Heat activation of Streptomyces viridochromogenes spores.

The lag period preceding germination of Streptomyces viridochromogenes spores during incubation in a defined germination medium was completely eliminated by a gentle heat shock. The rate of germination was not affected. The optimum pH for activation extended from 6.0 to 9.6. The time of heating required for maximum activation was 1 min at 60 C, 2 to 5 min at 55 C, 20 min at 50 C, and 40 to 50 min at 45 C. Activated spores had the same temperature and pH optima and nutritional requirements for germination as unactivated spores. Activated spores deactivated during incubation for 8 h at 25 C and were activated again by a second heat shock. Spores that had been aged for 4 weeks or longer did not germinate in the defined germination medium unless they were first heat activated.     

Streptomyces viridochromogenes spore germination initiated by calcium ions.

Initiation of germination of heat-activated Streptomyces viridochromogenes spore occurs in media containing only calcium ions and organic buffer. The calcium-induced initiation of germination was accompanied by a decrease in absorbance of the spore suspension, an increased rate of endogenous metabolism, the loss of spore carbon, and the loss of heat resistance. Calcium amounts to 0.28% of the dry weight of freshly harvested spores. The amount of calcium remained the same after incubation of spores in water after heat activation. The spore content of calcium doubled after incubation in 0.5 mM CaCl2 for 5 min at 4 degrees C and during calcium-induced germination. Nearly all of the calcim appears to be bound to sites external to the spore membrane, since the chelating agents (ethylenedinitrilo) tetraacetic acid and arsenazo III removed virtually all of the calcium ions. The calcium ions must be present during the entire initiation of germination period. Germination ceases after an (ethylenedinitrilo) tetraacetic acid wash and begins again immediately after addition of calcium ions.     

Hypochlorite injury of Clostridium botulinum spores alters germination responses.

Clostridium botulinum spores were sublethally damaged by exposure to 12 or 28 micrograms of available chlorine per ml for 2 min at 25 degrees C and pH 7.0. The damaging dose was 2.7 x 10(-6) to 3.1 x 10(-6) micrograms of available chlorine per spore. Damage was manifested by a consistent 1.6 to 2.4 log difference between the most probable number enumeration of spores (modified peptone colloid medium) and the colony count (modified peptone yeast extract glucose agar); this did not occur with control spores. Damaged spores could be enumerated by the colony count procedure. Germination responses were measured in several defined and nondefined media. Hypochlorite treatment altered the rate and extent of germination in some of the media. Calcium lactate (9 mM) permitted L-alanine (4.5 mM) germination of hypochlorite-treated spores in a medium containing 12 or 55 mM sodium bicarbonate, 0.8 mM sodium thiosulfate, and 100 mM Tris-hydrochloride (pH 7.0) buffer. Tryptose inhibited L-alanine germination of the spores. Treatments with hypochlorite and with hydrogen peroxide (7%, 25 degrees C, 2 min) caused similar enumeration and germination responses, indicating that the effect was due to a general oxidation phenomenon.     

Hypochlorite injury of Clostridium botulinum spores alters germination responses

Clostridium botulinum spores were sublethally damaged by exposure to 12 or 28 micrograms of available chlorine per ml for 2 min at 25 degrees C and pH 7.0. The damaging dose was 2.7 x 10(-6) to 3.1 x 10(-6) micrograms of available chlorine per spore. Damage was manifested by a consistent 1.6 to 2.4 log difference between the most probable number enumeration of spores (modified peptone colloid medium) and the colony count (modified peptone yeast extract glucose agar); this did not occur with control spores. Damaged spores could be enumerated by the colony count procedure. Germination responses were measured in several defined and nondefined media. Hypochlorite treatment altered the rate and extent of germination in some of the media. Calcium lactate (9 mM) permitted L-alanine (4.5 mM) germination of hypochlorite-treated spores in a medium containing 12 or 55 mM sodium bicarbonate, 0.8 mM sodium thiosulfate, and 100 mM Tris-hydrochloride (pH 7.0) buffer. Tryptose inhibited L-alanine germination of the spores. Treatments with hypochlorite and with hydrogen peroxide (7%, 25 degrees C, 2 min) caused similar enumeration and germination responses, indicating that the effect was due to a general oxidation phenomenon.     

