Exposure of Escherichia colito acid habituation conditions sensitizes it to alkaline stress

R.J. ROWBURY AND N.H. HUSSAIN. 1996. Escherichia coli transferred from pH 7.0 to pH 5.5 or 6.0 became alkali-sensitive by a rapidly induced phenotypic response. Alkali sensitization was reduced at pH 5.0 and virtually abolished at pH 6.5. The response was triggered by cytoplasmic rather than external or periplasmic acidification and de novo protein synthesis was needed. Alkali sensitivity failed to appear at pH 5.5 plus DNA gyrase inhibitors and was markedly reduced by himA, himD, hns, ompC and nhaA lesions. A tonB deletion mutant showed alkali sensitivity at pH 7.0. Alkali sensitivity induction was not subject to catabolite repression nor was it appreciably affected by a relA lesion. Acid-induced cells were more sensitive to alkali damage to both DNA and β-galactosidase and to alkali inhibition of β-galactosidase induction. Alkali sensitization induced at pH 5.5 may involve NhaB loss.     

Modification of Cell Lethality at Elevated Temperatures The pH Effect

The lethal response of Chinese hamster ovary cells to hyperthermia was determined at selected extracellular pH. Decreasing pH from 7.6 to 6.7 increased the lethal response of cells over the temperature range of 41 to 44°C. Cell viability was not effected over this pH range at 37°C. The pH sensitizing affect was most prominent at temperatures which were marginally lethal at normal pH (7.4). Four hours of exposure to 42°C decreased survival to 10% at pH 7.4 and 0.01% at pH 6.7. Enhanced cell killing was observed when the cells were exposed to reduced pH and elevated temperatures simultaneously. Prolonging the time of pH exposure before and after hyperthermia did not influence survival. High-density culturing increased the sensitivity of cells to hyperthermia. This affect was due to metabolic acidification of the medium and could be reversed by adjusting the pH.     

Novel acid sensitivity induced in Escherichia coli at alkaline pH

Transfer of pH 7.0-grown Escherichia coli to pH 9.0 led to rapid acid sensitivity induction (ASI), the response being fully accomplished within 15 min at 37°C in broth. Only a slight increase in acid sensitivity occurred at pH 8.2 but the response was substantial at pH 8.4 and complete at pH 9.0 with no further sensitization at pH 9.5–10.5. ASI was not prevented by lesions in rpoH, katF, ompR, relA, spoT, fur, phoU, phoM (CreC), phoB/R, unc(atp), phoP or cadA and was unaffected by nalidixic acid, L-leucine or iron starvation or excess. Full acid sensitivity was maintained for at least 2 h after a shift from pH 9.0 back to pH 7.0. ASI did not depend to a major extent on PhoE derepression and increased acid sensitivity of alkali-induced strain C75a ( phoE⁺ ) probably did not involve use of a new outer membrane proton pore.     

Effects of increased intracellular pH-buffering capacity on the light response of Limulus ventral photoreceptor.

Aspects of a possible involvement of hydrogen ions in the electrophysiological responses to light of Limulus ventral photoreceptors were investigated. A 1 M solution of either a zwitter-ionic pH buffer or a weakly-buffering control substance was pressure injected through a micropipette into a ventral photoreceptor cell. To estimate the amount injected, 35SO4 was included in the solution. Membrane currents induced by light flashes were measured by a voltage-clamp technique. The buffer-filled micropipette passed current and a 3M KCl filled micropipette monitored membrane voltage. The sensitivity (peak light-induced current/stimulus energy) was measured, after dark adaptation, before and after injection. Injections of buffers, pH 6.3-7.2, to intracellular concentrations of at least 40-200 mM produced only a small mean decrease in sensitivity, approximately equal to that caused by injections of control substances. Excitation, therefore, apparently is not mediated by a change in intracellular pH. Buffers with pH values 5.4-8.4 were also injected. The time to peak of the response depended on pH, being shortened by up to 20% at pH values below 7.7 and lengthened at higher pH values. The time to peak of the response appeared to be shortened by an increase in intracellular pH-buffering capacity even when there was no change in intracellular pH.     

