Development of Thermoactinomyces vulgaris on a synthetic medium]

A synthetic medium was used to obtain the dormant spores of Thermoactinomyces vulgaris. The fraction of the dormant spores depended on the amino acid composition of the growth medium. The rate of growth and development of the organism on the synthetic medium is lower as compared to the routinely employed complex medium.     

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 degrees C, inocula containing less than 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give greater than 10(8) 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 10(6). 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.     

Characteristics of the ionic composition and ultrastructure of Bordetella pertussis in controlled regulation of the mineral element content in a growth medium

The content of trace elements necessary for the normal growth of bacteria was found to have no effect on the intracellular concentration of Ca2+ and Al3+. The content of Cu2+, Fe3+, Mn2+, Mg2+ was considerably reduced. The addition of Mg2+ at different concentrations to this culture medium stimulated the capacity of cells for accumulating not only Mg2+, but also some other ions. Their maximum intracellular concentration was observed when the concentration of Mg2+ in the culture medium was 41 mM. The growth of microbial cells in the standard culture medium containing Mg2+ at a concentration of 4 mM was accompanied by the increased consumption of elements actively participating in redox reactions (Cu2+, Fe3+, Mn2+). Shifts in the ionic composition of microbial cells were manifested by the morphological features of B. pertussis, linked with the increased synthesis of crystalloid structures. The influence of Mn2+, Al3+, Zn2+ at different concentrations on the ionic composition and morphology of B. pertussis was studied.     

胁迫时间与非毒性离子对重金属抑制拟南芥种子发芽及幼苗生长的影响

测定了Hg²⁺、Cd²⁺、Cu²⁺、Pb²⁺单一重金属胁迫对拟南芥种子发芽和幼苗生长的影响.结果表明,重金属对幼苗生长的毒性大于对种子发芽的毒性,以抑制种子发芽的IC₅₀为指标,4种重金属的毒性顺序为Hg²⁺＞Cd²⁺＞Pb²⁺/Cu²⁺,以幼苗生长为指标,则毒性顺序为:Cu²⁺＞Hg²⁺＞Cd²⁺/Pb²⁺,并随着胁迫时间延长,种子萌发率下降.此外,不同重金属在不同发芽时段对种子的毒性也不尽相同,Cd²⁺的毒性在种子吸水后的0～12 h大于12～24 h,而Hg²⁺毒性在12～24 h大于0～12 h,其中,种皮对减轻重金属毒性起着十分重要的作用.通过非毒性离子(Ca²⁺、Mg²⁺、K⁺、Na⁺)与重金属离子(Hg²⁺、Cd²⁺、Cu²⁺、Pb²⁺)交互作用对拟南芥种子发芽及幼苗生长效应的研究发现, mmol·L^-1的Ca²⁺、Mg²⁺、K⁺、Na⁺可以增强Hg²⁺对种子发芽的毒性,但对Cd²⁺的毒性却没有影响.对于幼苗来说,Ca²⁺、Mg²⁺、K⁺、Na⁺可以显著增强Hg²⁺的毒性,Ca²⁺可以缓解Cd²⁺的毒性,但却增加Cu²⁺的毒性,K⁺可以缓解Pb²⁺对幼苗的毒害作用.最后,本文对重金属的毒害机理进行了探讨.     

Sporulation of several species of Streptomyces in submerged cultures after nutritional downshift

Streptomyces griseus ATCC 10137, S. griseus IMRU 3570, S. griseus JI 2212, S. acrimycini JI 2236 and S. albus G sporulated abundantly in several liquid media after nutritional downshift. Spores formed in submerged cultures were viable and as thermoresistant as aerial spores. Scanning electron microscopy showed that submerged spores are morphologically similar to aerial spores. The sporulation of the Streptomyces strains tested in complex medium appeared to be triggered by phosphate nutritional downshift, induced by addition of Ca2+ to the medium. Spore-shaped bodies were formed by S. lividans JI 1326 and S. coelicolor JI 2280 when grown in complex medium supplemented with Ca2+ and proline. The thermoresistance of these spore-shaped bodies differed from that of aerial spores.     

