Calorimetrie Analysis of the Effect of Streptomycin,Tetracycline and Chloramphenicol on the Growth of Escherichia coli

The effects of streptomycin, tetracycline and chloramphenicol on the growth of Escherichia coli were studied quantitatively in a conduction-type batch calorimeter at pH 6.2 and 37°C. Change in the growth thermograms with increasing drug concentrations in the medium were analyzed with a kinetic model of non-competitive inhibition.

The number of drug molecules needed to inactivate a unit viable cell, m, was estimated to be 1.2 + 0.1, 0.7 ±0.1 and 1.3 ±0.1 for streptomycin, tetracycline and chloramphenicol, respectively. With the assumption that the drug binding sites are all identical, the microscopic dissociation constant per binding site, K, for the drugs was found to be 0.19, 0.43 and 0.94μmoldm^-3 for streptomycin, tetracycline and chloramphenicol, respectively. With these m and K_{ values, drug potency curves were drawn for the three drugs.     

Reduction of Escherichia coli O157:H7 on radish seeds by sequential application of aqueous chlorine dioxide and dry‐heat treatment

Aims: To assess the effectiveness of sequential treatments of radish seeds with aqueous chlorine dioxide (ClO₂) and dry heat in reducing the number of Escherichia coli O157:H7. Methods and Results: Radish seeds containing E. coli O157:H7 at 5·5 log CFU g^?1 were treated with 500 μg ml^?1 ClO₂ for 5 min and subsequently heated at 60°C and 23% relative humidity for up to 48 h. Escherichia coli O157:H7 decreased by more than 4·8 log CFU g^?1 after 12 h dry‐heat treatment. The pathogen was inactivated after 48 h dry‐heat treatment, but the germination rate of treated seeds was substantially reduced from 91·2 ± 5·0% to 68·7 ± 12·3%. Conclusions: Escherichia coli O157:H7 on radish seeds can be effectively reduced by sequential treatments with ClO₂ and dry heat. To eliminate E. coli O157:H7 on radish seeds without decreasing the germination rate, partial drying of seeds at ambient temperature before dry‐heat treatment should be investigated, and conditions for drying and dry‐heat treatment should be optimized. Significance and Impact of the study: This study showed that sequential treatment with ClO₂ and dry‐heat was effective in inactivating large numbers of E. coli O157:H7 on radish seeds. These findings will be useful when developing sanitizing strategies for seeds without compromising germination rates.     

The Effect of Various Carbonate Sources on the Survival of Escherichia coli in Dairy Cattle Manure

Manure slurries (n = 3) prepared from the feces and urine of lactating dairy cattle (1 part urine, 2.2 parts feces, and 6.8 parts distilled water) had an initial pH of 8.6 ± 0.1; dissolved carbonate concentrations of 48 ± 4 mm, and Escherichia coli counts of 5.9 ± 0.7 logs per ml slurry. The pH of untreated slurries declined to pH 7.0 ± 0.1 by the 10th day of incubation, and the E. coli count increased approximately 10-fold (P < 0.05). When slurries were treated with Na₂CO₃, K₂CO₃, NaHCO₃ or Na₂CO₃·NaHCO₃ (0 to 16 g/kg slurry), the dissolved carbonates increased in a linear fashion, but only Na₂CO₃ and K₂CO₃ (8 g/kg or greater) or Na₂CO₃·NaHCO₃ (16 g/kg) ensured an alkaline pH. Even relatively low concentrations of Na₂CO₃ or K₂CO₃ (8 or 12 g/kg) caused a decrease in E. coli viability (P < 0.05), and E. coli could not be detected if 16 g/kg was added (day 5 or 10 of incubation). Na₂CO₃·NaHCO₃ also caused a decrease in E. coli viability, (P < 0.05), but some E. coli (approximately 10⁴ cells per g) were detected on day 10 even if the concentration was 16 g/kg. NaHCO₃ did not prevent the decrease in pH or cause a decrease in E. coli numbers (P > 0.05). Calculations based on the Henderson-Hasselbalch equation (pH and dissolved carbonates) indicated that little E. coli killing was noted until the dissolved carbonate anion concentrations (CO₃ ⁻²) were greater than 1 mm, but bicarbonate anion (HCO₃ ⁻) concentrations as high as 180 mm did not affect E. coli viability. These results are consistent with the idea that carbonate anion has antimicrobial properties and can kill E. coli in dairy cattle manure. Received: 20 December 2000 / Accepted: 7 February 2001     

