On the Nature of the Heat Resistance of Thermophilic Bacteria

The importance of inorganic ions for the heat stability of thermophilic bacteria was investigated. Cells of Bacillus stearothermophilus, strain 1503, were incubated at elevated temperatures in various media and the number of surviving organisms was determined at suitable intervals. The bacteria rapidly died at temperatures ordinarily employed for their cultivation if the surrounding medium lacked calcium ions. Besides calcium ions, potassium and phosphate ions and glucose, or some other energy source, seemed to be required for the heat stability of the cells. A chemically defined stabilizing medium with these components was developed for the above-mentioned strain. When any component of this medium was excluded, the heat resistance of this organism was lost. This medium had a stabilizing effect also on the cells of three other strains of B. stearothermophilus. These requirements suggest that the heat stability of thermophilic bacteria is attributable to an active transport of calcium ions from the environment into the cells.     

Further Investigations on the Nature of the Heat Resistance of Thermophilic Bacteria

When cells of Bacillus stearothermophilus, strain NCA 1503, were grown in tryptone starch broth and subsequently transferred to tris buffer, a fraction of the cells: rapidly died in ttie buffer. This fraction increased with increasing content of calcium chloride in the growth medium. The' addition of sodium, potassium or magnesium chloride to the growth medium had no such effect. The rapid dying of the cells in tris buffer was associated with a leakage of organic material and calcium ions from the cells. The results obtained are probably caused by a damage to the osmotic barrier of the cells during their contact with the buffer. Observations: made during the present investigation and a previous one (Ljunger 1970) indicate that the heat resistance of thermophilic bacteria depends on the maintenance of a high intracellular concentration of free calcium ions.     

嗜热酶的耐热机理

酶蛋白在高温下的不稳定性是影响其广泛应用的主要瓶颈,嗜热酶因为独特的性质而被作为热稳定研究的极好材料。了解嗜热酶的热稳定性机制,对于采用酶工程定向设计、改造酶具有重要的意义。嗜热酶的热稳定性并不是由单一因素决定的,氨基酸组成、氢键、离子对、二硫键等都是影响嗜热酶热稳定性的重要因素。相对于嗜温酶,嗜热酶更多地采用寡聚体的形式。     

嗜热微生物酶的嗜热机制及应用研究进展

从细胞膜组分、蛋白质结构、遗传物质、钨元素等方面阐述了嗜热微生物酶的嗜热机制 ,并简要介绍了其应用的研究进展。     

Heat Shock Response in the Thermophilic Enteric Yeast Arxiozyma telluris

Heat stress tolerance was examined in the thermophilic enteric yeast Arxiozyma telluris. Heat shock acquisition of thermotolerance and synthesis of heat shock proteins hsp 104, hsp 90, hsp 70, and hsp 60 were induced by a mild heat shock at temperatures from 35 to 40°C for 30 min. The results demonstrate that a yeast which occupies a specialized ecological niche exhibits a typical heat shock response.     

Inactivation and Heat Stabilization of Poliovirus by 2-Thiouracil

Treatment of poliovirus Type I with 10(-3)m 2-thiouracil (2-TU) resulted in the inactivation of greater than 90% of the virus infectivity and stabilization of approximately 50% of the residual virus to heat inactivation. These effects were due to a reaction with the protein moiety of the virus and could be blocked by pre-treatment of the virus with l-cystine or of the drug with cysteine. Both inactivation and stabilization occurred synchronously and reached equilibrium at the same time. Neither process was reversed by reducing agents. It is suggested that an oxidized form of 2-TU reacts with capsid sulfhydryl groups to form a product which is stable in either the inactive or heat-resistant form.     

