Chemotaxis of Spirochaeta aurantia: involvement of membrane potential in chemosensory signal transduction.

The effects of valinomycin and nigericin on sugar chemotaxis in Spirochaeta aurantia were investigated by using a quantitative capillary assay, and the fluorescent cation, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide was used as a probe to study effects of chemoattractants on membrane potential. Addition of a chemoattractant, D-xylose, to cells in either potassium or sodium phosphate buffer resulted in a transient membrane depolarization. In the presence of valinomycin, the membrane potential of cells in potassium phosphate buffer was reduced, and the transient membrane depolarization that resulted from the addition of D-xylose was eliminated. Although there was no detectable effect of valinomycin on motility, D-xylose taxis of cells in potassium phosphate buffer was completely inhibited by valinomycin. In sodium phosphate buffer, valinomycin had little effect on membrane potential or D-xylose taxis. Nigericin is known to dissipate the transmembrane pH gradient of S. aurantia in potassium phosphate buffer. This compound did not dissipate the membrane potential or the transient membrane depolarization observed upon addition of D-xylose to cells in either potassium or sodium phosphate buffer. Nigericin did not inhibit D-xylose taxis in either potassium or sodium phosphate buffer. This study indicates that the membrane potential but not the transmembrane pH gradient of S. aurantia is somehow involved in chemosensory signal transduction.     

Chemotaxis in Spirochaeta aurantia.

Cell of Spirochaeta aurantia M1 suspended in isotropic buffer solution swam in nearly straight lines and appeared to spin around their longitudinal axis. Occasionally, cells stopped and flexed, and then resumed translational motility, usually in a different direction. The average cell velocity was 26 micron/s. A quantitative assay for chemotaxis was used to test various chemicals for their ability to attract S. aurantia M1. The cells exhibited a tactic response toward 5 X 10(-2) M D-glucose between 10 and 35degree C; the optimum response was at 25degree C. At 5 degree C motility was not impaired, but D-glucose taxis was abolished. Chemotaxis toward D-glucose was stimulated by L-cysteine (2 X 10(-4) M). D-Glucose, 2-deoxy-D-glucose, alpha-methyl-D-glucoside, D-galactose, D-fucose, D-mannose, D-fructose, D-xylose, maltose, cellobiose, and D-glucosamine were effectve attractants for S. aurantia M1. D-Galactose taxis and D-fucose taxis were induced by the presence of D-galactose in the growth medium. The amino acids tested did not serve as attractants, tgrowing cells of S. aurantia M1 exhibited an aerotactic response.     

Inhibition of Spirochaeta aurantia chemotaxis by neurotoxins.

The effects of neurotoxic compounds on the chemotactic response of Spirochaeta aurantia were investigated. In the presence of neurotoxins that affect action potential generation and transmission in excitable eucaryotic cells, D-xylose taxis was inhibited by 69 to 93%. Inhibition of chemotaxis was not due to decreased viability or motility. This study supports the hypothesis that the molecular basis for sensory signal transduction in S. aurantia involves ion fluxes across the cytoplasmic membrane.     

Relationship between proton motive force and motility in Spirochaeta aurantia.

The effects of various metabolic inhibitors on the motility of Spirochaeta aurantia were investigated. After 15 min in sodium arsenate buffer, 90% of cells remained motile even though adenosine triphosphate levels dropped from 5.6 to 0.1 nmol/mg (dry weight) of cells. After 70 min in sodium arsenate, 5% of cells were motile. Addition of phenazine methosulfate plus ascorbate at this time resulted in motility of 95% of cells, but adenosine triphosphate levels remained at 0.1 nmol/mg of cell dry weight. Carbonyl cyanide-m-chlorophenyl hydrazone rapidly (within 1 min) and completely inhibited motility of metabolizing cells in potassium phosphate buffer. However, after 15 min in the presence of carbonyl cyanide m-chlorophenyl hydrazone the cellular adenosine triphosphate level was 3.4 nmol/mg (dry weight) of cells, and the rate of oxygen uptake was 44% of the rate measured in the absence of carbonyl cyanide m-chlorophenyl hydrazone. Cells remained motile under conditions where either the electrical potential or the pH gradient across the membrane of S. aurantia was dissipated. However, if both gradients were simultaneously dissipated, motility was rapidly inhibited. This study indicates that a proton motive force, in the form of either a transmembrane electrical potential or a transmembrane pH gradient, is required for motility in S. aurantia. Adenosine triphosphate does not appear to directly activate the motility system in this spirochete.     

Protonmotive force and motility of Bacillus subtilis.

