Initiation of Bacillus spore germination by hydrostatic pressure: effect of temperature.

Suspensions of Bacillus cereus T, B. subtilis, and B. pumilus spores in water or potassium phosphate buffer were germinated by hydrostatic pressures of between 325 and 975 atm. Kinetics of germination at temperatures within the range of 25 to 44 degrees C were determined, and thermodynamic parameters were calculated. The optimum temperature for germination was dependent on pressure, species, suspending medium, and storage time after heat activation. Germination rates increased significantly with small increments of pressure, as indicated by high negative deltaV values of -230 +/- 5 cm3/mol for buffered B. subtilis (500 to 700 atm) and B. pumilus (500 atm) spores and -254 +/- 18 cm3/mol for aqueous B. subtilis (400 to 550 atm) spores at 40 degrees C and -612 +/- 41 cm3/mol for B. cereus (500 to 700 atm) spores at 25 degrees C. The ranges of thermodynamic constants calculated at 40 degrees C for buffered B. pumilus and B. subtilis spores at 500 and 600 atm and for aqueous B. subtilis spores at 500 atm were: Ea = 181,000 to 267,000 J/mol; deltaH = 178,000 to 264,000 J/mol; deltaG = 94,000 to 98,300 J/mol; deltaS = 264 to 544 J/mol per degree K. These values are consistent with the concept that the transformation of a dormant to a germinating spore induced by hydrostatic pressure involves either hydration or a reduction in the visocosity of the spore core and a conformational change of an enzyme.     

Trehalase activity in extracts of Phycomyces blakesleeanus spores following the induction of germination by heat activation

The heat activation of trehalase in extracts of sporangiospores of Phycomyces blakesleeanus, following the induction of germination by heat activation and the gelatinization of potato starch granules were studied under different conditions in order to discriminate between several phenomena as possible triggers in the activation of trehalase.Short-chain alcohols (from methanol to pentanol) lower the activation temperature of trehalase while long-chain alcohols (from heptanol to nonanol) raise it. Short-chain alcohols also lower the gelatinization temperature of potato starch granules, while long-chain alcohols, hexanol and heptanol have hardly any influence on the gelatinization temperature. Octanol raises the gelatinization temperature. More polar phenols lower the activation temperature of trehalase, while more apolar phenols will raise it. The gelatinization temperature of starch granules is more lowered by the polar polyphenols than by the more apolar phenols.The effect of high pressure on starch gelatinization was investigated in order to compare data from such a model system with the data on trehalase activation.The gelatinization temperature of starch granules is shifted upwards with about 3–5 K/1000 atm (1.013×10⁵ kPa). Pressures higher than 1500 atm do not further increase the gelatinization temperature. However, no reversal of the effect, as occurs with protein conformational changes, is seen with pressure up to 2500 atm. Also for trehalase activation we find a continuous upward shift of the activation temperature with about 5–9 K/1000 atm. These data are in agreement with a thermal transition in a polysaccharide matrix, being the trigger in the heat activation of trehalase.     

Thermal inactivation kinetics of Bacillus subtilis spores suspended in buffer and in oils

The heat resistance of Bacillus subtilis 5230 and A spores freeze dried and suspended in buffer or oils was investigated. As expected, spores were more resistant to heat when suspended in oils than in buffer. This was ascribed to the low a _w of oils and to their content of free fatty acids. Linear survivor curves were obtained for spores suspended in buffer at 105°C or above and for B. subtilis A spores suspended in a vegetable oil. However, the survivor curves of the spores suspended in mineral oil (strain 5230) or olive oil (both strains) were concave upward with a characteristic tailing. The tailing could not be ascribed to spore clumping or to a specific heat injury that can be circumvented by Ca-dipicolinate. It is possibly due to another mechanism of injury or to the activation at high temperature of a normally dormant germination system.     

