Effect of Some Inhibitors Derived from Nitrite on Macromolecular Synthesis in Bacillus cereus

The effect of several inhibitors derived from nitrite on incorporation of [¹⁴C]uracil into ribonucleic acid of Bacillus cereus during outgrowth and vegetative growth has been determined. A heat-induced inhibitor presumably of the Perigo type was compared with the nitrosothiols of thioglycolate and β-mercaptoethanol. All were found to effectively inhibit uracil incorporation at all stages tested. Phase-contrast microscopy revealed that inhibition of morphological events occurred either before germination or during early outgrowth, depending on inhibitor concentration. It was also found that a precursor to the heat-induced inhibitor could be isolated from dialyzable tryptone (a pancreatic digest of casein), which chromatographed as a single species on diethylaminoethyl-cellulose and Sephadex G-25, with an apparent molecular weight of about 2,000.     

Inhibition of Bacillus cereus spore outgrowth by covalent modification of a sulfhydryl group by nitrosothiol and iodoacetate.

Nitrosothiols with the general structure RSN==O were studied as a model system of bacteriostatic action toward outgrowing bacterial spores. With a Taft plot analysis, the influence of the structure of the R group on the inhibitory effectiveness of a series of nitrosothiols showed that effectiveness as an inhibitor of Bacillus cereus T outgrowth correlated with the electron withdrawal of R, but that size, shape, charge, hydrophobicity, and transportability had little influence. This was interpreted to mean that nitrosothiols do not traverse the membrane to act. The Taft plot together with competition data between nitrosothiol and iodoacetate indicated that the mode of nitrosothiol action is the covalent modification of a sulfhydryl group, probably to form RSN(OH)--SX, where --SX is derived from a sensitive spore sulfhydryl group. Cooperativity effects indicated that outgrowth inhibition is accompanied by a conformational change occurring upon sulfhydryl group modification, which is communicated among at least three to five subunits. Uptake of label during spore germination indicated that most of the sulfhydryl groups which can be modified are associated with the inhibitory event. These data suggest that this sulfhydryl group may be sufficiently unique that inhibitors designed to interfere specifically with it could have value as bacteriostatic agents.     

Growth from Spores of Clostridium perfringens in the Presence of Sodium Nitrite

The method by which sodium nitrite may act to prevent germination or outgrowth, or both, of heat-injured spores in canned cured meats was investigated by using Clostridium perfringens spores. Four possible mechanisms were tested: (i) prevention of germination of the heat-injured spores, (ii) prior combination with a component in a complex medium to prevent germination of heat-injured spores, (iii) inhibition of outgrowth of heat-injured spores, and (iv) induction of germination (which would render the spore susceptible to thermal inactivation). Only the third mechanism was effective with the entire spore population when levels of sodium nitrite commercially acceptable in canned cured meats were used. Concentrations of 0.02 and 0.01% prevented outgrowth of heat-sensitive and heat-resistant spores, respectively. Nitrite-induced germination occurred with higher sodium nitrite concentrations.     

独脚金内酯调控侧枝发育的研究进展

植物通过内源激素或环境信号调控叶腋内腋芽的形成和发育,从而控制其分枝特性。独脚金内酯(strigolactones,SLs),一种产生于植物根部的类胡萝卜素衍生物,具有刺激寄生植物种子的萌发和促进丛枝菌根真菌菌丝分枝的作用,最近的研究表明,它还可以沿茎干向上运输,与生长素和细胞分裂素一起直接或间接抑制植物分枝,目前已经作为一种新的植物激素受到广泛认可。本文综述了独脚金内酯的结构、合成途径和生物活性,以及调控植物分枝的分子机理,并展望了其在抑制杂草或新型除草剂的研发、促进植物和有益真菌的共生,以及调控作物的分枝和株型等方面的应用前景。     

