the amino acids lysine, arginine and ε-aminocaproic acid. Bound plasmin was also eluted from the bacterial cell using the same amino acids. Bound plasmin was lost from the bacterium in a time- and temperature-dependent fashion, the rate of plasmin loss increased with increasing temperature over the range 4–55 °C, and the elution of plasmin from live and heat-killed bacteria was similar. Cell-bound plasmin was only partially inhibited by the physiological inhibitor α₂-antiplasmin whereas the serine protease inhibitor aprotinin, and the active site titrant p -nitrophenyl- p -guanidiniobenzoate, inhibited the activity of the cell-bound plasmin by more than 95%.     

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.     

Enhancement of Tryptophan Uptake by Divalent Cations in the Absence of Sodium Ions

Abstract: Nations were found to inhibit the uptake of L-tryptophan into synaptosomes with a shallow dose-response curve. Almost maximal inhibition was obtained with 10 mM-Na⁺. The divalent cations Ca²⁺ and Mg²⁺ were shown to be responsible for the increased uptake of L-tryptophan in the absence of Na⁺ ions. Other divalent cations also promoted tryptophan uptake under this condition (Ca²⁺ < Mg²⁺ < Mn²⁺ < Fe²⁺ < Zn²⁺ < Cu²⁺). It was concluded that monovalent chelate complexes were responsible for this enhancing effect. The measured L-tryptophan uptake was the net product of membrane bound and unbound tryptophan. Both bound and unbound tryptophan were increased in the presence of divalent cations. If no divalent cations were added to the incubation medium, Na⁺ ions decreased the unbound tryptophan but were without effect on bound tryptophan. Under these circumstances D-tryptophan had no effect on binding of the L-isomer and affected the transport of 1.-tryptophan only at very high does (100 x conc. L-tryptophan). These results suggest that I -tryptophan binds to a stereospecific transport carrier located in the synaptosomal membrane and that Na⁺ ions prevent the translocation of this carrier amino acid complex from the outer to the inner site of the neuronal membrane.     

Chlorophyll Formation in Euglena gracilis var. bacillaris: Effect of Chelating Agents

SYNOPSIS. 8-Hydroxyquinoline (8-HQ) and cysteine markedly inhibit the synthesis of chlorophylls in non-proliferating etiolated Euglena gracilis var. bacillaris on illumination. This is thought to be due, at least to some extent, to the binding of Mg²⁺ ions in the non-proliferation medium, but largely as a consequence of intracellular chelating action. The effect of 8-HQ could be reversed by the presence of metal ions higher in the Mellor-Malley series than Mg²⁺, e.g., Zn²⁺ and Co²⁺. Sodium diethyldithiocarbamate (Na-DDG) and ethylenediaminetetraacetic acid (EDTA) have no effect on chlorophyll synthesis in non-proliferation conditions. Growth in the light is more affected than growth in the dark by all these chelating agents, including Na-DDG and EDTA. This is due to the differential requirement of Mg²⁺ under these 2 growth conditions.     

Plasmalemma from the roots of cucumber: Isolation by two-phase partitioning and characterization

Plasmalemma was isolated from the roots of 2-week-old cucumber plants ( Cucumis sativus L. cv. Rhensk druv) by utilizing an aqueous polymer two-phase system with 6.5%:6.5% (w/w) Dextran T500 and polyethylene glycol (PEG) 3350 at pH 7.8. The plasmalemma fraction comprised ca 6% of the membrane proteins contained in the microsomal fraction. The specific activity of the plasma membrane marker enzyme (K⁺, Mg²⁺-ATPase) was 14- to 17-times higher in the upper (PEG-rich) than in the lower (Dextran-rich) phase, and the reverse was true for marker enzymes (cytochrome c oxidase, EC 1.9.3.1, and antimycin A-resistant NADPH cytochrome c reductase) of intracellular membranes. The ATPase was highly stimulated by the addition of detergent (Triton X-100), so that the isolated plasmalemma vesicles appear tightly sealed and in a right-side-out orientation. Further characterization of the ATPase activities showed a pH optimum at 6.0 in the presence of Mg²⁺. This optimum was shifted to pH 5.8 after addition of K⁺. K⁺ stimulated the ATPase activity below pH 6 and inhibited above pH 6. The ATPase activity was specific for ATP and sensitive to N,N-dicyclohexylcarbodiimide and sodium vanadate, with K⁺ enhancing the vanadate inhibition. The enzyme was insensitive to sodium molybdate, NO⁻₃, azide and oligomycin. No Ca²⁺-ATPase was detected, and even as little as 0.05 m M Ca²⁺ inhibited the Mg²⁺-ATPase activity.     

