Activation of Clostridium perfringens spores under conditions that disrupt hydrophobic interactions of biological macromolecules.

The effect of hydrophobic interactions on the activation of C. perfringens NCTC 8679 spores was examined by heating spores under conditions that modify the hydrophobic properties of biological macromolecules. After the activation treatment and a washing procedure, germination was determined by measuring the decrease in optical density of spores suspended in an enriched germination medium. Activation was inhibited for spores that were treated under conditions that strengthen hydrophobic interactions, i.e., a decrease in pH or the presence of structure-stabilizing neutral salts. Activation was enhanced by treatment under conditions that disrupt hydrophobic interactions, i.e., an increase in pH or the presence of urea, dibucaine, or denaturing neutral salts. A deactivation treatment with the antichaotropic salt (NH4)2SO4 reversed activation by the chaotropic salt CaCl2 and to a lesser extent reversed activation by sublethal heat (75 degrees C) or urea. Most treatments that enhanced activation increased spore injury at higher temperatures, which resulted in decreased germination. However, (NH4)2SO4 and a decrease in pH from 5.6 to 3.8, which inhibited activation, also favored injury. The results suggest that activation involves a conformational change of a spore protein(s) through weakening of hydrophobic molecular forces and that activation and injury occur at different spore sites.     

Activation and killing of Dictyostelium discoideum spores with urea.

The optimal conditions for activation of Dictyostellium discoideum spores are an 8 M urea treatment for 30 min. The lag between activation and swelling is 45 min. Lower concentrations of urea do not activate entire spore populations. Incubating spores in 8 M urea for 60 min or treatment with 10 M urea for 30 min results in a lengthening of the post-activation lag and a decrease in the final percentage of germination. Urea-activated spores can be deactivated by azide, cyanide, osmotic pressure, and low-temperature incubation. Activated spores do not germinate if incubated in 1 M urea for 24 h but will complete germination upon resuspension in urea-free buffer. Shocking spores at 45 degrees C in 8 M urea or incubating spores in 4-8 M urea for 10 h at 23.5 degrees C causes inactivation. When suspended in urea-free buffer, a larger percentage of these dead spores release spheroplasts through a longitudinal split in the spore case. Sequential enzyme treatment of spheroplasts with cellulase and pronase causes them to release lysable protoplasts. The data of these experiments suggest that shedding of the outer and middle wall layers during physiological spore swelling may be a physical process rather than an enzymatic one.     

REVERSIBLE ACTIVATION FOR GERMINATION AND SUBSEQUENT CHANGES IN BACTERIAL SPORES

Lee, W. H. (University of Illinois, Urbana) and Z. John Ordal. Reversible activation for germination and subsequent changes in bacterial spores. J. Bacteriol. 85:207-217. 1963.-It was possible to isolate refractile spores of Bacillus megaterium, from a calcium dipicolinate germination solution, that were activated and would germinate spontaneously in distilled water. Some of the characteristics of the initial phases of bacterial spore germination were determined by studying these unstable activated spores. Activated spores of B. megaterium were resistant to stains and possessed a heat resistance intermediate between that of dormant and of germinated spores. The spontaneous germination of activated spores was inhibited by copper, iron, silver, or mercury salts, saturated o-phenanthroline, or solutions having a low pH value, but not by many common inhibitors. These inhibitions could be partially or completely reversed by the addition of sodium dipicolinate. The activated spores could be deactivated and made similar to dormant spores by treatment with acid. Analyses of the exudates from the variously treated spore suspensions revealed that whatever inhibited the germination of activated spores also inhibited the release of spore material. The composition of the germination exudates was different than that of extracts of dormant spores. Although heavy suspensions of activated spores gradually became swollen and dark when suspended in solutions of o-phenanthroline or at pH 4, the materials released resembled those found in extracts of dormant spores rather than those of normal germination exudates.     

