Growth and proton-potassium exchange in the bacterium Enterococcus hirae: the effect of protonophore and the role of redox potential

The bacterium Enterococcus hirae is able to grow under anaerobic conditions during sugar fermentation (pH 8.0) in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone at a considerably lower specific rate. Bacterial growth was accompanied by acidification of the medium and a drop in its redox potential from positive to negative values (by −220 mV). We showed that the reducer dithiothreitol, which determines negative values of the redox potential, accelerates bacterial growth and enhances acidification of the medium, abolishing the effect of the protonophore without binding to dithiothreitol. Conversely, the nonpenetrating oxidant ferricyanide, which maintained positive values of the redox potential, suppressed bacterial growth. These results are indicative of the role of the proton-motive force and the importance of reductive processes in bacterial growth. The proton-potassium exchange is inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone and recovers in the presence of dithiothreitol. Dithiothreitol can substitute for the proton-motive force; however, both ferricyanide and dithiothreitol may have a direct effect on the bacterial membrane.     

Redox potential affects the measured heat resistance of Escherichia coli O157:H7 independently of oxygen concentration

Cells of Escherichia coli O157:H7 were heat-treated at 59 °C and enumerated in (i) anaerobic medium with a low redox potential, (ii) anaerobic media with the oxidizing agents potassium ferricyanide or 2,6-dichloroindophenol (DPIP) added to raise the redox potential, (iii) aerobic medium with a high redox potential and (iv) aerobic medium with the reducing agent dithiothreitol added to lower the redox potential. The measured heat-resistance was greatest when the enumeration medium was highly anaerobic due to the absence of oxygen and the presence of hydrogen and cysteine HCl. Measured heat resistance was influenced by the redox potential of the enumeration medium independently of the chemical used to adjust it and therefore, independently of the presence of oxygen. Sub-lethally heat-damaged cells regained their ability to grow in media of high redox potential at a similar rate whether the redox potential was increased by the addition of potassium ferricyanide, DPIP or oxygen.     

Effects of the thiol reagent dithiothreitol on the oxidation-reduction potential and the growth of Escherichia coli in anaerobic conditions at different pH

Changes in the oxidation-reduction potential of medium during the growth of Escherichia coli bacteria under anaerobic conditions in the pH range 5.5-7.5 were studied. It was shown that dithiothreitol, a reducer of thiol groups, at a concentration of 3 mM decreased the initial value of oxidation-reduction potential to negative values of -60 to -220 mV, increased the duration of the lag growth phase, decreased the growth rate, and lowered the acidification of the medium and accumulation of potassium ions independently of pH. The value of the potential in the presence of 3 mM dithiothreitol decreased by 15-20 mV both at alkaline and acidic pH. Dithiothreitol at a concentration of 10 mM stopped molecular hydrogen production at pH 5.5 only. The effects of dithiothreitol on the oxidation-reduction potential and bacterial growth were probably associated with changes in the state of thiol groups in membrane proteins, which affects the generation of membrane potential, proton secretion from cells, K+ accumulation in bacteria, and the activity of membrane-associated enzymes.     

Redox sensing by Escherichia coli: effects of dithiothreitol, a redox reagent reducing disulphides, on bacterial growth

Escherichia coli is able to grow with a high rate under anaerobic conditions upon decrease in redox potential (E(h)) both either in slightly alkaline (pH 7.5) or acidic (pH 5.5) medium. Upon transition of E. coli MC4100 culture to stationary growth phase a decrease in E(h) from the positive values of +120 to +160 mV to the negative ones of -380 to -550 mV, and the H(2) production are observed at various pH. A redox reagent dl-dithiothreitol (DTT) in a concentration of 3mM reduces E(h) to the negative values, and increases a latent (lag) growth phase duration, as well as delays a logarithmic growth phase independently of pH. At alkaline and acidic pH the changes in membrane potential (DeltaPsi) are observed in the presence of 3mM DTT. K(+) uptake is recovered. At pH 5.5 the H(2) production is suppressed by DTT only in a higher concentration of 10 mM. The results suggest DTT effects that are in addition to the effects of E(h). The mechanism of DTT action on bacterial growth might be intermediated through thiol group modulation of the membrane proteins, which is reflected as the generation of DeltaPsi as well as K(+) accumulation and the activity of the membrane-associated enzymes.     

