Sporulation, Heat Resistance, and Biological Properties of Clostridium perfringens

A sporulation medium for 134 Clostridium perfringens strains, including types A, B, C, D, E, and F, was devised according to Grelet's observation that sporulation occurred when cultural environment became limited in any nutritional requirement indispensable for the growth of the organism. Sporulation took place most prominently when 10% cooked-meat broth (pH 7.2) containing 3% Proteose Peptone and 1% glucose was used for the preculture and 2% Poli Peptone medium (pH 7.8) was used for the subculture medium. Sometimes, terminal spores could be observed. A correlation between sporulation and heat resistance was examined by use of C. perfringens strains isolated from samples heated at different temperatures. Almost all strains isolated from unheated samples and from those heated at lower temperatures gave rise to spores in our sporulation medium, but the spores were weakly heat-resistant, whereas strains isolated from samples heated at 100 C for 60 min were highly heat-resistant but sporulated poorly. A majority of these heat-resistant strains were non-gelatinolytic and definitely salicin-fermenting.     

Relation between inhibition of Bacilli sporulation and synthesis of lytic enzymes.

In two strains of Bacillus, the synthesis of two specific lytic enzymes was studied concomitantly with an inhibition of the sporulation: LD-carboxypeptidase synthesis was unaffected whereas γ-D-glutamyl-(L)meso-diaminopimelyl endopeptidase synthesis was shown to be closely related to sporulation. The endopeptidase production is totaly inhibited when netropsin inhibits sporulation in B. sphaericus and is low in B. subtilis Thy^?A when sporulation is inhibited by thymidine starvation. This enzyme seems directly connected with the sporulation sequence.     

Initiation of Dermatophyte Pleomorphic Strain Sporulation by Increased Aeration

A normally asporogenous pleomorphic strain of Microsporum gypseum was induced to sporulate by controlled aeration and dehydration. Aeration of the pleomorphic strain under optimal cultivation conditions caused the initiation of a sporulation cycle with equivalent growth parameters and percentage intracellular water loss as the wild-type strain. Initiation of sporulation was not due to alteration of the medium's nutrient concentration or consistency, concentration of fungal growth by-products, or removal of volatile „staling factors.” Macroconidia formed by the pleomorphic colonies were of characteristic wildtype morphology, but germinated to form typical pleomorphic colonies, indicating that the induced sporulation was strictly phenotypic and reversible. Other asporogenous pleomorphic strains from different dermatophyte genera also were induced to form macroconidia by aeration, suggesting a similarity in sporulation induction in Microsporum sp., Epidermophyton floccosum, and Trichophyton violaceum. Initiation of sporulation by aeration further suggested that the pleomorphic mutation was one which affected the sensitivity of the pleomorphic aerial hyphae to natural sporulation inducers (i.e., decreased humidity) and did not represent a loss in the ability to form fertile macroconidia.     

RNA synthesized during late sporulation is required for germ tube formation inBlastocladiella emersonii

Inhibition of RNA synthesis with actinomycin D as late as 210 min (T₂₁₀) afterBlastocladiella emersonii is induced to sporulate results in complete blockage of germ tube formation in the next generation. In agreement with other reports, actinomycin D added during germination did not block germ tube formation. Protein synthesis during germination is reduced by approximately one-half when actinomycin D is added atT₂₁₀ but remains at virtually control levels when actinomycin D is added during germination. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel analyses of the abundant proteins synthesizedin vivo during the first hour of germination revealed no qualitative differences in the proteins which accumulate when control cells are compared to cells treated with actinomycin D atT₂₁₀. Comparison of proteins synthesized from 20 to 40 min germination vs 40 to 60 min germination demonstrated that actinomycin D alters the temporal pattern of accumulation of some abundant proteins. The RNA synthesized afterT₂₁₀ is associated with polysomes, suggesting that an mRNA fraction made in late sporulation is required for a germ tube. The available data do not exclude the possibility that a regulatory RNA synthesized during late sporulation is required for germ tube formation.     

Function of glycolytic bypass and S-lactoylglutathione in sporulation of yeast cells

When vegetative cells of a yeast Saccharomyces cerevisiae were incubated on sporulation medium, the cells were sporulated and thereby activities of methylglyoxal synthase and glyoxalase I, both of which are glycolytic bypass enzymes responsible for the conversion of dihydroxyacetone phosphate into S-lactoylglutathione, were preferentially and markedly increased. Sporulation was also enhanced in the presence of S-lactoylglutathione. We propose a possibility that the glycolytic bypass regulates the yeast cell sporulation and S-lactoylglutathione has a function to enhance the sporulation of yeast cells.     