Changes in the hydrophobic characteristics of Clostridium perfringens spores and spore coats by heat

The hydrophobic characteristics of Clostridium perfringens NCTC 8679 spores were demonstrated by adherence to toluene in a toluene-aqueous partition system. Spores and spore coat preparations were hydrophobic. Vegetative cells and spores extracted with a dithiothreitol-sodium dodecyl sulfate treatment known to remove spore coats were not hydrophobic. A heat activation treatment (75 degrees C for 20 min) which promotes more rapid spore germination increased the hydrophobicity of intact spores and decreased that of isolated spore coats. The hydrophobic changes were reversed by washing and stabilized by 0.5% glutaraldehyde. Heat-induced hydrophobic changes were observed in spore coats prepared from spores that were preheated and washed before rupturing in a buffer containing glutaraldehyde. These results suggest the occurrence of a heat-induced change in the spore coat (possibly in the conformation of a macromolecule) which was stable only within the architectural confines of the intact spore.     

The physiological effects of restrictive environmental conditions on Dictyostelium discoideum spore germination.

Spores may be reversibly activated by the application of heat, dimethyl sulfoxide, urea, or ethylene glucol. Severe changes in four environmental variables (high osmotic pressure, low oxygen tension, low or high pH, and low or high temperature) interfere with the germination process. Spores at the end of the postactivation lag phase of germination were usually deactivated if exposed to severe environmental conditions and thus did not swell; spores in the swelling and oxygen uptake which began during spore activation was primarily attributable to a cyanide-sensitive pathway and secondarily to a salicylhydroxamic acid (SHAM) sensitive pathway. Inhibition of the SHAM-sensitive pathway did not cause spore deactivation while the addition of cyanide resulted in rapid spore deactivation. Treatment of activated spores with azide or environmental shifts also resulted in inhibition of oxygen uptake and spore deactivation. Deactivating spores did not demonstrate the amino acid incorporation, uridine incorporation, and expression of trehalase activity which is found in the later stages of germinating control spores. Protein synthesis inhibitors did not cause spore deactivation or a decrease in oxygen uptake but they inhibited amino acid incorporation and the expression trehalase activity in swollen spores. It is concluded that control of respiratory activity is involved in regulation of reversible activation.     

Effect of troponin C on the cooperativity in Ca2+ activation of cardiac muscle

The presence of protein kinase C (PKC), a key enzyme in signal transduction, has not been investigated in fungal cells. The phorbol ester TPA, an activator of PKC, may be used as an indicator of the presence and role of PKC in Phycomyces blakesleeanus spores. Activation of spore germination by acetate was prevented by 6 nM TPA. The TPA analog 4 alpha PDD, an ineffective activator of PKC, did not affect spore germination. 3 mM dbcAMP, on the other hand, reversed the inhibition of germination caused by TPA. TPA-stimulated protein kinase activity was detected in spores. The possible relationship between PKC and the increased levels of cAMP that accompany the induction of spore germination is discussed.     

Thermodynamic analysis of the effect of concentrated salts on protein interaction with hydrophobic and polysaccharide columns

An attempt was made to explain the effect of concentrated salts on protein interaction with hydrophobic columns. From the previously observed results of preferential interactions for salting-out salts with proteins, it was shown that the free energy of the protein is increased by addition of the salts and this unfavorable free energy is smaller for the proteins bound to the columns because of their smaller surface area exposed to solvent; i.e., the bound form of the proteins is thermodynamically more stable. This explains the protein binding to the hydrophobic columns at high salt concentrations and the elution by decreasing the salt concentration. The unfavorable interaction free energy was greater for Na2SO4 or (NH4)2SO4 than for NaCl, which explains the stronger effect of the former salts on the protein binding to the columns. The observed favorable interaction between KSCN or guanidine hydrochloride and the proteins explains the decreasing effect of these salts on the protein binding to the hydrophobic columns.     

The effects of alterations in the suspending medium on low temperature activation of spores of Bacillus stearothermophilus NGB101

The effects of eight different sodium salts on the activation of spores of Bacillus stearothermophilus NGB101 at 30°C were examined. Sodium nitrite was a potent activator spores of NGB101. Complete activation of spore populations was obtained after 6 h or less at 30°C. Activation of spores of NGB101 in solutions of sodium nitrite, like activation in distilled water, was temperature dependent, with optimal activation at 30°C. While a potent activator of spores of NGB101 at 30°C, sodium nitrite was ineffective as an initiator of germination at 65°C. Activation of spores of NGB101 produced marked increases in colony forming spores compared with nonactivated populations. Spore populations activated in solutions of sodium nitrite gave higher plate counts compared with spores activated in distilled water.     