Motility of Bacillus subtilis was inhibited within a few minutes by a combination of valinomycin and a high concentration of potassium ions in the medium at neutral pH. Motility was restored by lowering the concentration of valinomycin or potassium ions. The valinomycin concentration necessary for motility inhibition was determined at various concentrations of potassium ions and various pH's. At pH 7.5, valinomycin of any concentration did not inhibit the motility, when the potassium ion concentration was lower than 9 mM. In the presence of 230 mM potassium ion, the motility inhibition by valinomycin was not detected at pH lower than 6.1. These results are easily explained by the idea that the motility of B. subtilis is supported by the electrochemical potential difference of the proton across the membrane, or the protonmotive force. The electrochemical potential difference necessary for motility was estimated to be about -90 mV.     

Sodium-dependent transport of neutral amino acids by whole cells and membrane vesicles of Streptococcus bovis, a ruminal bacterium.

Streptococcus bovis JB1 cells were able to transport serine, threonine, or alanine, but only when they were incubated in sodium buffers. If glucose-energized cells were washed in potassium phosphate and suspended in potassium phosphate buffer, there was no detectable uptake. Cells deenergized with 2-deoxyglucose and incubated in sodium phosphate buffer were still able to transport serine, and this result indicated that the chemical sodium gradient was capable of driving transport. However, when the deenergized cells were treated with valinomycin and diluted into sodium phosphate to create both an artificial membrane potential and a chemical sodium gradient, rates of serine uptake were fivefold greater than in cells having only a sodium gradient. If deenergized cells were preloaded with sodium (no membrane potential or sodium gradient), there was little serine transport. Nigericin and monensin, ionophores capable of reversing sodium gradients across membranes, strongly inhibited sodium-dependent uptake of the three amino acids. Membrane vesicles loaded with potassium and diluted into either lithium or choline chloride were unable to transport serine, but rapid uptake was evident if sodium chloride was added to the assay mixture. Serine transport had an extremely poor affinity for sodium, and more than 30 mM was needed for half-maximal rates of uptake. Serine transport was inhibited by an excess of threonine, but an excess of alanine had little effect. Results indicated that S. bovis had separate sodium symport systems for serine or threonine and alanine, and either the membrane potential or chemical sodium gradient could drive uptake.     

Control of the chemotactic behavior of Bacillus subtilis cells

The effects of nigericin, valinomycin and some lipophilic cations on the motile behavior of non-starved and methionine-starved Bacillus subtilis cells were studied. For valinomycin and nigericin a quantitative relationship between the flux in the proton-motive force and the duration of the twiddle response was found. Lipophilic cations bind to the ion gate controlling the twiddle frequency and thereby cause the cells to swim smoothly. To explain the transmission of the chemotactic signal a model is given in which receptors, a hyperpolarizing wave, an ion gate and two methylation sites, viz. methyl-accepting chemotaxis proteins and a further methylation site (MT), play a role. For the transmission of the signal caused by an attractant both the hyperpolarizing wave and an interaction between receptor and methylation site (MT) are needed. The methyl-accepting chemotaxis proteins are involved in the adaptation/deadaptation to altered levels of attractant. Artificial changes in the proton-motive force act directly on the ion gate, which finally controlls the twiddle frequency of the cells.Abbreviations KT medium potassium taxis medium - NAT medium sodium taxis medium - HT medium acidic taxis medium - OHT medium alkaline taxis medium - ImT medium imidazole taxis medium - GT medium glycylgycine taxis medium - Di-S-C₃(5) 3,3-dipropyl-2,2-thiacarbocyanine iodide - TPAs⁺ tetraphenylarsonium ion - TPMP⁺ triphenylmethylphosphonium ion - DDA⁺ dibenzyldimethylammonium ion - TPB^- tetraphenylboron ion - pmf proton-motive force - MCP methyl-accepting chemotaxis protein - MT methylation site - membrane potential     

The motile and tactic behaviour of Pseudomonas aeruginosa in anaerobic environments

ATP generated by the anaerobic metabolism of L-arginine in Pseudomonas aeruginosa was used to maintain the membrane potential. Although both the ATP concentration and membrane potential were lower than in aerobically incubated bacteria, motility and chemotaxis were almost normal. Venturicidin stopped anaerobic motility by abolishing the membrane potential. The addition of venturicidin to aerobic bacteria caused an increase in the membrane potential, but a decrease in internal ATP concentration, resulting in bacteria which were motile but non-chemotactic. The membrane potential was the only requirement for continued motility but ATP was required in addition for chemotaxis.     

The proton motive force generated in Leuconostoc oenos by L-malate fermentation.

