Light-induced, carrier-mediated transport of tetracycline by Rhodopseudomonas sphaeroides.

Tetracycline accumulation by the phototrophic bacterium Rhodopseudomonas sphaeroides has been studied, using the fluorescence properties of the antibiotic and measuring uptake of [7- 3H]tetracycline. Accumulation was carrier mediated, with a Km of approximately 300 micronM. Efflux also appeared to be carried mediated, with a Km of 25 mM. Chlorotetracycline competitively inhibited tetracycline transport. The transport was energy dependent. Efflux occurred during the influx process, and an energy-requiring steady state was reached when influx balanced efflux. Transport was inhibited by metabolic inhibitors such as antimycin A, cyanide, and iodoacetate. Proton conductors such as carbonylcyanide m-chlorophenyl hydrazone were strongly inhibitory. Efflux was not energy dependent. Efflux is partially blocked by mercuric ions and completely blocked by an external pH of 9 to 11. Although efflux rates increased continuously with lowering of the pH, influx rates have a sharp maximum at pH 7.     

Blue light inactivates plasma membrane H(+)-ATPase in pulvinar motor cells of Phaseolus vulgaris L

Unilateral blue light irradiation induces bending of pulvini of Phaseolus vulgaris towards the source of light. The pulvinar bending is caused by a decrease in turgor pressure of motor cells that are irradiated with blue light. Decrease in the turgor pressure is caused by the net efflux of K(+) and counter anions, accompanying membrane depolarization. In the present study the effect of blue light on the activity of plasma membrane H(+)-ATPase was studied in relation to the membrane depolarization. The activity of the plasma membrane H(+)-ATPase was measured using protoplast suspensions prepared from laminar pulvini from primary leaves. A pulse of blue light under continuous red light irradiation induced both a transient increase in the external pH and transient inhibition of the vanadate-sensitive ATPase. Continuous blue light irradiation under continuous red light irradiation induced both a sustained increase in the external pH and sustained inhibition of the vanadate-sensitive ATPase. These results show that blue light inhibits the activity of the plasma membrane H(+)-ATPase. Inactivation of the plasma membrane H(+)-ATPase supports the membrane depolarization induced by the blue light irradiation.     

Fluorimetric pH measurement in whole cells of dark aerobic and anaerobic cyanobacteria

9-Aminoacridine and atebrin fluorescence quenching by dark aerobic and anaerobic suspensions of Anacystis nidulans, Plectonema boryanum and Gloeobacter violaceus was determined at external pH 7–9. Individual pH values in cytosol and thylakoid compartments were calculated from the simultaneously different intracellular enrichment factors of the monovalent and the divalent base. Concomitantly, at external pH 4–7 the cytosolic pH was measured with fluorescein diacetate which is taken up and decomposed in the cell by cytosolic hydrolases. The pH-dependent fluorescence of the free fluorescein, which remains trapped in the cell, monitors the cytosolic pH. The fact that the latter was higher in aerobic than in anaerobic cells, and insensitive to saturating concentrations of dicyclohexylcarbodiimide aerobically, was taken to support the concept of a proton-translocating respiratory chain in the plasma membrane.     

Flooding resistance of Rumex species strongly depends on their response to ethylene: Rapid shoot elongation or foliar senescence

In excised Zea maus L. coleoptiles incubated in aerated media at high fresh weight per volume ratios, indole-3-acetic acid induces transient drops of extracellular pH. Based on the quantitative dependency of the response on the initial auxin concentration we developed a novel auxin bioassay, which allows reliable estimation of IAA concentrations between 10⁻⁸⁵ and 10⁻⁵M. Using the bioassay and complementary concentration measurements by IAA fluorescence we found the transient IAA-induced pH response paralleled by a decrease of auxin activity and concentration in the medium. This decline is rapid and starts immediately upon auxin addition, and insofar differs from the well known IAA degradation by epiphytic bacteria in long-term auxin tests. We conclude that the transient character of the auxin pH response is due to rapid IAA metabolism. The effect occurs under those experimental conditions that are necessary for reliable estimations of auxin-induced shifts of cell wall pH, which considerably complicates the interpretation of the results.     

Tetracycline and Acne Vulgaris: A Clinical and Laboratory Investigation

A satisfactory clinical response to long-term oral tetracycline treatment was associated with a mean serum tetracycline of 1·98 μg/ml. The surface lipid showed an increased triglyceride, decreased free fatty acids, and decreased cholesterol, and the amount of keratin within the pilosebaceous duct was reduced. At this dose level there was no quantitative decrease in the bacterial flora though there was a decrease in the fatty acids. We believe that the latter was due to a direct inhibition by tetracycline on extracellular lipases.     