Glycerol formation after the breaking of dormancy of Phycomyces blakesleeanus spores. Role of an interconvertible glycerol-3-phosphatase

The breaking of dormancy of Phycomyces blakesleeanus spores by a heat shock was followed by a transient production of glycerol, which culminated within 5-10 min and was terminated at 20 min. Extracts of spores contained a magnesium-dependent glycerol-3-phosphatase active on both L-glycerol 3-phosphate and dihydroxyacetone phosphate but having more affinity for the first substrate than for the second. In extracts from dormant spores, the phosphatase was profoundly inhibited by physiological concentrations of inorganic phosphate, which induced cooperativity for the substrate, whereas the enzyme from heat-activated spores was much less inhibited and this difference in kinetic properties persisted after gel filtration of the enzymic preparation. When measured at 1 mM phosphate and 0.1 mM glycerol 3-phosphate, the phosphatase activity was undetectable in dormant spores, increased sharply during the heat treatment and the following 5 min at 25 degrees C, then fell again to a low value by 20 min. A similar transient activation of the enzyme was observed following the breaking of dormancy by incubation of the spores in the presence of 0.1 M ammonium acetate. Incubation of a cell-free extract or of the partially purified glycerol-3-phosphatase in the presence of ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase released the enzyme from inhibition by phosphate and endowed it with the same kinetic properties as did the heat treatment of the spores. It appears therefore most likely that phosphorylation of glycerol-3-phosphatase by cyclic-AMP-dependent protein kinase causes its activation and that this transient process explains the equally transient formation of glycerol by the spores after the heat shock.     

Activation and injury of Clostridium perfringens spores by alcohols

The activation properties of Clostridium perfringens NCTC 8679 spores were demonstrated by increases in CFU after heating in water or aqueous alcohols. The temperature range for maximum activation, which was 70 to 80 degrees C in water, was lowered by the addition of alcohols. The response at a given temperature was dependent on the time of exposure and the alcohol concentration. The monohydric alcohols and some, but not all, of the polyhydric alcohols could activate spores at 37 degrees C. The concentration of a monohydric alcohol that produced optimal spore activation was inversely related to its lipophilic character. Spore injury, which was manifested as a dependence on lysozyme for germination and colony formation, occurred under some conditions of alcohol treatment that exceeded those for optimal spore activation. Treatment with aqueous solutions of monohydric alcohols effectively activated C. perfringens spores and suggests a hydrophobic site for spore activation.     

Activation and injury of Clostridium perfringens spores by alcohols.

The activation properties of Clostridium perfringens NCTC 8679 spores were demonstrated by increases in CFU after heating in water or aqueous alcohols. The temperature range for maximum activation, which was 70 to 80 degrees C in water, was lowered by the addition of alcohols. The response at a given temperature was dependent on the time of exposure and the alcohol concentration. The monohydric alcohols and some, but not all, of the polyhydric alcohols could activate spores at 37 degrees C. The concentration of a monohydric alcohol that produced optimal spore activation was inversely related to its lipophilic character. Spore injury, which was manifested as a dependence on lysozyme for germination and colony formation, occurred under some conditions of alcohol treatment that exceeded those for optimal spore activation. Treatment with aqueous solutions of monohydric alcohols effectively activated C. perfringens spores and suggests a hydrophobic site for spore activation.     

Long chain fatty acid inhibition of sodium plus potassium-activated adenosine triphosphatase from rat heart.

Long chain fatty acids were found to inhibit (Na+ + K+)-ATPase prepared from rat heart. Unsaturated and polyunsaturated fatty acids were more inhibitory than saturated fatty acids with myristic acid being the most inhibitory saturated fatty acid tested and linoleic the most inhibitory unsaturated fatty acid. As an example of fatty acid modification of the enzyme, inhibition of (Na+ + K+)-ATPase by oleate was examined. When compared to ouabain, inhibition of (Na+ + K+)-ATPase by oleate was found to be similar in that both were dependent on K+ concentration, but, in contrast to the almost instantaneous inhibition by ouabain, oleate inhibition was a slow process requiring over 20 min incubation at 37 degrees to produce maximum inhibition. Inhibition of rat heart (Na+ + K+)-ATPase by oleate was found to be readily reversible by washout. In the presence of albumin an oleate/albumin molar ratio greater than 7.5 was required for inhibition to occur. The activity of rat heart (Na+ + K+)-ATPase had a temperature optimum above 40 degrees and a discontinuous Arrhenius' plot with a transition temperature of 25 degrees. In the presence of oleate, however, the enzyme's optimum temperature decreased to below 40 degrees, the activation energy of the reaction at temperatures below 25 degrees was lowered from 24.7 kcal/mol to 12.6 kcal/mol and the enzyme had a linear Arrhenius' plot. The possibility of in vivo inhibition of cardiac (Na+ + K+)-ATPase under conditions of elevated fatty acids is discussed.     

