Arthrobacter Sp. Strain MF-2 Induces High-Mg Calcite Formation: Mechanism and Implications for Carbon Fixation

To better understand the mechanism of formation of carbonate minerals by microbes, culture experiments with Arthrobacter sp. strain MF-2 were carried out using M2 medium without carbonate ions for 50 days. A series of sterile control experiments without bacteria were run simultaneously. During the incubation, cell density, the quantity of precipitate, the extracellular polysaccharide (EPS) content, the activity of carbonic anhydrase (CA), the low molecular weight organic acid concentration, the pH, the electrical conductivity, and the Ca²⁺ and Mg²⁺ concentrations of the medium were determined. The morphologies of the precipitated carbonates were observed using scanning electron microscopy, and their mineral species were determined by X-ray diffraction. The results demonstrated that the quantity of precipitate in the biotic experiments increased gradually with the incubation time; precipitate was not obtained in the abiotic experiments. The average precipitation rate correlated positively with the cell density and the EPS content, with r = 0.64 and 0.61, respectively. This suggests that bacterial cells and EPS effected carbonate precipitation. Carbonate ion incorporation into minerals results from carbon dioxide hydration, promoted by microbial secretion of CA by bacteria. These findings contribute to the ongoing search for feasible mechanisms for the sequestration of carbon dioxide in the subsurface, in this case mediated by microorganisms.     

Aragonite crystallization in a Christmas-tree model microfluidic device with the addition of magnesium ions

Aragonite is an important dimorph of calcium carbonate, industrially and biologically. However, aragonite is so thermodynamically unstable that it is difficult to understand its formation mechanism. A continuous microfluidic system was employed, in which crystallization was induced only by diffusion in a micron-scale channel. Calcium carbonate (CaCO₃) formed by liquid-liquid reaction and magnesium ions (Mg²⁺) were used as additives. To assess the influence of Mg²⁺ concentration, the Mg²⁺/Ca²⁺ molar ratio was set to 1, 3, and 5. Laminar streams flowed in the detection channel with different concentration gradients. The initial crystallization time (t_I.C) increased exponentially and the density of crystals decreased as the Mg²⁺ ion concentration increased. Following transformation of all particles into snowman or sphere shapes, they became spinose sphere-shaped crystals, which was the final form in this study.     

Nanoparticle Accumulation in Microbial Induced Carbonate Precipitation: The Crucial Role of Extracellular Polymeric Substance

Abstract

Microbes and their secreted extracellular polymeric substance (EPS) could regulate the mineral phases, morphologies and structure of the microbial induced carbonate precipitates (MICPs). Here, two groups of mineralization experiments were carried out respectively to investigate the mechanism of the microbial induced carbonate precipitation (MICP) using Arthrobacter sp. MF-2 strain and its EPS. X-ray diffraction (XRD) were used to characterize the mineral species and scanning electron microscopy (SEM) was used to investigate the morphologies of the precipitates. Besides, focused ion beam (FIB) was used to prepare calcified bacterial slice for transmission electron microscopy (TEM) analysis. Meanwhile, the temporal change of bacterial density, pH value, conductivity, weight of precipitates, Ca²⁺ concentration and EPS content were also recorded. The results of this paper showed EPS could serve as the stabilizer of unstable phases and the scaffold for the accumulation of nanoparticles.     

Preliminary study on the correlation between the trace Mn2+ and the calcite polymorph in gallstones containing calcium carbonate from the northeast China via electron spin resonance

Gallstones containing calcium carbonate (GCCC) from the northeast China were analyzed using X-ray diffraction (XRD), infrared spectroscopy (IR), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and electron spin resonance (ESR). The sextet signal arising from the allowed transitions of the trace Mn²⁺ ions in GCCC was found to be ESR-detectable and strong. The XRD technique revealed the crystal habit of calcite in GCCC. Of the three polymorphs of calcium carbonate, no calcite was present as a solitary crystallization form, accompanied by aragonite or vaterite or both. The sextet ESR signal and the (104) main XRD peak at 2θ = ∼29.4° were employed as two probes to explore the relationship between trace Mn²⁺ and calcite. The Mn content can be considered as an indicator of the amount of calcite in GCCC because of the existence of a correlation between Mn²⁺ and calcite. The correlation between Mn²⁺ and calcite, the relation between the levels of Mn²⁺ and the type of gallstones, the structural preference of Mn²⁺ to the calcite polymorph, and the influence of dietary habits on calcite in calcium carbonate gallstones are discussed.     