Mechanism and Mechanism-Based Inactivation of 4-Hydroxyphenylpyruvate Dioxygenase

Six substrate analogs of 4-hydroxyphenylpyruvate, specifically pentafluorophenylpyruvate, 4-hydroxytetrafluorophenylpyruvate,2-thienylpyruvate, 3-thienylpyruvate, thiophenol oxalate, and p-thiocresoloxalate were synthesized and their interactions with porcine liver 4-hydroxyphenylpyruvate dioxygenase investigated. Both pentafluorophenylpyruvate and thiophenol oxalate are competitive inhibitors of the enzyme with K_I values of 14 and 150 μM, respectively, but p-thiocresol oxalate has no effect on the enzymic activity. The other three substrate analogs are both substrates and mechanism-based inactivators of the enzyme with the following kinetic characteristics (compound, K_m, V_max, k_inact, K′, partition ratio) at pH 6.0, 37°C, and an air atmosphere: 4-hydroxytetrafluorophenylpyruvate, 50 μM, 1.9 mkat/kg, 1.5/min, 70 μM 4.2; 2-thienylpyruvate, 500 μM, 7.8 mkat/kg, 0.6/min, 400 μM, 41; 3-thienylpymvate, 250 μM, 2 9 mkat/kg, 0.6/min, 300 μM, 22. When inactivated, the dioxygenase was found to contain per mole of active enzyme, 0.78 mol of label from 3-thienyl-3[³H]pyruvate and 0.85 mol of label from 4-hydroxytetrafluorophenyl-3 [³H]pyruvate. The product formed from the enzyme-catalyzed oxidation of 3-thienylpyruvate was determined to be 3-carboxymethyl-3-thiolene-2-one. The implication of these results to the mechanism of the dioxygenase is considered,     

Mechanisms of Protection of Trehalase Against Heat Inactivation in Neurospora

The half-life of trehalase and invertase at 65 and 60 C was found to be much greater when intact ascospores of Neurospora tetrasperma were heated, as compared with extracts. By contrast, no protection was afforded these enzymes when they were heated in intact conidia and mycelium of N. crassa or N. tetrasperma. The protective effect of ascospores for trehalase was further investigated by heating ascospore extracts before and after dialysis. The removal of small molecules by dialysis lowered the heat resistance of trehalase significantly in such extracts. When the dialysate from extracts of mycelium, conidia, or ascospores was added to dialyzed enzyme extracts, that from ascospores was by far the most active. However, the same dialysates had only a small protective effect on invertase. The addition of ashed dialysates did not protect trehalase, and trehalose and glucose protected less effectively than the dialysate.     

Heat Inactivation of Mycobacterium avium subsp. paratuberculosis in Milk

The effectiveness of pasteurization and the concentration of Mycobacterium avium subsp. paratuberculosis in raw milk have been identified in quantitative risk analysis as the most critical factors influencing the potential presence of viable Mycobacterium paratuberculosis in dairy products. A quantitative assessment of the lethality of pasteurization was undertaken using an industrial pasteurizer designed for research purposes with a validated Reynolds number of 62,112 and flow rates of 3,000 liters/h. M. paratuberculosis was artificially added to raw whole milk, which was then homogenized, pasteurized, and cultured, using a sensitive technique capable of detecting one organism per 10 ml of milk. Twenty batches of milk containing 10³ to 10⁴ organisms/ml were processed with combinations of three temperatures of 72, 75, and 78°C and three time intervals of 15, 20, and 25 s. Thirty 50-ml milk samples from each processed batch were cultured, and the logarithmic reduction in M. paratuberculosis organisms was determined. In 17 of the 20 runs, no viable M. paratuberculosis organisms were detected, which represented >6-log₁₀ reductions during pasteurization. These experiments were conducted with very heavily artificially contaminated milk to facilitate the measurement of the logarithmic reduction. In three of the 20 runs of milk, pasteurized at 72°C for 15 s, 75°C for 25 s, and 78°C for 15 s, a few viable organisms (0.002 to 0.004 CFU/ml) were detected. Pasteurization at all temperatures and holding times was found to be very effective in killing M. paratuberculosis, resulting in a reduction of >6 log₁₀ in 85% of runs and >4 log₁₀ in 14% of runs.     

A Mathematical Analysis of Concomitant Virus Replication and Heat Inactivation

A mathematical analysis of virus production with accompanying heat inactivation, from which the rate of virus release and total virus production are readily calculated, is presented. Applications of this analysis for Sindbis and Chikungunya viruses are discussed.     

Heat Inactivation of In Vivo- and In Vitro-Grown Mycobacteria in Meat Products

Heat inactivation of mycobacteria from lesions and from culture was compared in meat products. In vivo-grown organisms were more easily heat inactivated than were in vitro-grown organisms.     