用疏水色谱法复性并同时纯化重组人粒细胞-巨噬细胞集落刺激因子

目的：建立一种简单、快速复性并同时纯化大肠杆菌表达的重组人粒细胞一巨噬细胞集落刺激因子（rhGM-CSF）的方法。方法：研究rhGM-CSF在疏水色谱（HIC）上的复性和纯化机理,并对固定相和流动相进行选择和优化,包括固定相配基、流动相中盐的种类、流动相pH值、流动相中还原型谷胱甘肽（GSH）和氧化型谷胱甘肽（GSSG）的比例,以及流动相中尿素的浓度。结果：优化后的固定相为PEG600,流动相中的盐为（NH4）2SO4,流动相pH值为7．0,流动相中添加2．0mol／L尿素、1．8mmol／LGSH和0-3mmol／LGSSG。在优化条件下,HIC可使rhGM-CSF在分离纯化的同时得到复性,比活达1．58×10^7U／mg,纯度为95．7％,质量回收率为56．8％。结论：建立的疏水色谱复性和纯化工艺可简化操作步骤,缩短生产周期。     

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.     

Progesterone analogs influence germination of Clostridium sordellii and Clostridium difficile spores in vitro

Clostridium sordellii and Clostridium difficile are closely related anaerobic Gram-positive, spore-forming human pathogens. C. sordellii and C. difficile form spores that are believed to be the infectious form of these bacteria. These spores return to toxin-producing vegetative cells upon binding to small molecule germinants. The endogenous compounds that regulate clostridial spore germination are not fully understood. While C. sordellii spores require three structurally distinct amino acids to germinate, the occurrence of postpregnancy C. sordellii infections suggests that steroidal sex hormones might regulate its capacity to germinate. On the other hand, C. difficile spores require taurocholate (a bile salt) and glycine (an amino acid) to germinate. Bile salts and steroid hormones are biosynthesized from cholesterol, suggesting that the common sterane structure can affect the germination of both C. sordellii and C. difficile spores. Therefore, we tested the effect of sterane compounds on C. sordellii and C. difficile spore germination. Our results show that both steroid hormones and bile salts are able to increase C. sordellii spore germination rates. In contrast, a subset of steroid hormones acted as competitive inhibitors of C. difficile spore germination. Thus, even though C. sordellii and C. difficile are phylogenetically related, the two species' spores respond differently to steroidal compounds.     

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.     

Ammonium sulfate modifies adenylate cyclase and the chemotactic receptor of Dictyostelium discoideum. Evidence for a G protein effect

(NH4)2SO4 was found to activate adenylate cyclase in Dictyostelium discoideum membranes. The effect of (NH4)2SO4 on the enzyme was observed after pretreatment of membranes but could not be observed if the salt was added to the assay mixture. Activation was seen when membranes were pretreated with 0.16 M (NH4)2SO4 and was maximal at 0.6-1.0 M. The maximal activation of the enzyme was observed within 3 min of pretreatment and was not readily reversible. The effect was specific for the NH+4 ion since pretreatment of membranes with other NH+4 salts could activate the enzyme, whereas pretreatment with NaCl or KCl could not. Pretreatment of plasma membranes with (NH4)2SO4 eliminated the sensitivity of the enzyme to the inhibitory effect of guanine nucleotides. (NH4)2SO4 pretreatment also significantly attenuated the inhibition by guanine nucleotides of cAMP binding to its plasma membrane receptor. The effect of (NH4)2SO4 on GTP inhibition of cAMP binding to its receptor was even more dramatic when the salt was present in the binding assay. (NH4)2SO4 also increased the ADP-ribosylation by cholera toxin of a 39,000-Da membrane protein. The data support the hypothesis that (NH4)2SO4-induced changes in adenylate cyclase and the cAMP receptor are due to an alteration of a putative G protein.     

