Iwao Kusaka

Calcium channel blockers inhibit bacterial chemotaxis

The effect of several Ca2+ channel blockers, which inhibit the voltage‐dependent Ca2+ uptake in Bacillus subtilis, on chemotactic behaviour of the bacterium was studied. Nitrendipine, verapamil, LACl3 and ω‐conotoxin were tested and these blockers inhibited chemotactic behaviour in the bacterium toward L‐alanine. Among these blockers, ω‐conotoxin was the most effective inhibitor of chemotaxis. EGTA was also as effective as ω‐conotoxin. In contrast, these blockers, did not inhibit the motility and the growth of the bacterium. These results suggest that internal Ca2+ plays an important role in the sensory system of bacterial chemotaxis.

Na+-driven Ca2+ transport in alkalophilic Bacillus.

Ca2+ transport was studied in membrane vesicles of alkalophilic Bacillus. When Na+-loaded membrane vesicles were suspended in KHCO3/KOH buffer (pH 10) containing Ca2+, rapid uptake of Ca2+ was observed. The apparent Km value for Ca2+ measured at pH 10 was about 7 microM, and the Km value shifted to 24 microM when measured at pH 7.4. The efflux of Ca2+ was studied with Ca2+-loaded vesicles. Ca2+ was released when Ca2+-loaded vesicles were suspended in medium containing 0.4 M Na+. Ca2+ was also transported in membrane vesicles driven by an artificial pH gradient and by a membrane potential generated by K+-valinomycin in the presence of Na+. These results indicate the presence of Ca2+/Na+ and H+/Na+ antiporters in the alkalophilic Bacillus A-007

Patch Clamp Studies on Ion Pumps of the Cytoplasmic Membrane ofEscherichia coli FORMATION, PREPARATION, AND UTILIZATION OF GIANT VACUOLE-LIKE STRUCTURES CONSISTING OF EVERTED CYTOPLASMIC MEMBRANE

Formation of giant protoplasts from normalEscherichia coli cells resulted in the formation of giant vacuole-type structures (which we designate as provacuoles) in the protoplasts. Electron microscopic observation revealed that these provacuoles were surrounded by a single membrane. We detected inner (cytoplasmic) membrane proteins in the provacuolar membrane but not outer membrane proteins. Biochemical analyses revealed that the provacuoles consist of everted cytoplasmic membranes. We applied the patch clamp method to the giant provacuoles. We have succeeded in measuring current that represents inward movement of H+because of respiration and to ATP hydrolysis by the FoF1-ATPase. Such current was inhibited by inhibitors of the respiratory chain or FoF1-ATPase. This method is applicable for analyses of ion channels, ion pumps, or ion transporters in E. coli or other microorganisms.

Secretory enzyme production and conidiation of Aspergillus oryzae in submerged liquid culture

SummaryThe production of α- and β-galactosidases, proteinase and α-amylase and also conidiation of Aspergillus oryzae were examined in liquid soybean meal culture. In a culture of soybean meal only, conidiation of the fungus was not induced and the production of the enzymes was not significant, although fungal growth was abundant. When phosphate was added to the medium at concentrations above 0.2 M, enzyme production was significantly increased and the cells formed conidiophores after enzyme production had attained maximum level. Increase in production of galactosidases was the most marked.K- or Na-salts other than phosphate were not effective stimulants for enzyme production, while conidiation was not induced under these growth conditions. Conidiation and production of enzymes were repressed by the addition of glucose or casamino acids to the soybean meal medium containing KH2PO4.Conidiation and enzyme production were also studied in modified Czapek media in which sucrose was replaced by other carbon sources. Lactose, lactulose, melibiose and polysaccharides composed of galactosyl linkage such as arabinogalactan induced both conidiation and production of the enzymes.

Characterization of a Ca2+ Uniporter from Bacillus Subtilis by Partial Purification and Reconstitution into Phospholipid Vesicles

Ca2+ was accumulated by right-side-out membrane vesicles of Bacillus subtilis following imposition of a diffusion potential, inside-negative, owing to K+-efflux via valinomycin. Uptake was dependent on the magnitude of the membrane potential. This voltage-dependent Ca2+ uptake was inhibited by Ca2+ channel blockers such as nitrendipine, verapamil and LaCl3, and was competitively inhibited by Ba2+ and Sr2+. The system showed saturation kinetics with an apparent Km for Ca2+ of about 250 microM. Proteins responsible for the voltage-dependent Ca2+ uptake were partially purified by preparative isoelectric focusing in a Sepharose bed. A fraction at pH 5.28-5.33 contained the activity. The characteristics of Ca2+ uptake in reconstituted proteoliposomes were the same as those in membrane vesicles (sensitive to Ca2+ channel blockers; inhibited by Ba2+ and Sr2+). In addition, uptake was not influenced by a pH gradient imposed on the vesicles. The apparent Km for Ca2+ in the reconstituted system was about 260 microM. The specific activity was increased about 50-fold by purification with isoelectric focusing.

