Morphological and physiological changes induced by high hydrostatic pressure in exponential- and stationary-phase cells of Escherichia coli: relationship with cell death

The relationship between a loss of viability and several morphological and physiological changes was examined with Escherichia coli strain J1 subjected to high-pressure treatment. The pressure resistance of stationary-phase cells was much higher than that of exponential-phase cells, but in both types of cell, aggregation of cytoplasmic proteins and condensation of the nucleoid occurred after treatment at 200 MPa for 8 min. Although gross changes were detected in these cellular structures, they were not related to cell death, at least for stationary-phase cells. In addition to these events, exponential-phase cells showed changes in their cell envelopes that were not seen for stationary-phase cells, namely physical perturbations of the cell envelope structure, a loss of osmotic responsiveness, and a loss of protein and RNA to the extracellular medium. Based on these observations, we propose that exponential-phase cells are inactivated under high pressure by irreversible damage to the cell membrane. In contrast, stationary-phase cells have a cytoplasmic membrane that is robust enough to withstand pressurization up to very intense treatments. The retention of an intact membrane appears to allow the stationary-phase cell to repair gross changes in other cellular structures and to remain viable at pressures that are lethal to exponential-phase cells.     

Morphological and Physiological Changes Induced by High Hydrostatic Pressure in Exponential- and Stationary-Phase Cells of Escherichia coli: Relationship with Cell Death

The relationship between a loss of viability and several morphological and physiological changes was examined with Escherichia coli strain J1 subjected to high-pressure treatment. The pressure resistance of stationary-phase cells was much higher than that of exponential-phase cells, but in both types of cell, aggregation of cytoplasmic proteins and condensation of the nucleoid occurred after treatment at 200 MPa for 8 min. Although gross changes were detected in these cellular structures, they were not related to cell death, at least for stationary-phase cells. In addition to these events, exponential-phase cells showed changes in their cell envelopes that were not seen for stationary-phase cells, namely physical perturbations of the cell envelope structure, a loss of osmotic responsiveness, and a loss of protein and RNA to the extracellular medium. Based on these observations, we propose that exponential-phase cells are inactivated under high pressure by irreversible damage to the cell membrane. In contrast, stationary-phase cells have a cytoplasmic membrane that is robust enough to withstand pressurization up to very intense treatments. The retention of an intact membrane appears to allow the stationary-phase cell to repair gross changes in other cellular structures and to remain viable at pressures that are lethal to exponential-phase cells.     

Role of membrane fluidity in pressure resistance of Escherichia coli NCTC 8164

The relationship among growth temperature, membrane fatty acid composition, and pressure resistance was examined in Escherichia coli NCTC 8164. The pressure resistance of exponential-phase cells was maximal in cells grown at 10 degrees C and decreased with increasing growth temperatures up to 45 degrees C. By contrast, the pressure resistance of stationary-phase cells was lowest in cells grown at 10 degrees C and increased with increasing growth temperature, reaching a maximum at 30 to 37 degrees C before decreasing at 45 degrees C. The proportion of unsaturated fatty acids in the membrane lipids decreased with increasing growth temperature in both exponential- and stationary-phase cells and correlated closely with the melting point of the phospholipids extracted from whole cells examined by differential scanning calorimetry. Therefore, in exponential-phase cells, pressure resistance increased with greater membrane fluidity, whereas in stationary-phase cells, there was apparently no simple relationship between membrane fluidity and pressure resistance. When exponential-phase or stationary-phase cells were pressure treated at different temperatures, resistance in both cell types increased with increasing temperatures of pressurization (between 10 and 30 degrees C). Based on the above observations, we propose that membrane fluidity affects the pressure resistance of exponential- and stationary-phase cells in a similar way, but it is the dominant factor in exponential-phase cells whereas in stationary-phase cells, its effects are superimposed on a separate but larger effect of the physiological stationary-phase response that is itself temperature dependent.     