Genome‐derived minimal metabolic models for Escherichia coli MG1655 with estimated in vivo respiratory ATP stoichiometry

Metabolic network models describing growth of Escherichia coli on glucose, glycerol and acetate were derived from a genome scale model of E. coli. One of the uncertainties in the metabolic networks is the exact stoichiometry of energy generating and consuming processes. Accurate estimation of biomass and product yields requires correct information on the ATP stoichiometry. The unknown ATP stoichiometry parameters of the constructed E. coli network were estimated from experimental data of eight different aerobic chemostat experiments carried out with E. coli MG1655, grown at different dilution rates (0.025, 0.05, 0.1, and 0.3 h^?1) and on different carbon substrates (glucose, glycerol, and acetate). Proper estimation of the ATP stoichiometry requires proper information on the biomass composition of the organism as well as accurate assessment of net conversion rates under well‐defined conditions. For this purpose a growth rate dependent biomass composition was derived, based on measurements and literature data. After incorporation of the growth rate dependent biomass composition in a metabolic network model, an effective P/O ratio of 1.49 ± 0.26 mol of ATP/mol of O, K_X (growth dependent maintenance) of 0.46 ± 0.27 mol of ATP/C‐mol of biomass and m_ATP (growth independent maintenance) of 0.075 ± 0.015 mol of ATP/C‐mol of biomass/h were estimated using a newly developed Comprehensive Data Reconciliation (CDR) method, assuming that the three energetic parameters were independent of the growth rate and the used substrate. The resulting metabolic network model only requires the specific rate of growth, µ, as an input in order to accurately predict all other fluxes and yields. Biotechnol. Bioeng. 2010;107: 369–381. © 2010 Wiley Periodicals, Inc.     

Metal chelates in the storage and transport of neurotransmitters: formation of mixed ligand chelates of Mg 2+ -ATP with biogenic amines

Abstract— Quantitative studies on the interactions of adenosine-triphosphate and several biogenic amines with magnesium ion have been carried out in an attempt to correlate the thermodynamic stabilities of the metal-binding of the amines with the in vivo affinities of the amines for granule-binding. Equilibrium data indicate that in each of the ternary chelate systems (viz. Mg²⁺-ATP-amine), the predominant reaction in the pH range 3.0–7.0 is the formation of a magnesium-ATP chelate with a stability constant, log K_ML=3.22 ± 0.02. Each of the biogenic amines coordinates with Mg²⁺-ATP system in the pH range 7.0–10.5 to form the mixed ligand chelate (or ternary chelate), Mg²⁺-ATP-amine(1:1:1). The stability constants for the binding of the amines with Mg²⁺-ATP are: (i) norepinephrine (NE) = 2.34 ± 0.32; (ii) epinephrine (E) = 2.95 ± 0.08; (iii) dopamine (DA) = 3.05 ± 0.06; (iv) octopamine (OA) = 1.93 ± 0.12; (v) 6-hydroxydopamine (6-HDA) = 2.42 ± 0.14; (vi) 3-methoxynorephedrine (MeN) =2.76 ± 0.09; (vii) amphetamine (AA) =2.09 ± 0.05; (viii) tyramine (TA) = 2.60 ± 0.04; (ix) phenylethylamine (PEA) = 0. A general correlation is indicated between the stability constants (binding strengths) of the amine chelates and the metal-binding functionalities of the amines on the one hand and their vesicular binding characteristics in in vivo systems on the other (Carlsson and Waldeck , 1966). The Mg²⁺-ATP-dependant amine storage mechanism of KIRSHNER (1962a;b) and Carlsson , Hillårp and Waldeck (1963) is discussed both in the light of the data on metal chelate stability and of a significant modification of metal coordination hypothesis.     

Preliminary characterization of a thermostable DNA polymerase I from a mesophilic Bacillus sphaericus strain C3-41

A thermostable DNA polymerase I from a mesophilic Bacillus sphaericus strain C3-41 was characterized in this study. The polI was cloned, sequenced and over-expressed in Escherichia coli. The expressed 110 kDa fusion protein of PolI was stable at 70°C for 1 h. Compared with DNA polymerase I of E. coli (TaKaRa), the relative polymerase activity of this PolI was 3.33 ± 0.1 RFU μl⁻¹ at 37°C using fluorescent quantitative analysis. It showed higher polymerase activity than E. coli PolI at higher temperature, with a relative activity of 3.75 ± 0.1 RFU μl⁻¹ at 70°C. The polI sequence analysis and the protein structure prediction indicated that this protein had a high similarly to other PolI from thermophilic micro-organisms. This information is of importance for future study for evolution of the house-keeping gene polI in entomopathogenic bacterium B. sphaericus.     