Preparation of artificial vesicles having an L-alanine uptake activity which requires NADH as energy source

The vesicles (proteoliposomes) were artificially prepared by sonic oscillation from hydrophobic protein, which was isolated from cytoplasmic membrane fraction of Bacillus subtilis W-23, and phospholipids or lipids, which were isolated from different organims; phospholipids from hen-egg yolk and soybean, and lipids from B . subtilis W-23. In the vesicles, remarkable activity of L-alanine uptake was demonstrated in the presence of NADH or ascorbate-phenazine methosulfate as an energy source. The uptake activity was temperature-dependent and inhibited by respiration inhibitor (KCN) and uncoupler (ClCCP). Replacement experiment was also performed.

Involvement of Autolysis of Cytoplasmic Membranes in the Process of Autolysis of Bacillus cereus

SUMMARY: When exponentially growing Bacillus cereus organisms were suspended in buffer or buffered hypertonic sucrose solution and incubated at 37°, a rapid decrease in turbidity of the suspension was observed (autolysis). During autolysis ultraviolet-absorbing substances leaked from the bacteria and over 50% of the total phospholipids was released. Of the amino sugars, main components of the cell walls of this organism, less than 20% was released. Phase-contrast microscopy showed the empty rod-shaped ghosts which increased in number, while the total counts were constant during autolysis. Mg2+ and Mn2+ inhibited the autolysis; these cations either prevented leakage of ultraviolet absorbing substances or release of phospholipids. Isolated cytoplasmic membranes autolysed when the membranes were incubated at 37° in buffer solution (pH 7.0 to 7.5). It could be assumed, therefore, that the cytoplasmic membranes of Bacillus cereus were lysed faster than the cell walls during autolysis and that the autolysis of the membranes was inhibited by Mg2+ and Mn2+.

Patch clamp analysis of the respiratory chain in Bacillus subtilis

Bacillus subtilis is a representative Gram-positive bacterium. In aerobic conditions, this bacterium can generate an electrochemical potential across the membrane with aerobic respiration. Here, we developed the patch clamp method to analyze the respiratory chain in B. subtilis. First, we prepared giant protoplasts (GPs) from B. subtilis cells. Electron micrographs and fluorescent micrographs revealed that GPs of B. subtilis had a vacuole-like structure and that the intravacuolar area was completely separated from the cytoplasmic area. Acidification of the interior of the isolated and purified vacuole-like structure, due to H(+) translocation after the addition of NADH, revealed that they consisted of everted cytoplasmic membranes. We called these giant provacuoles (GVs) and again applied the patch clamp technique. When NADH was added as an electron donor for the respiratory system, a significant NADH-induced current was observed. Inhibition of KCN and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) demonstrated that this current is certainly due to aerobic respiration in B. subtilis. This is the first step for more detailed analyses of respiratory chain in B. subtilis, especially H(+) translocation mechanism.

Guanine nucleotide-induced Ca2+ release in permeabilized murine thymocytes.

GTP and IP3 induced Ca2+ release from an internal store in permeabilized murine thymocytes loaded with Ca2+ by ATP. Ca2+ release was dependent on the concentration of GTP: half‐maximal release with 0.5 μM and maximal release with 10 μM. The GTP effect was completely abolished by 100 μM GTPγS, GMPPNP and UTP. None of the other nucleotides used except ITP induced Ca2+ release. When GTP was added after the effect of IP3 had virtually subsided, and vice versa, further Ca2+ release occurred, which led to the conclusion that the mechanism of GTP‐mediated Ca2+ release may be different from that of IP3‐mediated release.

H+- or K+-dependent transport systems of phosphate in alkalophilic Bacillus

Abstract Membrane vesicles isolated from an alkalophilic Bacillus accumulated phosphate when a K + or H + gradient (extravesicular high) was imposed across the membranes. K + - or H + -dependent uptake of phosphate was not influenced by the reverse gradient of H + or K + . Phosphate preloaded into the vesicles was released by an imposed H + or K + gradient (Intravesicular high) and the release was inhibited by the reverse gradient of these ions. The optimum pH of the uptake lay between 9–10 in H + and K + -dependent case. K + -dependent uptake was stimulated by valinomycin beyond pH 9, indicating the transport was electrogenic in the pH. The kinetic profile of the activities indicates these two transport mechanisms are different systems.