Redox properties of mesophilic and hyperthermophilic rubredoxins as a function of pressure and temperature.

The formal equilibrium reduction potentials of recombinant electron transport protein, rubredoxin (MW = 7500 Da), from both the mesophilic Clostridium pasteurianum (Topt = 37 degrees C) and hyperthermophilic Pyrococcus furiosus (Topt = 95 degrees C) were recorded as a function of pressure and temperature. Measurements were made utilizing a specially designed stainless steel electrochemical cell that easily maintains pressures between 1 and 600 atm and a temperature-controlled cell that maintains temperatures between 4 and 100 degrees C. The reduction potential of P. furiosus rubredoxin was determined to be 31 mV at 25 degrees C and 1 atm, -93 mV at 95 degrees C and 1 atm, and 44 mV at 25 degrees C and 400 atm. Thus, the reduction potential of P. furiosus rubredoxin obtained under standard conditions is likely to be dramatically different from the reduction potential obtained under its normal operating conditions. Thermodynamic parameters associated with electron transfer were determined for both rubredoxins (for C. pasteurianum, DeltaV degrees = -27 mL/mol, DeltaS degrees = -36 cal K-1 mol-1, and DeltaH degrees = -10 kcal/mol, and for P. furiosus, DeltaV degrees = -31 mL/mol, DeltaS degrees = -41 cal K-1 mol-1, and DeltaH degrees = -13 kcal/mol) from its pressure- and temperature-reduction potential profiles. The thermodynamic parameters for electron transfer (DeltaV degrees, DeltaS degrees, and DeltaH degrees ) for both proteins were very similar, which is not surprising considering their structural similarities and sequence homology. Despite the fact that these two proteins exhibit dramatic differences in thermostability, it appears that structural changes that confer dramatic differences in thermostability do not significantly alter electron transfer reactivity. The experimental changes in reduction potential as a function of pressure and temperature were simulated using a continuum dielectric electrostatic model (DELPHI). A reasonable estimate of the protein dielectric constant (epsilonprotein) of 6 for both rubredoxins was determined from these simulations. A discussion is presented regarding the analysis of electrostatic interaction energies of biomolecules through pressure- and temperature-controlled electrochemical studies.     

Inactivation of Geobacillus stearothermophilus spores by high-pressure carbon dioxide treatment

High-pressure CO2 treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO2 treatment at 30 MPa and 35 degrees C, to high-hydrostatic-pressure treatment at 200 MPa and 65 degrees C, or to heat treatment at 0.1 MPa and 85 degrees C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO2 treatment. We also investigated the influence of temperature on CO2 inactivation of G. stearothermophilus. Treatment with CO2 and 30 MPa of pressure at 95 degrees C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95 degrees C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95 degrees C for 120 min had little effect. The activation energy required for CO2 treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2 treatment of G. stearothermophilus spores, CO2 treatment at 95 degrees C was more effective than treatment at 95 degrees C alone.     

Characterization of spore germination of a thermoacidophilic spore-forming bacterium, Alicyclobacillus acidoterrestris

The germination behaviors of spores of Alicyclobacillus acidoterrestris, which has been considered to be a causative microorganism of flat sour type spoilage in acidic beverages, were investigated. The spores of A. acidoterrestris showed efficient germination and outgrowth after heat activation (80 degrees C, 20 min) in Potato dextrose medium (pH 4.0). Further, the spores treated with heat activation germinated in McIlvaine buffer (pH 4.0) in the presence of a germinative substance (L-alanine) and commercial fruit juices, although not in phosphate buffer (pH 7.0). Heat activation was necessary for germination. The spores of A. acidoterrestris, which easily survived the heat treatment in acidic conditions, lost their resistance to heat during germination. Our results suggest that the models obtained from spore germination of A. acidoterrestris might be beneficial to determine adequate thermal process in preventing the growth of potential spoilage bacteria in acidic beverages.     

