Features of consequences of osmotic modification at different steps of cell heating]

The influence of media with different osmotic pressure (NaCl water solutions) on survival and permeability of Escherichia coli B/r and Escherichia coli Bs-1 cells heated up to 50, 52 and 60 degrees C was investigated. Hypotonic media increased, while hypertonic media, within a certain range of sodium chloride concentrations, decreased the damaging action of heating independently of the temperature. The effectiveness of thermoprotection was seen to increase, and the range of osmolyte concentrations, at which the highest effect of protection takes place, to move markedly towards higher concentrations of NaCl with increase in heating temperature. A certain relationship is suggested between the observed phenomenon and the osmotic homeostasis system of microorganisms under condition of thermogenic and tonic stress.     

The effect of media tonicity on Escherichia coli resistance to heating with different gradient

The influence of NaCl water solutions and glycerine hypertonic concentration on the survival of bacteria Escherichia coli B/r heated with different values of heat drop was investigated. It was shown that the transfer of cell suspensions from isotonic conditions to media with raised osmotic pressure, preliminarily heated up to 60 degrees C, and the following heating at this temperature inhibited differences in cell sensitivity to heating at different heat drop. Unlike, it was found that the transfer of cell suspensions from isotonic conditions to hypertonic media before and after heating at 60 degrees C increased differences in resistance of these microorganisms to heating at different heat drop. It is proposed that different resistance of bacteria to damaging action of hyperthermia at different heat drop, and a modified influence of hypertonic solutions on these differences may be due to heat induced destabilization of cell osmotic homeostasis. The extent of expression of this destabilization may be determined by a quantitative ratio of osmotic pressure values in the cell-suspension medium system in particular temperature and tonic environmental conditions.     

The influence of cyclic heating and cooling on Escherichia coli survival

Repeated heating and cooling in lethal (2-52 degrees C) and nonlethal (2-37 degrees C) temperature ranges resulted in cell death of Escherichia coli B/r and E. coli B(S-1) suspended in 0.01 M phosphate buffer, pH 7.0 at varying osmotic pressure, but not in cow's milk. The lethal effect increased with the rate of heating and with increasing suspension media tonicity; it may be caused by the temperature destabilization of cellular osmotic homeostasis.     

Viability of Escherichia coli after combined osmotic and thermal treatment: a plasma membrane implication

This study investigates the influence of temperature (T) and osmotic pressure (Pi) on the viability of Escherichia coli K12 during an osmotic treatment. Osmotic shock (dehydration and rehydration within 1 s) in liquid media at different temperatures (4, 10, 30 and 37 degrees C) and different levels of osmotic pressure (26, 30, 35, 40, 82 and 133 MPa) were realized.Results show that a sudden dehydration, below 40 MPa, destroyed up to 80% of the bacterial population for each tested temperature, whereas viability was greater than 90% for an osmotic pressure less than 26 MPa. The influence of T and Pi on the membrane's physical structure is finally considered to explain the results in light of FTIR and electron microscopy study of the influence of temperature and osmotic pressure on E. coli membrane phospholipids conformation.     

Effect of suspension media on nonthermal inactivation of Escherichia coli

AIMS: To investigate the influence of suspension media on the survival of Escherichia coli M23 exposed to nonthermal, lethal stresses. METHODS AND RESULTS: Populations of E. coli M23 suspended in minimal medium (MM) or in different nutrient-rich broths were exposed to water activity 0.90 and/or pH 3.5 and inactivation was determined by culture-based enumeration. In response to the osmotic or acid challenges, E. coli M23 displayed enhanced survival in MM rather than in complex broth. That trend was reversed when populations were exposed to low water activity in combination with low pH. Comparison of microbial survival in three complex media indicated that even relatively small differences in composition influenced inactivation. In most media the combination of lethal stresses resulted in a synergism, which enhanced bacterial inactivation; however, an exception (tryptone soya broth) was observed. CONCLUSIONS: The suspension medium strongly influences the inactivation of E. coli M23 by osmotic and/or acid stresses. This should be considered when comparing studies of microbial survival that use different media and when broth-derived data are intended to represent specific environments (e.g. food matrices). SIGNIFICANCE AND IMPACT OF THE STUDY: The specific effects of synthetic media need to be appreciated when studying bacterial inactivation in conditions relevant to food-manufacturing regimes.     

