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All aerobic organisms cope with reactive oxygen species, such as superoxide anions, hydrogen peroxide, and hydroxyl radicals, which accumulate in cells as products of the incomplete reduction of molecular oxygen (10, 14, 15). Cells are equipped with several defense systems to protect them from the harmful effects of these reactive oxygen species (36). They include antioxidants, such as reduced glutathione (GSH; L--glutamyl-L-cysteinylglycine) and enzymes (catalases and superoxide dismutases). GSH is usually the most abundant intracellular low-molecular-weight thiol in Escherichia coli. It is a major component of all the cell systems involved in protection against oxidants and free-radical-mediated cell injuries, since the redox status (E₀' = 240 mV) is mainly maintained by thiol-disulfide equilibrium inside the bacterial cell (2, 24, 32). GSH is also indirectly implicated in the regulation of the OxyR transcription factor (38), SoxR (7), and SOS (26) systems.

Recently, the question was raised of the benefit of such a protective system for microorganisms in specific environments, such as drinking water distribution networks, in which the bacteria grow in a low-nutrient environment (17, 33) that contains variable concentrations of oxidative species, such as chlorine used for postdisinfection (4). Water distribution networks are continuously colonized by autochthonous bacteria which are impossible to eradicate with the usual disinfectants. The limited transfer of oxidants through biofilms and bacterial envelopes (6, 25, 30, 31) and genetically controlled resistance are the two major explanations for this situation. Glutathione homeostasis and a high intracellular GSH level were recently proposed as a potential resistance pathway of E. coli cells to chlorine (5).

In this study we tested the validity of the hypothesis that oxygenation (oxygenation of the growth medium from 10% to saturation) and starvation (24 h of starvation in minimal medium) of cultures of E. coli lead to significant changes in glutathione homeostasis (concentration of GSH and its precursor, the -glutamylcysteine Glucys) and hence modify E. coli sensitivity to further chlorine disinfection.

Bacterial models. Experiments were carried out with an isogenic pair of E. coli strains, since GSH was not detectable in indigenous biomass recovered from drinking water treatment plants. E. coli AB1157 and the GSH-deficient strain JTG10 (derived in Bruce Demple's laboratory, Harvard University, Cambridge, Mass.), reported to be deficient in GSH synthetase (13), were provided by Danièle Touati (Institut Jacques Monod, Paris, France). E. coli AB1157 and JTG10 were grown in completely mixed 5-liter chemostats (dilution rate, 0.05 h¹; 30 ± 1°C; regulated oxygen) and continuously fed with Luria-Bertani (LB) medium (Difco), and 50 µg of kanamycin monosulfate ml¹ (final concentration) was added every 24 h to the JTG10 culture. The steady-state bacterial density in the reactor was ca. 2 × 10⁹ ml¹ (determined by 4',6-diamidino-2-phenylindole [DAPI] staining) (31), including about 50% culturable bacteria (counted as CFU after dilution in sterile 0.9% NaCl solution and incubation for 48 h at 30 ± 2°C in LB agar [Difco] containing 50 µg of kanamycin ml¹). The E. coli parental strain (AB1157) produced GSH (ca. 6 µmol/10¹² cells, which is close to previously reported values) (13). A cellular GSH level was measurable in the mutant strain (JTG10), but the values were always low (5- to 35-fold lower than that of its parent strain) (Table 1). This fact could be explained by a mutation mechanism that was easily reversible even when the strain was cultured in the presence of kanamycin. Nevertheless, the difference between the GSH levels of the two strains was considered great enough to use them as an experimental model for the examination of the role of GSH in the response to stress factors.

Determination of thiol concentrations. Suspensions of bacteria (180 ml) were centrifuged at 10,000 × g for 20 min at 20°C. The pellets were washed twice by resuspending them in 0.9% NaCl and centrifuging them at 10,000 × g for 5 min at 20°C. Thiols were extracted from the bacteria by immediately suspending each pellet in 10 ml of 3.3% HClO₄ containing 2 mM disodium EDTA, with vigorous vortexing and sonication for 5 min in an ultrasound bath (Prolabo 670/H; power, 9) at 6°C. The suspensions were incubated for 10 min in an ice bath and centrifuged at 10,000 × g for 5 min at 4°C. The resulting supernatants were immediately frozen and stored in liquid nitrogen. The frozen samples were fast thawed in a water bath at 30°C and diluted with cold 0.1 M HCl containing 2 mM disodium EDTA (4°C), and a 50-µl aliquot was injected onto the column of the high-performance liquid chromatography system optimized for thiol measurement (18). This technique included reverse-phase separation, postcolumn derivatization with ortho-phthalaldehyde, and fluorimetric detection. The technique selectively measured the GSH and Glucys. Total GSH (GSH_t; GSH plus oxidized forms [symmetrical and mixed disulfides]) was measured by the same high-performance liquid chromatography technique, but the samples were reduced with 1,4-D,L-dithiothreitol. GSH_t was expressed as GSH equivalents. Thiol concentrations were expressed as micromoles (or micromolar concentrations) per 10¹² total cells.