Phosphorus-31 nuclear magnetic resonance analysis of internal pH during photosynthesis in the cyanobacterium Synechococcus

Phosphorus-31 nuclear magnetic resonance (31P NMR) spectra were obtained from actively photosynthesizing and darkened suspensions of the unicellular cyanobacterium Synechococcus. These spectra show intracellular resonances belonging to inorganic phosphate (Pi), a sugar phosphate (sugar-P), nucleotide di- and triphosphates, and poly-phosphates. The pH-dependent chemical shifts of Pi and sugar-P allowed the estimation of intracellular pH. When irradiated with high-intensity tungsten-halogen light (100 x 10(4) ergs . cm-2 . s-1, measured in the visible range), concentrated cell suspensions in the NMR spectrometer incorporated NaH14CO3 at approximately two-thirds the rate shown by a dilute suspension of cells at saturating light intensity. On the basis of NaH14CO3 incorporation, the effective light intensity obtained under NMR conditions would support growth at approximately one-fourth the maximum rate in dilute suspensions of cells. Irradiated cells maintained a cytoplasmic pH of 7.1--7.3 when exposed to an external pH from 6.4 to 8.3. At an external pH of 6.7, a darkness to light shift caused a 0.4 pH unit alkalinization of the cytoplasm. Treatment of cell suspensions with the uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), in light or darkness, collapsed the internal pH to the level of the external pH. The results suggest a strong light- or energy-dependent buffering of the cytoplasm over a range of external pH. The study demonstrates that 31P NMR can be used to investigate intracellular events in an actively photosynthesizing microorganism.     

pH of the cytoplasm and periplasm of Escherichia coli: rapid measurement by green fluorescent protein fluorimetry

Cytoplasmic pH and periplasmic pH of Escherichia coli cells in suspension were observed with 4-s time resolution using fluorimetry of TorA-green fluorescent protein mutant 3* (TorA-GFPmut3*) and TetR-yellow fluorescent protein. Fluorescence intensity was correlated with pH using cell suspensions containing 20 mM benzoate, which equalizes the cytoplasmic pH with the external pH. When the external pH was lowered from pH 7.5 to 5.5, the cytoplasmic pH fell within 10 to 20 s to pH 5.6 to 6.5. Rapid recovery occurred until about 30 s after HCl addition and was followed by slower recovery over the next 5 min. As a control, KCl addition had no effect on fluorescence. In the presence of 5 to 10 mM acetate or benzoate, recovery from external acidification was diminished. Addition of benzoate at pH 7.0 resulted in cytoplasmic acidification with only slow recovery. Periplasmic pH was observed using TorA-GFPmut3* exported to the periplasm through the Tat system. The periplasmic location of the fusion protein was confirmed by the observation that osmotic shock greatly decreased the periplasmic fluorescence signal by loss of the protein but had no effect on the fluorescence of the cytoplasmic protein. Based on GFPmut3* fluorescence, the pH of the periplasm equaled the external pH under all conditions tested, including rapid acid shift. Benzoate addition had no effect on periplasmic pH. The cytoplasmic pH of E. coli was measured with 4-s time resolution using a method that can be applied to any strain construct, and the periplasmic pH was measured directly for the first time.     

Acidification of culture media by isolated plant protoplasts

Summary We report the observation of a decrease in media pH caused by isolated protoplasts after alkalinization of the culture medium. Additions of other cations or anions did not produce a similar response. Dinitrophenol immediately terminated the response. The acidification response was larger in suspensions that were cultured in auxins. The responses of protoplasts to changes in external pH may provide a means for assessing viability of nondividing protoplasts.     

Green fluorescent protein as a novel indicator of antimicrobial susceptibility in Aureobasidium pullulans.

