A microtiter fluorometric assay to detect the germination of Bacillus anthracis spores and the germination inhibitory effects of antibodies

Bacillus anthracis spore germination is usually detected in vitro by alterations in spore refractility, heat resistance, and stainability. We developed a more quantitative, sensitive, and semi-automated procedure for detecting germination by using a microtiter kinetic reader for fluorescence spectrophotometry. The procedure was based on the increase in fluorescence of spores with time during their incubation in germination medium containing a fluorescent nucleic acid-binding dye which stained germinated B. anthracis but not ungerminated (UG) spores. Spore germination in the presence of several germinants was characterized. Although L-alanine and inosine alone stimulated rapid germination in this assay, a medium containing optimal concentrations of L-alanine, adenosine, and casamino acids gave low background fluorescence, stimulated germination completely, and at a reasonable rate. Suspensions of heat-activated, UG spores of B. anthracis strain Ames were preincubated with antibodies (Abs) against whole spores to assess their effect on germination. Analyses of the germination data obtained revealed significant differences between spores pretreated with these Abs and those treated with non-immune sera or IgG. Germination inhibitory activity (GIA) was detected for several polyclonal rabbit anti-spore Ab preparations. These included anti-Ames strain spore antisera, IgG purified from the latter, and spore affinity-purified Abs from antisera elicited against four strains of B. anthracis. Abs elicited against UG as well as completely germinated Ames spores inhibited germination. Abs were ranked according to their GIA, and those specific for UG spores usually exhibited greater GIA. Direct binding to spores of these Abs was detected by an ELISA with whole un-germinated Ames spores. Although specific binding to spores by the anti-spore Abs was shown, their titers did not correlate with their GIA levels. Current efforts are focused on identifying the spore antigens recognized by the anti-spore Abs, characterizing the role of these targeted antigens in disease pathogenesis, and evaluating the ability of specific anti-spore Abs to protect against infection with B. anthracis.     

Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi

The effects of nutrient medium composition and temperature on the germination of conidia of the fungi Beauveria bassiana (strain AlG) and Metarhizium anisopliae (strain M-99) and their entomopathogenic activity have been studied. It was demonstrated that the presence of carbohydrates alone was sufficient for the spores of M. anisopliae M-99 to germinate, whereas the germination of B. bassiana AlG spores was inhibited by carbohydrates. Addition of KJ, ZnSO4, or KBr into the Czapek medium increased the entomopathogenic activity of B. bassiana. The optimum temperature for spore germination was 20-35 degrees C in both fungal species.     

Effect of mechanical abrasion on the viability, disruption and germination of spores of Bacillus subtilis

AIMS: To elucidate the factors influencing the sensitivity of Bacillus subtilis spores in killing and disrupting by mechanical abrasion, and the mechanism of stimulation of spore germination by abrasion. METHODS AND RESULTS: Spores of B. subtilis strains were abraded by shaking with glass beads in liquid or the dry state, and spore killing, disruption and germination were determined. Dormant spores were more resistant to killing and disruption by abrasion than were growing cells or germinated spores. However, dormant spores of the wild-type strain with or without most coat proteins removed, spores of strains with mutations causing spore coat defects, spores lacking their large depot of dipicolinic acid (DPA) and spores with defects in the germination process exhibited essentially identical rates of killing and disruption by abrasion. When spores lacking all nutrient germinant receptors were enumerated by plating directly on nutrient medium, abrasion increased the plating efficiency of these spores before killing them. Spores lacking all nutrient receptors and either of the two redundant cortex-lytic enzymes behaved similarly in this regard, but the plating efficiency of spores lacking both cortex-lytic enzymes was not stimulated by abrasion. CONCLUSIONS: Dormant spores are more resistant to killing and disruption by abrasion than are growing cells or germinated spores, and neither the complete coats nor DPA are important in spore resistance to such treatments. Germination is not essential for spore killing by abrasion, although abrasion can trigger spore germination by activation of either of the spore's cortex-lytic enzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanisms of the killing, disruption and germination of spores by abrasion and makes the surprising finding that at least much of the spore coat is not important in spore resistance to abrasion.     