Control of Germination in Striga asiatica: Chemistry of Spatial Definition

Striga asiatica (Scrophulariaceae), a member of a heterogeneous group known as the parasitic plants, is totally dependent on host root attachment for survival. In agar, Striga seeds germinated in high percentages within 5 millimeters of a sorghum (Sorghum bicolor (L.) Moench) host root surface, and no germination was observed at distances greater than 1 centimeter. This spatially restricted germination may be explained by the chemistry of a single compound, 2-hydroxy-5-methoxy-3-[8′Z, 11′Z)-8′, 11′, 14′ -pentadecatriene]-p-hydroquinone, structure 1, which is exuded by sorghum roots. The presence of the compound was chemically imaged with pigments such as methylene blue. The use of methylene blue suggested that structure 1 was exuded along the entire surface of the root for long periods. This exudation and the inherent instability of structure 1 together establish an apparent steady state concentration gradient of the germination stimulant around the sorghum root. The Striga seed must be exposed to micromolar concentrations of 1 for ≥5 hours before high germination percentages were observed. Such a requirement for a long term exposure to a steady state concentration of an inherently labile, exuded compound would provide an extra degree of resolution to signal detection and host commitment in Striga parasitism.     

Effect of Sodium Nitrite, Sodium Chloride, and Sodium Nitrate on Germination and Outgrowth of Anaerobic Spores

The effects of meat-curing agents on germination and outgrowth of putrefactive anaerobe 3679h (PA 3679h) spores were studied in microcultures. Nitrite concentrations up to 0.06% at pH 6.0 or between 0.8 and 1% at pH 7.0 allowed emergence and elongation of vegetative cells but blocked cell division. The newly emerged cells then lysed. With more than 0.06% nitrite at pH 6.0 or more than 0.8 to 1% at pH 7.0, the spores lost refractility and swelled, but vegetative cells did not emerge. Even as much as 4% nitrite failed to prevent germination (complete loss of refractility) and swelling of the spores. Sodium chloride concentrations above 6% prevented complete germination (i.e., the spores retained a refractile core). In the presence of 3 to 6% sodium chloride, most of the spores germinated and produced vegetative cells, but cell division was often blocked. Sodium nitrate had no apparent effect on germination and outgrowth at concentrations up to 2%.     

The influence of gramicidin S on hydrophobicity of germinating Bacillus brevis spores

Gramicidin S is known to prolong the outgrowth stage of spore germination in the producing culture. Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to cell-surface hydrophobicity and germination of their spores. Parental spores were hydrophobic as determined by adhesion to hexadecane, whereas mutant spores showed no affinity to hexadecane. Addition of gramicidin S to mutant spores resulted in a high cell surface hydrophobicity and a delay in germination outgrowth. The hydrophobicity of parental spores was retained throughout most of the germination period. Hydrophobicity was lost as outgrowing spores entered into the stage of vegetative growth. The data indicate that gramicidin S is responsible for the hydrophobicity of B. brevis spores. It is suggested that in making spores hydrophobic, the antibiotic plays a role in concentrating the spores at interfaces where there is a higher probability of finding nutrients for germination and growth.Abbreviation GS Gramicidin S     

Promotive effects of alginate-derived oligosaccharide on maize seed germination

The influence of alginate-derived oligosaccharide (molecular weight 1445 Da) was tested on the maize seed germination at different concentrations. Assays of α-and β-amylase and protease activities showed the highest response at 0.75‰.Compared with the control, root growth on days 3 and 7 showed increases of 34% and18%, respectively; and shoot growth on day 7 an increase of 46%. In the case of protease activity, treatments with both 0.75‰ and 1.50‰ alginate-derived oligosaccharide gave higher activities than the control. These results indicate that the rate of seed germination was enhanced by increasing the activities of several enzymes beneficial for germination. This revised version was published online in August 2006 with corrections to the Cover Date.     

Induction of petite mutations during germination and outgrowth of Saccharomyces cerevisiae ascospores.

The germination and outgrowth of Saccharomyces cerevisiae ascospores were studied by determining the sensitivity of the ascospores to the action of chemical mutagens. Survival of the ascospores after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment was low during the first 2 h of germination and then increased and remained constant. Survival of the ascospores after 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino)acridine-2HC1 (ICR-170) treatment was constant from 0 to 5 h, but as the ascospores completed outgrowth at 6 h they became more sensitive to killing by ICR-170. Survival of the ascospores remained high during treatment with 2-methoxy-6-chloro-9-(3-[ethyl-2-hydroxyethyl]aminopropylamino)acridine-2HC1 (ICR-170-OH) or 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide. The main classes of mutations screened for were petites and auxotrophs. The induction of petites and auxotrophs by MNNG was independent of the stage of germination and outgrowth treated. Petite induction by ICR-170 was dependent upon the stage of germination and outgrowth treated. The early hours of germination (0 to 3 h) were not sensitive to petite induction. However, there was maximal petite induction at 5 h into germination and outgrowth, followed by a decline. During this same time period, ICR-170 induced less than 1% auxotrophic colonies. This finding is very unusual because ICR-170 induced 15% auxotrophic colonies in starved log-phase cultures of S. cerevisiae. The acridine ICR-170-OH induced no mutations during germination and outgrowth of the ascospores. Ethidium bromide induced petites, and the petite frequency became maximal at 5 h of germination and outgrowth, a result similar to that obtained with ICR-170.     