Effect of redox potential on methanogenesis by Methanosarcina barkeri

Concentrations of 0.5% O₂ immediately inhibited CH₄ production from methanol by Methanosarcina barkeri. Simultaneously, the redox potential of the medium increased to about +100 mV. However, the rates of CH₄ production were not significantly affected, when the redox potential of an anoxic medium was adjusted to values between -420 mV and +100 mV by addition of titanium (III) citrate, sodium dithionite, flavin adenine dinucleotide, or sodium ascorbate. When the redox potential was adjusted to values between -80 mV and +550 mV by means of mixtures of ferrocyanide and ferricyanide, CH₄ production was not inhibited until a redox potential of about +420 mV was reached. M. barkeri was able to reduce 0.5 mM ferricyanide solution at +430 mV within <30 min to a value of about +50 mV, and then to start CH₄ production. Higher ferricyanide concentrations were only partially reduced. The extent of reduction of ferricyanide was also dependent on the substrate concentration (methanol) and the density of the bacterial suspension. The results show that M. barkeri was able to generate to a certain extent by itself the redox environment which suited the production of CH₄. However, the bacteria probably have not enough reducing power to decrease the redox potential below the critical level of +50 mV, if O₂ is present at concentrations >0.005%.     

Transplasma-membrane ferricyanide reduction in sycamore cells. Characterization of the system and inhibition by some phenyl biscarbamates

Evidence is presented for the presence of a transplasma-membrane redox system in sycamore cells, which were found able to reduce external ferricyanide. This reduction induced a simultaneous acidification of the external medium, with a H⁺/e⁻ stoichiometry of 0.925. Ferricyanide reduction was accompanied by a slight reduction of potassium uptake (15% inhibition). The transmembrane electron transport was inhibited by oligomycin and quinacrine; the effect of the latter inhibitor was only temporary, owing to its progressive absorption by the cells.At 100 μM, several derivatives of the herbicide phenmedipham were able to inhibit the growth of sycamore cells. These compounds inhibited the external acidification induced by fusicoccin, without interfering with the integrity of the plasmalemma. They had no effect on the phosphohydrolase activity of a microsomal fraction enriched in plasmalemma ATPase. They were not uncouplers of oxidative phosphorylation, and appeared as poor inhibitors of mitochondrial electron transfer (I₅₀ > 100 μM). In intact cell suspension, they inhibited both the reduction of ferricyanide and the accompanying acidification of the external medium (I₅₀ = 32 μM). Moreover, they could induce a reversal of the functioning of the redox system, which consequently induced ferrocyanide oxidation.     

Effect of the medium redox potential on the growth and metabolism of anaerobic bacteria

Based on the available literature data on a decrease in the redox potential of medium to low negative values and a decrease in pH during the growth of sugar-fermenting anaerobic bacteria, it was concluded that these processes cannot be described by the theory of redox potential. A theory was developed according to which the regulation of bacterial metabolism is accomplished through changes in the redox potential. The theory considers the redox potential as a factor determining the growth of anaerobic bacteria, which is regulated by oxidizers and reducers. The assumption is put forward that, under anaerobic conditions, bacteria are sensitive to changes in the redox potential and have a redox taxis. The effect of the redox potential on the transport of protons and other substances through membranes and the activity of membrane-bound enzymes, including the proton F1-F0-ATPase, whose mechanisms of action involve changes in the proton conductance of the membrane, the generation of proton-driving force, and dithiol-disulfide transitions in proteins was studied.     

Fe(III) and Fe(II) ions different effects on Enterococcus hirae cell growth and membrane-associated ATPase activity

Enterococcus hirae is able to grow under anaerobic conditions during glucose fermentation (pH 8.0) which is accompanied by acidification of the medium and drop in its oxidation-reduction potential (E(h)) from positive values to negative ones (down to ～-200 mV). In this study, iron (III) ions (Fe(3+)) have been shown to affect bacterial growth in a concentration-dependent manner (within the range of 0.05-2 mM) by decreasing lag phase duration and increasing specific growth rate. While iron(II) ions (Fe(2+)) had opposite effects which were reflected by suppressing bacterial growth. These ions also affected the changes in E(h) values during bacterial growth. It was revealed that ATPase activity with and without N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F(0)F(1)-ATPase, increased in the presence of even low Fe(3+) concentration (0.05 mM) but decreased in the presence of Fe(2+). It was established that Fe(3+) and Fe(2+) both significantly inhibited the proton-potassium exchange of bacteria, but stronger effects were in the case of Fe(2+) with DCCD. Such results were observed with both wild-type ATCC9790 and atpD mutant (with defective F(0)F(1)) MS116 strains but they were different with Fe(3+) and Fe(2+). It is suggested that the effects of Fe(3+) might be due to interaction of these ions with F(0)F(1) or there might be a Fe(3+)-dependent ATPase different from F(0)F(1) in these bacteria that is active even in the presence of DCCD. Fe(2+) inhibits E. hirae cell growth probably by strong effect on E(h) leading to changes in F(0)F(1) and decreasing its activity.     