In cells of Leuconostoc oenos, the fermentation of L-malic acid generates both a transmembrane pH gradient, inside alkaline, and an electrical potential gradient, inside negative. In resting cells, the proton motive force ranged from -170 mV to -88 mV between pH 3.1 and 5.6 in the presence Of L-malate. Membrane potentials were calculated by using a model for probe binding that accounted for the different binding constants at the different pH values at the two faces of the membrane. The delta psi generated by the transport of monovalent malate, H-malate-, controlled the rate of fermentation. The fermentation rate significantly increased under conditions of decreased delta psi, i.e., upon addition of the ionophore valinomycin in the presence of KCl, whereas in a buffer depleted of potassium, the addition of valinomycin resulted in a hyperpolarization of the cell membrane and a reduction of the rate of fermentation. At the steady state, the chemical gradient for H-malate- was of the same magnitude as delta psi. Synthesis of ATP was observed in cells performing malolactic fermentation.     

Chemoattractants elicit methylation of specific polypeptides in Spirochaeta aurantia

On the basis of this investigation, chemotaxis in Spirochaeta aurantia correlates with methylation of specific polypeptides which are presumed to be analogous to the methyl-accepting chemotaxis proteins (MCPs) in bacteria such as Escherichia coli. The polypeptides exhibited apparent molecular weights in the range of 55,000 to 65,000. Generally, two major presumptive MCP bands and three minor bands were observed on sodium dodecyl sulfate-polyacrylamide gels. Upon addition of D-glucose to S. aurantia cells, methylation of the presumptive MCPs increased for 10 to 12 min to a level greater than 4 times the level of methylation in the absence of D-glucose. Removal of D-glucose resulted in a decrease in methylation of the presumptive MCPs to a level similar to that in unstimulated cells. All attractants tested, including a non-metabolizable attractant (alpha-methyl-D-glucoside) stimulated methylation of the presumptive MCPs (from 1.7 to 4.3 times the level of methylation in unstimulated cells). D-Mannitol, a metabolizable sugar which is not an attractant for S. aurantia, did not stimulate methylation. Stimulation of methylation by D-galactose occurred in cells induced for D-galactose taxis but not in uninduced cells. These data are indicative of an evolutionary relationship between the chemotaxis systems of spirochetes and of flagellated bacteria.     

Requirement of a transmembrane pH gradient for the entry of diphtheria toxin into cells at low pH

The effects of acidification of the cytosol and of electrical depolarization on the entry of diphtheria toxin were studied. Entry of the toxin from the cell surface was induced by low pH, and the presence of the toxin in the cytosol was monitored as toxin-induced inhibition of protein synthesis. To reduce the membrane potential the cells were incubated in a buffer containing a high concentration of potassium. The cytosol was acidified either by incubating the cells with acetic acid, by incubating them with ammonium chloride which was subsequently removed in the presence of amiloride to prevent pH regulation by the Na+/H+ exchanger, or by incubating the cells in isotonic KCl in the presence of nigericin and valinomycin. The results showed that when the cytosol was acidified by either method toxin entry was inhibited, while a reduction in the membrane potential did not strongly interfere with the entry. A pH gradient across the membrane of at least 1 pH unit was required for entry. Possibly this gradient acts as a driving force for diphtheria toxin entry.     

A novel method for the determination of electrical potentials across cellular membranes. I. Theoretical considerations and mathematical approach

A new method for the determination of electrical potentials across cellular membranes has been developed. In order to determine the membrane potential, cells were incubated in buffer solutions with increasing concentrations of KCl. Parallel experiments were performed with buffer solutions which additionally contained valinomycin. After sedimentation of the cells, the membrane potential was calculated from data which were obtained by simply measuring the wet mass, the dry mass and the potassium content of cell pellets by atomic absorption spectroscopy.     

Voltage clamp effects on bacterial chemotaxis

To examine whether or not sensory signaling in bacteria is by way of fluctuations in membrane potential, we studied the effect of clamping the potential on bacterial chemotaxis. The potential was clamped by valinomycin, a K+ -specific ionophore, in the presence of K+. Despite the clamped potential, sensory signaling did occur: both Escherichia coli and Bacillus subtilis cells were still excitable and adaptable under these conditions. It is concluded that signaling in the excitation and adaptation steps of chemotaxis is not by way of fluctuations in the membrane potential.     