Effects of increased intracellular pH-buffering capacity on the light response of Limulus ventral photoreceptor

Aspects of a possible involvement of hydrogen ions in the electrophysiological responses to light of Limulus ventral photoreceptors were investigated. A I M solution of either a zwitter-ionic pH buffer or a weakly-buffering control substance was pressure injected through a micropipette into a ventral photoreceptor cell. To estimate the amount injected, ³⁵SO₄ was included in the solution. Membrane currents induced by light flashes were measured by a voltage-clamp technique. The buffer-filled micropipette passed current and a 3 M KCl filled micropipette monitored membrane voltage. The sensitivity (peak light-induced current/stimulus energy) was measured, after dark adaptation, before and after injection. Injections of buffers, pH 6.3–7.2, to intracellular concentrations of at least 40–200 mM produced only a small mean decrease in sensitivity, approximately equal to that caused by injections of control substances. Excitation, therefore, apparently is not mediated by a change in intracellular pH. Buffers with pH values 5.4–8.4 were also injected. The time to peak of the response depended on pH, being shortened by up to 20% at pH values below 7.7 and lengthened at higher pH values. The time to peak of the response appeared to be shortened by an increase in intracellular pH-buffering capacity even when there was no change in intracellular pH.     

Urea sensor with single poly(propylene) membrane

Urease was immobilized on the plasma-aminated surface of a hyfrophobic poly(propylene) (PP) membrane. This membrane, with urease matrix on one side while maintaining its original hydrophobic property on the other, was used to construct the urea sensor. The new urea sensors had response sensitivities ranged from 19 mV/decade to 30 mV/decade depending on the conditions of the plasma reaction. The enzyme electrode using single membrane gave a shorter response time as compared to the corresponding conventional electrode employing two seperate PP membranes. The sensitivity of the enzyme electrode increased with increasing buffer pH and reached a maximal level (40 mV/decade) at pH 7.6. The response sensitivity of the electrode was not affected by the change of buffer strength. Deamination of the plasma-modified hydrophobic PP membrane did not occur in aqueous environment judging from the stability of the urea electrode up to 12 days of operation. (c) 1992 John Wiley & Sons. Inc.     

Predictive models of the combined effects of curvaticin 13, NaCl and pH on the behaviour of Listeria monocytogenes ATCC 15313 in broth

Thirty-three strains of Listeria monocytogenes belonging to different serotypes were tested for their sensitivity to curvaticin 13, an antilisterial bacteriocin produced by Lactobacillus curvatus SB13, using the well diffusion method in Institut Pasteur agar plates at 37 degrees C. No relationship between serotype and sensitivity was observed. The sensitivity of this species was strain-dependent and a large variation in tolerance to curvaticin 13 was observed. The combined effects of curvaticin 13 (0-160 AU ml-1), NaCl (0-6% w/v), pH values (5.0-8.2) and incubation time (0-24 h) were investigated on L. monocytogenes ATCC 15313 in trypcase soy-yeast extract broth at 22 degrees C. For this study, two Doehlert matrices were used in order to investigate the main effects of these factors and their different interactions. The results were analysed using the Response Surface Methodology. Curvaticin 13 had a major inhibitory effect and the response was NaCl concentration-, time- and pH-dependent. This inhibitory activity was the same at pH values between 6.6 and 8.2. Curvaticin 13 was bactericidic at acidic pH values, but the surviving cells resumed growth. For a short incubation time (12 h), the effectiveness of curvaticin 13 was maximal in the absence of NaCl. For longer incubation times (12-48 h), with high NaCl (6%) and curvaticin 13 concentrations (160 AU ml-1), the inhibition of L. monocytogenes was greater than that observed with NaCl or curvaticin 13 alone.     

Excitatory signaling in bacterial probed by caged chemoeffectors.

Chemotactic excitation responses to caged ligand photorelease of rapidly swimming bacteria that reverse (Vibrio alginolyticus) or tumble (Escherichia coli and Salmonella typhimurium) have been measured by computer. Mutants were used to assess the effects of abnormal motility behavior upon signal processing times and test feasibility of kinetic analyses of the signaling pathway in intact bacteria. N-1-(2-Nitrophenyl)ethoxycarbonyl-L-serine and 2-hydroxyphenyl 1-(2-nitrophenyl) ethyl phosphate were synthesized. These compounds are a 'caged' serine and a 'caged' proton and on flash photolysis release serine and protons and attractant and repellent ligands, respectively, for Tsr, the serine receptor. The product quantum yield for serine was 0.65 (+/- 0.05) and the rate of serine release was proportional to [H+] near-neutrality with a rate constant of 17 s-1 at pH 7.0 and 21 degrees C. The product quantum yield for protons was calculated to be 0.095 on 308-nm irradiation but 0.29 (+/- 0.02) on 300-350-nm irradiation, with proton release occurring at > 10(5) s-1. The pH jumps produced were estimated using pH indicators, the pH-dependent decay of the chromophoric aci-nitro intermediate and bioassays. Receptor deletion mutants did not respond to photorelease of the caged ligands. Population responses occurred without measurable latency. Response times increased with decreased stimulus strength. Physiological or genetic perturbation of motor rotation bias leading to increased tumbling reduced response sensitivity but did not affect response times. Exceptions were found. A CheR-CheB mutant strain had normal motility, but reduced response. A CheZ mutant had tumbly motility, reduced sensitivity, and increased response time to attractant, but a normal repellent response. These observations are consistent with current ideas that motor interactions with a single parameter, namely phosphorylated CheY protein, dictate motor response to both attractant and repellent stimuli. Inverse motility motor mutants with extreme rotation bias exhibited the greatest reduction in response sensitivity but, nevertheless, had normal attractant response times. This implies that control of CheY phosphate concentration rather than motor reactions limits responses to attractants.     