Microcyst Germination in Myxococcus xanthus

Germination of glycerol-prepared microcysts of Myxococcus xanthus was studied. The sequence of morphological events during germination resembled that of germinating fruiting body-microcysts. The turbidity drop of a culture of germinating microcysts could be described by McCormick's formula derived for germinating Bacillus spores. The rate of uptake of labeled glycine and acetate did not change during germination. Temperature, aeration, and pH optima for germination were the same as for vegetative cell growth. Germination was induced by protein hydrolysates and the individual amino acids glycine, alanine, valine, aspartic acid, and glutamic acid. A number of organic compounds, including sugars, alcohols, aldehydes, ketones, organic acids, and chelating agents, did not induce germination. The inorganic ions HPO(4) (2-), Mg(++), Ca(++), and NH(4) (+) induced germination, although ionic strength was not a factor. Microcysts incubated in distilled water at concentrations greater than about 10(9) cells/ml germinated; supernatant fluid from such suspensions (germination factor) induced germination of less concentrated suspensions. The activity of germination factor was resistant to boiling, but was lost on charring and dialysis. Germination of microcysts and growth of vegetative cells was equally sensitive to a variety of metabolic inhibitors, including penicillin and chloramphenicol. Germination was more resistant than vegetative growth to inhibition by antibiotics of the streptomycin family and by actinomycin D.     

The influence of gelling agent purity and ion additions on in vitro tomato pollen germinability and pollen tube growth on semi-solid substrates

The physicochemical properties and inherent ion content of the gelling agents used for the preparation of semi-solid substrates significantly affect the germination of tomato pollen in vitro. The addition of Ca, K and Mg to semi-solid, agar-based, substrates improved the germination of tomato pollen when the inherent Ca content of the agar was low, but was without effect or even inhibitory when the Ca level was high. However, Κ and/or Mg addition was beneficial irrespective of the agar source. When agarose replaced agar and K, Mg and Ca were added individually or in combination, pollen germination and pollen tube growth were affected differently by each ion but were optimal only in the presence of all three ions, reflecting the absence of these ions in agarose. An involvement of Na was also implicated since reduction of the inherently high Na content of agar by washing improved germination, with or without the addition of Κ, Mg and Ca. Since >3 mM Ca in the semi-solid substrate impairs tomato pollen germination, the gelling agent must be of high purity, which in the case of agar may entail washing, followed by the addition of K, Mg and Ca. The adoption of such a medium would permit the standardization of semi-solid substrates for in vitro tomato pollen germination studies.     

In vitro growth and organogenesis of Prosopis farcta plantlets (Fabaceae, Mimosoideae) in culture medium supplemented with various concentrations of Ca++ and Na+

The objective of this study was to vary the mineral composition of the culture medium of Prosopis farcta seedlings per addition of Na+ and Ca++ ions with the aim to identify the culture media which support the growth and/or the expression of the in vitro plant organogenesis. The Na+ and Ca++ ions were added in the culture medium in various concentrations by taking the Gamborg medium, in which macroelements were diluted 10 times, as the basic one. After two months of culture, parameters relating to the vegetative development of plant seedlings and to the various expressions of organogenesis were measured. Weak concentrations in sodium and calcium ions as well as a weak concentration in Ca++ (0.1 mM) with 50 mM in Na+ support the best vegetative development of the plantlets. The most important percentage of plant seedlings presenting a bud initiation was obtained on a medium containing 0.1 mM of Na+ and 50 mM of Ca++. Our study defined several media likely to support in vitro development of Prosopis farcta plantlets allowing the selection of salt tolerant plants or cellular lines. Some other media were chosen for improving micropropagation of the species without adding growth substances.     

ATP synthesis catalyzed by the purified erythrocyte Ca-ATPase in the absence of calcium gradients

The Ca2+-transporting ATPase of erythrocytes was isolated by calmodulin affinity chromatography. The backward reaction of the ATPase was investigated. The phosphorylation of the solubilized enzyme by Pi required Mg and was inhibited by Ca and vanadate in the micromolar concentration range. Significant amounts of phosphoenzyme could be obtained only in a medium containing high dimethyl sulfoxide concentrations (greater than 25%) in order to diminish water activity at the phosphorylation site. The phosphoenzyme formed in this way could not phosphorylate ADP. However, upon addition of Ca2+ ions and dilution of dimethyl sulfoxide in the phosphorylated preparation (water activity jump), a highly reactive phosphoenzyme species was obtained which could transfer phosphate in nearly stoichiometric amounts to ADP to form ATP.     