Stimulation of germination of unactivated Bacillus cereus spores by ammonia

Inclusion of ammonia in germinant mixtures containing L-alanine and inosine stimulated germination of unactivated Bacillus cereus spores at rates equal to those obtained using heat-activated spores without ammonia. D-Alanine had little effect on germination of heat-activated spores, but severely inhibited germination of unactivated spores in the presence of ammonia. Ammonia did not replace the requirement for either L-alanine or inosine: all three compounds were required for rapid germination. Kinetic analysis suggested that the functions of ammonia and L-alanine were more closely related than the functions of ammonia and inosine. With rate-saturating concentrations of L-alanine and inosine, germination rates showed saturation kinetics for ammonia with a Km for NH4Cl of 5 mM. Comparisons of the effects of salts, amines and pH on germination rates suggested that NH4OH rather than NH+4 was the rate-limiting form of ammonia. In comparisons of various strains of B. cereus, stimulation of germination by ammonia occurred in all cases, although spores of most soil isolates germinated more rapidly than B. cereus T spores in the absence of ammonia.     

Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus

Foerster, Harold F. (The University of Texas, Austin), and J. W. Foster. Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus. J. Bacteriol. 91:1333-1345. 1966.-Spores were produced by washed vegetative cells suspended in deionized water supplemented with CaCl(2), SrCl(2), or BaCl(2). Normal, refractile spores were produced in each case; a portion of the barium spores lost refractility and darkened. Thin-section electron micrographs revealed no apparent anatomical differences among the three types of spores. Analyses revealed that the different spore types were enriched specifically in the metal to which they were exposed during sporogenesis. The calcium content of the strontium and the barium spores was very small. From binary equimolar mixtures of the metal salts, endotrophic spores accumulated both metals to nearly the same extent. Viability of the barium spores was considerably less than that of the other two types. Strontium and barium spores were heat-resistant; however, calcium was essential for maximal heat resistance. Significant differences existed in the rates of germination; calcium spores germinated fastest, strontium spores were slower, and barium spores were slowest. Calcium-barium and calcium-strontium spores germinated readily. Endotrophic calcium and strontium spores germinated without the prior heat activation essential for growth spores. Chemical germination of the different metal-type spores with n-dodecylamine took place at the same relative rates as physiological germination. Heat-induced release of dipicolinic acid occurred much faster with barium and strontium spores than with calcium spores. The washed "coat fraction" from disrupted spores contained little of the spore calcium but most of the spore barium. The metal in this fraction was released by dilute acid. The demineralized coats reabsorbed calcium and barium at neutral pH.     

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.     

Analysis of damage due to moist heat treatment of spores of Bacillus subtilis

Aims:  To determine conditions for generation and recovery of Bacillus subtilis spore populations heavily damaged by moist heat treatment.
Methods and Results:  Bacillus subtilis spores were treated with moist heat and spore viability was assessed on different media. A rich medium and several minimal media gave similar spore recoveries after moist heat treatment, but lack of glucose in minimal media greatly decreased spore recovery. High NaCl levels also greatly decreased the recovery of moist heat-treated spores on minimal media, and addition of good osmoprotectants reversed this effect. Moist heat treatment did not decrease spore recovery on minimal media with high salt through DNA damage or by eliminating spore germination, but by affecting spore outgrowth.
Conclusions:  Conditions for generating B. subtilis spore populations with high levels of conditional moist heat damage have been determined. The major conditional damage appears to be in spore outgrowth, perhaps because of damage to one or more important metabolic enzymes.
Significance and Impact of the Study:  This work has provided new insight into the mechanism of B. subtilis spore killing by moist heat.     