Background-subtraction of fast-scan cyclic staircase voltammetry at protein-modified carbon-fiber electrodes

Background-subtraction techniques were applied to the voltammetry of nicotinamide adenine dinucleotide (NADH) at protein-modified carbon-fiber microelectrodes. The background currents at carbon-fiber electrodes were stable and voltammetric scans immediately before or after the analyte were effectively used for background subtraction. Digital step-potential waveforms were used to excite these carbon-fiber electrodes, where the resulting voltammetric analysis assessed the optimal switching and initial potentials and the electrochemical response time was determined. The initial potential was 0.0 V and the switching potential 1.1 V (versus Ag/AgCl) and the response time was approximately 300 ms. Some sensitivity to NADH was lost and voltammetric prescans were required at protein-modified electrodes to obtain a stable baseline. Current versus time was assessed by the average current of the faradaic region from each voltammogram and by differential current; the average current minus the current from a non-faradaic potential range. Differential current assessments discriminated against artifacts caused by pH (as high as 1.0 pH unit) and ionic strength flux (100 mM). These background-subtraction techniques allowed the faradaic information to be obtained quickly and conveniently while maximizing sensitivity and maintaining selectivity.     

Species-dependent changes in stomatal sensitivity to abscisic acid mediated by external pH

The direct effects of pH changes and/or abscisic acid (ABA) on stomatal aperture were examined in epidermal strips of Commelina communis L. and Arabidopsis thaliana. Stomata were initially opened at pH 7 or pH 5. The stomatal closure induced by changes in external pH and/or ABA (10 microM or 10 nM) was monitored using video microscopy and quantified in terms of changes in stomatal area using image analysis software. Measurements of aperture area enabled stomatal responses and, in particular, small changes in stomatal area to be quantified reliably. Both plant species exhibited a biphasic closure response to ABA: an initial phase of rapid stomatal closure, followed by a second, more prolonged, phase during which stomata closure proceeded at a slower rate. Changes in stomatal sensitivity to ABA were also observed. Comparison of these effects between C. communis and A. thaliana demonstrate that this differential sensitivity of stomata to ABA is species-dependent, as well as being dependent on the pH of the extracellular environment.     

Factors influencing the properties of voltammetric carbon fibre electrodes: the importance of the pH of the medium used for the electrical treatment and of the resin coating of the fibres

Electrical treatment of resin-coated voltammetric carbon fibre electrodes with triangular voltage at low (1.1) pH resulted in electrodes almost insensitive to ascorbic acid (AA) and dihydroxyphenylacetic acid (DOPAC) while their response to 5-hydroxyindoleacetic acid (5-HIAA) was practically the same as after treatment in the usually employed pH 7.4 medium. Electrodes treated at high pH (12.0), on the other hand, were more sensitive to AA and DOPAC than those treated at pH 7.4 and less sensitive to 5-HIAA. Exposing resin-coated electrodes to the treatment media without electrical treatment was not sufficient to obtain the same results as with the application of the current. Electrodes without resin coating were sensitive to AA without electrical treatment while coated electrodes were not. Electrical treatment increased the sensitivity of non-coated electrodes and rendered coated electrodes even more sensitive than non-coated ones. Treatment of coated electrodes for a maximum sensitivity to 5-HIAA was found to require less time than to obtain maximum sensitivity to the other compounds. Present results suggest that it is possible to prepare selective voltammetric electrodes by choosing the right parameters for their electrical pretreatment.     