Mechanism of thermoresistance in Escherichia coli cells exposed to temperature elevation

The influence of media with different osmotic pressure (NaCl water solution) and chloramphenicol (10 micrograms/ml) on the survival, permeability, and survival curve shape of Escherichia coli B/r and E. coli Bs-1 cells, heated up to 50, 52, and 60 degrees C was investigated. As shown, the survival curve of cells heated up to 60 degrees C in isotonic conditions was characterized by exponential shape, while the survival curves of cells heated up to 50 and 52 degrees C consisted of two components characterizing thermosensitive and thermoresistant parts of cell population. Hypertonic conditions of heat at 52 degrees C decreased cell lethality and permeability. In this case, survival curves were characterized by exponential shape. Chloramphenicol was shown to protect against damaging action of heat at 50 degrees C and not to affect the viability of cells heated at 52 and 60 degrees C. It is proposed that the increase of cell thermoresistance with heat dose elevation at 50 and 52 degrees C in isotonic conditions, which is accompanied by the appearance of thermotolerant components on survival curves, may be associated with accommodational cell reactions. The essence of these reactions consists in stabilization of the osmotic cell homeostasis.     

Relationship between membrane damage and cell death in pressure-treated Escherichia coli cells: differences between exponential- and stationary-phase cells and variation among strains

The relationship between membrane damage and loss of viability following pressure treatment was examined in Escherichia coli strains C9490, H1071, and NCTC 8003. These strains showed high, medium, and low resistance to pressure, respectively, in stationary phase but similar resistance to pressure in exponential phase. Loss of membrane integrity was measured as loss of osmotic responsiveness or as increased uptake of the fluorescent dye propidium iodide. In exponential-phase cells, loss of viability was correlated with a permanent loss of membrane integrity in all strains, whereas in stationary-phase cells, a more complicated picture emerged in which cell membranes became leaky during pressure treatment but resealed to a greater or lesser extent following decompression. Strain H1071 displayed a very unusual pressure response in stationary phase in which survival decreased to a minimum at 300 MPa but then increased at 400 to 500 MPa before decreasing again. Membranes were unable to reseal after treatment at 300 MPa but could do so after treatment at higher pressures. Membrane damage in this strain was thus typical of exponential-phase cells under low-pressure conditions but of stationary-phase cells under higher-pressure conditions. Heat shock treatment of strain H1071 cells increased pressure resistance under low-pressure conditions and also allowed membrane damage to reseal. Growth in the presence of IPTG (isopropyl-beta-D-thiogalactopyranoside) increased resistance under high-pressure conditions. The mechanisms of inactivation may thus differ at high and low pressures. These studies support the view that membrane damage is an important event in the inactivation of bacteria by high pressure, but the nature of membrane damage and its relation to cell death may differ between species and phases of growth.     

Effect of Ca2+ and K+ on the intracellular pH of an Escherichia coli L-form.

The L-form NC7, derived from Escherichia coli K12, grew in a complex medium containing 0.2 M-CaCl2 as osmotic stabilizer, but not at pH values above 7.8. The cessation of growth at alkaline pH was not due to cell death. In complex media containing K+ or Na+, the L-form grew ove a wide pH range. Growth at alkaline pH was inhibited by 1 mM-amiloride, indicating that Na+/H+ antiport activity was required for growth at alkaline pH. The internal pH (pHi) of the L-form in media containing K+, Na+ or Ca2+ was constant at about 7.8 to 8.0 at external pH (pHo) values of 7.2 and 8.2. The rates of O2 consumption by intact cells, lactate oxidation by membrane vesicles from cells grown in Ca(2+)-containing medium, and cell division were all strongly repressed under alkaline conditions.     