Fluoride inhibition of yeast enolase: Crystal structure of the enolase–Mg2+–F−–Pi complex at 2.6 Å resolution

Enolase in the presence of its physiological cofactor Mg²⁺ is inhibited by fluoride and phosphate ions in a strongly cooperative manner (Nowak, T, Maurer, P. Biochemistry 20:6901, 1981). The structure of the quaternary complex yeast enolase–Mg²⁺–F^?–P_i has been determined by X-ray diffraction and refined to an R = 16.9% for those data with F/σ(F) ≥ 3 to 2.6 Å resolution with a good geometry of the model. The movable loops of Pro-35-Ala-45, Val-153-Phe-lo9, and Asp-255-Asn-266 are in the closed conformation found previously in the precatalytic substrate–enzyme complex. Calculations of molecular electrostatic potential show that this conformation stabilizes binding of negatively charged ligands at the Mg²⁺ ion more strongly than the open conformation observed in the native enolase. This closed conformation is complementary to the transition state, which also has a negatively charged ion, hydroxide, at Mg²⁺. The synergism of inhibition by F^? and P_i most probably is due to the requirement of P_i, for the closed conformation. It is possible that other Mg²⁺-dependent enzymes that have OH^? ions bound to the metalion in the transition state also will be inhibited by fluoride ions. © Wiley-Liss, Inc.     

Ouabain receptor interactions in (Na + K)-ATPase preparations. III. On the stability of the ouabain receptor against physical treatment, hydrolases and SH reagents

The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant (K_D) of 13 nM was found in the presence of (Mg²⁺ + P_i) and (Na⁺ + Mg²⁺ + ATP). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg²⁺, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K⁺ and its congeners, by Na⁺ in the presence of (Mg²⁺ + P_i), and by ATP analogs (ADP-C-P, ATP-OCH₃). Ca²⁺ antagonized the action of K⁺ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (R_α) and a high affinity conformational state (R_β). The equilibrium between both states is influenced by ligands of (Na⁺ + K⁺)-ATPase. With 3 mM Mg²⁺ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot).     

Ca2+ and Mg2+ ions counteract the reduction by fosetyl-Al (aluminum tris[ethyl phosphonate]) of peroxidase activity from suspension-cultured grapevine cells

Grapevine (Vitis vinifera cv. Monastrell) cell suspension cultures were treated with 1.5 mM fosetyl-Al, a frequently used systemic fungicide for grapevine diseases caused by oomycetes. These cells showed a reduction in the level of peroxidase activity secreted into the culture media when compared to non-treated cells, the effect being mainly related to a decrease in the level of the basic B₁ peroxidase isozyme. The effect of fosetyl-Al on peroxidase was analogous to that observed with the Ca²⁺-channel blockers Co²⁺, Cd²⁺ and La³⁺, and was counteracted by Ca²⁺ ions, but was not reversed when the Ca²⁺-ionophore A23187 was added to the culture media. Moreover, the effect of fosetyl-Al on peroxidase activity and peroxidase isozymes was also partially reversed by Mg²⁺ ions but not by Sr²⁺, and was accentuated by Ba²⁺ ions. These results suggested that Ca²⁺ and Mg²⁺ ions specifically overcome the inhibitory effect of fosetyl-Al on peroxidase. In this context, an apoplastic Ca²⁺/Mg²⁺-displacement hypothesis is proposed for the mechanism of action of fosetyl-Al on peroxidase from grapevine cells.     

The crystallographic study of left-handed Z-DNA d(CGCGCG)2 and thermine complexes crystallized at various temperatures and at various concentration of cations

In crystals of complexes of thermine and d(CGCGCG)₂ molecules grown at 4, 10, and 20 °C, the numbers of thermine molecules connected to the DNA molecule were dependent on the temperature of the crystallization. Two molecules of thermine and one Mg²⁺ ion were connected to DNA molecule when thermine and d(CGCGCG)₂ were co-crystallized at 4 and at 20 °C. When an increased concentration of magnesium and thermine molecules were co-crystallized with d(CGCGCG)₂ molecules at 10 °C, three Mg²⁺ ions and only one thermine molecule were bound with a d(CGCGCG)₂ molecule. The number of polyamines and of Mg²⁺ ions connected to DNA was dependent on the atomic values of the polyamine and of the metal ion. The binding of more Mg²⁺ ions occurred when the atomic value of Mg²⁺ exceeded that of the corresponding mono- or polyamine, and when the Mg²⁺ ion concentration was elevated. Furthermore, this study is the first documentation of a naturally occurring polyamine bound to the minor groove of DNA in a crystal structure.     