Effects of potassium sorbate and other antibotulinal agents on germination and outgrowth of Clostridium botulinum type E spores in microcultures.

The effects of potassium sorbate, sodium hypophosphite, sodium tripolyphosphate, sodium nitrite, and linoleic acid on the germination and outgrowth of Clostridium botulinum type E spores were studied in microcultures. At pH 5.8 to 6.0 in liver veal agar, the germination rate was decreased to nearly zero with 1.0, 1.5, or 2.0% sorbate. At pH 7.0 t 7.2, these levels of sorbate afforded germination and outgrowth of abnormally shaped cells that were defective in cell division. At the high pH range, 0.5 or 1.0% hypophosphite had effects similar to those of sorbate. The use of 0.05% sodium nitrite with sorbate enhanced the lysis of outgrowing cells at pH 7.2 or lower. Emergence and elongation were inhibited by 0.05% linoleic acid with or without 1.0% sorbate at pH 7.0 to 7.2. The addition of 0.5% tripolyphosphate to media containing 1.5% sorbate at pH 7.1 prevented normal cell growth to an extent greater than with sorbate alone.     

Carbohydrate Composition during Germination and Outgrowth of Ascospores of Saccharomyces cerevisiae

Single spores of Saccharomyces cerevisiae were examined to distinguish changes in the synthesis and degradation of intracellular and wall carbohydrates during germination and outgrowth. Intracellular carbohydrate was fractionated into trehalose and glycogen. Trehalose degradation occurred during germination and outgrowth. The intracellular glycogen was degraded during germination and then synthesized during outgrowth. Wall carbohydrate was fractionated into glycogen, glucan and mannan. The wall glycogen and the KOH-soluble glucan were degraded during germination and then synthesized prior to and during outgrowth, respectively. The major component of the KOH-insoluble glucan in the wall is β-1,3-glucan. The glucan and mannan were synthesized during outgrowth.

The study revealed that the development of a vegetative cell from a spore follows rapid decreases in the amounts of trehalose, glycogen and KOH-soluble glucan during germination, and great increases in the amounts of glycogen, β-1,3-glucan and mannan during outgrowth.     

Non-variable sources of pure water and the germination and outgrowth of Bacillus subtilis spores

Variable germination and outgrowth occurred when Bacillus subtilis NCTC 8236 spores were inoculated into nutrient broth prepared with distilled water. More reproducible findings were achieved when the medium was prepared with Elgastat water and the greatest reproducibility occurred with Elgastat water as vehicle combined with a rigorous acid-washing of all glassware. This combined procedure also produced optimum and reproducible results for the synchronous growth of two B. subtilis 168 strains in casein medium supplemented with appropriate amino acids, a technique of value in monitoring the development of resistance to antibacterial agents during sporulation. The levels of aluminium in distilled water were higher than those of other elements; however, the incorporation of aluminium sulphate into broth prepared with Elgastat water had no effect on germination, and outgrowth was reduced (but not eliminated) only at high concentrations of this salt.     

The Effect of Sporulation Temperature on the Resistance of Bacillus subtilis to a Chemical Inhibitor

The ability of spores of Bacillus subtilis to germinate at 50° in sublethal concentrations of chlorocresol is related to sporulation temperature as is the resistance of the subsequent outgrowth at 50° to this substance. The degree of germination, age of spores and amount of outgrowth produced are of minor importance in determining resistance of the outgrowth.     