Presently there is no method available that allows noninvasive and real-time monitoring of fungal susceptibility to antimicrobial compounds. The green fluorescent protein (GFP) of the jellyfish Aequoria victoria was tested as a potential reporter molecule for this purpose. Aureobasidium pullulans was transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, BioTechniques 23:686-690, 1997). The transformed strain Ap1 gfp showed bright fluorescence that was amenable to quantification using fluorescence spectrophotometry. Fluorescence levels in Ap1 gfp blastospore suspensions were directly proportional to the number of viable cells determined by CFU plate counts (r(2) > 0.99). The relationship between cell viability and GFP fluorescence was investigated by adding a range of concentrations of each of the biocides sodium hypochlorite and 2-n-octylisothiozolin-3-one (OIT) to suspensions of Ap1 gfp blastospores (pH 5 buffer). These biocides each caused a rapid (< 25-min) loss of fluorescence of greater than 90% when used at concentrations of 150 microg of available chlorine ml(-1) and 500 microg ml(-1), respectively. Further, loss of GFP fluorescence from A. pullulans cells was highly correlated with a decrease in the number of viable cells (r(2) > 0.92). Losses of GFP fluorescence and cell viability were highly dependent on external pH; maximum losses of fluorescence and viability occurred at pH 4, while reduction of GFP fluorescence was absent at pH 8.0 and was associated with a lower reduction in viability. When A. pullulans was attached to the surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly than in cell suspensions, with > 95% loss of fluorescence after 27 h. This technique should have broad applications in testing the susceptibility of A. pullulans and other fungal species to antimicrobial compounds.     

Enzyme Basis for pH Regulation of Citrate and Pyruvate Metabolism by Leuconostoc oenos

Citrate and pyruvate metabolism by nongrowing cells of Leuconostoc oenos was investigated. (sup13)C nuclear magnetic resonance (NMR) spectroscopy was used to elucidate the pathway of citrate breakdown and to probe citrate or pyruvate utilization, noninvasively, in living cell suspensions. The utilization of isotopically enriched substrates allowed us to account for the end products derived from the metabolism of endogenous reserves. The effect of environmental parameters, e.g., pH, gas atmosphere, and presence of malate, on the end products of citrate utilization was studied. Approximately 10% of the citrate supplied was converted to aspartate which remained inside the cells. A metabolic shift with pH was observed, with acetoin production being favored at pH 4, whereas lactate and acetate production increased significantly at higher pH values. The information obtained with NMR was complemented with studies on the relevant enzyme activities in the metabolic pathway of citrate breakdown. The intracellular pH of the cells was strongly dependent on the external pH; this result, together with the determination of the pH profile of the enzymic activities, allowed us to establish the basis for pH regulation; lactate dehydrogenase activity was optimal at pH 7, whereas the acetoin-forming enzymes displayed maximal activities below pH 5. Citrate utilization was also monitored in dilute cell suspensions for comparison with NMR experiments performed with dense suspensions.     

Role of external calcium in homeostasis of intracellular pH in the cyanobacterium Anabaena sp. strain PCC7120 exposed to low pH

The role of external Ca²⁺ in the homeostasis of intracellular pH (pHi) of Anabaena sp. strain PCC7120 in response to a decrease in the external pH (pHex) has been studied in cell suspensions. Increase in cytoplasmic pH after acid shock is dependent on the presence of Ca²⁺ in the medium. The observed Ca²⁺-mediated alkalization of the cytoplasm depends on the extent of the shift in external pH. Acid pH shifts resulted in an increased permeability of the cytoplasmic membrane to protons, which could be reversed by increasing the concentration of Ca²⁺ in the medium. Thus, the ability of Ca²⁺ to increase cytoplasmic pH might be correlated with an inhibition of net proton uptake by increasing concentrations of external Ca²⁺ under these conditions. This combined response resulted in the generation and maintenance of a larger pH gradient (ΔpH) at acid external pH values. All Ca²⁺ channel blockers tested, such as verapamil and LaCl₃, inhibited the observed Ca²⁺-mediated response. On the other hand, the Ca ionophore calcimycin (compound A23187) was agonistic, and stimulated both cytoplasmic alkalization and inhibition of net proton uptake. The protonophorous uncoupler carbonylcyanide m -chlorophenyl hydrazone, inhibited this Ca²⁺-mediated response, whereas monensin, an inhibitor of the Na⁺/H⁺ antiporter, had no significant effect. The results of the present study suggest that an influx of Ca²⁺ from the extracellular space is required for the regulation of cytoplasmic pH in Anabaena sp. strain PCC7120 exposed to low external pH values.     