Metabolism of endogenous trehalose by Streptomyces griseus spores and by spores or cells of other actinomycetes.

The disaccharide trehalose is accumulated as a storage product by spores of Streptomyces griseus. Nongerminating spores used their trehalose reserves slowly when incubated in buffer for several months. In contrast, spores rapidly depleted their trehalose pools during the first hours of germination. Extracts of dormant spores contained a high specific activity of the enzyme trehalase. The level of trehalase remained relatively constant during germination or incubation in buffer. Nongerminating spores of Streptomyces viridochromogenes, Streptomyces antibioticus, and Micromonospora echinospora and nongrowing spherical cells of Arthrobacter crystallopoietes and Nocardia corallina also maintained large amounts of trehalose and active trehalase. These trehalose reserves were depleted during spore germination or outgrowth of spherical Arthrobacter and Nocardia cells into rods.     

Role of carbon dioxide in germination of spores of Streptomyces viridochromogenes

CO₂ in required continuously during germination of Streptomyces viridochromogenes spores. Spores incubated in a defined germination medium in the absence of CO₂ remain phase bright and do not release spore carbon. In the presence of CO₂, the spores initiate germination accompanied by loss of refractility and spore carbon. The CO₂ requirement is replaced by oxaloacetate or a mixture of tricarboxylic acid cycle (TCA) intermediates. Labeled CO₂ is taken up by germinating spores, and is incorporated into protein and RNA. TCA cycle intermediates and related amino acids contain most of the acid-soluble label following short term exposures of germinating spores to ¹⁴CO₂. TCA cycle inhibitors repress germination and ¹⁴CO₂ uptake whereas folic acid antagonists do not. The results indicate that CO₂ is incorporated into oxaloacetate which is converted to biosynthetic intermediates required for germination. Operation of the TCA cycle appears to be essential for spore germination. The conclusion is reached that CO₂ is required during germination in order to maintain the cycle by an anaplerotic reaction.Abbreviations SN sucrose-nitrate medium - TX buffer Trisbuffer pH 7.3 containing-Triton X-100 - DGM defined germination medium - TX salts TX buffer plus Mg and Ca ions - TA trichloroacctic acid - TCA tricarboxylic acid     

Effects of intracellular trehalose content on Streptomyces griseus spores.

The disaccharide trehalose is accumulated as a storage product by spores of Streptomyces griseus. Growth on media containing excess glucose yielded spores containing up to 25% of their dry weight as trehalose. Spores containing as little as 1% of their dry weight as trehalose were obtained during growth on media containing a limiting amount of glucose. Spores containing low levels of trehalose accumulated this sugar when incubated with glucose. The increase in trehalose content coincided with increases in spore refractility, heat resistance, desiccation resistance, and the time required for spore germination in complex media. Trehalose is accumulated by a wide variety of actinomycetes and related bacteria and may be partially responsible for their resistance properties.     

Factors Affecting the Rate of Heat-induced Spore Germination in Dictyostelium discoideum

Washed spores of Dictyostelium discoideum, strains NC-4H, NC-4D, and V-12, germinated rapidly after being heat shocked at or near 45.0 C for 30 min. Cultures of the slime molds were grown in association with Escherichia coli B/r as the host bacterium; spores taken from plates of synthetic medium had a higher final germination value than spores from complex medium containing peptone and yeast extract. Young spores germinated more rapidly than older spores. Optimal germination occurred between pH 6.0 and 7.0, and, of the buffers tested, potassium phosphate allowed the most rapid germination. After heat shocking, spores were diluted into fresh oxygenated buffer to provide enough oxygen for completion of germination. Germination occurred most rapidly between incubation temperatures of 22 and 25 C.     