Intracellular accumulation of potassium and glutamate specifically enhances survival of Escherichia coli in seawater.

The high resistance of Escherichia coli grown in saline media to seawater was suppressed by an osmotic down-shock. The shock released several molecules into the medium, including potassium, glutamate, and glycine betaine when cells were previously grown in the presence of this osmolyte. Incubation of such sensitized cells in a solution containing K+ (80 mM) and glutamate (50 mM) at pH 7.4 restored their resistance to seawater up to a level close to that observed initially. The protective effect was partly due to the rapid accumulation of K+; a significant exponential relationship between intracellular concentration of K+ and resistance to seawater was observed. Glutamate was accumulated more slowly and progressively completed the action of K+. These data emphasize the specific influence of potassium glutamate on osmotically stressed E. coli cells. They confirm that regulation of osmotic pressure and, probably, of intracellular pH strongly enhances survival of E. coli in seawater. Osmotic fluctuations in waters carrying enteric bacteria from intestines to seawater, together with variations in their K+ and amino acid contents, could modify the ability of cells to survive in marine environments. These results demonstrate the need to strictly control conditions (K+ content, temperature) used to wash cells before their transfer to seawater microcosms. They suggest that the K+ and glutamate contents of media in which E. coli cells are transported to the sea can influence their subsequent survival in marine environments.     

Intracellular accumulation of potassium and glutamate specifically enhances survival of Escherichia coli in seawater.

The high resistance of Escherichia coli grown in saline media to seawater was suppressed by an osmotic down-shock. The shock released several molecules into the medium, including potassium, glutamate, and glycine betaine when cells were previously grown in the presence of this osmolyte. Incubation of such sensitized cells in a solution containing K+ (80 mM) and glutamate (50 mM) at pH 7.4 restored their resistance to seawater up to a level close to that observed initially. The protective effect was partly due to the rapid accumulation of K+; a significant exponential relationship between intracellular concentration of K+ and resistance to seawater was observed. Glutamate was accumulated more slowly and progressively completed the action of K+. These data emphasize the specific influence of potassium glutamate on osmotically stressed E. coli cells. They confirm that regulation of osmotic pressure and, probably, of intracellular pH strongly enhances survival of E. coli in seawater. Osmotic fluctuations in waters carrying enteric bacteria from intestines to seawater, together with variations in their K+ and amino acid contents, could modify the ability of cells to survive in marine environments. These results demonstrate the need to strictly control conditions (K+ content, temperature) used to wash cells before their transfer to seawater microcosms. They suggest that the K+ and glutamate contents of media in which E. coli cells are transported to the sea can influence their subsequent survival in marine environments.     

Magnitude and kinetics of rehydration influence the viability of dehydrated E. coli K-12

The influence of rehydration conditions on the recovery of Escherichia coli K-12 was studied. The results showed that the osmotic pressure gradient of rehydration shock realized before plating greatly affected cell viability. When rehydration occurred quickly from an hyperosmotic level of 133 MPa in glycerol solution before slow rehydration by plating on an agar surface to reach initial osmotic pressure (1.4 MPa), bacterial viability was strongly related to the intensity of the hypo-osmotic gradient used. Rehydration to 107 MPa resulted in a survival ratio of 41%, whereas strong rehydration to 1.4 MPa resulted in only 0.7% survival. These studies also demonstrated the influence of the rehydration kinetic on cell recovery. An optimal rehydration rate of 0.136 MPa x s(-1) increased cell recovery by a factor of 40 when compared with the faster and slower rates of 131.6 MPa x s(-1) and 0.006 MPa x s(-1), respectively.     