Coral acid rich protein selects vaterite polymorph in vitro

Corals and other biomineralizing organisms use proteins and other molecules to form different crystalline polymorphs and biomineral structures. In corals, it’s been suggested that proteins such as Coral Acid Rich Proteins (CARPs) play a major role in the polymorph selection of their calcium carbonate (CaCO₃) aragonite exoskeleton. To date, four CARPs (1–4) have been characterized: each with a different amino acid composition and different temporal and spatial expression patterns during coral developmental stages. Interestingly, CARP3 is able to alter crystallization pathways in vitro, yet its function in this process remains enigmatic. To better understand the CARP3 function, we performed two independent in vitro CaCO₃ polymorph selection experiments using purified recombinant CARP3 at different concentrations and at low or zero Mg²⁺ concentration. Our results show that, in the absence of Mg²⁺, CARP3 selects for the vaterite polymorph and inhibits calcite. However, in the presence of a low concentration of Mg²⁺ and CARP3 both Mg-calcite and vaterite are formed, with the relative amount of Mg-calcite increasing with CARP3 concentration. In all conditions, CARP3 did not select for the aragonite polymorph, which is the polymorph associated to CARP3 in vivo, even in the presence of Mg²⁺ (Mg:Ca molar ratio equal to 1). These results further emphasize the importance of Mg:Ca molar ratios similar to that in seawater (Mg:Ca equal to 5) and the activity of the biological system in a aragonite polymorph selection in coral skeleton formation.     

Recent Advances in the Structural Biology of Mg2+ Channels and Transporters

Magnesium ions (Mg²⁺) are the most abundant divalent cations in living organisms and are essential for various physiological processes, including ATP utilization and the catalytic activity of numerous enzymes. Therefore, the homeostatic mechanisms associated with cellular Mg²⁺ are crucial for both eukaryotic and prokaryotic organisms and are thus strictly controlled by Mg²⁺ channels and transporters. Technological advances in structural biology, such as the expression screening of membrane proteins, in meso phase crystallization, and recent cryo-EM techniques, have enabled the structure determination of numerous Mg²⁺ channels and transporters. In this review article, we provide an overview of the families of Mg²⁺ channels and transporters (MgtE/SLC41, TRPM6/7, CorA/Mrs2, CorC/CNNM), and discuss the structural biology prospects based on the known structures of MgtE, TRPM7, CorA and CorC.     

Effects of Divalent Cations,Trypsin, and Phospholipases on the Passive Permeability to Sodium of Inside-Out Vesicles From Human Red Cells

Inside-out vesicles (IOV) were prepared from human red blood cells. Steady-state uptake of ²²Na was observed to generally follow an exponential time course with a rate constant of 1.57 ± 0.09 h^?1 (SE). One week of cold storage (0–4°C) increased the rate constant to 2.50 ± 0.12 h ^?1 (SE). Mg²⁺, Ca²⁺, or Sr²⁺ decreased the rate of ²²Na uptake with no observable differences between the three divalent cations when tested at concentrations of 50 μM. Mg²⁺ was shown to decrease the rate of ²²Na uptake at concentrations as low as 5 μM with maximal effect at 50 to 100 μM. The decrease in rate of ²²Na uptake induced by Mg²⁺ could be enhanced by exposure of IOV to Mg²⁺ for longer periods of time. Trypsin treatment of IOV increased the rate of uptake of ²²Na and was dependent on the concentration of trypsin added between 5 to 25 μg/ml (treated for 5 min at 25°C). The ability of Mg²⁺ (50 μM) to decrease the rate of ²²Na uptake was still observed after maximal trypsin treatment. Phospholipase A₂ or phospholipase C treatment of IOV increased the rate of ²²Na uptake and was dependent on the amount of phospholipase A₂ (0.1 to 1.0 units/ml) or phospholipase C (0.25 to 2.5 units/ml) added (treated for 5 min at 25°C). After phospholipase A₂ treatment, the observed decrease in the rate of ²²Na uptake induced by Mg²⁺ (50 μM) was generally greater than controls. After phospholipase C treatment, the observed decrease in rate of ²²Na uptake induced by Mg²⁺ (50 μM) was less or absent when compared with controls. Phospholipase C treatment was less effective in preventing the Mg²⁺ effect the longer IOV were exposed to Mg²⁺. The results suggest that Mg²⁺ binds to phospholipid head-groups to reduce Na permeability perhaps by inducing a change in bilayer structure or phospholipid association.     