Thermal inactivation of Bacillus anthracis spores in cow's milk

Decimal reduction time (time to inactivate 90% of the population) (D) values of Bacillus anthracis spores in milk ranged from 3.4 to 16.7 h at 72 degrees C and from 1.6 to 3.3 s at 112 degrees C. The calculated increase of temperature needed to reduce the D value by 90% varied from 8.7 to 11.0 degrees C, and the Arrhenius activation energies ranged from 227.4 to 291.3 kJ/mol. Six-log-unit viability reductions were achieved at 120 degrees C for 16 s. These results suggest that a thermal process similar to commercial ultrahigh-temperature pasteurization could inactivate B. anthracis spores in milk.     

Biochemistry of spore germination in Phycomyces

Abstract The constitutionally dormant spores of Phycomyces blakesleeanus can be activated by heat shock or treatment with several monocarboxylic acids. Activation is followed first by a general stimulation of metabolism, e.g. respiration, protein-, RNA- and cell-wall synthesis, and subsequently by nuclear division and germ-tube emergence. Initial germination is not dependent on RNA synthesis and can even start without protein synthesis. The first common effect of different activating treatments is a transient rise in cyclic AMP (cAMP) content, caused by a change in phosphodiesterase activity after heat activation, and by unknown factors during activation by acids. cAMP transiently activates trehalase and glycerol-3-phosphatase in the spores. The activation of these enzymes causes a quick turnover of trehalose into glycerol. During the same period, the water status of the cells is altered so dramatically that perhaps this may explain at least part of the stimulation of metabolism in the germinating spore.     

Initiation of Germination and Inactivation of Bacillus pumilus Spores by Hydrostatic Pressure

The effect of hydrostatic pressures as high as 1,700 atm at 25 C on the heat and radiation resistance of Bacillus pumilus spores was studied. Phosphate-buffered spores were more sensitive to compression than spores suspended in distilled water. Measurements of the turbidity of suspensions, the viability, refractility, stainability, dry weight, and respiratory activity of spores, and calcium and dipicolinic acid release were made for different pressures and times. Initiation of germination occurred at pressures exceeding 500 atm and was the prerequisite for inactivation by compression. The rate of initiation increased with increasing pressure at constant temperature. This result is interpreted as a net decrease in the volume of the system during initiation as a result of increased solvation of the spore components.     

Heat-induced temperature sensitivity of outgrowing Bacillus cereus spores

Inactivation of Bacillus cereus spores during cooling (10 degrees C/h) from 90 degrees C occurred in two phases. One phase occurred during cooling from 90 to 80 degrees C; the second occurred during cooling from 46 to 38 degrees C. In contrast, no inactivation occurred when spores were cooled from a maximum temperature of 80 degrees C. Inactivation of spores at a constant temperature of 45 degrees C was induced by initial heat treatments from 80 to 90 degrees C. The higher temperatures accelerated the rate of inactivation. Germination of spores was required for 45 degrees C inactivation to occur; however, faster germination was not the cause of accelerated inactivation of spores receiving higher initial heat treatments. Repair of possible injury was not observed in Trypticase soy broth (BBL Microbiology Systems), peptone, beef extract, starch, or L-alanine at 30 or 35 degrees C. Microscopic evaluation of spores outgrowing at 45 degrees C revealed that when inactivation occurred, outgrowth halted at the swelling stage. Inhibition of protein synthesis by chloramphenicol at the optimum temperature also stopped outgrowth at swelling; thus protein synthesis may play a role in the 45 degree C inactivation mechanism.     

Heat-induced temperature sensitivity of outgrowing Bacillus cereus spores.

Inactivation of Bacillus cereus spores during cooling (10 degrees C/h) from 90 degrees C occurred in two phases. One phase occurred during cooling from 90 to 80 degrees C; the second occurred during cooling from 46 to 38 degrees C. In contrast, no inactivation occurred when spores were cooled from a maximum temperature of 80 degrees C. Inactivation of spores at a constant temperature of 45 degrees C was induced by initial heat treatments from 80 to 90 degrees C. The higher temperatures accelerated the rate of inactivation. Germination of spores was required for 45 degrees C inactivation to occur; however, faster germination was not the cause of accelerated inactivation of spores receiving higher initial heat treatments. Repair of possible injury was not observed in Trypticase soy broth (BBL Microbiology Systems), peptone, beef extract, starch, or L-alanine at 30 or 35 degrees C. Microscopic evaluation of spores outgrowing at 45 degrees C revealed that when inactivation occurred, outgrowth halted at the swelling stage. Inhibition of protein synthesis by chloramphenicol at the optimum temperature also stopped outgrowth at swelling; thus protein synthesis may play a role in the 45 degree C inactivation mechanism.     