Effects of pH, temperature and Ca2+ content on the conformation of alpha-lactalbumin in a medium modelling physiological conditions

Data obtained by the intrinsic protein fluorescence technique showed that, in addition to Ca2+ and Mg2+ ions, bovine alpha-lactalbumin also binds physiologically significant Na+ and K+ ions, the nucleotides ATP, ADP, UTP, UDP and UDP-galactose. The release of the bound Ca2+ ions from the protein in a medium modelling physiological conditions (containing Mg2+, Na+, K+, ATP and ADP in physiological concentrations) induced a transition of the protein from the native state of the Ca2+-loaded form to a state which is a mixture of native and and thermally changed states of the apo- and metal bound forms. Any variations in temperature result in changes in the populations of these states. This may be associated with some Ca2+ and temperature dependent regulation of the protein function. Variations of pH within the physiological limits had little influence on the conformation of both Ca2+-loaded and Ca2+-free alpha-lactalbumin.     

Crystal structure of Mg2+- and Ca2+-bound Gla domain of factor IX complexed with binding protein

Factor IX is an indispensable protein required in the blood coagulation cascade. It binds to the surface of phospholipid membrane by means of a gamma-carboxyglutamic acid (Gla) domain situated at the N terminus. Recently, we showed that physiological concentrations of Mg2+ ions affect the native conformation of the Gla domain and in doing so augment the biological activity of factor IXa and binding affinity with its binding protein even in the presence of Ca2+ ions. Here we report on the crystal structures of the Mg2+/Ca2+-bound and Ca2+-bound (Mg2+-free) factor IX Gla domain (IXGD1-46) in complex with its binding protein (IX-bp) at 1.55 and 1.80 A resolutions, respectively. Three Mg2+ and five Ca2+ ions were bound in the Mg2+/Ca2+-bound IXGD1-46, and the Mg2+ ions were replaced by Ca2+ ions in Mg2+-free IXGD1-46. Comparison of Mg2+/Ca2+-bound with Ca2+-bound structures of the complexes showed that Mg2+ ion, which formed a bridge between IXGD1-46 and IX-bp, forced IXGD1-46 to rotate 4 degrees relative to IX-bp and hence might be the cause of a more tight interaction between the molecules than in the case of the Mg2+-free structure. The results clearly suggest that Mg2+ ions are required to maintain native conformation and in vivo function of factor IX Gla domain during blood coagulation.     

Lethal germination of spores ofBacillus megaterium 9885

Washed spore suspensions germinated promptly without prior heat shock in a basal germination solution containingl-leucine.Germination was inhibited by dipicolinic acid. The inhibition was reversed by eitherl-leucine or phosphate.Phosphate accelerated the rate and increased the extent of germination, which was accompanied by an uncommonly large fall in the optical density of the suspension, but phosphate also caused a massive lysis after germination. This was accompanied by a sudden shedding of the spore coats. The suspensions consisted of shrivelled, cellular walls and membranes attached to the empty spore coats.Lysis of the germinated cells was prevented by fairly high concentrations of Ca or Mg.During germination, exogenous Ca we used Ca⁴⁵ was absorbed by the cells. Both cells and sonically disrupted cellular particles firmly retained the calcium, and evidence suggested that much of the Ca was bound in the cytoplasmic membranes.The cations contained in plain agar enabled spores which germinated on tryptone soya agar plates to develop into colonies; in the corresponding broth medium these spores lysed upon germination.Hypertonic sucrose delayed but did not prevent lysis.     

The role of Ca++ in regulation of oxoglutarate dehydrogenase complex activity from bovine adrenal cortex

Binding of Ca2+ ions by EGTA is established to increase the oxoglutarate dehydrogenase complex K'm for 2-oxoglutarate up to 25 mM in spite of the Mg2+ ions presence in the medium. The maximum reaction rate remains unchanged. Addition, besides EGTA of an equimolar Ca2+ amount to the medium induces a more than 100-fold decrease in K'm. A positive effect of Ca2+ ions is intensified by ADP. Ca2+ counteracts the inhibition of the oxoglutarate dehydrogenase complex activity by NADH. When chelating Ca2+ by EGTA with NADH available, a nonhyperbolic dependence of the reaction rate on the 2-oxoglutarate concentration is observed. When Ca2+ is absent, signs of a positive cooperative interaction of the enzyme with ADP and NADH are observed under conditions when 2-oxoglutarate concentration is much lower than the saturating one.     