Regulation of divalent cations of the membrane-bound pyrophosphatase of Rhodospirillum rubrum,as shown by the hydrolysis of tripositive-pyrophosphate complexes

Tripositive-pyrophosphate [M(III)-PPi] complexes were used to investigate the role of free divalent cations on the membrane-bound pyrophosphatase. Divalent cations remain free and the M(III)-PPi complexes were employed as substrates. Formation of a La-PPi complex was studied by fluorescence, and the fact that Zn²⁺ and Mg²⁺ remain free in the solution was validated. Hydrolysis of La-PPi is stimulated by the presence of fixed concentrations of free Mg²⁺ or Zn²⁺ and this stimulation depends on the concentration of the cations when the La-PPi complex is fixed. The divalent cation stimulation order is Zn²⁺ > Co²⁺ > Mg²⁺ > Mn²⁺ > Ca²⁺ (at 0.5 mm of free cation). With different M(III)-PPi complexes, Zn²⁺ produces the same K _m, for all the complexes and Mg²⁺ stimulates with a different K _m. The results suggest that both Mg²⁺ and Zn²⁺ activate the membrane-bound pyrophosphatase but through different mechanisms.     

The role of magnesium and calcium ions in the glucose dehydrogenase activity of Klebsiella pneumoniae NCTC 418

Magnesium-limited chemostat cultures of Klebsiella pneumoniae NCTC 418 with 20 M CaCl₂ in the medium showed a low rate of gluconate plus 2-ketogluconate production relative to potassium- or phosphate-limited cultures. However, when the medium concentration of CaCl₂ was increased to 1 mM, the glucose dehydrogenase (GDH) activities also increased and became similar to those observed in potassium- or phosphate limited cultures. It is concluded that this is due to Mg²⁺ and Ca²⁺ ions being involved in the binding of pyrroloquinoline quinone (PQQ) to the GDH apoenzyme. There seems to be an absolute requirement of divalent cations for proper enzyme functioning and in this respect Ca²⁺ ions could replace Mg²⁺ ions. The high GDH activity which has been found in cells grown under Mg^2–-limited conditions in the presence of higher concentrations of Ca²⁺ ions, is compatible with the earlier proposal that GDH functions as an auxiliary energy generating system involved in the maintenance of high transmembrane ion gradients.Abbreviations PQQ pyrroloquinoline quinone - GDH glucose dehydrogenase (EC 1.1.99.17) - GaDH gluconate dehydrogenase (EC 1.1.99.3) - CAP chloramphenicol - WB Wurster's Blue [1,4-bis-(dimethylamino)-benzene perchlorate]     

Mobile loop mutations in an archaeal inositol monophosphatase: Modulating three‐metal ion assisted catalysis and lithium inhibition

The inositol monophosphatase (IMPase) enzyme from the hyperthermophilic archaeon Methanocaldococcus jannaschii requires Mg²⁺ for activity and binds three to four ions tightly in the absence of ligands: K_D = 0.8 μM for one ion with a K_D of 38 μM for the other Mg²⁺ ions. However, the enzyme requires 5–10 mM Mg²⁺ for optimum catalysis, suggesting substrate alters the metal ion affinity. In crystal structures of this archaeal IMPase with products, one of the three metal ions is coordinated by only one protein contact, Asp38. The importance of this and three other acidic residues in a mobile loop that approaches the active site was probed with mutational studies. Only D38A exhibited an increased kinetic K_D for Mg²⁺; D26A, E39A, and E41A showed no significant change in the Mg²⁺ requirement for optimal activity. D38A also showed an increased K_m, but little effect on k_cat. This behavior is consistent with this side chain coordinating the third metal ion in the substrate complex, but with sufficient flexibility in the loop such that other acidic residues could position the Mg²⁺ in the active site in the absence of Asp38. While lithium ion inhibition of the archaeal IMPase is very poor (IC₅₀～250 mM), the D38A enzyme has a dramatically enhanced sensitivity to Li⁺ with an IC₅₀ of 12 mM. These results constitute additional evidence for three metal ion assisted catalysis with substrate and product binding reducing affinity of the third necessary metal ion. They also suggest a specific mode of action for lithium inhibition in the IMPase superfamily.     