大肠杆菌生长温度、膜脂肪酸组成和压力抗性之间的关系

通过对大肠杆菌生长温度、膜脂肪酸组成和压力抗性之间关系研究发现,10℃培养,对数期细胞有最大的压力抗性,随着培养温度的升高直到4 5℃,压力抗性呈下降的趋势;相反,10℃培养,稳定期的细胞对压力最敏感,随着培养温度的升高,压力抗性呈增加趋势,30～37℃时达到最大,之后到4 5℃有下降。对数期和稳定期细胞膜脂中不饱和脂肪酸的组成随温度的上升而下降,这与从全细胞中抽提的磷脂的熔点密切相关。因此,对数期细胞压力抗性随着膜流动性的增大而升高;但稳定期细胞,膜流动性与压力抗性之间不存在简单的对应变化关系     

Role of rpoS in the development of cell envelope resilience and pressure resistance in stationary-phase Escherichia coli

This work investigated the role of rpoS in the development of increased cell envelope resilience and enhanced pressure resistance in stationary-phase cells of Escherichia coli. Loss of both colony-forming ability and membrane integrity, measured as uptake of propidium iodide (PI), occurred at lower pressures in E. coli BW3709 (rpoS) than in the parental strain (BW2952). The rpoS mutant also released much higher concentrations of protein under pressure than the parent. We propose that RpoS-regulated functions are responsible for the increase in membrane resilience as cells enter stationary phase and that this plays a major role in the development of pressure resistance. Strains from the Keio collection with mutations in two RpoS-regulated genes, cfa (cyclopropane fatty acyl phospholipid synthase) and osmB (outer membrane lipoprotein), were significantly more pressure sensitive and took up more PI than the parent strain, with cfa having the greatest effect. Mutations in the bolA morphogene and other RpoS-regulated lipoprotein genes (osmC, osmE, osmY, and ybaY) had no effect on pressure resistance. The cytoplasmic membranes of the rpoS mutant failed to reseal after pressure treatment, and strains with mutations in osmB and nlpI (new lipoprotein) were also somewhat impaired in the ability to reseal their membranes. The cfa mutant, though pressure sensitive, was unaffected in membrane resealing, implying that the initial transient permeabilization event is critical for loss of viability rather than the failure to reseal. The enhanced pressure sensitivity of polA, recA, and xthA mutants suggested that DNA may be a target of oxidative stress in pressure-treated cells.     

Induction of oxidative stress by high hydrostatic pressure in Escherichia coli

Using leaderless alkaline phosphatase as a probe, it was demonstrated that pressure treatment induces endogenous intracellular oxidative stress in Escherichia coli MG1655. In stationary-phase cells, this oxidative stress increased with the applied pressure at least up to 400 MPa, which is well beyond the pressure at which the cells started to become inactivated (200 MPa). In exponential-phase cells, in contrast, oxidative stress increased with pressure treatment up to 150 MPa and then decreased again, together with the cell counts. Anaerobic incubation after pressure treatment significantly supported the recovery of MG1655, while mutants with increased intrinsic sensitivity toward oxidative stress (katE, katF, oxyR, sodAB, and soxS) were found to be more pressure sensitive than wild-type MG1655. Furthermore, mild pressure treatment strongly sensitized E. coli toward t-butylhydroperoxide and the superoxide generator plumbagin. Finally, previously described pressure-resistant mutants of E. coli MG1655 displayed enhanced resistance toward plumbagin. In one of these mutants, the induction of endogenous oxidative stress upon high hydrostatic pressure treatment was also investigated and found to be much lower than in MG1655. These results suggest that, at least under some conditions, the inactivation of E. coli by high hydrostatic pressure treatment is the consequence of a suicide mechanism involving the induction of an endogenous oxidative burst.     

Induction of Oxidative Stress by High Hydrostatic Pressure in Escherichia coli

Using leaderless alkaline phosphatase as a probe, it was demonstrated that pressure treatment induces endogenous intracellular oxidative stress in Escherichia coli MG1655. In stationary-phase cells, this oxidative stress increased with the applied pressure at least up to 400 MPa, which is well beyond the pressure at which the cells started to become inactivated (200 MPa). In exponential-phase cells, in contrast, oxidative stress increased with pressure treatment up to 150 MPa and then decreased again, together with the cell counts. Anaerobic incubation after pressure treatment significantly supported the recovery of MG1655, while mutants with increased intrinsic sensitivity toward oxidative stress (katE, katF, oxyR, sodAB, and soxS) were found to be more pressure sensitive than wild-type MG1655. Furthermore, mild pressure treatment strongly sensitized E. coli toward t-butylhydroperoxide and the superoxide generator plumbagin. Finally, previously described pressure-resistant mutants of E. coli MG1655 displayed enhanced resistance toward plumbagin. In one of these mutants, the induction of endogenous oxidative stress upon high hydrostatic pressure treatment was also investigated and found to be much lower than in MG1655. These results suggest that, at least under some conditions, the inactivation of E. coli by high hydrostatic pressure treatment is the consequence of a suicide mechanism involving the induction of an endogenous oxidative burst.     