pH值对香蕉枯萎病菌4号生理小种生长的影响

【背景】香蕉枯萎病菌4号生理小种(镰刀菌)是香蕉产业的致命威胁。已有研究表明土壤pH值越高,香蕉枯萎病发病率越低,但是现有pH值对镰刀菌影响的研究大都是用强酸强碱调节pH值,pH值没有缓冲体系保护,而且尚未检测试验终点时介质的pH值。此外,关于pH值对香蕉枯萎病菌4号生理小种(Foc4)影响的研究尚不系统,难以用于指导生产实践。【目的】为系统地了解土壤酸碱度对Foc4生长的影响。【方法】在pH 3.0-11.0之间设定9个pH值梯度,模拟酸性到碱性土壤pH值条件,于室内培养条件下系统研究pH值对Foc4生长、产孢、孢子萌发的影响及其生长过程对环境pH值的影响。【结果】弱酸性至中性环境(pH 5.0-7.0)最适宜于香蕉枯萎病菌的生长、产孢和孢子萌发。弱碱性处理(pH8.0和pH9.0)孢子平均萌发率较弱酸性环境处理(pH5.0和pH6.0)下降了73.1%。与pH 6.0酸性处理相比,pH 8.0和pH 9.0处理的产孢量分别下降了52.3%和68.1%。【结论】香蕉枯萎病菌Foc4生长和萌发过程会产酸,但是在缓冲体系液体培养基中,除了pH 9.0和pH10.0处理终点培养液pH值分别下降了0.34和0.27个单位外,其它处理起始和终点的pH值无差异。说明在缓冲体系液体培养基中的研究结果可以反映环境pH值对Foc4生长和萌发的影响。在作物可以生长的pH值范围内(pH5.0-9.0),碱性和微碱性条件(pH8.0-9.0)能明显抑制Foc4生长、产孢和孢子萌发。     

Mapping interactions between germinants and Clostridium difficile spores

Germination of Clostridium difficile spores is the first required step in establishing C. difficile-associated disease (CDAD). Taurocholate (a bile salt) and glycine (an amino acid) have been shown to be important germinants of C. difficile spores. In the present study, we tested a series of glycine and taurocholate analogs for the ability to induce or inhibit C. difficile spore germination. Testing of glycine analogs revealed that both the carboxy and amino groups are important epitopes for recognition and that the glycine binding site can accommodate compounds with more widely separated termini. The C. difficile germination machinery also recognizes other hydrophobic amino acids. In general, linear alkyl side chains are better activators of spore germination than their branched analogs. However, L-phenylalanine and L-arginine are also good germinants and are probably recognized by distinct binding sites. Testing of taurocholate analogs revealed that the 12-hydroxyl group of taurocholate is necessary, but not sufficient, to activate spore germination. In contrast, the 6- and 7-hydroxyl groups are required for inhibition of C. difficile spore germination. Similarly, C. difficile spores are able to detect taurocholate analogs with shorter, but not longer, alkyl amino sulfonic acid side chains. Furthermore, the sulfonic acid group can be partially substituted with other acidic groups. Finally, a taurocholate analog with an m-aminobenzenesulfonic acid side chain is a strong inhibitor of C. difficile spore germination. In conclusion, C. difficile spores recognize both amino acids and taurocholate through multiple interactions that are required to bind the germinants and/or activate the germination machinery.     

Levels of H+ and other monovalent cations in dormant and germinating spores of Bacillus megaterium.

Previous investigators using the extent of uptake of the weak base methylamine to measure internal pH have shown that the pH in the core region of dormant spores of Bacillus megaterium is 6.3 to 6.5. Elevation of the internal pH of spores by 1.6 U had no significant effect on their degree of dormancy or their heat or ultraviolet light resistance. Surprisingly, the rate of methylamine uptake into dormant spores was slow (time for half-maximal uptake, 2.5 h at 24 degrees C). Most of the methylamine taken up by dormant spores was rapidly (time for half-maximal uptake, less than 3 min) released during spore germination as the internal pH of spores rose to approximately 7.5. This rise in internal spore pH took place before dipicolinic acid release, was not abolished by inhibition of energy metabolism, and during germination at pH 8.0 was accompanied by a decrease in the pH of the germination medium. Also accompanying the rise in internal spore pH during germination was the release of greater than 80% of the spores K+ and Na+. The K+ was subsequently reabsorbed in an energy-dependent process. These data indicate (i) that between pH 6.2 and 7.8 internal spore pH has little effect on dormant spore properties, (ii) that there is a strong permeability barrier in dormant spores to movement of charged molecules and small uncharged molecules, and (iii) that extremely early in spore germination this permeability barrier is breached, allowing rapid release of internal monovalent cations (H+, Na+, and K+).