Prevalence and Antimicrobial Resistance of Thermophilic Campylobacter spp. from Cattle Farms in Washington State

The prevalence of thermophilic Campylobacter spp. was investigated in cattle on Washington State farms. A total of 350 thermophilic Campylobacter isolates were isolated from 686 cattle sampled on 15 farms (eight dairies, two calf rearer farms, two feedlots, and three beef cow-calf ranches). Isolate species were identified with a combination of phenotypic tests, hipO colony blot hybridization, and multiplex lpxA PCR. Breakpoint resistance to four antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, and doxycycline) was determined by agar dilution. Campylobacter jejuni was the most frequent species isolated (34.1%), followed by Campylobacter coli (7.7%) and other thermophilic campylobacters (1.5%). The most frequently detected resistance was to doxycycline (42.3% of 350 isolates). Isolates from calf rearer facilities were more frequently doxycycline resistant than isolates from other farm types. C. jejuni was most frequently susceptible to all four of the antimicrobial drugs studied (58.8% of 272 isolates). C. coli isolates were more frequently resistant than C. jejuni, including resistance to quinolone antimicrobials (89.3% of isolates obtained from calves on calf rearer farms) and to erythromycin (72.2% of isolates obtained from feedlot cattle). Multiple drug resistance was more frequent in C. coli (51.5%) than in C. jejuni (5.1%). The results of this study demonstrate that C. jejuni is widely distributed among Washington cattle farms, while C. coli is more narrowly distributed but significantly more resistant.     

Protection of Vaccinia from Heat Inactivation by Nucleotide Triphosphates

The presence of adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, or uridine triphosphate reduced the rate of inactivation of vaccinia when heated at 50 C. The virus-associated nucleoside triphosphate phosphohydrolases (adenosine triphosphatase, guanosine triphosphatase, cytosine triphosphatase, and uridine triphosphatase) and ribonucleic acid polymerase were also protected from heat inactivation by these compounds. These obervations are best explained by postulating that ribonucleoside triphosphates bind to enzymes in the virus particle, and that these enzyme-substrate complexes are more resistant to thermal denaturation than are the enzymes without their substrates. The kinetics of heat inactivation of the vaccinia ATP phosphohydrolase activity is biphasic, suggesting that there are two proteins in the vaccinia particle that have this enzyme activity but they have different kinetics of heat inactivation. Any of the vaccinia-associated nucleotide phosphohydrolase activities are protected from heat inactivation by the presence of any one of the respective nucleoside triphosphates. This observation suggests that there is a single enzymatic site in vaccinia that is able to react with any ribonucleoside triphosphate.     

