Characterization of the binding of valinomycin to bacteriorhodopsin

Circular dichroism spectroscopy has been used to investigate the binding of valinomycin to bacteriorhodopsin in purple membrane suspensions. Addition of valinomycin to purple membrane suspensions obtained from Halobacterium halobium causes the circular dichroism spectrum to shift from an aggregate spectrum to one resembling a monomer spectrum, indicating a loss of chromophore-chromophore interactions. By observing the spectral change upon titration of valinomycin, an apparent dissociation constant of 30–40 M for valinomycin binding was determined. Kinetics of dark adaptation for valinomycin-treated purple membrane are comparable to those for monomeric bacteriorhodopsin. Centrifugation studies demonstrate that valinomycin-treated purple membrane sediments the same as untreated purple membrane suspensions. These results are consistent with a model in which valinomycin binds specifically to bacteriorhodopsin without disrupting the purple membrane fragments.Abbreviations BR bacteriorhodopsin - CD circular dichroism - Tricine N-[tris-(hydroxymethyl) methyl] glycine     

Glucose transport into the extremely halophilic archaebacteria

Penetration of glucose into cells of several extremely halophilic archaebacteria of the Halobacterium and Haloferax genera (Halobacterium saccharovorum and Halobacterium salinarium, Haloferax volcanii and Haloferax mediterranei) has been studied. Some characteristics of transport systems of carbohydrate-utilizing halobacteria Halobacterium saccharovorum, Haloferax mediterranei and Haloferax volcanii (pH and temperature optima, stereospecificity, kinetic parameters) have been determined. Inability of H. salinarium cells for active glucose transport has been shown. The dependence of glucose transport on the Na⁺ ions gradient (on the whole cells and membrane vesicles) has been demonstrated. Cells or membrane vesicles of carbohydrate-utilizing halobacteria grown in media containing this sugar indicated the activation of glucose transport, whereas cells grown in media without sugars did not. This fact has allowed us to conclude that corresponding transport systems are inducible.     

A theory of electrical conductivity of non-equivalent molten salts

The electrical conductivity of non-equivalent molten salts has been theoretically investigated based on the linear response theory, which is an extension from the expression for equivalent molten salts as we have developed in the previous papers. The velocity correlation functions are expressed in terms of pair potentials and pair distribution functions. Using these functions and Langevin equations for ions, the relations among the conductivity coefficients are obtained. It is shown that the ratio of partial conductivities σ⁺/σ^?  is equal to |z ⁺|m ^? /|z ^? |m ⁺, which is an extensive expression from the inverse mass ratio in the case of equivalent molten salts. Numerical results for molten CaCl₂ and AlF₃ are presented, which agree well with the results of experiments and molecular dynamics simulations.     

A study of light-induced conductivity in bacteriorhodopsin

Measurements have been made of light-induced conductivity changes and the associated kinetics of the relaxation processes in aqueous suspensions and sonicated liposomes containing bacteriorhodopsin (bR). Aqueous suspensions exhibit a single relaxation time of 1 to 2 ms. The addition of D₂O to the aqueous suspension slows down the relaxation time, fourfold. Similar behaviour is seen in sonicated liposomes with a relaxation time of 2 to 3 ms. Activation energies of approximately 14 and 6 kJM^-1 are obtained for the effect in sonicated liposomes and aqueous suspension containing bR, respectively. These relaxation processes with lifetime of 1 to 2 ms suggest conformational changes in the protein moiety of bR which most probably may be associated with protonation-deprotonation processes or less likely the release and binding of small ions.     

The effect of anions on K+-binding in aHalobacterium species

Summary Trinitrocresolate (TNC) at a concentration of 2×10^–3 m brings about rapid loss of K from starvingHalobacterium cells. Higher concentrations of other anions such as salicylate, thiocyanate, and perchlorate produce a similar effect. The TNC-induced K loss is not significantly reversed when TNC is removed from the ambient medium. The rate of K loss in the presence of 2×10^–3 m TNC is only slightly increased by the temperature in the ranges of 30 to 40°C and 0 to 20°C; between 20 and 30°C, however, the rate increases 10-fold. The K loss was partly replaced by Na⁺. These data are interpreted in terms of the hypothesis that K is retained in starvingHalobacterium sp. not by active transport, but rather by selective binding on loci which are modified by TNC.     