Inactivation of Escherichia coli by citral

Aims: The aim was to evaluate (i) the resistance of Escherichia coli BJ4 to citral in a buffer system as a function of citral concentration, treatment medium pH, storage time and initial inoculum size, (ii) the role of the sigma factor RpoS on citral resistance of E. coli, (iii) the role of the cell envelope damage in the mechanism of microbial inactivation by citral and (iiii) possible synergistic effects of mild heat treatment and pulsed electric fields (PEF) treatment combined with citral. Methods and Results: The initial inoculum size greatly affected the efficacy of citral against E. coli cells. Exposure to 200 μl l^?1 of citral at pH 4·0 for 24 h at 20°C caused the inactivation of more than 5 log₁₀ cycles of cells starting at an inoculum size of 10⁶ or 10⁷ CFU ml^?1, whereas increasing the cell concentration to 10⁹ CFU ml^?1 caused <1 log₁₀ cycle of inactivation. Escherichia coli showed higher resistance to citral at pH 4·0 than pH 7·0. The rpoS null mutant strain E. coli BJ4L1 was less resistant to citral than the wild‐type strain. Occurrence of sublethal injury to both the cytoplasmic and outer membranes was demonstrated by adding sodium chloride or bile salts to the recovery media. The majority of sublethally injured cells by citral required energy and lipid synthesis for repair. A strongly synergistic lethal effect was shown by mild heat treatment combined with citral but the presence of citral during the application of a PEF treatment did not show any advantage. Conclusions: This work confirms that cell envelope damage is an important event in citral inactivation of bacteria, and it describes the key factors on the inactivation of E. coli cells by citral. Significance and Impact of the Study: Knowledge about the mechanism of microbial inactivation by citral helps establish successful combined preservation treatments.     

Thermal Analysis of Bacterial Ribosomes

Ribosomal particles of E. coli were examined by using a heat leakage scanning calorimeter. Remarkable changes were observed in thermograms of 70S ribosomes and their subunits when the Mg²⁺ concentration was raised from 1 mm to 10 mm. It was suggested that ribosomal subunits exist in more than one conformation, and changes in their conformation might be the primary cause of the association-dissociation process of ribosomes. Comparisons of thermograms of RNase- and chymotrypsin-treated, as well as non-treated SOS and 30S subunits suggest that conformational changes in each subunit may be ascribed to changes in rRNA.     

Molecular characterization of Alr1105 a novel arsenate reductase of the diazotrophic cyanobacterium Anabaena sp. PCC7120 and decoding its role in abiotic stress management in Escherichia coli

This paper constitutes the first report on the Alr1105 of Anabaena sp. PCC7120 which functions as arsenate reductase and phosphatase and offers tolerance against oxidative and other abiotic stresses in the alr1105 transformed Escherichia coli. The bonafide of 40.8 kDa recombinant GST+Alr1105 fusion protein was confirmed by immunoblotting. The purified Alr1105 protein (mw 14.8 kDa) possessed strong arsenate reductase (Km 16.0 ± 1.2 mM and Vmax 5.6 ± 0.31 μmol min^?1 mg protein^?1) and phosphatase activity (Km 27.38 ± 3.1 mM and Vmax 0.077 ± 0.005 μmol min^?1 mg protein^?1) at an optimum temperature 37 °C and 6.5 pH. Native Alr1105 was found as a monomeric protein in contrast to its homologous Synechocystis ArsC protein. Expression of Alr1105 enhanced the arsenic tolerance in the arsenate reductase mutant E. coli WC3110 (?arsC) and rendered better growth than the wild type W3110 up to 40 mM As (V). Notwithstanding above, the recombinant E. coli strain when exposed to CdCl₂, ZnSO₄, NiCl₂, CoCl₂, CuCl₂, heat, UV-B and carbofuron showed increase in growth over the wild type and mutant E. coli transformed with the empty vector. Furthermore, an enhanced growth of the recombinant E. coli in the presence of oxidative stress producing chemicals (MV, PMS and H₂O₂), suggested its protective role against these stresses. Appreciable expression of alr1105 gene as measured by qRT-PCR at different time points under selected stresses reconfirmed its role in stress tolerance. Thus the Alr1105 of Anabaena sp. PCC7120 functions as an arsenate reductase and possess novel properties different from the arsenate reductases known so far.     