High Salinity Alters the Germination Behavior of Bacillus subtilis Spores with Nutrient and Nonnutrient Germinants

The effect of high NaCl concentrations on nutrient and nonnutrient germination of Bacillus subtilis spores was systematically investigated. Under all conditions, increasing NaCl concentrations caused increasing, albeit reversible, inhibition of germination. High salinity delayed and increased the heterogeneity of germination initiation, slowed the germination kinetics of individual spores and the whole spore population, and decreased the overall germination efficiency, as observed by a variety of different analytical techniques. Germination triggered by nutrients which interact with different germinant receptors (GRs) was affected differently by NaCl, suggesting that GRs are targets of NaCl inhibition. However, NaCl also inhibited GR-independent germination, suggesting that there is at least one additional target for NaCl inhibition. Strikingly, a portion of the spore population could initiate germination with l-alanine even at NaCl concentrations near saturation (∼5.4 M), suggesting that spores lack a salt-sensing system preventing them from germinating in a hostile high-salinity environment. Spores that initiated germination at very high NaCl concentrations excreted their large depot of Ca²⁺-pyridine-2,6-dicarboxylic acid and lost their heat resistance, but they remained in a phase-gray state in the phase-contrast microscope, suggesting that there was incomplete germination. However, some metabolic activity could be detected at up to 4.8 M NaCl. Overall, high salinity seems to exert complex effects on spore germination and outgrowth whose detailed elucidation in future investigations could give valuable insights on these processes in general.     

Mechanism of the Heat Sensitization of Bacillus subtilis Spores by Ethidium Bromide

Pretreatment with ethidium bromide (5 μg/ml) followed by a water wash had no effect on unheated Bacillus subtilis spores, but the viability of these spores after heating was much lower than that of similarly heated spores exposed to water alone. The fate of water- or ethidium bromide-treated spores, unheated or heated, was followed by allowing them to germinate and outgrow in a minimal or a complex liquid medium. Spores exposed to ethidium bromide and then heated (85°C, 10 min) exhibited a developmental block during germination and outgrowth. Many of them were blocked at the stage when the bacterium emerged from the germinated spore. When 0.35 μg of ethidium bromide per ml was added to heated spores in the germination-growth medium, the outgrowth of heated spores was inhibited to the same extent as were pretreated spores. Ethidium bromide acted in the first hour of germination of heated spores since addition after this time was ineffective in inhibiting recovery events. Repair of heat-damaged spore DNA was detected during the first 2 h of germination. The addition of ethidium bromide (final concentration, 0.35 μg/ml) inhibited DNA repair during early outgrowth. Increased sensitivity of spores to heat after pretreatment with sublethal concentrations of ethidium bromide was due to the inhibition of the repair of heat-damaged DNA.     

Influence of cellular differentiation on ultraviolet induced DNA damage and its repair mechanisms in B. cereus

Susceptibility to UV irradiation of B. cereus BIS-59 spores undergoing germination at various stages-dormant spores to vegetative cell stage and their ability to recover from radiation damage were studied. For a given dose of radiation, the number of spore photoproducts (SPP) formed in the DNA of dormant spores was about 5-times greater than that of thymine dimers (TT) formed in the DNA of vegetative cells. At intermediate stages of the germination cycle, there was a rapid decline in the UV radiation-induced SPP formed in DNA with a concomitant increase in the UV radiation-induced TT formed in DNA. Bacterial spores undergoing germination (up to 3 hr) in the low nutrient medium (0.3% yeast extract) displayed much higher resistance to UV radiation than those germinating in the rich nutrient medium, even though there was no discernible difference under the two incubation conditions in respect of the extent of germination and the time at which the outgrowth stage appeared (3 hr). This was due to the formation TT in the DNA of spores germinating in the low nutrient as compared to that of spores germinating in the rich-nutrient medium. In UV-irradiated dormant spores, SPP formed in the spore DNA did not disappear even after prolonged incubation in the non-germinating medium. However, when the UV-irradiated dormant spores were germinated in low or rich nutrient medium, a significant proportion of SPP in DNA was eliminated. The dormant spores incubated in either of the germinating media for 15 min and then UV-irradiated were capable of eliminating SPP (presumably by monomerization) even by incubation in a non-germinating medium and in the complete absence of protein synthesis (buffer holding recovery), thereby implying that spore-repair enzymes were activated in response to initial's germination. The acquisition of photo-reactivation ability appeared in spores subjected to germination only in the rich-nutrient medium at the outgrowth stage and required de novo synthesis of the required enzymes.     

