The requirement for Mn2+ and Ca2+ in nitrogen fixation by the photosynthetic bacterium Rhodospirillum rubrum

Abstract: The role of Ca²⁺ and Mn²⁺ in Rhodospirillum rubrum grown under different conditions with respect to nitrogen source has been studied. The results show that this phototroph does not have an absolute requirement for these cations. In vitro studies of one of the enzymes operative in the metabolic regulation of nitrogenase in Rsp. rubrum have shown that Mn²⁺ or Fe²⁺ is required for activity. This investigation indicates that Mn²⁺ is not required in vivo for the function of this enzyme, suggesting that either Fe²⁺ is functional or that the enzyme has other properties when active in the cell.     

The regulation of Mn2+ and Cu2+ uptake in cells of the yeast Candida utilis grown in continuous culture

Abstract Transport of Mn²⁺ was repressed in Candida utilis cells grown in continuous culture in high-Mn²⁺ (100 μM Mn²⁺) medium as compared to cells grown in basic (0.45 μM Mn²⁺) and low-Mn²⁺ (< 0.05 μM Mn²⁺) media. In contrast, no repression of Cu²⁺ uptake occurred in high-Cu²⁺-grown (25 μM Cu²⁺) cells as compared to cells grown in basic medium (0.54 μM Cu²⁺). Cu²⁺-limited cells did not hyperaccumulate Cu²⁺ and there was not significant difference in initial uptake rates for all 3 Cu²⁺ conditions. Mn²⁺ uptake appears to be regulated by a mechanism sensitive to the external Mn²⁺ concentration, whereas Cu²⁺ transport is not governed in this way by the external Cu²⁺.     

Binding of Cd2+, Ni2+, Cu2+ and Zn2+ by isolated envelopes of Pseudomonas fluorescens

Abstract Cell envelopes of Pseudomonas fluorescens , cytoplasmic membrane, peptidoglycan and outer membrane were obtained from a fractionation procedure and tested for their metal binding capacity. Isolated envelopes (cytoplasmic membrane, peptidoglycan and outer membrane) were chemically modified and functional carboxyl groups transformed to electropositive amine groups, using carbodiimide ethylenediamine. Transformation of carboxyl groups was evaluated by measuring total amine groups in all fractions (modified or not). Using equilibrium dialysis and Scatchard plots for the data, we have established that isolated unmodified cell envelopes (cytoplasmic membrane, peptidoglycan and outer membrane) possess at least two types of metal binding sites with different association constants ( K _a and K '_a). Introduction of positive charges into the bacterial envelopes resulted in the disappearance of one type of metal binding site which had the highest association constant value for Ni²⁺, Cu²⁺ and Zn²⁺. All fractions, modified or not, always presented at least two types of binding sites with different association constants for Cd²⁺.     

Stimulation of proton extrusion by K+ and divalent cations (Ni2+, Co2+ Zn2+) in maize root segments

Entry of the divalent cations Ni²⁺, Co²⁺ and Zn²⁺ into cells of maize ( Zea mays L. cv. Dekalb XL 85) root tissue is accompanied by an acidification of the incubation medium, a decrease in both the pH of the cell sap and the level of malate in the cells, and by an inhibition of dark fixation of CO₂. K⁺, on the contrary, induces only a very low acidification of the incubation medium, does not change either the pH of the cell sap or the malate level in the cells, and induces an increase in CO₂ dark fixation. Different mechanisms are postulated for the stimulation of proton extrusion by divalent cations and K⁺.     