Kinetic Study and Mathematical Modeling of Chromium(VI) Reduction and Microorganism Growth Under Mixed Culture

The kinetics of chromium(VI) reduction by Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) was studied under both pure and mixed cultures. Initially, the study of kinetics was performed in pure culture. It was observed that the growth of the two bacteria was both inhibited in the presence of chromium(VI). The maximum specific growth rate (μ _m ) of P. aeruginosa decreased from 2.3942 h^?1 (without Cr(VI)) to 1.8551 h^?1 (with Cr(VI)). Under the mixed culture, the growth of E. coli was inhibited by P. aeruginosa. The maximum specific growth rate (μ _m ) of E. coli decreased form 0.871 h^?1 (in pure culture) to 0.153 h^?1 (in mixed culture). When the concentration of each bacterium was 4.5 × 10⁸ cells ml^?1, the half-velocity reduction rate constant (K _C) and the maximum specific reduction rate constant (v _max) of chromium(VI) were 80.05 mg chromium(VI) l^?1 and 3.674 mg chromium(VI) cells^?1 h^?1, respectively. The results showed that the simulation appeared in good agreement with the experimental data, supporting the series of mathematical models represented the bacteria growth and chromium(VI) reduction in both pure and mixed cultures usefully.     

Optimal conditions for hepatitis B core antigen production in shaked flask fermentation

The effects of various environmental factors such as pH (5, 6, 7, 8 and 9), temperature (30, 37 and 40°C) and rotational speed (150, 200 and 250 rpm) on the growth and the hepatitis B core antigen (HBcAg) production ofEscherichia coli W3110IQ were examined in the present study. The highest growth rate is achieved at PH 7, 37°C and at a rotational speed of 250 rpm which is 0.927 h⁻¹. The effect of pH on cell growth is more substantial compared to other parameters; it recorded a 123% different between the highest growth rate (0.927 h⁻¹) at pH 7 and lowest growth at pH 5. The highest protein yield is achieved at pH 9, rotational speed of 250 rpm and 40°C. The yield of protein at pH 7 is 154% higher compared to the lowest yield achieved at pH 5. There is about 28% different of the protein yield for theE. coli cultivated at 250 rpm compared to that at 150 rpm which has the lowest HBcAg yield. The yield of protein at 40°C is 38% higher compared to the lowest yield achieved, at 30°C.     

Open-loop control of specific growth rate in fed-batch cultures of recombinant E.coli

Summary A computer-based algorithm was used for the open-loop control of specific growth rate in fed-batch cultures of recombinant E.coli.. The control of nutrient feed rate to an exponential trajectory resulted in growth of the culture at a constant specific growth rate. Stable specific growth rates between 0.08 and 0.4 h^–1 were achieved.     

Disruption of glpF gene encoding the glycerol facilitator improves 1,3-propanediol production from glucose via glycerol in Escherichia coli

Engineered Escherichia coli has recently been applied to produce 1,3-propanediol (1,3-PDO) from glucose. A metabolic intermediate in the production pathway, glycerol, is partially secreted into the extracellular of E. coli through a glycerol facilitator encoded by glpF, and this secretion consequently decreases 1,3-PDO production. Therefore, we aimed to determine whether disrupting the glpF gene would improve 1,3-PDO production in E. coli. The intracellular glycerol concentration in a glpF-disruptant was 7·5 times higher than in a non-disruptant. The glpF-disrupted and non-disrupted E. coli strains produced 0·26 and 0·09 g l⁻¹ of 1,3-PDO, respectively, from 1% glucose after 72 h of cultivation. The specific growth rate (μ) and the 1,3-PDO yield from glucose (Y_P/S) in the disruptant were higher than those in the non-disruptant (ΔglpF, μ = 0·08 ± 0·00 h⁻¹, Y_P/S = 0·06 mol mol-glucose⁻¹; BW25113, μ = 0·06 ± 0·00 h⁻¹, Y_P/S = 0·02 mol mol-glucose⁻¹). Disruption of the glpF gene decreased the production of the by-product, acetic acid. These results indicated that disruption of glpF increased the intracellular concentration of glycerol and consequently increased 1,3-PDO production in E. coli.     