DNA-DNA homology studies among strains ofHaloferax and other halobacteria

Deoxyribonucleic acid (DNA)-DNA hybridization was used to determine the relationships of the type strains of the three species of the genusHaloferax to 21 halobacterial strains representing isolates from hypersaline habitats and collection strains. Two genotypic groups with high DNA homology were obtained, belonging to the speciesHaloferax gibbonsii andHaloferax mediterranei. The highest DNA relatedness (74%) was obtained in theHalobacterium salinarium group.Halobacterium denitrificans showed very low DNA homology with the other halobacteria investigated. The G+C content of the eight strains ofHaloferax tested ranged between 59.1 and 65.5 mol%.     

Relationship between growth of parsley and soybean cells in suspension cultures and changes in the conductivity of the culture medium

Summary Changes in conductivity and pH during the growth cycle of cell suspensions derived from parsley (Petroselinum hortense) and soybean (Glycine max) have been investigated. Measurement of the conductivity of the medium represents a simple, rapid, and reliable method for the precise determination of the growth phase of a culture. The accuracy of this method has been tested by using phenylalanine ammonia-lyase, an enzyme that has a characteristically short, distinct period of activity during the growth cycle of soybean cell suspensions. It is suggested that an automatic regulation of the conductivity of the medium might be employed for growing plant cells in a continuous culture at a defined stage of growth.     

Mechanisms of acetate formation and acetate activation in halophilic archaea

The halophilic archaea Halococcus (Hc.) saccharolyticus, Haloferax (Hf.) volcanii, and Halorubrum (Hr.) saccharovorum were found to generate acetate during growth on glucose and to utilize acetate as a growth substrate. The mechanisms of acetate formation from acetyl-CoA and of acetate activation to acetyl-CoA were studied. Hc. saccharolyticus, exponentially growing on complex medium with glucose, formed acetate and contained ADP-forming acetyl-CoA synthetase (ADP-ACS) rather than acetate kinase and phosphate acetyltransferase or AMP-forming acetyl-CoA synthetase. In the stationary phase, the excreted acetate was completely consumed, and cells contained AMP-forming acetyl-CoA synthetase (AMP-ACS) and a significantly reduced ADP-ACS activity. Hc. saccharolyticus, grown on acetate as carbon and energy source, contained only AMP-ACS rather than ADP-ACS or acetate kinase. Cell suspensions of Hc. saccharolyticus metabolized acetate only when they contained AMP-ACS activity, i.e., when they were obtained after growth on acetate or from the stationary phase after growth on glucose. Suspensions of exponential glucose-grown cells, containing only ADP-ACS but not AMP-ACS, did not consume acetate. Similar results were obtained for the phylogenetic distantly related halophilic archaea Hf. volcanii and Hf. saccharovorum. We conclude that, in halophilic archaea, the formation of acetate from acetyl-CoA is catalyzed by ADP-ACS, whereas the activation of acetate to acetyl-CoA is mediated by an inducible AMP-ACS.Abbreviations. Hc. Halococcus - Hf. Haloferax - Hr. Halorubrum - Hb. Halobacterium An erratum to this article can be found at     

The distribution of tetramethylammonium ions on the surface of purple membranes

The two-dimensional distribution of deuterated tetramethylammonium (TMA⁺) ions on the surface of purple membranes of Halobacterium halobium was determined by neutron diffraction. The measurements were performed on stacks of these membranes with a high concentration of TMA⁺ molecules in the water layer between the membranes. A difference Fourier analysis of samples with deuterated and undeuterated ions showed an excess of 8.5 TMA⁺ ions per elementary cell in the lipid areas compared to the protein areas. A total number of 90 ions per elementary cell in the intermembrane space was estimated from the preparation procedure. The excess in the lipid domains may result from the higher affinity of TMA⁺ ions for the lipid head groups and/or from the fact that the protein (bacteriorhodopsin) protrudes slightly out of the lipid surface.Abbreviations BR bacteriorhodopsin - TMA Tetramethylammonium     

Determination of membrane potential with lipophilic cations: correction of probe binding