A new regulatory function of the A-factor--stimulation of the streptomyces spore germination

Spore germination in streptomycetes was shown to be stimulated by exogenously added A-factor. Agar medium either containing or not containing A-factor was inoculated with spore suspensions of three strains differing in their ability to produce regulators of the A-factor group: Streptomyces griseus 773, which produces A-factor and two its lower homologs, S. coelicolor A3(2), which forms six AcL-factors (A-factor analogues), and S. avermitilis JCM5070, which fails to form regulators of this group. The count of the grown colonies showed that exogenous A-factor stimulated spore germination in strains that were themselves able to synthesize regulators of the A-factor group. In S. griseus 773, the number of germinated spores increased by 67% on average after the addition A-factor to the medium in an amount 10 micrograms/ml. In strain S. coelicolor A3 (2), the number of germinated spores increased by 75% after the addition of 1 microgram/ml of A-factor. During germination of the S. avermitilis JCM5070 spores, no changes in the CFU number was observed after the addition of A-factor.     

Colonization of arbuscular-mycorrhizal fungi on Ri T-DNA transformed roots in synthetic medium

Hairy root culture of tomato (Lycopersicon esculantum L.) was induced with three strains of Agrobacterium rhizogenes namely A4, ATCC 15834 and LBA 9402. The best response in terms of growth of hairy root was observed with A. rhizogenes strain A4 and LBA 9402 followed by ATCC 15834. Hairy roots were maintained on Murashige and Skoog (MS) medium but it could also grow on minimal (M) medium. Spores of Gigaspora margarita were isolated by wet sieving and decanting method and further recovered by sucrose density gradient method. A new method for surface sterilization of spores has been described which is simpler than the methods described earlier. Surface sterilized spores of G. margarita were used for inoculation of transformed roots grown on M medium as it was found more favourable for germination and growth of spores. During co-cultivation, mycorrhizal spore germination and its penetration into root cortex were observed. Inter and intracellular mycelial spread and formation of arbuscules were also observed in the cortical region of transformed roots of this plant.     

Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi Beauveria bassiana and Metarhizium anisopliae

The effects of nutrient medium composition and temperature on the germination of conidia of the fungi Beauveria bassiana (strain AlG) and Metarhizium anisopliae (strain M-99) and their entomopathogenic activity have been studied. It was demonstrated that the presence of carbohydrates alone was sufficient for the spores of M. anisopliae M-99 to germinate, whereas the germination of B. bassiana AlG spores was inhibited by carbohydrates. Addition of KJ, ZnSO₄, or KBr into the Czapek medium increased the entomopathogenic activity of B. bassiana. The optimum temperature for spore germination was 20–35°C in both fungal species.     

Germination and physiological properties of Frankia spores

Spores from four Frankia strains were isolated and purified to homogeneity. The purified spores were biochemically and physiologically characterized and compared to vegetative cells. Frankia spores exhibited low levels of endogenous respiration that were at least ten-fold lower than the endogenous respiration rate of vegetative cells. The macromolecular content of purified spores and vegetative cells differed. One striking difference among the Frankia spores was their total DNA content. From DAPI staining experiments, only 9% of strain ACN1^AG spore population contained DNA. With strains DC12 and EuI1c, 92% and 67% of their spore population contained DNA. The efficiency of spore germination was correlated to the percentage of the spore population containing DNA. These results suggest that the majority of strain ACN1^AG spores were immature or nonviable. The presence of a solidifying agent inhibited the initial stages of spore germination, but had no effect once the process had been initiated. The optimal incubation temperature for spore germination was 25°C and 30°C for strains DC12 and EuI1c, respectively. A mild heat shock increased the efficiency of spore germination, while root extracts also stimulated spore germination. These results suggest that strains DC12 and EuI1c may be suitable strains for further germination and genetic studies.     

Germination of concentrated suspensions of spores from Aspergillus niger

Summary Germination of condiospores fromA.niger in very concentrated suspension was required to inoculate solid fermentation media, but a germination self-inhibitor was detected in spores. It was found that the inhibitory effect depended on the composition of the medium and was reduced when glucose was used as a carbon source. The effect of the self-inhibitor was eliminated by washing the spores and using glucose and a protein nitrogen source in the germination medium. By this method it was possible to increase about 100 times (10⁶ to 10⁸) spore concentration, keeping more than 90% germination.     

Production of large amounts of acetate during germination of Bacillus megaterium spores in the absence of exogenous carbon sources.