Characterization of a Listeria monocytogenes Scott A isolate with high tolerance towards high hydrostatic pressure

An isolate of L. monocytogenes Scott A that is tolerant to high hydrostatic pressure (HHP), named AK01, was isolated upon a single pressurization treatment of 400 MPa for 20 min and was further characterized. The survival of exponential- and stationary-phase cells of AK01 in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer was at least 2 log units higher than that of the wild type over a broad range of pressures (150 to 500 MPa), while both strains showed higher HHP tolerance (piezotolerance) in the stationary than in the exponential phase of growth. In semiskim milk, exponential-phase cells of both strains showed lower reductions upon pressurization than in buffer, but again, AK01 was more piezotolerant than the wild type. The piezotolerance of AK01 was retained for at least 40 generations in rich medium, suggesting a stable phenotype. Interestingly, cells of AK01 lacked flagella, were elongated, and showed slightly lower maximum specific growth rates than the wild type at 8, 22, and 30 degrees C. Moreover, the piezotolerant strain AK01 showed increased resistance to heat, acid, and H(2)O(2) compared with the wild type. The difference in HHP tolerance between the piezotolerant strain and the wild-type strain could not be attributed to differences in membrane fluidity, since strain AK01 and the wild type had identical in situ lipid melting curves as determined by Fourier transform infrared spectroscopy. The demonstrated occurrence of a piezotolerant isolate of L. monocytogenes underscores the need to further investigate the mechanisms underlying HHP resistance of food-borne microorganisms, which in turn will contribute to the appropriate design of safe, accurate, and feasible HHP treatments.     

Suppression of ATP in Candida albicans by imidazole and derivative antifungal agents

Several antifungal agents, at concentrations of 10 micrograms/ml, were shown to suppress ATP concentrations very rapidly in intact cells and spheroplasts of Candida albicans. The highest ATP-suppressing activity was shown by the highly lipophilic imidazole derivatives difonazole, clotrimazole, econazole, isoconazole, miconazole, oxiconazole and tioconazole, which all caused a reduction of cellular ATP content of more than 50% in 10 min. Relatively hydrophilic imidazole derivatives such as ketoconazole were essentially inactive in the test, as were the triazole derivatives fluconazole, ICI 153066, itraconazole and terconazole, and 5-fluorocytosine. Amphotericin B and terbinafine possessed intermediate ATP-suppressing activity, and the dose-response and pH-response curves for these compounds suggested their mechanism of ATP suppression differed from that of the active imidazole derivatives. ATP suppression by azole antifungals did not involve leakage of ATP from the cells and the effect was entirely abrogated by the presence of serum. Intact cells and spheroplasts of yeast-form and hyphal-form C. albicans were generally equally sensitive to ATP suppression, but stationary-phase cells of both morphological forms were less sensitive than exponential-phase cells. The extent of ATP suppression was significantly reduced in stationary-phase yeast cells of a C. albicans strain with known resistance to azole antifungals, but exponential-phase cells of resistant and susceptible strains were equally sensitive. The effect is tentatively ascribed to membrane damage caused directly by the antifungals.     

Survival of low-pH stress by Escherichia coli O157:H7: correlation between alterations in the cell envelope and increased acid tolerance.