Effect of Available Water on Thermal Resistance of Three Nonsporeforming Species of Bacteria

Cells of Escherichia coli, Pseudomonas fluorescens, and Staphylococcus aureus, previously grown in Trypticase Soy Broth (TSB) at a high level of available moisture (a(w) 0.994) and at low levels produced by addition of NaCl or glucose, were heated in neutral phosphate buffer, and in this buffer adjusted to low levels of available moisture by means of NaCl or glucose. Glucose in the heating medium was more protective than NaCl for E. coli and P. fluorescens, but hastened the thermal destruction of S. aureus. Added protection was given P. fluorescens during heating in glucose-buffer solution at a(w) 0.97 by previous growth in TSB adjusted to that a(w) value with glucose. Added protection was given E. coli during heating in NaCl-buffer solution at a(w) 0.98 by previous growth in TSB adjusted to that value with NaCl. With S. aureus, however, previous growth in TSB plus NaCl or glucose had little effect on heat resistance, but the solute in the heating medium had great influence, in that NaCl was very protective and glucose destructive. Opportunity may have been given during tempering of the cell suspension at 30 C in the heating medium prior to heating for the NaCl and glucose to diffuse into the staphylococcal cells.     

Recovery of heat-injured Citrobacter freundii cells

The influence of various factors in the recovery process of heat-injured cells of Citrobacter freundii has been studied. In particular the temperature of the liquid recovery medium and the residence time of the cells in this medium are important. Cells heated in media with a high osmotic pressure are better recovered in low osmotic liquid recovery media. The influence of the temperature of the solid recovery medium on the decimal reduction time for Ctbt. freundii cells strongly suggests that the number of critical sites to be inactivated before the cell wall be lethally injured also depends on the recovery conditions.     

Proteins induced by high osmotic pressure in Escherichia coli

Abstract The protein composition of Escherichia coli grown under conditions of high and low osmotic pressure and following shifts from one condition to the other was examined by two-dimensional gel electrophoresis. Upon shift to high osmotic pressure there is a rapid induction of a group of proteins whose synthesis persists during prolonged growth at elevated osmotic pressure. An osmotic down-shift results in the repression of these proteins. Neither shift induced the heat shock regulon.     

Effects of NaCl and iso-osmotic PEG stress on growth,osmolytes accumulation and antioxidant defense in cultured sugarcane cells

In order to discriminate between the ionic and osmotic components of salt stress, sugarcane (Saccharum officinarum L. cv. Co 86032) calli were cultured on media containing NaCl or polyethylene glycol (PEG) 8000 that exerted the same osmotic pressure (−0.7 MPa). PEG stress exposure for 15 days led to significant growth reduction and loss in water content than salt stressed and control tissues. Osmotic adjustment (OA) was observed in callus tissues grown on salt, but was not evident in callus grown on PEG. Oxidative damage to membranes, estimated in terms of accumulation of thiobarbituric acid reactive substances-TBARS and electrolytic leakage was significantly higher in both the stressed calli than the control however, the extent of damage was more in the PEG stressed calli. The stressed callus tissues showed inhibition of ascorbate peroxidase activity, while catalase activity was increased. These results indicate sensitivity of cells to PEG-mediated stress than salt stress and differences in their OA to these two stress conditions. The sensitivity to the osmotic stress indicate that expression of the stress tolerance response requires the coordinated action of different tissues in a plant and hence was not expressed at the cellular level.     

进化代谢选育高渗透压耐受型产琥珀酸大肠杆菌

在以碳酸钠为酸中和剂的大肠杆菌两阶段发酵产琥珀酸的过程中,由于Na+的积累造成发酵体系中渗透压的提高,严重抑制了琥珀酸的产物浓度。为了增强大肠杆菌对渗透压的耐受性,考察了利用进化代谢方法筛选高渗透压耐受型高产琥珀酸大肠杆菌菌株的可行性。进化代谢系统作为一种菌株突变装置,可以使菌体在连续培养条件下以最大的生长速率生长。以NaCl为渗透压调节剂,通过在连续培养装置中逐步提高NaCl浓度使菌体在高渗透压条件下快速生长,最终得到了一株高渗透压耐受型琥珀酸生产菌株Escherichia coli XB4。以碳酸钠为酸中和剂,在7 L发酵罐中利用Escherichia coli XB4进行两阶段发酵,厌氧培养60 h后,琥珀酸产量达到了69.5 g/L,琥珀酸生产速率达到了1.81 g/(L.h),分别比出发菌株提高了18.6%和20%。     