Dynamic Model of Heat Inactivation Kinetics for Bacterial Adaptation

The Weibullian-log logistic (WeLL) inactivation model was modified to account for heat adaptation by introducing a logistic adaptation factor, which rendered its “rate parameter” a function of both temperature and heating rate. The resulting model is consistent with the observation that adaptation is primarily noticeable in slow heat processes in which the cells are exposed to sublethal temperatures for a sufficiently long time. Dynamic survival patterns generated with the proposed model were in general agreement with those of Escherichia coli and Listeria monocytogenes as reported in the literature. Although the modified model''s rate equation has a cumbersome appearance, especially for thermal processes having a variable heating rate, it can be solved numerically with commercial mathematical software. The dynamic model has five survival/adaptation parameters whose determination will require a large experimental database. However, with assumed or estimated parameter values, the model can simulate survival patterns of adapting pathogens in cooked foods that can be used in risk assessment and the establishment of safe preparation conditions.Combined with heat transfer data or models, microbial survival kinetics, especially of bacteria or spores, is extensively used to determine the safety of industrial heat preservation processes like canning, extant or planned. The same is true for milder heat processes such as milk and fruit pasteurization. However, survival models are also a valuable tool to assess the safety of prepared foods, especially those made of raw meats, poultry, and eggs, where surviving pathogens can be a public health issue.The heat resistance of a bacterium, or any other microorganism, is almost always determined from a set of its isothermal survival curves, recorded at several lethal temperatures. The kinetic models, which define the heat resistance parameters, may vary, but the calculation procedure itself is usually the same. First, the experimental isothermal survival data are fitted with what is known as the “primary model.” Once fitted, the temperature dependence of this primary model''s coefficients is described by what is known as the “secondary model.” When combined with a temperature profile expression, T(t), and incorporated into the inactivation rate equation, the result is a “tertiary model,” which enables its user to predict the organism''s survival curve under any static or dynamic (i.e., nonisothermal) conditions.The traditional log-linear (“first-order kinetic”) model is the best-known primary survival model, and it is still widely used in sterility calculations in the food, pharmaceutical, and other industries. Traditionally, it has been assumed that the D value calculated with this model has a log-linear temperature dependence or, alternatively, that the temperature effect on the exponential rate constant, k, the D value''s reciprocal, follows the Arrhenius equation. However, accumulating experimental evidence in recent years indicates that bacterial heat inactivation only rarely follows the first-order kinetics and that there is no reason that it should (3, 18, 29). Nonlinear survival curves can be described by a variety of mathematical models (6). Perhaps the most frequently used in recent years is the Weibullian model, of which the traditional log-linear model is a special case—see below.Regardless of the log-linearity issue, none of the above-mentioned models accounts for adaptation, the ability of certain bacterial cells to adjust their metabolism in response to stress in order to increase their survivability (2, 10, 26, 27, 28). A notable example is Escherichia coli. Its cells can produce “heat shock proteins,” which help them to survive mild heat treatments (1, 11). Other organisms, Salmonella enterica and Bacillus cereus among them, can also develop defensive mechanisms that help them to survive in an acidic environment (8, 9, 13). Whether adaptation allows the cells to avoid injury or to repair damage once it has occurred, or both, should not concern us here. (Injury and recovery, although related, are a separate issue, one which is amply discussed in the literature. Their quantitative aspects and mathematical modeling are discussed elsewhere [5].)The cells'' ability to augment their resistance is not unlimited, and it takes time for the cells to activate the protective system and synthesize its chemical elements (10, 12). Consequently, the effect of heat adaptation on an organism''s survival pattern becomes measurable only at or at slightly above what''s known as the “sublethal” temperature range. Under dynamic conditions, therefore, adaptation can be detected only when the heating rate is sufficiently low to allow the cells to respond metabolically to the heat stress prior to their destruction.Several investigators have reported and discussed the quantitative aspects of adaptation (25, 27, 28). When it occurs, adaptation is noticed as a gap between survival curves determined at low heating rates and those predicted by kinetic models whose parameters had been determined at high lethal temperatures (7, 8, 9, 27, 28). The question is how to modify the inactivation kinetic model so that it can properly account for adaptation at low heating rates while maintaining its predictive ability at high rates and clearly lethal temperatures. Stasiewicz et al. (25) have recently given a partial answer to this question. They started with the Weibullian inactivation model (see below) and assumed that its rate parameter''s temperature dependence follows a modified version of the Arrhenius equation. Using this model and experimental data for Salmonella bacteria, they showed that a “pathway-dependent model” is more reliable than a “state-dependent model.”The objectives of our work were to develop a variant of the Weibullian-log logistic (WeLL) inactivation model to account for dynamic adaptation and to demonstrate its applicability with reported adaptive survival patterns exhibited by Escherichia coli and Listeria monocytogenes, two organisms of food safety concern.     

高温胁迫对紫花苜蓿的影响及其适应机制的相关研究

紫花苜蓿是优质的多年生豆科牧草,喜温暖半干旱气候。高温能使其植株生长缓慢、枯萎甚至死亡,病虫害增多,严重影响紫花苜蓿的产量和品质,是限制紫花苜蓿推广利用的一个重要环境因子之一。紫花苜蓿在高温胁迫后,细胞膜流动性、光合作用及呼吸效率等发生改变,产生大量的渗透调节物质、活性氧和热激蛋白等,提高了紫花苜蓿的耐热性,形成了适应高温应激的适应机制。本研究在分析紫花苜蓿特征特性的基础上,从高温对紫花苜蓿的影响、紫花苜蓿适应高温胁迫的机制及其耐热性评价指标的选择和可靠性评价的方法等方面综述了紫花苜蓿耐热性的研究现状,为开展进一步的研究工作提供参考。