Evidence that H+ efflux stimulated by redox activity is independent of plasma membrane ATPase activity

Proton efflux from mesophyll cells of Asparagus sprengeri Regel was inhibited completely by diethylstilbestrol (DES) and NN'-dicyclohexylcarbodiimide (DCCD), known inhibitors of the plasma membrane ATPase. At the concentrations of inhibitors employed, fusicoccin did not reactivate proton efflux. Subsequent addition of ferricyanide however resulted in significant rates of acidification of the medium and reduction of ferricyanide. Similar results were obtained in the light and in the dark. Thus, medium acidification in response to redox activity appears to be independent of the ATP-dependent acidification process.     

Dynamics of the redox potential and rh of the rumen fluid of goats

The redox potential (Eh) of the rumen fluid of goats varied from -145 to -190 mV and the corresponding rH values from 6.3 to 8.6. The redox potential values of the rumen fluid were influenced by changes in pH. The most oxidizing Eh values --and at the same time the lowest rH and pH values--were observed after a feeding ration containing readily available carbohydrates. No relationship was found between the fermentation rate and the redox potential. The association between the oxidation reduction state and the metabolic activity is best expressed by the rH values. In in vitro experiments, a higher pH or the addition of cysteine or sodium sulphide moved the redox potential of the rumen fluid towards more reducing values. A shift towards more oxidizing values occurred after acidification of the medium, or after the action of heavy metal ions or atmospheric O2. Various other compounds, including bubbling of the rumen fluid with hydrogen, had little or no effect. SH-groups probably play an important role in the formation of the negative redox potential in rumen fluid.     

Relationship between Electron Transport across the Plasmalemma and a pH Decrease in the Bulk Medium

The hypothesis is tested that acidification of the bulk medium during transplasmalemma electron transport to ferricyanide is due solely to a requirement for charge balance. According to this hypothesis, reduction of the trivalent anion, ferricyanide, to the tetravalent anion, ferrocyanide, results in a charge difference that is balanced by protons. A coulometric device is used that rapidly and efficiently reoxidizes ferrocyanide to ferricyanide, thus maintaining a constant charge in the bulk medium. Oat (Avena sativa L. cv Garry) mesophyll protoplasts are chosen as experimental material to facilitate ferricyanide reduction and the concomitant ferrocyanide reoxidation by the coulometric device. The kinetics of ferricyanide reduction and proton excretion by protoplasts are similar to those of other cell types and tissues. Rates of net proton excretion are identical regardless of whether the ferrocyanide is simultaneously reoxidized. We conclude that acidification may occur during transplasmalemma electron transport when there is no change in negative charge of the bulk medium.     

Isolation and characterization of sulphur-oxidizing Thiomonas sp. and its potential application in biological deodorization

AIMS: To isolate and characterize a sulphur-oxidizing bacterial strain from activated sludge and to evaluate its potential application in biological deodorization. METHODS AND RESULTS: A dominant sulphur-oxidizing bacterial strain, designated as strain SS, was isolated from an enrichment culture using thiosulphate as a sole energy source and CO2 as a sole carbon source. The cells of this organism were aerobic, rod-shaped, Gram-negative and motile. Strain SS could grow autotrophically, heterotrophically as well as mixotrophically. Autotrophic growth was observed at pH values ranging from 2.3 to 9.0. Phylogenetic analyses revealed that strain SS belonged to Group 1 of the genus Thiomonas, closely related to Thiomonas perometabolis and Thiomonas intermedia. The thiosulphate oxidation rates of strain SS at different pH values were evaluated in terms of oxygen uptake using a Micro-Oxymax respirometer. The results showed that the maximum oxidation rate of 5.65 mg l(-1) h(-1) occurred at 56 h of growth and pH 6.0. Continuous H2S removal study demonstrated that strain SS could remove more than 99% of H2S when the inlet concentration was below 58.6 ppm. Further increase of the inlet concentration to 118 ppm gave rise to a decline in the removal efficiency to ca 90%. CONCLUSIONS: The strong acidification of the culture medium during the later period could result in the deterioration of the growth activity and the metabolism activity of strain SS. In practical application, the problems caused by the end-product inhibition and the acidification can be alleviated by periodical replacement of culture medium with fresh medium. Given the physiological flexibility and the ability to remove H2S rapidly and efficiently, strain SS could be a good 'deodorizing' candidate. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that Thiomonas species has been reported for biological deodorization application.     

Proton motive force is not obligatory for growth of Escherichia coli.