Kinetic and structural characterization of manganese(II)-loaded methionyl aminopeptidases

Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated. Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 6 +/- 0.5 and 1 +/- 0.5 microM for EcMetAP-I and PfMetAP-II, respectively. These K(d) values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 +/- 0.2 and 1.4 +/- 0.2 microM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25-85 degrees C. The activation energy for the Mn(II)-loaded PfMetAP-II hydrolysis of MGMM was found to be 25.7 kJ/mol while the remaining thermodynamic parameters calculated at 25 degrees C are DeltaG+ = 50.1 kJ/mol, DeltaH+ = 23.2 kJ/mol, and DeltaS++ = -90.2 J x mol(-1) x K(-1).     

Inactivation of bacillus spores in dry systems at low and high temperatures.

A plot of the thermal resistance of Bacillus subtilis var. niger spores (log D value) against temperature was linear between 37 and 190 degrees C (z = 23 degrees C), provided that the relative humidity of the spore environment was kept below a certain critical level. The corresponding plot for Bacillus stearothermophilus spores was linear in the range 150 to 180 degrees C (z = 29 degrees C) but departed from linearity at lower temperatures (decreasing z value). However, the z value of 29 degrees C was decreased to 23 degrees C if spores were dried before heat treatment. The straight line corresponding to this new z value was consistent with the inactivation rate at a lower temperature (60 degrees C). The data indicate that bacterial spores which are treated in dry heat at an environmental relative humidity near zero are inactivated mainly by a drying process. By extrapolation of the thermal resistance plot obtained under these conditions for B. subtilis var. niger spores, the D value at 0 degrees C would be about 4 years.     

Thermodynamic dependence of DNA/DNA and DNA/RNA hybridization reactions on temperature and ionic strength

The thermodynamics of 5'-ATGCTGATGC-3' binding to its complementary DNA and RNA strands was determined in sodium phosphate buffer under varying conditions of temperature and salt concentration from isothermal titration calorimetry (ITC). The Gibbs free energy change, DeltaG degrees of the DNA hybridization reactions increased by about 6 kJ mol(-1) from 20 degrees C to 37 degrees C and exhibited heat capacity changes of -1.42 +/- 0.09 kJ mol(-1) K(-1) for DNA/DNA and -0.87 +/- 0.05 kJ mol(-1) K(-1) for DNA/RNA. Values of DeltaG degrees decreased non-linearly by 3.5 kJ mol(-1) at 25 degrees C and 6.0 kJ mol(-1) at 37 degrees C with increase in the log of the sodium chloride concentration from 0.10 M to 1.0 M. A near-linear relationship was observed, however, between DeltaG degrees and the activity coefficient of the water component of the salt solutions. The thermodynamic parameters of the hybridization reaction along with the heat capacity changes were combined with thermodynamic contributions from the stacking to unstacking transitions of the single-stranded oligonucleotides from differential scanning calorimetry (DSC) measurements, resulting in good agreement with extrapolation of the free energy changes to 37 degrees C from the melting transition at 56 degrees C.     

Thermal analysis of the spores of Bacillus cereus with special reference to heat activation.

The heat activation of bacterial spores was studied by means of differential thermal analysis in the temperature range 30-110 degrees C using the spores of Bacillus cereus. The thermogram showed three endothermic peaks at 56, 95, and 103 degrees C with one exothermic peak at 105 degrees C during the heating process. The spore coat separated from the native spores also showed a peak at 56 degrees C on its heating thermogram. The peak at 56 degrees C was reversible for both native spores and the spore coat. It was suggested that this peak at 56 degrees C might be related to the heat-activation process that takes place in the spore-coat region. It seems that the peak is due to the denaturation or the structural change of the spore-coat protein that might facilitate either the permeation of germination stimulators or the release of some germination inhibitor into or out of the spores.     

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.