Survival of a nontoxigenic isolate of Escherichia coli O157:H7 at low pH (pH 3.0) was examined over prolonged time periods for each of three population types: exponential-phase cells, stationary-phase cells, and acid-adapted exponential-phase cells. In each population, approximately 5 x 10(4) CFU ml-1 were detected after a 24-h incubation at pH 3.0. Even after 3 days at pH 3.0, significant numbers of survivors from each of the three populations could be detected. The high level of acid tolerance exhibited by these survivors was found to be quickly lost once they were transferred to conditions which permitted growth to resume, indicating that they were not mutants. Proton flux measurements on the three populations of cells revealed that the initial rates of viability loss at pH 3.0 correlated well with net proton accumulation. Cells showing a high initial rate of viability loss (exponential-phase cells) accumulated protons at the highest rate, whereas resistant populations (adapted or stationary-phase cells) accumulated protons only slowly. Differences in the protein composition of the cell envelope between the three populations were studied by two-dimensional polyacrylamide gel electrophoresis. Complex differences in the pattern of proteins expressed by each population were uncovered. The implications of these findings are discussed in the context of a possible model accounting for acid tolerance in this important food-borne pathogen.     

Survival of Low-pH Stress by Escherichia coli O157:H7: Correlation between Alterations in the Cell Envelope and Increased Acid Tolerance

Survival of a nontoxigenic isolate of Escherichia coli O157:H7 at low pH (pH 3.0) was examined over prolonged time periods for each of three population types: exponential-phase cells, stationary-phase cells, and acid-adapted exponential-phase cells. In each population, approximately 5 × 10⁴ CFU ml⁻¹ were detected after a 24-h incubation at pH 3.0. Even after 3 days at pH 3.0, significant numbers of survivors from each of the three populations could be detected. The high level of acid tolerance exhibited by these survivors was found to be quickly lost once they were transferred to conditions which permitted growth to resume, indicating that they were not mutants. Proton flux measurements on the three populations of cells revealed that the initial rates of viability loss at pH 3.0 correlated well with net proton accumulation. Cells showing a high initial rate of viability loss (exponential-phase cells) accumulated protons at the highest rate, whereas resistant populations (adapted or stationary-phase cells) accumulated protons only slowly. Differences in the protein composition of the cell envelope between the three populations were studied by two-dimensional polyacrylamide gel electrophoresis. Complex differences in the pattern of proteins expressed by each population were uncovered. The implications of these findings are discussed in the context of a possible model accounting for acid tolerance in this important food-borne pathogen.     

Multiple periplasmic catalases in phytopathogenic strains of Pseudomonas syringae.

Phytopathogenic strains of Pseudomonas syringae are exposed to plant-produced, detrimental levels of hydrogen peroxide during invasion and colonization of host plant tissue. When P. syringae strains were investigated for their capacity to resist H2O2, they were found to contain 10- to 100-fold-higher levels of total catalase activity than selected strains belonging to nonpathogenic related taxa (Pseudomonas fluorescens and Pseudomonas putida) or Escherichia coli. Multiple catalase activities were identified in both periplasmic and cytoplasmic fluids of exponential- and stationary-phase P. syringae cells. Two of these activities were unique to the periplasm of P. syringae pv. glycinea. During the stationary growth phase, the specific activity of cytoplasmic catalases increased four- to eightfold. The specific activities of catalases in both fluids from exponential-phase cells increased in response to treatment with 0.25 to 10 mM H2O2 but decreased when higher H2O2 concentrations were used. In stationary-growth phase cultures, the specific activities of cytoplasmic catalases increased remarkably after treatment with 0.25 to 50 mM H2O2. The growth of P. syringae into stationary phase and H2O2 treatment did not induce synthesis of additional catalase isozymes. Only the stationary-phase cultures of all of the P. syringae strains which we tested were capable of surviving high H2O2 stress at concentrations up to 50 mM. Our results are consistent with the involvement of multiple catalase isozymes in the reduction of oxidative stress during plant pathogenesis by these bacteria.     

Multiple periplasmic catalases in phytopathogenic strains of Pseudomonas syringae.