Modelling of Mg2+, ATP-dependent mitochondrial Ca ions transport in smooth muscle cells using protonophore CCCP-sensitive fluorescent tetracycline

Mg2+, ATP-dependent Ca2+ accumulation in the rat myometrial mitochondria was investigated in complex experiment using Ca2+ isotope (45Ca2+) and Ca(2+)-sensitive label tetracycline. Monotonous increase of the fluorescence signal, insensitive to thapsigargin (100 nM) was observed with following establishing the stationary state of incubation at 2 min. which correlates with results obtained using isotope technique. Experiments with isotope label signify, that protonophore CCCP, ruthenium red and sodium azide, in concentration 1 microM, 10 microM and 10 mM respectively, totally inhibits the accumulation of the Ca ions in mitochondria. At the same time, in conditions of Mg2+, ATP-dependent Ca2+ accumulation modeling in these cellular structures, CCCP and sodium azide, used in the same concentration, diminished tetracycline fluorescence signal increase. In the same conditions, the introduction of the CCCP (1 mM) into the incubation medium at 75 sec. after initiation of the transport process induced reversible quenching of the tetracycline fluorescence signal to the level, observed in case of initial CCCP presence in the medium. According to data obtained in the experiment, using Ca2+ isotope, Ca(2+)-ionophore A-23187 induces both the reversible release of previously accumulated Ca ions, and cause reversible quenching of the tetracycline fluorescence signal to the level, observed in case of initial CCCP (1 mM) and sodium azide (10 mM) presence in the incubation medium. Conclusion was drawn that the thapsigargin-insensitive and CCCP, sodium azide and A-23187-sensitive tetracycline fluorescence increasing in case of modeling of Mg2+, ATP-dependent Ca2+ accumulation in myometrial mitochondria reflect the Ca2+ uniporter functioning in those subcellular structures.     

Germination of Heat- and Alkali-Altered Spores of Clostridium perfringens Type A by Lysozyme and an Initiation Protein

The normal system functioning in the utilization of metabolizable germinants by both heat-sensitive and heat-resistant spores of Clostridium perfringens was inactivated by heat or by treatment of the spores with alkali to remove a soluble coat protein layer. Altered spores were incapable of germination (less than 1%) and outgrowth (less than 0.0005%) in complex media without the addition of either lysozyme or an initiation protein produced by C. perfringens. The addition of either of these agents permitted, in the case of alkali-treated spores, both 90 to 95% germination and outgrowth, as measured by colony formation. In the case of heat-damaged spores, only 50% germination and 2% outgrowth resulted from addition of the initiation protein, whereas lysozyme permitted 85% germination and 8% outgrowth. Alteration of the spores by heat or alkali apparently inactivated the normal lytic system responsible for cortical degradation during germination. Kinetics of production of the initiation protein and conditions affecting both its activity and that of lysozyme on altered spores are described.     

Collapse of cortex expansion during germination of Bacillus megaterium spores

When spores of Bacillus megaterium ATCC 12872 were incubated with CdCl2, they germinated without decomposition of the cortex. Moreover, the volume ratio of cortex to protoplast-plus-cortex, C/(P+C), of the CdCl2-germinated spores was reduced. Incubation of isolated cortex with the divalent compounds Cd2+, Ca2+, and Mg2+ reduced the gel volume to about 1/5 but incubation with a nonionic compounds, glucose, did not. The spores with reduced C/(P+C) were observed in the early period of glucose-induced germination. The time required for a 50% change in cortex morphology to occur was 2.5 min, which corresponds well with the time for 50% loss of heat resistance. This time was shorter than that necessary for release of peptidoglycan fragments and hydrolysis of cortex glycan chains. These data indicate that cortex hydrolysis is not related to the initiation of germination. 50% of the dipicolinic acid, calcium and magnesium were released at 3.4, 4.0, and 2.4 min, respectively. These results suggest that collapse of cortex expansion by the interaction of cortex with dipicolinic acid and cations released from the core, or exogenous ionic germinants is an important step in the initiation of germination.     

金属离子对地衣芽孢杆菌合成多聚γ-谷氨酸的影响

多聚γ 谷氨酸 [γ Ｐｏｌｙ(ｇｌｕｔａｍｉｃａｃｉｄ) ,γ ＰＧＡ]是由某些杆菌 (Ｂａｃｉｌｌｕｓ)合成的一种细胞外水溶性高分子氨基酸聚合物 ,是由Ｌ 谷氨酸、Ｄ 谷氨酸两种构型的单体通过γ 酰胺键聚合形成的[1 ] 。γ ＰＧＡ具有极佳的成膜性、成纤维性 ,阻氧性、可塑性、粘结性、保湿性和可生物降解等许多独特的理化和生物学特性[2 ,3] 。因此 ,γ ＰＧＡ可以被广泛用于医药制造 ,食品加工 ,蔬菜、水果、海产品防冻、保鲜 ,化妆品工业 ,烟草、皮革制造工业和植物种子保护等许多领域 ,是一种有极大开发价值和前景的多功能新型生物制…     