When 50 microM carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), a protonophore, was added to growth medium containing glucose at pH 7.5, Escherichia coli TK1001 (trkD1 kdpABC5) started exponential growth after 30 min; the generation time was 70 min at 37 degrees C. Strain AS1 (acrA), another strain derived from E. coli K-12, also grew in the presence of 50 microM CCCP under the same conditions, except that the lag period was ca. 3 h. When this strain was grown in the presence of 50 microM CCCP and then transferred to fresh medium containing 50 microM CCCP, cells grew without any lag. Neither a membrane potential nor a pH gradient was detected in strain AS1 cells growing in the presence of CCCP. When either succinate or lactate was substituted for glucose, these strains did not grow in the presence of 50 microM CCCP. Thus, it is suggested that E. coli can grow in the absence of a proton motive force when glucose is used as an energy source at pH 7.5.     

Antioxidants protect the yeast Saccharomyces cerevisiae against hypertonic stress

Yeast (Saccharomyces cerevisiae) mutants lacking CuZnSOD have been reported to be hypersensitive to hypertonic media and to show increased oxidative damage. This study demonstrates that hypertonic medium (containing 0.8 M NaCl) increases the generation of superoxide and other reactive species in yeast cells. Other sequelae of exposure to hypertonic medium include oxidation of cellular low-molecular weight thiols and decrease in total antioxidant capacity of cellular extracts. deltasod1 mutant is more sensitive than a wild-type strain to colony growth inhibition on a hypertonic medium. Anaerobic conditions, ascorbate, glutathione, cysteine and dithiothreitol are able to ameliorate this growth inhibition but a range of other antioxidants does not protect. The protective ability of the antioxidants does not correlate with the rate of their reactions with superoxide but seems to be conditioned by low redox potential for one-electron oxidation of free radicals of the antioxidants. It suggests that repair of low-redox potential targets rather than prevention of their damage by superoxide is important in the antioxidant protection against oxidative stress induced by hypertonic conditions.     

Reversible inactivation of Saccharomyces cerevisiae glutathione reductase under reducing conditions

Glutathione reductase from Saccharomyces cerevisiae was rapidly inactivated following aerobic incubation with NADPH, NADH, and several other reductants, in a time- and temperature-dependent process. The inactivation had already reached 50% when the NADPH concentration reached that of the glutathione reductase subunit. The inactivation was very marked at pH values below 5.5 and over 7, while only a slight activity decrease was noticed at pH values between these two values. After elimination of excess NADPH the enzyme remained inactive for at least 4 h. The enzyme was protected against redox inactivation by low concentrations of GSSG, ferricyanide, GSH, or dithiothreitol, and high concentrations of NAD(P)+; oxidized glutathione effectively protected the enzyme at concentrations even lower than GSH. The inactive enzyme was efficiently reactivated after incubation with GSSG, ferricyanide, GSH, or dithiothreitol, whether NADPH was present or not. The reactivation with GSH was rapid even at 0 degree C, whereas the optimum temperature for reactivation with GSSG was 30 degrees C. A tentative model for the redox interconversion, involving an erroneous intramolecular disulfide bridge, is put forward.     

Plasmalemma redox activity and h extrusion: I. Activation of the h-pump by ferricyanide-induced potential depolarization and cytoplasm acidification

Ferricyanide reduction by Elodea densa leaves, in the dark, is associated with: (a) acidification of the medium; (b) decrease (about 0.2-0.3 units) of intracellular pH (measured in cell sap, cytoplasm, and vacuole); (c) depolarization of the transmembrane potential; (d) net efflux of K⁺ to the medium. Ferricyanide-induced acid secretion is markedly increased by the presence of fusicoccin (FC), and this effect is severely inhibited by the proton pump inhibitors erythrosine B and vanadate. In the presence of ferricyanide FC-induced H⁺ extrusion no longer requires the presence of K⁺ in the medium. The (ferricyanide reduced)/(H⁺ extruded) ratio varies from about 2, in the absence of FC, to about 1 when the toxin is present, and to more than 4, when ATP-driven H⁺ extrusion is inhibited by erythrosine B or by vanadate. Fusicoccin markedly reduces K⁺ release to the medium. The ratio (ferricyanide reduced)/(H⁺ extruded + K⁺ released) approaches unity under all of the three conditions considered. These results indicate that ferricyanide reduction depends on a plasmalemma system transporting only electrons to the extracellular acceptor, with consequent potential depolarization and cytoplasm acidification. Most of the protons released in the cytoplasm would be secondarily extruded by the ATP-driven pump, stimulated by both intracellular acidification and depolarization. K⁺ efflux would depend on potential depolarization.     