Evidence for Arylamine N-Acetyltransferase Activity in the Escherichia coli

N-Acetyltransferase activities with p-aminobenzoic acid and 2-aminofluorene as substrates were determined in isolates of the bacterium Escherichia coli. The N-acetyltransferase activity was determined by an acetyl CoA recycling assay and high pressure liquid chromatography. The N-acetyltransferase activities from a number of E. coli isolates were found to be 0.67 ± 0.04 nmole/min/mg protein for 2-aminofluorene, and 0.46 ± 0.02 nmole/min/mg protein for p-aminobenzoic acid. The apparent K _m and V _max values obtained were 2.85 ± 0.65 mM and 7.51 ± 0.86 nmol/min/mg protein, respectively, for 2-aminofluorene, and 2.35 ± 0.39 mM and 9.43 ± 0.78 nmol/min/mg protein, respectively, for p-aminobenzoic acid. The optimal pH value for the enzyme activity was 7.0 for both substrates tested. The optimal temperature for enzyme activity was 37°C for both substrates. The N-acetyltransferase activity was inhibited by iodoacetamide: at 0.25 mM iodoacetamide, activity was reduced 50%, and at 1.0 mM, more than 90%. Among a series of divalent cations and salts, Cu²⁺ and Zn²⁺ were demonstrated to be the most potent inhibitors. This report is the first demonstration of acetyl CoA:arylamine N-acetyltransferase activity in E. coli. Received: 29 April 1997 / Accepted: 2 July 1997     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides

Aims: Analysis of the physiology and metabolism of Escherichia coli arcA and creC mutants expressing a bifunctional alcohol‐acetaldehyde dehydrogenase from Leuconostoc mesenteroides growing on glycerol under oxygen‐restricted conditions. The effect of an ldhA mutation and different growth medium modifications was also assessed. Methods and Results: Expression of adhE in E. coli CT1061 [arcA creC(Con)] resulted in a 1·4‐fold enhancement in ethanol synthesis. Significant amounts of lactate were produced during micro‐oxic cultures and strain CT1061LE, in which fermentative lactate dehydrogenase was deleted, produced up to 6·5 ± 0·3 g l^?1 ethanol in 48 h. Escherichia coli CT1061LE derivatives resistant to >25 g l^?1 ethanol were obtained by metabolic evolution. Pyruvate and acetaldehyde addition significantly increased both biomass and ethanol concentrations, probably by overcoming acetyl‐coenzyme A (CoA) shortage. Yeast extract also promoted growth and ethanol synthesis, and this positive effect was mainly attributable to its vitamin content. Two‐stage bioreactor cultures were conducted in a minimal medium containing 100 μg l^?1 calcium d ‐pantothenate to evaluate oxic acetyl‐CoA synthesis followed by a switch into fermentative conditions. Ethanol reached 15·4 ± 0·9 g l^?1 with a volumetric productivity of 0·34 ± 0·02 g l^?1 h^?1. Conclusions: Escherichia coli responded to adhE over‐expression by funnelling carbon and reducing equivalents into a highly reduced metabolite, ethanol. Acetyl‐CoA played a key role in micro‐oxic ethanol synthesis and growth. Significance and Impact of the Study: Insight into the micro‐oxic metabolism of E. coli growing on glycerol is essential for the development of efficient industrial processes for reduced biochemicals production from this substrate, with special relevance to biofuels synthesis.     

betaine modulates intracellular thiol and potassium levels in Escherichia coli in medium with high osmolarity and alkaline pH

Glycine betaine stimulates the growth rate of various bacteria in high osmolarity medium. In our studies, glycine betaine stimulated the growth rate of Escherichia coli K 12 in minimal medium with normal osmolarity at alkaline pH (pH 8.2). Betaine also caused a reduction in the intracellular pools of K⁺ and low molecular weight thiols in E. coli growing both in medium with high osmolarity and at alkaline pH. These effects of betaine were absent at pH 7.0. In cells growing in high osmolarity medium, 10 mM sodium acetate or 10 M N-ethylmaleimide reduced expression of the osmosensitive gene proU to the same extent as treatment with betaine; however, under these conditions, sodium acetate and N-ethylmaleimide did not stimulate the growth of E. coli. It is proposed that low molecular weight thiols and intracellular pH may participate in the response of E. coli to betaine.     