Phytopathogenic strains of Pseudomonas syringae are exposed to plant-produced, detrimental levels of hydrogen peroxide during invasion and colonization of host plant tissue. When P. syringae strains were investigated for their capacity to resist H2O2, they were found to contain 10- to 100-fold-higher levels of total catalase activity than selected strains belonging to nonpathogenic related taxa (Pseudomonas fluorescens and Pseudomonas putida) or Escherichia coli. Multiple catalase activities were identified in both periplasmic and cytoplasmic fluids of exponential- and stationary-phase P. syringae cells. Two of these activities were unique to the periplasm of P. syringae pv. glycinea. During the stationary growth phase, the specific activity of cytoplasmic catalases increased four- to eightfold. The specific activities of catalases in both fluids from exponential-phase cells increased in response to treatment with 0.25 to 10 mM H2O2 but decreased when higher H2O2 concentrations were used. In stationary-growth phase cultures, the specific activities of cytoplasmic catalases increased remarkably after treatment with 0.25 to 50 mM H2O2. The growth of P. syringae into stationary phase and H2O2 treatment did not induce synthesis of additional catalase isozymes. Only the stationary-phase cultures of all of the P. syringae strains which we tested were capable of surviving high H2O2 stress at concentrations up to 50 mM. Our results are consistent with the involvement of multiple catalase isozymes in the reduction of oxidative stress during plant pathogenesis by these bacteria.     

Variation in resistance of natural isolates of Escherichia coli O157 to high hydrostatic pressure, mild heat, and other stresses

Strains of Escherichia coli O157 isolated from patients with clinical cases of food-borne illness and other sources exhibited wide differences in resistance to high hydrostatic pressure. The most pressure-resistant strains were also more resistant to mild heat than other strains. Strain C9490, a representative pressure-resistant strain, was also more resistant to acid, oxidative, and osmotic stresses than the pressure-sensitive strain NCTC 12079. Most of these differences in resistance were observed only in stationary-phase cells, the only exception being acid resistance, where differences were also apparent in the exponential phase. Membrane damage in pressure-treated cells was revealed by increased uptake of the fluorescent dyes ethidium bromide and propidium iodide. When strains were exposed to the same pressure for different lengths of time, the pressure-sensitive strains took up stain sooner than the more resistant strain, which suggested that the differences in resistance may be related to susceptibility to membrane damage. Our results emphasize the importance of including stress-resistant strains of E. coli O157 when the efficacy of a novel or mild food preservation treatment is tested.     

Characterization of a Listeria monocytogenes Scott A Isolate with High Tolerance towards High Hydrostatic Pressure

An isolate of L. monocytogenes Scott A that is tolerant to high hydrostatic pressure (HHP), named AK01, was isolated upon a single pressurization treatment of 400 MPa for 20 min and was further characterized. The survival of exponential- and stationary-phase cells of AK01 in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer was at least 2 log units higher than that of the wild type over a broad range of pressures (150 to 500 MPa), while both strains showed higher HHP tolerance (piezotolerance) in the stationary than in the exponential phase of growth. In semiskim milk, exponential-phase cells of both strains showed lower reductions upon pressurization than in buffer, but again, AK01 was more piezotolerant than the wild type. The piezotolerance of AK01 was retained for at least 40 generations in rich medium, suggesting a stable phenotype. Interestingly, cells of AK01 lacked flagella, were elongated, and showed slightly lower maximum specific growth rates than the wild type at 8, 22, and 30°C. Moreover, the piezotolerant strain AK01 showed increased resistance to heat, acid, and H₂O₂ compared with the wild type. The difference in HHP tolerance between the piezotolerant strain and the wild-type strain could not be attributed to differences in membrane fluidity, since strain AK01 and the wild type had identical in situ lipid melting curves as determined by Fourier transform infrared spectroscopy. The demonstrated occurrence of a piezotolerant isolate of L. monocytogenes underscores the need to further investigate the mechanisms underlying HHP resistance of food-borne microorganisms, which in turn will contribute to the appropriate design of safe, accurate, and feasible HHP treatments.     

Variation in Resistance of Natural Isolates of Escherichia coli O157 to High Hydrostatic Pressure, Mild Heat, and Other Stresses

Strains of Escherichia coli O157 isolated from patients with clinical cases of food-borne illness and other sources exhibited wide differences in resistance to high hydrostatic pressure. The most pressure-resistant strains were also more resistant to mild heat than other strains. Strain C9490, a representative pressure-resistant strain, was also more resistant to acid, oxidative, and osmotic stresses than the pressure-sensitive strain NCTC 12079. Most of these differences in resistance were observed only in stationary-phase cells, the only exception being acid resistance, where differences were also apparent in the exponential phase. Membrane damage in pressure-treated cells was revealed by increased uptake of the fluorescent dyes ethidium bromide and propidium iodide. When strains were exposed to the same pressure for different lengths of time, the pressure-sensitive strains took up stain sooner than the more resistant strain, which suggested that the differences in resistance may be related to susceptibility to membrane damage. Our results emphasize the importance of including stress-resistant strains of E. coli O157 when the efficacy of a novel or mild food preservation treatment is tested.     