The structural peculiarities of condensed DNA micro- and nanoparticles formed in PCR

Studies of DNA condensation have opened new perspectives in biotechnology and medicine. DNA condensation induced by polyamines or trivalent metal ions in vitro at room temperature has been investigated in detail. Our recent studies have demonstrated Mg²⁺-mediated formation of DNA condensates during the PCR. In this study, we report the unique morphology and fine structure of PCR-generated condensed DNA particles using electron and atomic force microscopy. The principal morphologies of studied DNA condensates are 3D particles of micrometer dimensions, oval microdisks of nanometer thickness, filaments, and compact nano-sized particles. SEM examinations have revealed a new structural type of spherical and elliptical 3D microparticles formed by numerous definitely oriented microdisks and their segments. AFM revealed a granular structure of the microdisk surface and the smallest nano-sized disks and thinnest nanofibrils – that appear to be the primary products of DNA condensation during the PCR. We suggest that the formation of DNA nanofibrils and nanodisks in PCR occurs due to Mg²⁺ – mediated intermolecular (lateral) and intramolecular condensation of ssDNA. Aggregation of elementary nanodisks in the course of thermal PCR cycles, occurring both by magnesium cations and via complementary interactions, give a rise to large nano-sized aggregates and more complex microparticles.     

Effect of monovalent and divalent ions upon the activity of nisin againstMicrococcus flavus

Effect of monovalent and divalent ions on the activity of nisin againstMicrococcus flavus has been studied and Mg²⁺ ions have been found to reverse the effect of nisin. The protection by Mg²⁺ ions has been found to be due to complex formation with the antibiotic.     

Ca-stimulated, Mg-independent ATP hydrolysis and the high affinity Ca-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m = 0.25 μM, V_max = 24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.     

Screening of flocculant-producing microorganisms and some characteristics of flocculants

Summary 27 strains of microorganisms including moulds, bacteria, actinomyces and yeasts, which produced substances that flocculated a kaolin clay, were screened. One strain, identified as Arthrobacter sp., secreted bioflocculant during growth. The flocculant (MF-6) was purified to electrophoretic homogeneity by precipitation with ethanol and gel chromatography. Its molecular weight was higher than 10⁶ Da. It could efficiently flocculate all suspended solids tested in aqueous solution and colored water like dye solution, and its flocculating ability was significantly enhanced by adding Ca²⁺, Mn²⁺, Mg²⁺, etc.     

Vasopressin-sensitive kidney adenylate cyclase: Modulation of the hormonal response

Vasopressin-sensitive pig kidney adenylate cyclase is sensitive to several effectors, such as Mg²⁺, other divalent cations, and guanyl nucleotides. The purpose of the present study was to compare the main characteristics of adenylate cyclase activation by vasopressin, Mg²⁺, and GMPPNP, respectively. Mg^2+·ions were shown to exert at least three different effects on adenylate cyclase. The substrate of the adenylate cyclase reaction is the Mg-ATP complex. Mg²⁺ interacts with an enzyme regulatory site. Finally, Mg²⁺ can modulate the hormonal response, with Mg²⁺ions affecting the coupling function–that is, the quantitative relationship between receptor occupancy and adenylate cyclase activation. At all the magnesium concentrations tested, from 0.25 mM to 16 mM, adenylate cyclase activation was not a direct function of receptor occupancy. At low Mg²⁺ concentrations, adenylate cyclase activation dose-response curve to the hormone tended to be superimposable to the hormone dose-binding curve. These results suggest a role of magnesium at the coupling step between the hormone-receptor complex and adenylate cyclase response. Cobalt, but not calcium, ions could exert the same effects as Mg²⁺ ions on this coupling step. GMPPNP induced considerable adenylate cyclase activation (15 to 35 times the basal value). Activation by GMPPNP was highly time and temperature dependent. At 30° C, a 20 to 60 min preincubation period in the presence of GMPPNP was needed to obtain maximal activation. The higher the dose of GMPPNP in the medium, the longer it took to reach equilibrium. At 15° C, activation was still increasing with time after 3 hr preincubation in the presence of the nucleotide. GMPPNP was active in a 10^?8 M to 10^?5 M concentration range. Unlike the results obtained with lysine vasopressin, the kinetic characteristics of dose-dependent adenylate cyclase activation curves by GMPPNP were unaffected by varying Mg²⁺ concentrations except for the increase in velocity when raising Mg²⁺ concentration. It was not clear whether or not the activation processes by the hormone and by GMPPNP had common mechanisms.