The CLS2 gene encodes a protein with multiple membrane-spanning domains that is important Ca2+ tolerance in yeast

Genetic screening of Saccharomyces cerevisiae mutants defective in Ca²⁺ homeostasis identified cls2, which exhibits a specific Ca²⁺-sensitive growth phenotype. We describe here the CLS2 gene and a multicopy suppressor (named BCL21, for bypass of CLS2) of the cls2 mutation. The CLS2 gene encodes a polypeptide of 410 amino acid residues, and its hydropathy profile indicates that the predicted Cls2 protein (Cls2p) contains ten putative membrane spanning regions. Immunofluorescent staining of the yeast cells expressing epitopetagged Cls2p suggests that Cls2p is localized to endoplasmatic reticulum (ER) membrane. A cls2 disruption strain is viable, but shows a Ca²⁺-sensitive phenotype like the original cls2 mutants. BCL21 suppresses the cls2 disruption mutation, indicating that the multicopy suppression does not require the Cls2p. Suppression of cls2 was observed even after introduction of a singlecopy plasmid harboring BCL21. The BCL21 gene encodes a protein of 382 amino acid residues and is identical to the SUR1 gene. sur1 was originally isolated as a suppressor of rvs161, which has reduced viability in nutrient starvation conditions. Possible mechanisms of the multicopy suppression are discussed.     

Effects of interrupted K+ supply on growth and uptake of K+, Ca2+, Mg2+ and Na+ in spring wheat

Effects of interrupted K⁺ supply on different parameters of growth and mineral cation nutrition were evaluated for spring wheat (Triticum aestivum L. cv. Svenno). K⁺ (2.0 mM) was supplied to the plants during different periods in an otherwise complete nutrient solution. Shoot growth was reduced before root growth after interruption in K⁺ supply. Root structure was greatly affected by the length of the period in K⁺ -free nutrient solution. Root length was minimal, and root branching was maximal within a narrow range of K⁺ status of the roots. This range corresponded to cultivation for the last 1 to 3 days, of 11 in total, in K⁺ -free nutrient solution, or to continuous cultivation in solution containing 0.5 to 2 mM K⁺. In comparison, both higher and lower internal/external K⁺ concentrations had inhibitory effects on root branching. However, the differing root morphology probably had no significant influence on the magnitude of Ca²⁺, Mg²⁺ and Na⁺ uptake. Uptake of Ca²⁺ and especially Mg²⁺ significantly increased after K⁺ interruption, while Na⁺ uptake was constant in the roots and slowly increased in the shoots. The two divalent cations could replace K⁺ in the cells and maintain electroneutrality down to a certain minimal range of K⁺ concentrations. This range was significantly higher in the shoot [110 to 140 μmol (g fresh weight)^?1] than in the root [20 to 30 μmol (g fresh weight)^?1]. It is suggested that the critical K⁺ values are a measure of the minimal amount of K⁺ that must be present for physiological activity in the cells. At the critical levels, K⁺ (⁸⁶Rb) influx and Ca²⁺ and Mg²⁺ concentrations were maximal. Below the critical K⁺ values, growth was reduced, and Ca²⁺ and Mg²⁺ could no longer substitute for K⁺ for electrostatic balance. In a short-term experiment, the ability of Ca²⁺ to compete with K⁺ in maintaining electroneutrality in the cells was studied in wheat seedlings with different K⁺ status. The results indicate that K⁺, which was taken up actively and fastest at the external K⁺ concentration used (2.0 mM), partly determines the size of Ca²⁺ influx.     

Hydrolysis of glucuronide-based substrates mediated by tungsten, Cu2+, Fe2+, and Zn2+

A variety of metal microprojectiles are currently used for carrying foreign DNA into living cells via particle-acceleration techniques. While developing a microprojectile-mediated protocol for transforming cells of sugarbeet ( Beta vulgaris L.), formation of a blue precipitate was observed with the indigoqenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-gluc) in the absence of gusA DNA encoding β-D-glucuronidase (GUS). Tungsten microcarriers, but not gold or silicon carbide, proved capable of catalyzing the cleavage of the glucuronide residue from three histochemical substrates evaluated: X-gluc, salmon X-gluc and magenta X-gluc. Indigo-stained sugarbeet cells were observed following bombardment with tungsten in the absence of DNA. Addition of oxidative catalysts to tungsten microcarriers during substrate incubation had no apparent effect on the metal-mediated catalysis. Treatment of microcarriers with Proteinase K and heat ruled out the presence of enzymes. Microbiological evaluation indicated the absence of contaminating microbes. Similarly, metal-catalyzed hydrolysis of the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronic acid (4-MUG) was observed in the presence of tungsten spheres but not with gold or silicon carbide particles. With this substrate, hydrolysis also occurred with millimolar concentrations of Cu²⁺, Fe²⁺ and Zn²⁺ ions. Consequently, careful monitoring of DNA-minus controls and avoidance of millimolar concentrations of Cu²⁺, Fe²⁺ and Zn²⁺ ions are recommended in microprojectile bombardment experiments where transient assays for gusA expression are performed.     