Germination of Heat- and Alkali-Altered Spores of Clostridium perfringens Type A by Lysozyme and an Initiation Protein

The normal system functioning in the utilization of metabolizable germinants by both heat-sensitive and heat-resistant spores of Clostridium perfringens was inactivated by heat or by treatment of the spores with alkali to remove a soluble coat protein layer. Altered spores were incapable of germination (less than 1%) and outgrowth (less than 0.0005%) in complex media without the addition of either lysozyme or an initiation protein produced by C. perfringens. The addition of either of these agents permitted, in the case of alkali-treated spores, both 90 to 95% germination and outgrowth, as measured by colony formation. In the case of heat-damaged spores, only 50% germination and 2% outgrowth resulted from addition of the initiation protein, whereas lysozyme permitted 85% germination and 8% outgrowth. Alteration of the spores by heat or alkali apparently inactivated the normal lytic system responsible for cortical degradation during germination. Kinetics of production of the initiation protein and conditions affecting both its activity and that of lysozyme on altered spores are described.     

Chloride dependence of endospore germination in Halobacillus halophilus

The ion requirement for germination and outgrowth of endospores from the moderately halophilic salt marsh bacterium Halobacillus halophilus was studied. Germination and outgrowth of endospores plated onto nutrient broth was dependent on the salt concentration in the artificial seawater used as the source of ions. Maximal germination and outgrowth were observed when double-concentrated artificial seawater was used. Replacement of chloride salts in the artificial seawater by other salts resulted in a complete loss of germination and outgrowth that was restored upon addition of chloride. To analyze the role of chloride more directly and quantitatively, a defined growth medium was used in which the artificial seawater was substituted by a solution of magnesium sulfate and sodium chloride. Spore germination and outgrowth were strictly dependent on the chloride concentration; maximal germination and outgrowth were observed at ≈ 1.3 M Cl^–. Chloride could be substituted by bromide, but not by sulfate or nitrate. Microscopic examinations of single spores clearly showed that germination is the chloride-dependent step. This first report on chloride dependence of spore germination in any endospore-forming bacterium adds another function to chloride in H. halophilus apart from its being essential for the physiology of the vegetative cell. Received: 21 May 1999 / Accepted: 26 July 1999     

Involvement of the spore coat in germination of Bacillus cereus T spores

Bacillus cereus T spores were prepared on fortified nutrient agar, and the spore coat and outer membrane were extracted by 0.5% sodium dodecyl sulfate-100 mM dithiothreitol in 0.1 M sodium chloride (SDS-DTT) at pH 10.5 (coat-defective spores). Coat-defective spores in L-alanine plus adenosine germinated slowly and to a lesser extent than spores not treated with SDS-DTT, as determined by decrease in absorbance and release of dipicolinic acid and Ca2+. Spores germinated in calcium dipicolinate only after treatment with SDS-DTT. Biphasic and triphasic germination kinetics were observed with normal and coat-defective spores, respectively, in an environment with temperature increasing from 20 to 65 degrees C at a rate of 1 degree C/min. Therefore, the physical and biochemical processes involved in germination are modified by coat removal. The data suggest that a portion of the germination apparatus located interior to the coat may be protected by the coat and outer membrane or that the coat and outer membrane otherwise enhance germination in L-alanine plus adenosine. When coat-defective spores were heat activated with the dialyzed (12,000-Mr cutoff) components extracted from the spores, germination of the SDS-DTT-treated spores was enhanced; thus, one or more components located in the spore coat or outer membrane with a molecular weight greater than 12,000 were essential for fast germination.     

A low molecular weight factor from sea urchin eggs elevates sperm cyclic nucleotide concentrations and respiration rates.

A factor associated with sea urchin eggs that increases sperm cyclic nucleotide concentrations and respiration rates was identified as having a low molecular weight. The factor was more potent at elevating cyclic GMP concentrations than cyclic AMP concentrations, and represents the first demonstration of a factor associated with eggs that is capable of causing elevations of sperm cyclic GMP. Concentration-response curves of the crude mixture of egg factors to increase sperm cyclic AMP and cyclic GMP concentrations and respiratory rates were very similar, and comparable losses of these three activities were observed after extensive dialysis and heat treatment of the crude egg factors. The factor was partly purified by ethanol precipitation of a large molecular weight egg jelly component, and by charcoal adsorption and LH-20 chromatography of the resultant ethanol-soluble material. The factor was not extracted into a variety of organic solvents and had an apparent molecular weight of between 1000 and 2000, as estimated by gel filtration.