31P NMR studies of Clostridium thermocellum. Mechanism of end product inhibition by ethanol

31P NMR studies of intact cells and perchloric acid extracts are used to investigate the effect of ethanol on the bioenergetics and glycolysis of Clostridium thermocellum, an anaerobic bacterium potentially useful for the single step conversion of biomass to ethanol. Whole cells suspended in phosphate buffer and given a carbon source (cellobiose) at 60 degrees C rapidly establish a pH gradient across the membrane that can be monitored by the chemical shifts of inorganic phosphate in the exterior buffer and in the cytoplasm. Peak intensities can be related to phosphate active transport rates. Wild type bacteria and cells grown in inhibiting concentrations of ethanol establish similar pH gradients, but with slower kinetics and slower phosphate transport rates for the cells adapted to growth in ethanol. Direct addition of ethanol does not affect the rate of pH gradient formation or phosphate transport. Thus, while ethanol does not directly affect processes for energy conservation carried out by the membrane, adaptation to ethanol does alter membrane functions such as phosphate transport. 31P NMR spectra of perchloric acid extracts show that when wild type cells are adapted to grow in inhibiting concentrations of ethanol and then energized with cellobiose, sugar phosphate content is increased and the steady state distribution of glycolytic intermediates is altered. Nucleotide triphosphate/nucleotide diphosphate ratios are unaltered in these cells. These results strongly indicate that in C. thermocellum growth inhibition by ethanol is related to a blockage in glycolysis.     

Anaerobic 109Cd accumulation by cadmium-resistant and -sensitive Staphylococcus aureus

Washed cells of the cadmium-sensitive Staphylococcus aureus 17810S accumulated 109Cd under anaerobic conditions via the Mn2+ porter down delta psi in 1 or 100 mM phosphate buffer, pH 7; in washed cells of the cadmium-resistant S. aureus 17810R 109Cd accumulation was highly reduced. Nigericin did not stimulate anaerobic Cd2+ accumulation by strain 17810R in 100 mM phosphate buffer, suggesting that delta psi could energize Cd2+ efflux. In 1 mM phosphate buffer nigericin restored Cd2+ accumulation via the Mn2+ porter down delta psi in strain 17810R, indicating involvement of delta pH in Cd2+ extrusion. Increase of phosphate buffer concentration from 1 to 100 mM and addition of energy source at steady-state caused delta psi-dependent Cd2+ efflux from the nigericin-pretreated cells of strain 17810R. This suggests that the Cd2+ efflux system in S. aureus may require energy of both ATP and delta mu H+.     

The effect of ionophores on proton flux in the ruminal bacterium,streptococcus bovis

NonenergizedStreptococcus bovis cells, which were washed in potassium-phosphate buffer and incubated in Tris buffer containing 200mm potassium chloride (pH 6.5), did not take up tetraphenylphosphonium ion (TPP⁺), but the same cells took up TPP⁺ when they were incubated in Tris buffer lacking potassium. This result indicated that passive potassium diffusion was creating an electrical potential () across the cell membrane. Neither cells took significant amounts of 9-aminoacridine (9-AA), an intracellular pH marker. Cells that were incubated in Tris buffer and treated with carbonyl cyanidem-chlorophenylhydrazone (CCCP) took up 9-AA, and this result indicated that this protonophore was facilitating proton influx. The ionophores monensin and lasalocid also caused 9-AA uptake, and it appeared that they were responsible for or responsive to potassium/proton antiport. However, there was also a rapid accumulation of 9-AA when the cells were treated with valinomycin, a potassium uniporter that cannot translocate protons. This latter result indicated that potassium efflux was associated with another avenue of proton influx (e. g., potassium/proton symport). Because cells treated with dicyclohexyl carbodiimide (DCCD) also exhibited valinomycin-dependent 9-AA uptake, it is unlikely that the F₁F₀ATPase or ATP formation was responsible for proton flux across the cell membrane.     

Comparative analysis of antimicrobial activities of valinomycin and cereulide, the Bacillus cereus emetic toxin

Cereulide and valinomycin are highly similar cyclic dodecadepsipeptides with potassium ionophoric properties. Cereulide, produced by members of the Bacillus cereus group, is known mostly as emetic toxin, and no ecological function has been assigned. A comparative analysis of the antimicrobial activity of valinomycin produced by Streptomyces spp. and cereulide was performed at a pH range of pH 5.5 to pH 9.5, under anaerobic and aerobic conditions. Both compounds display pH-dependent activity against selected Gram-positive bacteria, including Staphylococcus aureus, Listeria innocua, Listeria monocytogenes, Bacillus subtilis, and Bacillus cereus ATCC 10987. Notably, B. cereus strain ATCC 14579 and the emetic B. cereus strains F4810/72 and A529 showed reduced sensitivity to both compounds, with the latter two strains displaying full resistance to cereulide. Both compounds showed no activity against the selected Gram-negative bacteria. Antimicrobial activity against Gram-positive bacteria was highest at alkaline pH values, where the membrane potential (ΔΨ) is the main component of the proton motive force (PMF). Furthermore, inhibition of growth was observed in both aerobic and anaerobic conditions. Determination of the ΔΨ, using the membrane potential probe DiOC(2)(3) (in the presence of 50 mM KCl) in combination with flow cytometry, demonstrated for the first time the ability of cereulide to dissipate the ΔΨ in sensitive Gram-positive bacteria. The putative role of cereulide production in the ecology of emetic B. cereus is discussed.     