Poly (l-lysine) based semi-interpenetrating polymer network as pH-responsive hydrogel for controlled release of a model protein drug streptokinase

With the aim of developing a pH-sensitive controlled drug release system, a poly (L-lysine) (PLL) based cationic semi-interpenetrating polymer network (semi-IPN) has been synthesized. This cationic hydrogel was designed to swell at lower pH and de-swell at higher pH and therefore be applicable for achieving regulated drug release at a specific pH range. In addition to the pH sensitivity, this hydrogel was anticipated to interact with an ionic drug, providing another means to regulate the release rate of ionic drugs. This semi-IPN hydrogel was prepared using a free-radical polymerization method and by crosslinking of the polyethylene glycol (PEG)-methacrylate polymer through the PLL network. The two polymers were penetrated with each other via interpolymer complexation to yield the semi-IPN structures. The PLL hydrogel thus prepared showed dynamic swelling/de-swelling behavior in response to pH change, and such a behavior was influenced by both the concentrations of PLL and PEG-methacrylate. Drug release from this semi-IPN hydrogel was also investigated using a model protein drug, streptokinase. Streptokinase release was found to be dependent on its ionic interaction with the PLL backbones as well as on the swelling of the semi-IPN hydrogel. These results suggest that a PLL semi-IPN hydrogel could potentially be used as a drug delivery platform to modulate drug release by pH-sensitivity and ionic interaction.     

Cr(VI) quantification using an amperometric enzyme-based sensor: interference and physical and chemical factors controlling the biosensor response in ground waters

The development of an amperometric enzyme-based sensor for chromate (CrO(4)(2-)) quantification in ground waters was investigated. Crucial physical and chemical factors characterising ground waters were tested for their influence or interference on chromate quantification: pH (7.6-8.5), temperature (9-25 degrees C), ionic strength (0-0.2M), oxygen, metals, bicarbonate and sulphate. The biosensor's response was dependent on temperature and pH as sensitivity increased with temperature and was higher at pH 7.6 than at pH 8.5. Sensitivity decreased with ionic strength until 0.1M, and was stable for higher values. Dissolved oxygen did not allow chromate quantification when it was present, but O(2) could be eliminated by adding Na(2)SO(3) or bubbling nitrogen gas into the solution. Bicarbonate did not interfere with chromate quantification by the biosensor. Sulphate was detected with a detection threshold 80 times higher than that of chromate and a lower sensitivity. Several metals (V(V), W(VI), Mn(VII), Mo(VI)) similar to chromate due to their oxidative properties and structure (oxyanions) were tested as possible interfering compounds. The sensitivity of the biosensor for these metals was low and the detection level was 30 times higher than that of chromate. These metal concentrations are usually weaker than chromate concentration in polluted ground waters so that dilution of the sample should allow chromate quantification by the biosensor. This study shows that the cytochrome c(3)-based sensor can detect compounds other than chromate but with a lower sensitivity. Although non-specific for the detection of chromate, it can however be adapted and used for the quantification of chromate in ground waters containing low sulphate concentration.     

Age differences in responsiveness of brainstem chemosensitive neurons to extracellular pH changes

The sensitivity of neurons in the caudal chemosensitive area on the ventrolateral surface of the medulla oblongata (VMS) to extracellular pH changes was examined in newborn and young developing kittens and compared to that of adult cats. The pH was varied by superfusion of the VMS with mock cerebrospinal fluid (CSF) of pH 7.4 (control), 7.0 (acid) and 7.8 (alkaline). A total of 97 neuronal units in the three age groups changed their firing rates inversely in response to extracellular fluid (ECF) pH changes. The greatest sensitivity was found in the adult group where acid superfusion caused an increase in neuronal activity. The least sensitivity was observed in the newborn group (1-6 days old), whereas the young kitten group (4-6 weeks old) exhibited an intermediate sensitivity. Neurons of kittens older than 7 weeks of age demonstrated a response pattern characteristic of the adult group. Neurons of neonates older than seven days, exhibited a response pattern characteristic of the young kitten group.     

Physicochemical Effects of Long Chain Fatty Acids on Bacterial Cells and their Protoplasts

S ummary . Fatty acids of chain length > C₁₀ induced lysis of protoplasts at pH 7·4 when the concentration was nearly bactericidal. At pH 6, lauric and linoleic acids produced lysis above bactericidal concentrations but, at pH 8, lysis was produced by the same acids below bactericidal concentrations. The lysis was immediate at pH 8, but at pH 6 the effect was preceded by contraction of protoplasts. At pH 7·4 the order of lytic activity between individual fatty acids was similar to that of bactericidal activity and the response of protoplasts of Bacillus megaterium relative to those of Micrococcus lysodeikticus reflected differences in bactericidal sensitivity though whole cells were much less sensitive to fatty acid-induced leakage effects than protoplasts. Reversal agents antagonized the lysis of protoplasts by fatty acids. A physicochemical basis for the action of fatty acids and reversal agents on protoplasts and whole cells is discussed.     