When Bacillus megaterium spores germinate in the absence of an exogenous carbon source, the first minutes of germination are accompanied by production of large amounts (approximately 70 nmol/mg of dry spores) of acetate and much smaller amounts of pyruvate and lactate. The majority of these compounds are excreted into the medium. Exogenous pyruvate and alanine are also converted to CO2 and acetate by germinating spores, presumably by using the pyruvate dehydrogenase that is present in dormant spores. These data suggest that the 3-phosphoglyceric acid stores in the dormant spore and alanine generated by proteolysis early in germination can be catabolized to acetate during germination with production of large amounts of reduced nicotinamide adenine dinucleotide, acetyl coenzyme A, and adenosine 5'-triphosphate.     

Effects of temperature on the spores of thermophilic actinomycetes

The effects of temperature on the germination properties of spores of thermophilic actinomycetes were examined. Temperatures above and below the growth temperature of 55° C were found to produce marked changes in the germination properties of spores. High temperatures caused reductions in the germinative activities of spores. However, heated spore populations regained original germinative activities after maintaining them for suitable periods of time at 25°C. Recovery from the effects of heat on spore germination was also observed at 4°C, but at a much slower rate compared with 25°C. Spores of two strains of thermophilic actinomycetes, grown and prepared at 55°C, failed to germinate. Storage of dormant (nonactivated) spore populations at different temperatures demonstrated a low temperature requirement for the activation of these spores; while little or no activation occurred at 55°C, rapid activation took place at 25°C. Heating the spores at 80°C for 30 min slightly delayed the activation (rates) of spores at 25°C. The requirement of low temperature for spore activation was strain dependent and was influenced by the composition of the germination medium.     

Spore lytic enzyme released from Clostridium perfringens spores during germination.

The exudate of fully germinated spores of Clostridium perfringens was found to contain a large amount of a spore lytic enzyme which acted directly on alkali-treated spores of the organism to cause germination. Although no detectable amount of the enzyme was found in dormant spores during germination in a KCl medium, the enzyme was produced rapidly and released into the medium. The optimal conditions for enzyme activity were pH 6.0 and 45 degrees C. Maximum activity occurred in the presence of various univalent cations at a concentration of 50 mM. The enzyme was readily inactivated by several sulfhydryl reagents. A strong reducing condition was generated in the ionic germination of the spores, a minimum Eh level of -350 mV being reached 30 min after initiation of germination. Furthermore, adenosine triphosphate-dependent pyruvate:ferredoxin oxidoreductase (EC 1.2.7.1) was identified in both dorman and germinated spores. The relationship between the release of active enzyme and the generation of reducing conditions during germination is discussed.     

Green mould of oranges caused by Penicillium digitatutn Sacc.; effect of additives on spore germination and infection

Water extracts of rind, essential oil and juice from oranges, also citrus pectin and citric acid promoted the formation of lesions when spores of Penicillium digitatum were placed in wounds 1·0 mm deep in flavedo of oranges; fructose, glucose and sucrose had little effect. Rind extracts were less effective in wounds 0·5 mm deep but orange juice and pectin still increased infection. None of the substances allowed the parasite to infect fruit through unwounded surfaces. Germination of spores in water increased as spore concentration decreased but was poor even at low concentrations. Almost all spores germinated in aqueous extracts of flavedo, albedo or whole rind, or in wounds on the surface of fruit. Fructose, glucose, sucrose and xylose were less effective but still caused over two-thirds of spores to germinate but only in the presence of phosphate buffer. Without buffer, germination was little different from that in water. Arabinose and galactose stimulated germination to a lesser extent but with the same phosphate effect. Carboxymethylcellulose and pectin did not affect germination. A variety of substances containing nitrogen increased germination but to different degrees, decreasing in the order, casamino acids, yeast extract, ammonium salts, nitrate. Thiamin and to a lesser extent biotin were also effective. Volatile substances from rind infected with P. digitatum stimulated spore germination and growth of germ tubes. The significance of these results is discussed in relation to infection.     