Flow injection chemiluminescence study of acridinium ester stability and kinetics of decomposition

Decomposition of phenyl acridinium-9-carboxylate is monitored using electrogenerated chemiluminescence in a flow system. The formation of the pseudobase from the acridinium ester [AE] is described by rate = k′₁[AE] + k″₁[AE][OH^?]^0.5, where k′₁ = 0.020 ± 0.006 s^?1 and k″₁ = 2.1 ± 0.8 (L/mol)^?0.5 s^?1. Irreversible decomposition of the pseudobase is described by rate = k′₂[AE][OH^?], where k′₂ = 20.1 ± 3.8 (L/mol s). These kinetic equations, plus measurement of variation in emission intensity for constant acridinium ester concentration, are used to predict the resulting emission intensity v. pH behaviour given various contact times (in the 0.25 to 25 s range) for the acridinium ester to be in an alkaline solution prior to initiation of the chemiluminescence reaction.     

Kinetics of oxidation of tyrosine by a model alkoxyl radical

Abstract

Oxidation of tyrosine moieties by radicals involved in lipid peroxidation is of current interest; while a rate constant has been reported for reaction of lipid peroxyl radicals with a tyrosine model, little is known about the reaction between tyrosine and alkoxyl radicals (also intermediates in the lipid peroxidation chain reaction). In this study, the reaction between a model alkoxyl radical, the tert-butoxyl radical and tyrosine was followed using steady-state and pulse radiolysis. Acetone, a product of the β-fragmentation of the tert-butoxyl radical, was measured; the yield was reduced by the presence of tyrosine in a concentration- and pH-dependent manner. From these data, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 6?±?1 × 10⁷ M^?1 s^?1 at pH 10. Tyrosine phenoxyl radicals were also monitored directly by kinetic spectrophotometry following generation of tert-butoxyl radicals by pulse radiolysis of solutions containing tyrosine. From the yield of tyrosyl radicals (measured before they decayed) as a function of tyrosine concentration, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 7?±?3 × 10⁷ M^?1 s^?1 at pH 10 (the reaction was not observable at pH 7). We conclude that reaction involves oxidation of tyrosine phenolate rather than undissociated phenol; since the pK_a of phenolic hydroxyl dissociation in tyrosine is ～ 10.3, this infers a much lower rate constant, about 3 × 10⁵ M^?1 s^?1, for the reaction between this alkoxyl radical and tyrosine at pH 7.4.     

Evaluation of thermal inactivation of Escherichia coli using microelectrode ion flux measurements with osmotic stress

Aims: To elucidate the potential use of microelectrode ion flux measurements to evaluate bacterial responses to heat treatment. Methods and Results: Escherichia coli K12 was used as a test bacterium to determine whether various heat treatments (55–70°C for 15 min) affected net ion flux across E. coli cell membranes using the MIFE? system to measure net K⁺ fluxes. No difference in K⁺ fluxes was observed before and after heat treatments regardless of the magnitude of the treatment. Applying hyperosmotic stress (3% NaCl w/v) during flux measurement led to a net K⁺ loss from the heat‐treated E. coli cells below 65°C as well as from nonheated cells. In contrast, with E. coli cells treated at and above 65°C, hyperosmotic stress disrupted the pattern of K⁺ flux observed at lower temperatures and resulted in large flux noise with random scatter. This phenomenon was particularly apparent above 70°C. Although E. coli cells lost the potential to recover and grow at and above 62°C, K⁺ flux disruption was not clearly observed until 68°C was reached. Conclusions: No changes in net K⁺ flux from heat‐stressed E. coli cells were observed directly as a result of thermal treatments. However, regardless of the magnitude of heat treatment above 55°C, loss of viability indicated by enrichment culture correlated with disrupted K⁺ fluxes when previously heated cells were further challenged by imposing hyperosmotic stress during flux measurement. This two‐stage process enabled evaluation of the lethality of heat‐treated bacterial cells within 2 h and may be an alternative and more rapid method to confirm the lethality of heat treatment. Significance and Impact of the Study: The ability to confirm the lethality of thermal treatments and to specify minimal time/temperature combinations by a nonculture‐dependent test offers an alternative system to culture‐based methods.