Stimulation of the salt receptor of the blowfly. I. NaCl

Application of NaCl solutions to the tip of a labellar sensillum of the blowfly elicited a repetitive neural response from the salt receptor. The response was examined with respect to reproducibility and adaptation. A threshold was observed for tests with dilute solutions. Above this, the response increased linearly with the logarithm of the molarity. The response was not significantly affected by the pH of stimulating solutions, buffered or not, between 3 and 10. Beyond this range, it was reversibly inhibited until, at greater extremes of pH, atypical stimulation independent of the presence of salt was seen. Receptor sensitivity increased with fly age. The results presented here may be due to effects at sites in the sensillum other than the receptor membrane.     

The use of co-immobilization of Trichosporon cutaneum and Bacillus licheniformis for a BOD sensor

The microorganisms Trichosporon cutaneum and Bacillus licheniformis were used to develop a microbial biochemical oxygen demand (BOD) sensor. It was found that T. cutaneum gave a greater response to glucose, whereas B. licheniformis gave a better response to glutamic acid. Hence, co-immobilized T. cutaneum and B. licheniformis were used to construct a glucose and glutamic acid sensor with improved sensitivity and dynamic range. A membrane loading of T. cutaneum at 1.1x10(8 )cells ml(-1) cm(-2) and B. licheniformis at 2.2x10(8) cells ml(-1) cm(-2) gave the optimum result: a linear range up to 40 mg BOD l(-1) with a sensitivity of 5.84 nA mg(-1) BOD l. The optimized BOD sensor showed operation stability for 58 intermittent batch measurements, with a standard deviation of 0.0362 and a variance of 0.131 nA. The response time of the co-immobilized microbial BOD sensor was within 5-10 min by steady-state measurement and the detection limit was 0.5 mg BOD l(-1). The BOD sensor was insensitive to pH in the range of pH 6.8-7.2.     

A new amperometric enzyme electrode for alcohol determination

A new enzyme electrode for the determination of alcohols was developed by immobilizing alcohol oxidase in polvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of enzymatically generated H(2)O(2) was measured at a constant potential of +0.70 V versus SCE. The effects of substrate, buffer and enzyme concentrations, pH and temperature on the response of the electrode were investigated. The optimum pH was found to be pH 8.0 at 30 degrees C. The steady-state current of this enzyme electrode was reproducible within +/-5.0% of the relative error. The sensitivity of the enzyme electrode decreased in the following order: methanol>ethanol>n-butanol>benzyl alcohol. The linear response was observed up to 3.7 mM for methanol, 3.0 mM for ethanol, 6.2 mM for n-butanol, and 5.2 mM for benzyl alcohol. The apparent Michaelis-Menten constant (K(Mapp)) value and the activation energy, E(a), of this immobilized enzyme system were found to be 5.78 mM and 38.07 kJ/mol for methanol, respectively.     

Effects of pH on feeding responses in the apple maggot fly,Rhagoletis pomonella (Diptera: Tephritidae)

Phagostimulatory effects of pH values of sucrose on Rhagoletis pomonella adults were studied in the laboratory. Flies were standardized for age, diet and food deprivation. Two presentation schemes were employed. The first varied pH value (3.0-10.0) with sucrose concentration kept constant at 40%. The second varied both sucrose concentration (8%, 24% and 40%) and pH value (5.0-8.0). Fly feeding propensity was evaluated by observation of fly acceptance or rejection of sucrose and duration of feeding. When tested on red wooden spheres treated with 40% sucrose, fly feeding acceptance was significantly greater when pH ranged from 5.0 to 8.0, and duration of feeding was significantly longer at pH 6.0-7.0. At pH /=8.0, feeding propensity was significantly reduced. Decrease in sucrose concentration significantly increased fly sensitivity to pH. Males were more responsive to varying pH than females. The sucrose pH shown to stimulate maximal feeding response was 6.4. Such information is relevant to formulation improvement of a coating mixture of sucrose and insecticide applied to red spheres as part of apple maggot fly control programs.