Effect of oxygen on liquid holding recovery of Bacteroides fragilis.

Liquid holding recovery (LHR) in ultraviolet-irradiated Bacteroides fragilis cells occurred under aerobic conditions but was inhibited by anaerobic conditions. The increase in survival after aerobic LHR resulted in an increase in the shoulder regions of the ultraviolet survival curves. Maximum LHR was obtained after holding the cells for 2 to 3 h. LHR was temperature dependent, and in stationary-phase cells LHR was independent of nutrients. Higher levels of LHR occurred in exponential-phase cells than in stationary-phase cells, and LHR was affected by nutrients in exponential-phase cells. Sublethal concentrations of caffeine and acriflavine inhibited LHR. In addition to LHR, minimal medium recovery also occurred in the concentration of [3H]thymine-containing dimers in the acid-insoluble fraction of the cells. A corresponding increase in [3H]thymine-containing dimers was observed in the acid-soluble fraction after LHR. Although a small proportion of irradiated cells produced filaments, this phenomenon was not directly related to LHR in B. fragilis.     

Synergistic killing of Escherichia coli by near-UV radiation and hydrogen peroxide: distinction between recA-repairable and recA-nonrepairable damage.

Wild-type cells and six DNA repair-deficient mutants (lexA, recA, recB, recA, recB, polA1, and uvrA) of Escherichia coli K-12 were treated with near-ultraviolet radiation plus hydrogen peroxide (H2O2). At low H2O2 concentrations (6 X 10(-6) to 6 X 10(-4) M), synergistic killing occurred in all strains except those containing a mutation in recA. This RecA-repairable damage was absent from stationary-phase cells but increased in logarithmic cells as a function of growth rate. At higher H2O2 concentrations (above 6 X 10(-4) M) plus near-ultraviolet radiation, all strains, including those with a mutation in recA, were synergistically killed; thus, at high H2O2 concentrations, the damage was not RecA repairable.     

Density-dependent sorting of physiologically different cells of Vibrio parahaemolyticus

A pure bacterial culture is composed of clonal cells in different physiological states. Separation of those subpopulations is critical for further characterization and for understanding various processes in the cultured cells. We used density-dependent cell sorting with Percoll to separate subpopulations from cultures of a marine bacterium, Vibrio parahaemolyticus. Cells from cultures in the exponential and stationary phases were fractionated according to their buoyant density, and their culturability and ability to maintain culturability under low-temperature and low-nutrient stress (stress resistance) were determined. The buoyant density of the major portion of the cells decreased with culture age. The culturability of stationary-phase cells increased with increasing buoyant density, but that of exponential-phase cells did not. Stress resistance decreased with increasing buoyant density regardless of the growth phase. The results indicate that density-dependent cell sorting is useful for separating subpopulations of different culturabilities and stress resistances. We expect that this method will be a powerful tool for analyzing cells in various physiological states, such as the viable but nonculturable state.     

Cold Shock Lethality and Injury in Clostridium perfringens

Several observations have been made in regard to cold shock lethality of Clostridium perfringens: (i) loss of viability was not consequence of exposure of the cells to air; (ii) stationary-phase cells were much more resistant to cold shock at 4 C than exponential-phase cells; (iii) at 4 C 96% of an initial population of exponential-phase cells was killed upon cold shock and 95% of the remaining population was killed within 90 min of continued exposure at 4 C; (iv) the minimal temperature differential for detectable cold shock lethality was between 17 and 23 C, and the maximum beyond which lethality was not appreciably increased was between 28 and 33 C. Up to 75% of viable cold-shocked cells were injured, as demonstrated by cold shocking late exponential-phase cells at 10 C and using differential plating procedure for recovery. Repair of injury was temperature dependent, and occurred in a complex medium and 0.1% peptone but not water. Nalidixic acid, chloramphenicol, and rifampin did not inhibit repair of injury.