Energy coupling of facilitated transport of inorganic ions in Rhodopseudomonas sphaeroides.

Within the scope of a study on the effects of changes in medium composition on the proton motive force in Rhodopseudomonas sphaeroides, the energy coupling of sodium, phosphate, and potassium (rubidium) transport was investigated. Sodium was transported via an electroneutral exchange system against protons. The system functioned optimally at pH 8 and was inactive below pH 7. The driving force for the phosphate transport varied with the external pH. At pH 8, Pi transport was dependent exclusively on delta psi (transmembrane electrical potential), whereas at pH 6 only the delta pH (transmembrane pH gradient) component of the proton motive force was a driving force. Potassium (rubidium) transport was facilitated by a transport system which catalyzed the electrogenic transfer of potassium (rubidium) ions. However, in several aspects the properties of this transport system were different from those of a simple electrogenic potassium ionophore such as valinomycin: (i) accumulated potassium leaked very slowly out of cells in the dark; and (ii) the transport system displayed a threshold in the delta psi, below which potassium (rubidium) transport did not occur.     

Phototaxis and membrane potential in the photosynthetic bacterium Rhodospirillum rubrum.

Cells of the photosynthetic bacterium Rhodospirillum rubrum cultivated anaerobically in light show phototaxis. The behavior of individual cells in response to the phenomenon is reversal(s) of the swimming direction when the intensity of the light available to them abruptly decreases. The tactic response was inhibited by antimycin, an inhibitor of the photosynthetic electron transfer system. The inhibitory effect of antimycin was overcome by phenazine methosulfate. Motility of the cells was not impaired by antimycin under aerobic conditions. Valinomycin plus potassium also inhibited their phototactic response; however, valinomycin or potassium alone had no effect. A change in membrane potential of the cells was measured as an absorbance change of carotenoid. Changes in the membrane potential caused by "on-off" light were prevented by antimycin and by valinomycin plus potassium, but not by antimycin plus phenazine methosulfate nor valinomycin or potassium alone. The results indicated that the phototactic response of R. rubrum is mediated by a sudden change in electron flow in the photosynthetic electron transfer system, and that the membrane potential plays an important role in manifestation of the response.     

Active transport of alanine by thermostable membrane vesicles isolated from a thermophilic bacterium.

1. Thermostable membrane vesicles which were capable of active transport of alanine dependent on either respiration or an artificial membrane potential were isolated from the thermophilic aerobic bacterium PS3. 2. Uptake of alanine was dependent on the oxidation of ascorbate-phenazine methosulfate or on generated or exogenous NADH, but succinate and malate failed to drive the uptake. The optimum temperature for respiration-driven uptake of alanine was 45 to 60 degrees. 3. Potassium ion-loaded vesicles were prepared by incubating vesicles at 55 degrees in 0.5 M potassium phosphate. The addition of valinomycin elicited rapid and transient uptake of alanine under the test conditions. Uptake of alanine in response to valinomycin was progressively enhanced by the addition of dicylohexylcarbodiimide, but was completely abolished in the presence of a proton conductor or synthetic permeable cation. The effect of dicyclohexylcarbodiimide was dependent on its concentration and was maximal at a concentration of 0.4 mM. 4. The proton permeability of membrane vesicles was reduced by the addition of dicyclohexylcarbodiimide. A small but significant difference was found in the initial rates of proton uptake in the presence of dicyclohexylcarbodiimide with and without alanine. The results suggest that protons alanine are transported simultaneously in a stoichiometric ratio of 1 : 1. 5. The uptake of alanine was also driven by a pH gradient induced by an instantaneous pH drop in a suspension of alkali-loaded vesicles. Thus, alanine accumulation was driven not only by an electrical potential but also by a pH gradient. 6. Addition of ATP resulted in the inhibition of alanine uptake dependent on artificial membrane potential. ATP hydrolysis by membrane ATPase created a membrane potential which was inside-positive, and this might decrease the effective membrane potential (generated by K+ efflux mediated by valinomycin) available to drive alanine uptake.