Competitiveness of a nif− Bradyrhizobium japonicum mutant against the wild-type strain

Abstract By mixed inoculation experiments, the competitive ability of a nifD ::Tn 5 mutant of Bradyrhizobium japonicum was compared to its effective, isogenic parent strain. When the strains were inoculated in a 1:1 ratio at high concentration, the mutant was found to colonize almost as many nodules as the wild type. Thus, lack of expression of a functional nitrogenase system does not severely reduce competitiveness. In such experiments the majority of the nodules (> 60%) were infected by both wild-type and mutant strains. From statistical analysis it was concluded that a mean number of 2–4 bacteria have successfully elicited one nodule under the described conditions. Visual and microscopic observations of sections from mixed infected nodules revealed separated sectors containing effective or ineffective bacteroids.     

The gene sll0273 of the cyanobacterium Synechocystis sp. strain PCC6803 encodes a protein essential for growth at low Na+/K+ ratios

A mutant of Synechocystis sp. strain PCC6803 was obtained by random cartridge mutagenesis, which could not grow at low sodium concentrations. Genetic analyses revealed that partial deletion of the sll0273 gene, encoding a putative Na ⁺ /H ⁺ exchanger, was responsible for this defect. Physiological characterization indicated that the sll0273 mutant exhibited an increased sensitivity towards K ⁺ , even at low concentrations, which was compensated for by enhanced concentrations of Na ⁺ . This enhanced Na ⁺ demand could also be met by Li ⁺ . Furthermore, addition of monensin, an ionophore mediating electroneutral Na ⁺ /H ⁺ exchange, supported growth of the mutant at unfavourable Na ⁺ /K ⁺ ratios. Measurement of internal Na ⁺ and K ⁺ contents of wild‐type and mutant cells revealed a decreased Na ⁺ /K ⁺ ratio in mutant cells pre‐incubated at a low external Na ⁺ /K ⁺ ratio, while it remained at the level of the wild type after pre‐incubation at a high external Na ⁺ /K ⁺ ratio. We conclude that the Sll0273 protein is required for Na ⁺ influx, especially at low external Na ⁺ concentrations or low Na ⁺ /K ⁺ ratios. This system may be part of a sodium cycle and may permit re‐entry of Na ⁺ into the cells, if nutrient/Na ⁺ symporters are not functional or operating.     

The MgtE Mg2+ transport protein is involved in Aeromonas hydrophila adherence

Aeromonas hydrophila AH-3 strains carrying mutations in mgtE, which encodes a Mg2+ and Co2+ transport system, showed a 50% reduction of in vitro adherence to HEp-2 cells, a reduction in swarming in semisolid swarming agar, and decrease in biofilm formation of over 60% in comparison to the wild-type strain. The cloned A. hydrophila mgtE expressed from a plasmid complements a Salmonella typhimurium strain deleted for all Mg2+ transporters both phenotypically and by measurement of 57Co2+ uptake. Likewise, plasmid-borne mgtE was able to complement the changes observed in A. hydrophila mgtE mutants. We suggest that MgtE and thus Mg2+ and possibly Co2+ have a role in A. hydrophila related to their swarming ability and related consequences such as adherence and biofilm formation.     