Short-term thermal acclimation induces adaptive changes in the inner mitochondrial membranes of fish skeletal muscle

In the oxidative muscles (musculi laterales superficiales) of crucian carp Carassius carassius acclimated for 6 weeks to either 5 or 25° C, the volume density and the surface density of fibres per tissue did not differ significantly between the control and experimental groups. The correlation ratio (μ²) for these values was below 50, 39·3 and 43·9 respectively. After acclimation to 5° C, the surface density of outer mitochondrial membrane per fibre increased significantly from 0·93 to 1·23m² cm⁻³ in the summer population but dropped from 0·94 to 0·67 m² cm⁻³ in the winter population. The surface density of outer mitochondrial membrane per mitochondrion increased from 3·24 to 4·52 m² cm⁻³ in summer fish. After acclimation to 25° C, the surface density of inner mitochondrial membranes per muscle fibre decreased from 4·04 to 1·79 m² cm⁻³ in summer fish and from 3·86 to 1·07 m² cm⁻³ in winter fish. The surface density of inner mitochondrial membranes per mitochondrion increased from 14·17 to 15·60 m²cm⁻³ in summer fish but dropped from 13·91 to 10·67 m² cm⁻³ in winter fish. Correlation matrices demonstrate a negative correlation of the surface density of outer mitochondrial membrane per mitochondrion with the volume density of mitochondria per fibre and temperature, suggesting cold-induced proliferation of small mitochondria. It was concluded that short-term cold acclimation increased surface area of the inner mitochondrial membranes in summer fish.     

Ontogenesis of Ca++ and Mg++ Contents of Embryonic Chick Heart

The Ca⁺⁺ and Mg⁺⁺ contents of embryonic chick heart were studied by atomic absorption spectrophotometry during a period from 48 h of foetal development until 2-3 days post-hatching. The hearts were isolated and incubated for 40 min at 22°C in three different media aerated with 95% 0₂-5% C0₂. The media included: normal Ringer's; Ca⁺-free Ringer's with 3 mM EGTA; and Ca⁺⁺-free Ringer's with 3 mM EDTA. At 48 h, the tubular myocardium contained 7-3 mM Ca⁺⁺ per wet weight which decreased rapidly to 1-2 mM by 10 days of development and remained between 0-9 and 1-1 mM until hatching. The Ca⁺⁺ content paralleled the changes in Na⁺ content reported earlier. Treatment with excess chelators, EGTA or EDTA, resulted in removal of 65-75% of the Ca⁺⁺ content throughout development until the time of hatching, when 50% of the Ca⁺⁺ became firmly bound. In contrast to the results with Ca⁺⁺, myocardial Mg⁺⁺ content rose rapidly from an initial value of 3.2 mM at 48 h to 6.7 mM by the 5th day of development, and then gradually declined throughout the remaining foetal development to 4.8 mM 2-3 days post-hatching. The Mg⁺⁺ contents closely paralleled changes in K⁺ content during development, which were reported earlier. Treatment with EGTA and EDTA removed 13-22% and 19-28% of the myocardial Mg⁺⁺, respectively, during development until just prior to hatching, when only 10-12% could be removed by chelation.     

The Effect of Divalent Cations on Aggregation of Dictyostelium discoideum

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20–40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl₂ accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 × 10⁻⁵ M progesterone considerably delays it These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg²⁺ or the ionophore and greatly decreased accumulation in the case of the steroid.
Treatment with EDTA and Mg²⁺ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn²⁺ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg²⁺ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca²⁺ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca²⁺.
These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

Cell Envelopes of Gram negative Bacteria: Composition, Response to Chelating Agents and Susceptibility of Whole Cells to Antibacterial Agents

The effects of ethylenediamine tetraacetic acid (EDTA) and related chelating agents on the sensitivity of isolated cell envelopes of some β-lactamase +ve and -ve strains of Gram negative bacteria have been investigated. Envelopes from Pseudomonas aeruginosa (especially strain NCTC 1999) contained the greatest amounts of Mg²⁺ and were the most sensitive to these agents in terms of (i) lysis, (ii) release of cations, (iii) release of readily extractable lipid. Cyclohexane—1,2, -diamine-tetraacetic acid was the most effective chelator, followed by EDTA and N -hydroxy-ethylethylenediamine triacetic acid, with nitriloacetic acid and iminodiacetic acid having little effect. A lysozyme–Tris–EDTA system also caused lysis of P. aeruginosa envelopes. The sensitivity of whole cells of the various strains to some β-lactam antibiotics and other antibacterial agents has been carried out and the basis of sensitivity or resistance in relation to drug destruction and the above envelope composition discussed.     

The effect of abscisic acid on epidermal cells: protoplast swelling and ATPase activity

Isolated epidermal protoplasts of Commelina communis L. increase in volume in the presence of KCl. Since this swelling is an osmotic phenomenon it reflects K⁺ influx. ATP slightly decreased the volume of the protoplasts, pointing towards the possibility that K⁺ uptake is passive. On the other hand abscisic acid (ABA) and sodium orthovanadate increased the swelling, and their effect was reversed by ATP. This may support the suggestion that ABA inhibits the active and ATPase-mediated relase of K⁺ from epidermal cells. Mg²⁺-dependent, K⁺-stimulated ATPase activity was found in the microsomal fraction from epidermal cells. This activity was vandadate sensitive. ABA increased the basal activity in the presence of Mg²⁺ but inhibited the K⁺ stimulation.     

Effect of calcium and other metal ions on pectic activities from autolysed cultures of Alternaria alternata

Abstract The effect of increasing concentrations of Ca²⁺, Mg²⁺, Cu²⁺, Zn²⁺, Na⁺ and EDTA on the pectic enzymic activities (polymethylgalacturonase, endopectinase and pectin-lyase) present in the autolytic complex from Alternaria alternata has been studied. In all cases the divalent metal ions and EDTA produced an increased inhibition correlated with increasing concentration of each ion. An opposite effect was shown by the Na⁺ ion, which produced an increase in pectic enzymic activities, principally at low concentrations.     

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.