Thermal Activation and Dry-heat Inactivation of Spores of Bacillus subtilis MD2 and Bacillus subtilis var. niger

Spores of Bacillus subtilis MD2 and Bacillus subtilis var. niger were heat activated for different times at 60° and 80°C. Strain MD2 required considerable heat activation while B. subtilis var. niger did not. Maximum germination rates increased with heat activation dose and declined subsequently without loss of germinability. Germination rates and percentages were considerably greater in tryptone glucose extract (TGE) than in nutrient broth. The addition of 2°° dimethyl sulphoxide did not increase germination in nutrient broth. The spores of var. niger are more resistant to dry-heat than MD2 although they are less resistant to moist heat. Survivor curves in the dry-heat range 140°-170°C gave D-values from 4–123 to 0.106 min for MD2 and 5.679 to 0.233 min for var. niger recovered on TGE agar. D-values were lower on poorer media. The z-values for MD2 and var. niger on TGE were 18.7°C and 21.25C respectively.     

Germination initiation and outgrowth of spores ofBacillus brevis strain Nagano and its gramicidin S-negative mutant

Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to germination of their spores produced in several media. Germination initiation occurred in the presence of nutrient broth orL-alanine but not with inosine, glucose, glycerol or fructose; the process was activated by heat. Parental and mutant spores behaved similarly in these experiments. During outgrowth, parental spores remained in this phase of germination much longer than did mutant spores, but only when the parental spores had been harvested from a sporulation medium where significant gramicidin S synthesis had occurred. When parental spores were extracted or treated with an enzyme that hydrolyzes gramicidin S, rapid outgrowth occurred. Adding exogenous gramicidin S or the extract from parental spores to mutant spores lengthened the outgrowth in a dose-dependent manner. The uptake of labeledL-alanine by parental spores was delayed compared to mutant spores in the presence or absence of chloramphenicol. These data suggest a mechanism of action for gramicidin S whereby it interferes in membrane function, such as transport or energy metabolism, in outgrowing spores.Abbreviations GS Gramicidin S - CFU colony-forming units     

The Germination of Vavraia culicis Spores1

Buffered solutions of KCl and NaCl were tested for their stimulatory effect on the germination of variously-aged spores of Vavraia culicis. Germination was optimal in 0.2 M KCl, pH 6.5 for one isolate, and, for another isolate, peaks of germination occurred at pH 7.0 and 9.5. Spores incubated for several hours in suboptimal solutions became unable to germinate under optimal conditions. After being returned to water, they regained their ability to germinate. Calcium chloride, magnesium chloride, and ammonium chloride inhibited germination. After ingestion by mosquito larvae, spores germinated near the posterior end of the midgut.     

Properties of the germination inhibitor of Streptomyces viridochromogenes spores

Germinating spores of Streptomyces viridochromogenes excreted a substance into the surrounding medium which inhibited germination of another sample of the spores. The germination inhibitor (GI) was produced during submerged culture after exponential growth had ceased. The GI was purified 51-fold following extraction from growth liquor with chloroform. It was soluble in alcohol and water and had a molecular weight of less than 1000. The GI blocked growth and respiration of some Gram-positive bacteria and was an inhibitor of the membrane bound, but not solubilized, calcium-dependent ATPase of germinated spores and mycelia of the producing organism. Several sodium-potassium activated ATPases were also inhibited. All four activities (respiration, growth, germination inhibition, ATPase) co-purified during column and thin-layer chromatography. The GI activities released during germination and produced during growth were identical. A role for the GI antibiotic in regulation of dormancy of spores of the producing organism is discussed.     

Axenic germination of Scutellospora erythropa and Scutellospora nigra in in vitro conditions

Spores of Scutellospora erythropa and Scu. nigra isolated from neem rhizosphere soils from coastal regions of Chennai were tested for axenic germination in in vitro conditions. They showed positive results in media of different composition using root exudates, soil extract, thiamine HCl and inositol. The combined medium increased the spore germination in Scu. erythropa and in Scu. nigra over water agar control. The germ tube often grew up to 3.8 cm on combined media but no vegetative spores and extramatrical auxillary cells were observed during the experiment. There was significant increase in hyphal growth when the roots were introduced into the medium, 3 days after spore germination.     

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.     