The binding of lipophilic ions to the membrane of envelope vesicles from Halobacterium halobium was examined in the absence and presence of membrane potential. The lipophilic ions used constitute a homologous series of (Phe)₃-P⁺-(CH₂)_n-CH₃ (n = 0–4) and tetraphenylphosphonium (TPP⁺). In the absence of membrane potential, the amounts of binding were proportional to the probe concentration in the medium when the concentration is dilute. Upon illumination, interior negative membrane potential is generated which induces the uptake of phosphonium cation probe. 2 μM were employed as the initial probe concentration. The real membrane potential was evaluated by means of extrapolation to the state of no binding: The values of for various probes are plotted against the binding coefficient. Here, C_i^app is the apparent intra-vesicular concentration of the probes which is calculated without consideration of bound probes. The ordinate intercept of the plot gives the true concentration ratio, and from this the membrane potential is evaluated. The membrane potential-dependent binding was analysed with a model: the membrane is split into two halves, outer and inner half, and the amounts of bound probes in each region are governed by the concentration in the contiguous solution. We obtained a formula which describes amounts of binding as a function of the membrane potential.     

Continuous dielectrophoretic separation of cell mixtures

Use of stream-centered dielectrophoresis (1–4) produced continuous separations on three cell mixtures (1)Chlorella vulgaris withNetrium digitus, (2)Ankistrodesmus falcatus withStaurastrum gracile, and (3)Saccharomyces cerevisiae withNetrium digitus. Maximal separations were obtained for these mixtures of live cells at 100 kHz, 600 kHz, and 2.0 MHz, respectively. The technique was restricted to a frequency range of 0.01–32 MHz, and to suspensions of low conductivity in which microorganisms such as these algae and yeast are tolerant. Extension, however, to cellular organisms requiring higher osmolarity is readily feasible through the use of nonionic solutes such as sucrose, mannose, glycine, etc.     

Abrupt onset of large scale nonproton ion release in purple membranes caused by increasing pH or ionic strength.

The abrupt onset of large scale nonproton ion release by photo-excited purple membrane suspensions has been observed near neutral pH using transient conductivity measurements. At pH 7 and low ionic strength, the conductivity transients due to proton and nonproton ions are of comparable magnitude but of opposite sign: fast proton release and ion uptake, followed by slow proton uptake and ion release. By increasing either the pH or the NaCl concentration, the amplitude of the conductivity transient increases sharply and the signal is then dominated by nonproton ion release. These results can be understood in terms of light-induced changes in the population of counterions condensed at the purple membrane surface caused by changes in the surface charge density. The critical charge density required for condensation to occur is evidently achieved near neutral pH by ionizing dissociable groups on the membrane by either titration (increasing the pH) or shifting their pKs (increasing the ionic strength).     

A limnological reconnaissance of Lake Tebenquiche,Salar de Atacama,Chile

A number of expeditions to the area of Salar de Atacama, Chile, 68° 15'W, 20° 30'S, have involved studies of the biological and chemical features of Lake Tebenquiche, situated in the interior of the salar. Chemically, Tebenquiche is hypersaline, with practically anoxic waters dominated by sodium and chloride ions but with high concentrations of sulphate also. The lake is surrounded and invaded by macrophytes, dominated by Scirpus olmeyi and Juncus, which provide organic material for the formation of bacterial mats. The fauna of limnetic crustaceans is almost exclusively of Artemia salina. The most important genera of bacteria are: Marinomonas, Halobacterium, Acinetobacter and the sulphur reductors Vibrio and Bacillus. The Cyanobacteria are represented exclusively by Oscillatoria.     

Paramagnetism in melanins: pH dependence

Changes in the paramagnetic properties of aqueous suspensions of melanin polymers have been monitored over a pH range from 1 to 12. Distinct changes in spin concentration and electron spin resonance spectral parameters (effective g value and line shape) are shown to occur. These data are interpreted in terms of pH- and temperaturedependent equilibria between diamagnetic and paramagnetic units on the melanin polymer, which can be partly or completely quenched if the melanin is precipitated by lowering the pH or by increasing the salt concentration. The heterogeneity of these units and possible chemical structures are discussed.     

Electric field effects in bacteriorhodopsin.