Excretion of ions (Cd2+, Li+, Na+ and Cl−) by Tamarix aphylla

Excretion of minerals by the NaCl-resistant and comparatively cadmium-resistant tree Tamarix aphylla (L.) Karst, was investigated. Cd²⁺ was excreted by plants exposed for 1–10 days to 9 or 45 μ M Cd²⁺ solutions. Excretion of this toxic ion increased considerably with time but was less than 5% of the quantities that had been accumulated in the shoots. Excretion of Na⁺ and Cl⁻ was positively correlated with NaCl concentration (1.5, 10, 50 m M ) of the medium. The Na⁺/Cl⁻ ratios of the excrete were positively correlated with the concentration of the treatment solution. Ca²⁺ excretion decreased with increasing NaCl concentrations of the solution. Excretion of K⁺ and Mg²⁺ was only little affected by NaCl. Excretion of Li⁺ occurred whenever this element was supplied in the uptake solution; daily excretion rates of Li⁺ increased with time. The ecological significance of excretion is discussed in relation to the low selectivity of the mechanism in T. aphylla .     

The action potential in Chara: Ca2+ release from internal stores visualized by Mn2+‐induced quenching of fura‐dextran

The action potential (AP) in Chara is associated with a transient elevation in the concentration of cytoplasmic-free Ca²⁺ ([Ca²⁺]_cyt). The quenching properties of the fluorescent Ca²⁺ indicator dye fura-dextran, in combination with Mn²⁺, was used to investigate whether this [Ca²⁺]_cyt transient is due to Ca²⁺ release from internal stores or to Ca²⁺ influx across the plasma membrane. Adding Mn²⁺ to the external medium or pre-injection of Mn²⁺ into the vacuole caused no perceivable quenching of the fura fluorescence, during an AP. This makes it unlikely that Ca²⁺ influx across the plasma membrane or the tonoplast contributes significantly to the [Ca²⁺]_cyt transient in an excited cell. When cells were pre-incubated in external solutions containing Mn²⁺ from 25 to 30 mM APs evoked a transient quenching of fura fluorescence in Mn²⁺-free solutions. Under these conditions, the quenching must be attributed to an AP-associated release of Mn²⁺ from internal stores. Based on the finding that exposing cells to millimolar concentrations of Mn²⁺ caused a progressive quenching of the fura fluorescence in non-excited cells, it can be assumed that some Mn²⁺ enters the cells during pre-incubation and is loaded into internal stores. During excitation, this stored Mn²⁺ is released together with Ca²⁺.     

Alteration of Ca2+ Fluxes in Brain Microsomes by K+ and Na+: Modulation by Sulfated Polysaccharides and Trifluoperazine

Abstract: Rat brain microsomes accumulate Ca²⁺ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K⁺, Na⁺, or Li⁺. Both the Ca²⁺ uptake and the Ca²⁺-dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half-maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 µg/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca²⁺ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca²⁺ pump of brain microsomes. At low concentrations (20–80 µM) it stimulates the rate of Ca²⁺ influx, and at concentrations >100 µM it inhibits both the Ca²⁺ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca²⁺-ATPase; for the Ca²⁺-ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration-dependent inhibition of Ca²⁺ uptake. Passive Ca²⁺ efflux from brain microsomes preloaded with Ca²⁺ is increased by trifluoperazine (50–150 µM), and this effect is potentiated by heparin (10 µg/ml), even in the presence of KCl. It is proposed that the Ca²⁺-ATPase isoform from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.     

The effect of Ca2+-stress on fluxes of Ca2+ and K+ in wheat and cucumber

Betula papyrifera Marsh, seedlings adapted very poorly to flooding for up to 60 days. Responses to flooding included increased ethylene production; stomatal closure; leaf senescence; drastic inhibition of shoot growth, cambial growth, and root growth; decay of roots, and death of many seedlings. Flooding inhibited growth of leaves that formed prior to flooding, inhibited formation of new leaves, and induced abscission of old leaves. As a result of extensive leaf abscission, fewer leaves were present after flooding than before flooding was initiated. The drastic reduction in leaf area was associated with greatly decreased growth of the lower stem and roots. No evidence was found of adaptive morphological changes to flooding. The data indicate that intolerance of B. papyrifera seedlings to flooding is an important barrier to regeneration of the species on sites subject to periodic inundation.