Antioxidants protect the yeast Saccharomyces cerevisiae against hypertonic stress

Yeast (Saccharomyces cerevisiae) mutants lacking CuZnSOD have been reported to be hypersensitive to hypertonic media and to show increased oxidative damage. This study demonstrates that hypertonic medium (containing 0.8?M NaCl) increases the generation of superoxide and other reactive species in yeast cells. Other sequelae of exposure to hypertonic medium include oxidation of cellular low-molecular weight thiols and decrease in total antioxidant capacity of cellular extracts. Δsod1 mutant is more sensitive than a wild-type strain to colony growth inhibition on a hypertonic medium. Anaerobic conditions, ascorbate, glutathione, cysteine and dithiothreitol are able to ameliorate this growth inhibition but a range of other antioxidants does not protect. The protective ability of the antioxidants does not correlate with the rate of their reactions with superoxide but seems to be conditioned by low redox potential for one-electron oxidation of free radicals of the antioxidants. It suggests that repair of low-redox potential targets rather than prevention of their damage by superoxide is important in the antioxidant protection against oxidative stress induced by hypertonic conditions.     

Differential Effects of Oxygen and Oxidation Reduction Potential on the Multiplication of Three Species of Anaerobic Intestinal Bacteria

The sensitivity of three strains of anaerobic intestinal bacteria, Clostridium perfringens, Bacteroides fragilis, and Peptococcus magnus, to the differential effects of oxygen and adverse oxidation-reduction potential was measured. The multiplication of the three organisms was inhibited in the presence of oxygen whether the medium was at a negative oxidation-reduction potential (Eh of -50 mV), poised by the intermittent addition of dithiothreitol, or at a positive oxidation-reduction potential (Eh of near +500 mV). However, when these organisms were cultured in the presence of oxygen, no inhibition was observed, even when the oxidation-reduction potential was maintained at an average Eh of +325 mV by the addition of potassium ferricyanide. When the cultures were aerated, the growth patterns of the three organisms demonstrated different sensitivities to oxygen. P. magnus was found to be the most sensitive. After 2 h of aerobic incubation, no viable organisms could be detected. B. fragilis was intermediately sensitive to oxygen with no viable organisms detected after 5 h of aerobic incubation. C. perfringens was the least sensitive. Under conditions of aerobic incubation, viable organisms survived for 10 h. During the experiments with Clostridium, no spores were observed by spore staining.     

Effect of titanium (III) citrate as reducing agent on growth of rumen bacteria.

We compared the growth of 10 strains of rumen bacteria in an anaerobic medium reduced with cysteine hydrochloride, dithiothreitol, or titanium (III) citrate. The redox potential of medium reduced with cysteine hydrochloride was -167.8 mV; with dithiothreitol it was -175.8 mV; and with titanium(III) citrate it was -302.4 mV at a concentration of 5 X 10(-4) M titanium and -403.9 mV at 2 X 10(-3) M titanium. Maximum growth of the strains was generally lower with dithiothreitol or titanium(III) citrate than with cysteine hydrochloride, although growth was greater than in medium lacking an added reducing agent. Strains for which cysteine was required or markedly stimulatory grew only poorly with titanium(III) citrate. No strain grew in medium with sodium citrate as the energy source. Titanium(III) citrate could be used to reduce anaerobic media for some rumen bacteria if the exclusion of a sulfur-containing reducing agent is required.     

Modelling the effect of the redox potential and pH of heating media on Listeria monocytogenes heat resistance

Aims:  Study the effect of redox potential and pH of the heating media on Listeria monocytogenes heat resistance and model its action at fixed temperature.
Methods and Results:  The heat resistance of Listeria monocytogenes at 58°C was studied in Brain Heart Infusion broth as a function of pH (from 5·0 to 7·0) and redox potential ( E _h7). The media redox was adjusted with nitrogen gas, potassium ferricyanide and dithiothreitol. A Weibull model was used to fit survival curves. The heat resistance parameter (δ_58°C) was estimated from each inactivation curve. A major effect of pH was observed. Bigelow model was used to describe the effect of redox potential on the apparent L. monocytogenes heat resistance. The highest δ_58°C values have been obtained at pH 7·0 and oxidizing conditions.
Conclusions:  The developed model indicates that the E _h7 has a significant effect and varied depending on the pH of the heating media. The z _redox values, calculated from δ_58°C allowed quantifying the influence of heating media redox potential on L. monocytogenes thermal inactivation.
Significance and Impact of the Study:  The obtained model shows the action of redox potential on L. monocytogenes thermal destruction and might be useful to take into account in food thermal processes.