Chlorination assay. The washed bacterial suspension (20 ml) to be tested was filtered through a 5-µm-pore-size filter (Millipore) to remove or break up the bacterial aggregates. The filtered suspension was aseptically placed in 500-ml brown flasks (previously cleaned of trace organic matter by heating them to 550°C for 4 h) containing 400 ml of sterile phosphate-buffered saline (PBS). Sodium hypochlorite (20 mg liter¹) was added to the diluted E. coli suspension (approximately 10⁶ cells ml¹) to obtain final concentrations of from 2 × 10³ to 0.1 mg of Cl₂ liter¹ (one flask used as a control was not spiked with chlorine). Solutions of sodium hypochlorite (Sigma Chemical Co.) (20 mg liter¹) were prepared daily in sterile bacterium-free distilled water and adjusted to pH 7.0 with dilute HCl. The sodium hypochlorite concentration was determined by the N,N-diethyl-p-phenylene diamine method (1), and the results were expressed in milligrams of chlorine liter¹. The flasks were incubated for 30 min at 20°C in the dark with gentle shaking (160 rpm); then traces of residual chlorine were neutralized by adding sterile sodium thiosulfate (final concentration, 0.02%) to all flasks. The culturable bacteria were then counted, and the number of surviving bacteria was determined as CFU per milliliter. Plate counts (CFU/milliliter) were log₁₀ transformed, and the means and standard deviations of the means were calculated. The calculated means for each assay corresponding to each chlorine concentration tested were compared by one-way analysis of variance (P = 0.05) (11). Differences in the chlorine sensitivities of the two strains and the influence of oxygenation rates or starvation were then analyzed. All data were evaluated by analysis of variance testing with StatView F.4.5. (Abacus Concepts, Inc., Berkeley, Calif.).

Effect of oxygenation on E. coli cultures. The E. coli cultures were oxygenated with pure oxygen at 10, 50, 95, and 100% oxygen saturation of the medium (n = 1 for each value). One hundred milliliters of steady-state AB1157 and JTG10 cultures was washed twice by centrifugation at 10,000 × g for 5 min at 20°C and resuspended in 200 ml of sterile PBS (pH 6.5) (the washing protocol did not decrease the viable CFU [data not shown]). Samples (20 ml) were used for disinfection assays, and 180 ml was used to measure thiol concentrations. Changing the oxygenation rate of the parental E. coli growth cultures (AB1157) from 10 to 100% (Table 1) drastically increased its GSH content (threefold). There was a marked conversion of GSH to the oxidized form, as indicated by the decrease in the GSH/GSH_t ratio from 99 to 80%. The concentration of the GSH precursor, Glucys, also increased when the oxygenation rate was increased from 10 to 50% and decreased when the oxygenation rate reached 100%. The E. coli mutant (JTG10) was unable to restore its internal redox potential by overproducing GSH, especially when the oxygenation rate increased. The GSH content of JTG10 remained stable (ca. 1 µmol/10¹² cells) and dropped at the highest oxygenation rate (Table 1). The toxicity of molecular oxygen in the culture medium and its reactive species for the mutant strain was particularly dramatic, and the mutant was unable to adapt to this oxidative environment, as shown by a drop in CFU (2.5-fold) and in the total number of cells (1.6-fold) (data not shown).

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Sensitivity of the E. coli parental strain (A) and GSH mutant (B) to exposure to chlorine for 30 min at 20°C. Cultures of parental (AB1157) and mutant (JTG10) strains were grown in a reactor with oxygen regulation at 10% (), 50% (×), 95% (), or 100% () oxygen saturation of the medium (n = 1).

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Sensitivity to chlorine of dilute suspensions of GSH-replete (A) and GSH-deficient (B) E. coli cells grown in LB medium (open symbols) and after 24 h (solid symbols) of starvation in phosphate buffer (pH 6.5) at 30°C. The number of surviving bacteria represent the average of three separate experiments.

Effect of starvation on E. coli cultures. Cells were starved by incubating 200 ml of the washed AB1157 and JTG10 suspensions (obtained with a 95% oxygenation rate at 30°C) with gentle shaking (160 rpm) in 1-liter sterile flasks for 24 h. The starved cells were then washed by centrifugation at 10,000 × g for 5 min at 20°C and resuspended in 200 ml of PBS (pH 6.5), and the chlorination assays and thiol analysis were performed. All assays were carried out in triplicate. The populations of bacteria showed a slight decrease in the number of culturable bacteria (from 5.4 × 10⁸ to 3.0 × 10⁸ CFU per ml), while the total number of cells did not decrease (around 14.5 × 10⁸ per ml). Lack of nutrients for a relatively long period led to a major reduction in GSH and to an increased fraction of its oxidized form and, hence, a decrease in the GSH/GSH_t ratio (Table 2). The starved parental strain was more resistant to chlorine up to 0.05 mg/liter than the unstarved population (P > 99.9%), in spite of a threefold decrease in GSH (Fig. 2A). The sensitivity of the mutant strain to chlorine did not change after starvation (P < 95%) (Fig. 2B).