Exposure of aqueous suspensions of fragments of the purple membrane of Halobacterium halobium to electric field pulses leads to transient linear dichroism phenomena. The effects are interpreted in terms of field-induced alignments of the bacteriorhodopsin chromophore. Two observed relaxation times (tau) are attributed to rotation of the whole membrane fragments (tau s approximately 100 ms), and to a much faster reorientation of the chromophore within membrane (tau f approximately 260 microns).     

Optimization, production, and partial characterization of an alkalophilic amylase produced by sponge associated marine bacterium Halobacterium salinarum MMD047

An endosymbiont Halobacterium salinarum MMD047, which could produce high yields of amylase, was isolated from marine sponge Fasciospongia cavernosa, collected from the peninsular coast of India. Maximum production of enzyme was obtained in minimal medium supplemented with 1% sucrose. The enzyme was found to be produced constitutively even in the absence of starch. The optimum temperature and pH for the enzyme production was 40°C and 8.0, respectively. The enzyme exhibited maximum activity in pH range of 6∼10 with an optimum pH of 9.0. The enzyme was stable at 40°C and the enzyme activity decreased dramatically above 50°C. Based on the present findings, the enzyme was characterized as relatively heat sensitive and alkalophilic amylase which can be developed for extensive industrial applications.     

Formation and regeneration ofHalobacterium spheroplasts

Spheroplasts ofHalobacterium cutirubrum were formed upon suspension of cell pellets in 0.1M MES buffer, pH 7.0, containing 0.5M sucrose, 0.25M NaCl, and 0.01M MgCl₂. The spheroplasts regenerated into rod-shaped bacteria when plated on a complex medium containing 15% (wt/vol) sucrose, undergoing several divisions as spherical bodies before the rod shape developed. The frequency of regeneration was approximately 5% of the total spheroplasts plated. The yield of regenerants was increased significantly (to approximately 35%) when bovine serum albumin was present in the spheroplasting buffer and dilution media. The conditions for spheroplast formation and regeneration inH. cutirubrum were also found effective forHalobacterium salinarium but not forHalobacterium halobium.NRCC Paper no. 23080.     

Archaebacterial class I and class II aldolases from extreme halophiles

Both, class I (Schiff-base forming) and class II (metal requiring) fructose biphosphate aldolases were found to be distributed among halophilic archaebacteria. The aldolase activity fromHalobacterium halobium, H. salinarium, H. cutirubrum, H. mediterranei andH. volcanii exhibited properties of a bacterial class II aldolase as it was metal-dependent for activity and therefore inhibited by EDTA. In contrast, aldolase fromH. saccharovorum, Halobacterium R-113, H. vallismortis andHalobacterium CH-1 formed a Schiff-base intermediate with the substrate and therefore resembled to eukaryotic class I type. The type of aldolase did not vary by changes in the growth medium.     

Apparent cooperativity of amino acid transport in Halobacterium halobium: Effect of electrical potential

Active serine accumulation in cell envelope vesicles from Halobacterium halobium proceeds by co-transport with Na⁺ and can be induced by either transmembrane electrical potential or transmembrane Na⁺ concentration difference. It was shown earlier that in the former case the initial transport rate is a fourth-power function of the magnitude of the electrochemical potential difference of sodium ions, and in the latter, a second-power function. A possible interpretation of this finding is cooperativity of sodium-transporting sites in the transport carrier. When both kinds of driving force are imposed simultaneously on the vesicles, fourth-power dependence on the total potential difference of sodium ions is obtained, suggesting that the transport carrier is regulated by the electrical potential. Heat treatment of the vesicles at 48 ° partially inactivates transport and abolishes this effect of the electrical potential.     

Archaeal promoter-directed expression of the Halobacterium salinarum catalase-peroxidase gene

The Halobacterium salinarum catalase-peroxidase gene was subcloned into shuttle vectors pWL102 and pWL202 and expressed under the control of different archaeal promoters. When Hbt. salinarum was transformed with the catalase-peroxidase gene under the control of its own promoter, catalase-peroxidase activity increased twofold. Catalase-peroxidase activity increased threefold when Hbt. salinarum was transformed with the catalase-peroxidase gene under the control of a tRNA promoter. This bifunctional enzyme in Hbt. salinarum was not induced by environmental stresses such as H₂O₂, intense light, darkness, high temperature, low temperature, redox inhibitors, heavy metals, or ions. Received: May 5, 2000 / Accepted: August 28, 2000