Intracellular pH changes induced by calcium influx during electrical activity in molluscan neurons

Simultaneous measurements of electrical activity and light absorbance have been made on nerve cell bodies from Archidoris monteryensis injected with indicator dyes. pH indicators, phenol red and bromocresol purple, and arsenazo III, which under normal conditions is primarily a calcium indicator have been employed. Voltage clamp pulses which induced calcium influx caused an absorbance decrease of the pH dyes indicating an internal acidification. The onset of the pH drop lagged the onset of Ca2+ influx by 200-400 ms, and pH continued to decrease for several seconds after pulse termination which shut off Ca2+ influx. Trains of action potentials also produced an internal pH decrease. Recovery of the pH change required periods greater than 10 min. The magnitude of the pH change was largely unaffected by external pH in the range 6.8-8.4. The voltage dependence of the internal p/ change was similar to the voltage dependence of calcium influx determined by arsenazo III, and removal of calcium from the bathing saline eliminated the pH signal. In neurons injected with EGTA (1-5 mM), the activity- induced internal Ca2+ changes were reduced or eliminated, but the internal pH drop was increased severalfold in magnitude. After the injection of EGTA, voltage clamp pulses produced a decrease in arsenazo III absorbance instead of the normal increase. Under these conditions the dye was responding primarily to changes in internal pH. Injection of H+ caused a rise in internal free calcium. The pH buffering capacity of the neurons was measured using three different techniques: H+ injection, depressing intrinsic pH changes with a pH buffer, and a method employing the EGTA-calcium reaction. The first two methods gave similar measurements: 4-9 meq/unit pH per liter for pleural ganglion cells and 13-26 meq/unit pH per liter for pedal ganglion cells. The EGTA method gave significantly higher values (20-60 meq/unit pH per liter) and showed no difference between pleural and pedal neurons.     

Evidence for a plasmalemma redox system in sugarcane

A plasmalemma-bound NADH-dependent redox system has been identified in protoplasts isolated from cell suspensions of sugarcane. This system oxidized NADH as well as NADPH, increased O₂ consumption 3-fold, and increased the pH of the external medium while the cytoplasmic pH was decreased. In the presence of NADH, ferricyanide was rapidly reduced and the external medium was acidified. The uptake rates of K⁺, 3-O-methylglucose, leucine, and arginine were all decreased in the presence of NADH.     

Dependence of gluconeogenesis, urea synthesis, and energy metabolism of hepatocytes on intracellular pH

The relationship of intracellular pH to extracellular pH has been measured in suspensions of isolated hepatocytes at 25 degrees C. The internal pH was found to be a linear function of external pH and it changed by 0.45 pH unit per 1.0 unit change in external pH. The internal [H+] was equal to the external [H+] at approximately pH 7.1. Gluconeogenesis, urea synthesis, and oxidative phosphorylation showed different dependencies on the intracellular pH. Gluconeogenesis was the most sensitive to changes in [H+] and it declined by 80% when the intracellular pH decreased from 7.1 to 6.9. Urea synthesis was less pH-dependent, decreasing by about 30% for the same change in the intracellular [H+] whereas respiratory rate showed very little dependence on pH at this temperature. Intracellular [ATP]/[ADP] decreased linearly from 8.5 to 1.5 as the intracellular pH increased from 6.8 to 7.6, while intracellular [Pi] was essentially constant at 3.2 nmol/mg of cells, wet weight. Cytochrome c became more reduced with increasing intracellular pH, from less than 10% at pH 6.8 to 35% at pH 7.7. The calculated free energy of hydrolysis of ATP was nearly independent of pH as was the free energy of electron transfer from the intramitochondrial NAD couple (calculated from the [acetoacetate]/[3-OH-butyrate] ratio) to cytochrome c.     

The etiolated cucumber hypocotyl weight-growth test: A sensitive,easy and ultrafast bioassay for IAA

A sensitive, easy and fast bioassay is described for the detection of IAA. The bioassay consists of measuring the weight increase of hypocotyl sections from etiolated cucumber seedlings incubated in a simple medium containing 2 mM KCl, 0.1 mM CaCl and 10 mg/l chloramphenicol (pH 7). The sensitivity of the test is comparable to that of the Avena first internode test, exhibiting a significant response at an IAA concentration as low as 1 ng/ml. The bioassay requires only 3 h, and is easy to perform. The slope of the log-linear concentration-response curve is moderately steep giving good accuracy. The assay is insensitive to the pH of the media between 4 to 7 and can be performed in the presence or absence of buffer. The growth of the etiolated hypocotyl sections is insensitive to GA and to kinetin except at high concentrations.     

Quantitative 31P nuclear magnetic resonance analysis of metabolite concentrations in Langendorff-perfused rabbit hearts.