Effect of individual amino acids and glucose on activation and germination of Rhizopus oligosporus sporangiospores in tempe starter

AIM: To understand the conditions promoting activation and germination of spores, and to contribute to the control of tempe starters. METHODS AND RESULTS: Using microscopic counts of fluorescent labelled spores, the following results were obtained: (1) L-alanine plays an important role (of the same order as that of peptone) in stimulation of germination of dormant spores. Alanine can satisfy the requirements of carbon as well as nitrogen for spore germination; (2) L-proline, on the other hand, inhibits alanine uptake presumably by blocking/congesting transporters of spore cells, resulting in apparent low viability on agar media; (3) L-leucine and L-isoleucine slightly favour spore germination while L-arginine and L-lysine do not have any stimulating effect; (4) The stimulatory role of glucose was only evident in the presence of phosphate (in minimal medium); when glucose is used in the absence of phosphate, either alone or in combination with single amino acids its role is hardly distinguishable; (5) Phosphate plays a facilitating role in spore germination. CONCLUSIONS: Glucose and amino acids play important roles in activation and germination of sporangiospores of Rhizopus oligosporus in tempe starter (stored for 12 months). The ability and rate of germination of dormant/old sporangiospores of R. oligosporus, depend on their ability for uptake of individual amino acids and/or glucose. SIGNIFICANCE AND IMPACT OF STUDY: New light was shed on the counteractive role of proline and the stimulating effect of phosphate. Soybeans subjected to traditional preparation for tempe making are heavily leached; germination of starter spores on such beans is sub-optimal, and bean processing could be optimized.     

Characterization of L-leucine-induced germination of Trichophyton mentagrophytes microconidia

l-Leucine and several amino acids were effective germination inducers of microconidia of Trichophyton mentagrophytes. During germination, phase-darkening and swelling occurred concomitantly with the loss of resistance to heat and stain, reduction of dry weight and specific gravity, and development of active glucose utilization. Germination induced by l-leucine was significantly stimulated by a pretreatment of the spores with sublethal doses of heat. No nucleosides or nucleotides were stimulatory to the l-leucine-induced germination of the microconidia. d-Leucine was almost an equally effective germinant as its l form. No carbohydrates, salts, vitamins, or other compounds tested induced germination of the fungus spore. Other factors that affected l-leucine-induced germination of the microconidia included the concentration of leucine, the inoculum size of the spores, temperature, and pH. The anaerobic condition and the presence or absence of carbon dioxide had no significant effects on the germination. Short germ tubes usually developed when the germinated spores were further incubated either in the presence or absence of l-leucine. The cytological study of the germinating microconidia revealed that the fragmentation of lipid granules was the major structural change associated with the germination.     

Nutritional and physiological factors affecting germination of heterothallic Saccharomyces cerevisiae ascospores.

Saccharomyces cerevisiae strain AP-3 was examined with respect to those nutritional requirements and physiological conditions which influence its germination rate. It was found that glucose as a carbon source supported the most rapid rate of germination for this heterothallic strain. In contrast, strain AP-3 spore germination was supported the least by the carbon sources potassium acetate and lactose. Of the nitrogen sources tested in culture medium containing glucose, the complex nitrogen sources peptone and casein hydrolysate appeared to be capable of stimulating germination better than a control culture containing ammonium sulphate. None of the amino acids screened were found to stimulate strain AP-3 germination compared with ammonium sulphate. The optimal culture medium pH for ascospore germination was 4.5 although spore germination could still be initiated by glucose between pH 3.0 and pH 7.5. Germination initiation by glucose was observed over a temperature range from 25 degrees C to 50 degrees C, but the optimal temperature appeared to be 40 degrees C.     

The Role of Osmotic Pressure in the Germination of Nosema algerae Spores1

ABSTRACT. Both the lag period and the time required for the filament and sporoplasm to emerge from Nosema algerae spores were prolonged when germination occurred under hyperosmotic conditions. Polyethylene glycol (PEG) and sucrose inhibited germination, first by preventing eversion of the filament, and then at higher concentrations by preventing stimulation. The size of the spore cases decreased by about 21% following germination, indicating an elastic spore wall and turgor pressure in the dormant spores. Increased pressure during germination was indicated by less osmotically-induced shrinkage in stimulated than in dormant spores and by higher concentration of solutes in the homogenates of germinated than ungerminated spores. These results are consistent with the hypothesis of a pressure increase during germination that is caused by an endogenous increase in solute concentration.     