Use of a novel method to characterize the response of spores of non-proteolytic Clostridium botulinum types B, E and F to a wide range of germinants and conditions

AIMS: Limited information is available on the germination triggers for spores of non-proteolytic Clostridium botulinum. An automated system was used to study the effect of a large number of potential germinants, of temperature and pH, and aerobic and anaerobic conditions, on germination of spores of non-proteolytic Cl. botulinum types B, E and F. METHODS AND RESULTS: A Bioscreen analyser was used to measure germination by decrease in optical density. Results were confirmed by phase-contrast light microscopy. Spores of strains producing type B, E and F toxin gave similar results. Optimum germination occurred in L-alanine/L-lactate, L-cysteine/L-lactate and L-serine/L-lactate (50 mmol l(-1) of each). A further 12 combinations of factors induced germination. Sodium bicarbonate, sodium thioglycollate and heat shock each enhanced germination, but were not essential. Germination was similar in aerobic and anaerobic conditions. The optimum pH range was 5.5-8.0, germination occurred at 1-40 degrees C, but not at 50 degrees C, and was optimal at 20-25 degrees C. CONCLUSIONS: The automated system enabled a systematic study of germination requirements, and provided an insight into germination in spores of non-proteolytic Cl. botulinum. SIGNIFICANCE AND IMPACT OF THE STUDY: The results extend understanding of germination of non-proteolytic Cl. botulinum spores, and provide a basis for improving detection of viable spores.     

CTAB法提取野野村菌基因组DNA

针对用常规方法难以提取野野村菌基因组DNA的问题,通过选用添加甘氨酸的不同培养基和不同培养时间获得的菌丝体,采用液氮研磨结合CTAB法提取野野村菌DNA,电泳检测及计算OD260/OD280值。结果表明,在添加0.3%甘氨酸的麦芽汁-酵母膏(YE)培养基中振荡培养培养3d的菌丝体适合于DNA提取,用CTAB法获得的基因组DNA,长度约为20kb,且OD260/OD280在1.8左右,达到基因组DNA-DNA杂交的要求。     

THE GERMINATION AND ENZYMIC ACTIVITIES OF BACILLUS SPORES AT LOW TEMPERATURES

SUMMARY: Well washed spore preparations of Bacillus cereus and B. subtilis were suspended in various nutrient broths, soil extracts, autoclaved soil of various moisture contents, and in two inorganic solutions, phosphate buffer, pH 7·2 and Ringer's solution. These were incubated at 8°, 5°, 1° and 0° for periods up to 270 days. Periodic total and spore counts on plates indicated a progressive decrease in each, associated with germination taking place in all conditions except in the two inorganic media. Enzymic tests indicated secretion and activity of nitratase and gelatinase and, with spores of B. pasteurii , urease, as a result of germination. B. subtilis germinated to a greater extent than B. cereus in each of the nutrient media. Germination of both organisms at 5° was also observed in L - and D -alanine: in the latter it was probably the result of racemization to the L -form.     

Regulation of formation of aerial mycelia and spores of Streptomyces viridochromogenes.

Growth of Streptomyces viridochromogenes on a solid glycerol-NH4NO3 salts medium was accompanied by the formation of aerial mycelia and spores. Adding 0.5% or more casein hydrolysate to the medium stimulated growth while completely repressing the formation of aerial mycelia and spores. This repression was temporary, as evidenced by the fact that transfer of the organisms to media not containing casein hydrolysate resulted in the appearance of aerial mycelia and spores. The effects of individual amino acids were tested. Glycine retarded growth and repressed formation of both aerial mycelia and spores. L-Aspartic acid, L-glutamic acid, and L-histidine stimulated or had little effect on growth and repressed formation of spores but not aerial mycelia. Repression by casein hydrolysate could not be attributed to the carbon/nitrogen ratio or the pH of the medium. Adding 1.25 to 2.5 mM adenine to the medium caused a reversal of the casein hydrolysate repression of aerial mycelium formation but did not reverse repression of sporulation. Dimethyladenine and 8-azaguanine had an effect similar to that of adenine, but a variety of other purine or pyrimidine derivatives had no effect on casein hydrolysate repression. The repression of aerial mycelium and spore formation by casein hydrolysate occurred only in media containing 15 mM or more phosphate. Aerial mycelia and spores were formed in media containing casein hydrolysate and 3 mM or less phosphate.