The quantitative analysis of the mobile high-energy phosphorus metabolites in isovolumic Langendorff-perfused rabbit hearts has been performed by 31P NMR utilizing rapid pulse repetition to optimize sensitivity. Absolute quantification required reference to an external standard, determination of differential magnetization saturation and resonance peak area integration by Lorentzian lineshape analysis. Traditionally accepted hemodynamic indices (LVDP, dp/dt) and biochemical indices (lactate, pyruvate) of myocardial function were measured concomitantly with all NMR determinations. Hemodynamically and biochemically competent Langendorff-perfused rabbit hearts were found to have intracellular PCr, ATP, GPC, and Pi concentrations of 14.95 +/- 0.25, 8.08 +/- 0.13, 5.20 +/- 0.58 and 2.61 +/- 0.47 mM respectively. Intracellular pH was 7.03 +/- 0.01. Cytosolic ADP concentration was derived from a creatine kinase equilibrium model and determined to be approximately 36 microM. Reduction of perfusate flow from 20 to 2.5 ml/min demonstrated statistically significant decreases in PCr, ATP, and pH as well as an increase in Pi that correlated closely with the independent hemodynamic and biochemical indices of myocardial function. The decrease in ATP and PCr concentrations precisely matched the increase in Pi during reduced flow. These results constitute the first quantitative determination of intracellular metabolite concentrations by 31P NMR in intact rabbit myocardium under physiologic and low flow conditions.     

Bioassay of Mildiomycin and a Rapid, Cost-Effective Agar Plug Method for Screening High-yielding Mutants of Mildiomycin

Summary A simple, reliable and low-cost agar diffusion bioassay for quantitative determination of mildiomycin was developed using a strain of Rhodotorula rubra AS 2.166 as the indicator organism and potato dextrose agar at pH 7.0 as the test medium. With equivalent precision and accuracy to HPLC analysis, this method was applied to analyse mildiomycin in complex culture broth during the fermentation process. A modified agar plug method based on the bioassay was constructed for rapid and efficient screening of high-yielding mutants of mildiomycin. Within four weeks, a high production strain, the mildiomycin productivity of which was 75.5% higher than the parent strain, was obtained from 15,000 mutants.     

Acid-base response of bacterial suspensions

The response of suspensions of non-proliferating bacteria to external pH ranging from 3.6 to 9.9 was examined. The acid-base response is affected by the number of viable cells in the suspensions and culture conditions (aerobiosis or anaerobiosis) but not by culture time, composition of the culture medium or the age of suspensions. The results with carbonylcyanide m-chlorophenylhydrazone-and N, N′- dicyclohexylcarbodiimide-treated cells indicated that passive proton conductance and proton translocation by ATP could be involved in this response. pH profiles of suspensions of 18 chemoheterotrophic Gram-negative and Gram-positive bacteria support the hypothesis that the proton gradient generated by an alkali or an acid shift could be dissipated by bacteria with respiratory metabolism. Received 2 June 1998/ Accepted in revised form 6 August 1998     

Evidence for negative gating charges in Myxicola axons

In Myxicola giant axons, the total amount of intramembrane charge available to move over the range -80 to + 120 mV decreases by 23% when the external pH is reduced from 7.3 to 5.5. The remaining charge moves more slowly at the onset of depolarizing pulse, but the rate of charge movement at the end of the pulse is unchanged. In contrast to acidic external pH, intramembrane charge movements are insensitive to alkaline external pH or any change in the internal pH. The results are consistent with a hypothesis in which a portion of the initial outward gating current consists of titratable negative charges moving inward.     

Influence of Pseudomonas syringae culture conditions on initiation of the hypersensitive response of culture tobacco cells.

The inhibitor sensitivity and timing of the ionic response of suspension-cultured tobacco cells were used as a bioassay for the Pseudomonas syringae signal that elicits the hypersensitive response in resistant plants. The ionic response of tobacco cell suspensions inoculated with P. syringae pv. syringae 61 and P. syringae pv. pisi grown in rich media was inhibited by rifampin, tetracycline, and streptomycin during a 2- to 2.5-h induction stage. Coculturing the bacteria with tobacco cells for 3 h or more before inoculating fresh tobacco cells specifically abolished the sensitivity of the ionic response to these inhibitors and reduced the response time of the tobacco cells from 3 to 1 h. The apparent activation of the bacteria during coculture was not dependent on the plant cells and could be achieved by incubating the bacteria in a nitrogen-deficient medium containing a metabolizable carbon source. Addition of proteose peptone and Casamino Acids to this medium suppressed activation of the bacteria. The results suggest that the hypersensitive response-eliciting signal forms late in the induction stage, perhaps as a result of the derepression of some of the P. syringae genes functional in elicitation of the hypersensitive response. The nature of the activated state remains elusive but is consistent with the accumulation of protein(s) whose activity indirectly elicits the ionic response.