Effects of temperatures above the maximum for germination on the endospore of Bacillus cereus

Knaysi, Georges (Cornell University, Ithaca, N.Y.). Effects of temperatures above the maximum for germination on the endospore of Bacillus cereus. J. Bacteriol. 87:1129-1136. 1964.-This is chiefly a study of heat activation and of the instability observed in spores of Bacillus cereus exposed to high temperatures. Temperatures up to 80 C for periods up to 6 hr were used. The spores were in colonies grown on collodion films, and were observed with the phase microscope, in dark contrast, for any evidence of change in their optical properties, the structure of their spodograms, and any other evidence of germination or growth. Exposure of the endospores to 80 C for 4 hr or more, whether in distilled water or in glucose broth, entirely eliminated stage II (i.e. the germcell stage, when the germinating spore begins to grow), and also tended to render the spores unstable and to produce uniformly dark spodograms. This instability involves some destructive processes and is not equivalent to normal entry into stage I (the initial stage). True heat-activation seemed to take place at a stage prior to the one which yielded uniformly dark spodograms. It was shown to consist of two factors: accumulation of germination-promoting components of the medium and activation of some spore components involved in germination. Activation of the medium components by preheating did not stimulate the spores of the strain employed. Unstable or activated spores can not be instantaneously stabilized, deactivated, or their changes arrested by exposure to acid buffers. It is concluded that the optimal temperature for activation, without danger of injury to the spore, in a given strain, is just above the maximum for stage I in that strain.     

Spore germination and phytochrome biosynthesis in the fern Lygodium japonicum as affected by gabaculine and cycloheximide

We investigated whether the gradual increase in phytochrome content in the fern Lygodium japonicum (Thunb.) Sw. during dark imbibition results from hydration or from biosynthesis of phytochrome. Addition of gabaculine or cycloheximide to the culture medium caused inhibitions of both red light-induced spore germination and of the appearance of phytochrome in the spores. Fifty percent inhibition of both red light-induced germination and of the appearance of phytochrome in the spores occurred at ca 10⁻7 M cycloheximide. Red light-induced germination and phytochrome appearance were markedly inhibited by 10⁻4 M and completely by 10⁻3 M gabaculine, but germination induced by gibberellic acid was unaffected. Phytochrome was not detected in spores after forced hydration. These results suggest that the increase in phytochrome during imbibition was mainly due to de novo synthesis of the phytochrome apoprotein and to synthesis of the chromophore and/or proteins required for phytochrome formation, rather than to hydration of preexisting phytochrome molecules.     

Nutritional conditions for the germination of streptomyces galbus 5ME-13 spores

The nutritional conditions for the germination of spores of Streptomyces galbus 5ME-13 were determined under laboratory conditions. The germination of the spores was intiated by the emergence of 1–2 germ tubes after the second hour of incubation and attained its maximum at the sixth hour. This was accompanied by a steady rise in the optical density of the germinating spore suspension. A malt-extract yeast-extract medium was found to be the best medium for the germination of the spores. Glycerol as the sole source of carbon was the best supporter for spore germination while, as N source, L-alanine was preferred. The optimum pH and temperature for spore germination were 7.2 and 30°C, respectively.     

Stimulation of yeast ascospore germination and outgrowth by S-adenosylmethionine.

The supplementation of S-adenosylmethionine (SAM) to germination medium stimulated the accumulation of [14C]uracil from the medium into germinating cells, as well as its incorporation into ribonucleic acid during germination and outgrowth of ascospores of Saccharomyces cerevisiae. In addition to uracil, the accumulation of leucine, cytosine, serine, and methionine was also stimulated by the extracellular addition of this sulfonium compound. The SAM-stimulatory effect was dose dependent; half-maximal stimulation was observed at about 50 muM. The effect exerted by SAM supplementation appeared to be specific for SAM and for germination and outgrowth. In the absence of SAM biosynthesis (in the presence of cycloleucine), spores were inhibited in their ability to accumulate label, whereas the supplementation of SAM completely reversed the cycloleucine-induced inhibition of accumulation. In addition to accumulation and incorporation, the kinetics of bud formation during outgrowth were also stimulated by exogenous SAM. The stimulation of budding by SAM was amplified in an ethionine-resistant strain. These observations suggest that SAM may be essential for the initiation of cell division during the breaking of spore dormancy.