Peptides related to the calcium binding domains II and III of calmodulin. Synthesis and calmodulin-like features

Three hexadecapeptides which correspond to the putative Ca2+ binding domains II and III of calmodulin were synthesized employing solid phase methodology. One of the peptides contained an internal cystine bridge which was formed while the corresponding linear peptide was still attached to the polymeric carrier. The interaction of the synthetic peptides with calcium ions was investigated using Tb3+-mediated fluorescence. Binding was of the order Ca12 greater than Ca13 greater than Ca13C (Fig. 1) with binding constants KTb3+ = 0.68 X 10(-5), 0.54 X 10(-5), and 0.21 X 10(-5) M-1 respectively. Biological activity of the compounds was assessed by measuring their stimulatory effect on erythrocyte membrane (Ca2+ + Mg2+)-ATPase activity. For 50% activity as compared with CaM, the concentration of peptides required was for Ca12, Ca13 and Ca13C, 50, 100 and 167 times higher than CaM, respectively. The results suggest that the three synthetic peptides possess certain calmodulin-like features.     

Magnesium permeability of sarcoplasmic reticulum. Mg2+ is not countertransported during ATP-dependent Ca2+ uptake by sarcoplasmic reticulum

Magnesium transport across sarcoplasmic reticulum (SR) vesicles was investigated in reaction mixtures of various composition using antipyrylazo III or arsenazo I to monitor extravesicular free Mg2+. The half-time of passive Mg2+ efflux from Mg2+-loaded SR was 100 s in 100 mM KCl, 150 S in 100 mM K gluconate, and 370 S in either 100 mM Tris methanesulfonate or 200 mM sucrose solutions. The concentration and time course of Mg2+ released into the medium was also measured during ATP-dependent Ca2+ uptake by SR. In reaction mixtures containing up to 3 mM Mg2+, small changes in free magnesium of 10 microM or less were accurately detected without interference from changes in free Ca2+ of up to 100 microM. Three experimental protocols were used to determine whether the increase of free [Mg2+] in the medium after an addition of ATP was due to Mg2+ dissociated from ATP following ATP hydrolysis or to Mg2+ translocation from inside to outside of the vesicles. 1) In the presence of ATP-regenerating systems which maintained constant ATP to ADP ratios and normal rates of active Ca2+ uptake, the increase of Mg2+ in the medium was negligible. 2) Mg2+ released during ATP-dependent Ca2+ uptake by SR was similar to that observed during ATP hydrolysis catalyzed by apyrase, in the absence of SR. 3) In SR lysed with Triton X-100 such that Ca2+ transport was uncoupled from ATPase activity, the rate and amount of Mg2+ release was greater than that observed during ATP-dependent Ca2+ uptake by intact vesicles. Taken together, the results indicate that passive fluxes of Mg2+ across SR membranes are 10 times faster than those of Ca2+ and that Mg2+ is not counter-transported during active Ca2+ accumulation by SR even in reaction mixtures containing minimal concentrations of membrane permeable ions that could be rapidly exchanged or cotransported with Ca2+ (e.g. K+ or Cl-).     

Flocculation and adsorption of enzymes during growth of a moderate halophile, Micrococcus varians var. Halophilus.

Flocculation of a moderate halophile, Micrococcus varians ATCC 2197, occurred during growth in complex medium containing 3 M NaCl and a concentration of MgSO4 and KH2PO4 greater than 40 and 14 mM, respectively. Extracellular nuclease activity was absent in the flocculated cultures. Repeated washing of flocs by Mg2+-free Tris buffer containing 3 M NaCl, lowering of pH value of floc suspension below 6.3, or addition of ethylenediaminetetraacetic acid resulted in complete dissociation of the flocs and release of Mg2+ ions as well as nuclease and amylase. Inhibition of extracellular enzyme production accompanied by flocculation appeared to be the result of adsorption of enzyme proteins to surfaces of the flocs, but not of inhibition of biosynthesis. Floc formation could also occur in media containing 18 mM CaCl2 and 3.0 mM KH2PO4, but the Ca flocs were not deflocculated by washing with Ca2+-free buffer, suggesting that the affinity of Ca2+ for cell envelopes was stronger than that of Mg2+. It was also observed that most halophilic Planococcus and Micrococcus flocculated in the presence of MgSO4 and phosphate but halophilic Pseudomonas, Acinetobacter, and Bacillus did not.