Phosphorylation as a method of regulating inorganic pyrophosphatase activity in E. coli. II. Identification of the types of chemical bonds between phosphate and the enzyme

A bond formed by phosphate with Pi-phosphorylated pyrophosphatase from E. coli was found to be labile in acidic and alkaline media and to be rapidly cleaved by hydroxylamine at neutral pH. N-Methylhydroxylamine modifies also activated carboxyl groups of the enzyme. Interaction of inorganic pyrophosphatase with ATP produces an alkali-resistant phosphoamide bond. A phosphorylated amino acid, identified as phosphohistidine, was isolated from the alkaline hydrolyzate of the ATP-phosphorylated pyrophosphatase.     

The influence of cooling rates and medium tonicity on Escherichia coli B/r survival after heating with different lethal temperatures

Cell thermosensitivity of Escherichia coli B/r increases with the cooling rise, especially in hypertonic conditions after heating at 50, 55 and 60 degrees C. A certain relationship is suggested between the observed phenomenon and the osmotic homeostasis system of microorganisms under condition of thermogenic and tonic stress.     

Genotype-by-environment interactions influencing the emergence of rpoS mutations in Escherichia coli populations

Polymorphisms in rpoS are common in Escherichia coli. rpoS status influences a trade-off between nutrition and stress resistance and hence fitness across different environments. To analyze the selective pressures acting on rpoS, measurement of glucose transport rates in rpoS+ and rpoS bacteria was used to estimate the role of F(nc), the fitness gain due to improved nutrient uptake, in the emergence of rpoS mutations in nutrient-limited chemostat cultures. Chemostats with set atmospheres, temperatures, pH's, antibiotics, and levels of osmotic stress were followed. F(nc) was reduced under anaerobiosis, high osmolarity, and with chloramphenicol, consistent with a reduced rate of rpoS enrichment in these conditions. F(nc) remained high, however, with alkaline pH and low temperature but rpoS sweeps were diminished. Under these conditions, F(sp), the fitness reduction due to lowered stress protection, became significant. We also estimated whether the fitness need for the gene was related to its regulation. No consistent pattern emerged between the level of RpoS and the loss of rpoS function in particular environments. This dissection allows an unprecedented view of the genotype-by-environment interactions controlling a mutational sweep and shows that both F(nc) and F(sp) are influenced by individual stresses and that additional factors contribute to selection pressure in some environments.     

表达耐辐射异常球菌IrrE蛋白对产丁二酸大肠杆菌耐渗透压的影响

高渗透压胁迫是降低生物法制备丁二酸生产效率的关键因素之一。为提高丁二酸生产菌株对高渗透压胁迫的耐受性能,本研究考察了外源引入全局调控蛋白IrrE提高大肠杆菌耐高渗透压胁迫性能的可行性。试验结果表明,在不同浓度Na+胁迫下,重组菌生长和发酵性能明显提升。在5 L罐发酵中,重组菌最大细胞干重、糖耗和丁二酸产量比对照菌分别提高了15.6%、22%和23%,表明引入IrrE蛋白可提高菌株对高渗透压胁迫的耐受能力。进一步比较重组菌和对照菌胞内相容性物质海藻糖和甘油的浓度后发现,重组菌胞内海藻糖和甘油浓度明显提高,其最大积累量分别是对照菌的1.3和3.8倍,推测IrrE可通过增加胞内相容性物质的积累提高菌株对高渗透压胁迫的耐受性。