Laboratory transmission and in vivo incubation of Entomophthora muscae (Entomophthorales: Entomophthoracae) in the onion fly,Delia antiqua (Diptera: Anthomyiidae)

A high-moisture infection chamber was used for the in vivo transmission of Entomophthora muscae within laboratory populations of the onion fly, Delia antiqua. This cadaver-to-fly transmission procedure provided an average experimental infection rate well above 95%. Laboratory infection and temperature-dependent incubation rates of E. muscae were further examined in adult populations of D. antiqua. The time from initial exposure until host death and pathogen sporulation was accurately predicted using a second-order function of the incubation temperature. A developmental base temperature of approximately 5°C was estimated, with 105 degree-days being the average number of heat units required between host infection and death. E. muscae transmission between D. antiqua and D. platura, two insect pests typically associated with Michigan onion production, was verified under laboratory conditions.     

Visualization of Penicillin-Binding Proteins during Sporulation of Streptomyces griseus

We used fluorescein-tagged β-lactam antibiotics to visualize penicillin-binding proteins (PBPs) in sporulating cultures of Streptomyces griseus. Six PBPs were identified in membranes prepared from growing and sporulating cultures. The binding activity of an 85-kDa PBP increased fourfold by 10 to 12 h of sporulation, at which time the sporulation septa were formed. Cefoxitin inhibited the interaction of the fluorescein-tagged antibiotics with the 85-kDa PBP and also prevented septum formation during sporulation but not during vegetative growth. The 85-kDa PBP, which was the predominant PBP in membranes of cells that were undergoing septation, preferentially bound fluorescein-6-aminopenicillanic acid (Flu-APA). Fluorescence microscopy showed that the sporulation septa were specifically labeled by Flu-APA; this interaction was blocked by prior exposure of the cells to cefoxitin at a concentration that interfered with septation. We hypothesize that the 85-kDa PBP is involved in septum formation during sporulation of S. griseus.     

Effect of actinomycin D on viability,sporulation and nucleotide pool ofBacillus megaterium

A transient 7-fold rise of ppGpp concentration, 2-3-fold increase of pppGpp concentration and 50 % drop of the concentration of GTP inBacillus megaterium cells immediately after their transfer to the sporulation medium were observed. Actinomycin D, in concentrations inhibiting RNA synthesis by 95%, blocked the rise of the (p)ppGpp pool and caused an instant several-fold increase of the GTP level. When the cells were exposed to actinomycin D in the sporulation medium for a 1-h period (time 0–1 h, 1–2 h or 2.20–3.20-h), they were able to form colonies on nutrient agar after being kept, in addition for 1–2 h in the sporulation medium free of the antibiotic. The ability of sporulation was, however, markedly limited. The share of cells that could sporulate increased when the irreversible sporulation phase was reached.     

Role of acetate metabolism in sporulation ofSaccharomyces carlsbergensis

Several aspects of the role of acetate metabolism in the sporulation ofSaccharomyces carlsbergensis were investigated. Experiments in which the development of the respiratory system was either stimulated by growth on sugars to which the cells have to adapt, or inhibited by chloramphenicol suggested a correlation between respiratory development and sporulation. In cells in which the respiratory system has been repressed during growth, mitochondrial protein synthesis and derepression are prerequisites for sporulation. When derepression is complete, sporulation no longer depends on mitochondrial protein synthesis. Incorporation experiments with acetate showed that this compound is an important source of intermediates for biosynthetic processes that occur during sporulation. Its incorporation into macromolecular fractions is tightly coupled to sporulation.     

Sporulation of Bacillus stearothermophilus

A broth medium containing tryptone and manganese sulfate supported heavy sporulation of Bacillus stearothermophilus ATCC 7953 (NCA 1518) and four isolates identified as B. stearothermophilus. Maximal spore yields were obtained by use of inocula grown anaerobically in a medium containing glucose with aeration of sporulation medium via bubbling. After an extended stationary period, sporulation occurred concurrently with vegetative growth between 6 and 8 hr of incubation at 60 C. Omission of glucose from the inoculum or use of a “young” (2 hr) inoculum abolished the stationary period, but decreased spore yields. A requirement of oxygen for rapid vegetative growth and sporulation was demonstrated. Manganese (15 to 30 ppm) stimulated sporulation but did not enhance cell growth.     

Effect of pH on Adenine and Amino Acid Uptake During Sporulation in Saccharomyces cerevisiae

During the early stages of sporulation in Saccharomyces cerevisiae, the pH of the acetate sporulation medium rises to values of 8.0 or higher. Associated with this rise in pH is a reduced cell permeability to certain precursors of ribonucleic acid (RNA), deoxyribonucleic acid or protein. Uptake of adenine, alanine, and leucine was optimal at pH 5.6 to 6.0, but sporulation was inhibited when the sporulation medium was buffered below pH 7.0. Cellular impermeability can be largely overcome by adjusting the acetate sporulation medium to pH 6.0 for optimal uptake of ¹⁴ C-adenine during short pulses without any apparent effect on sporulation. Sporulating cells pulse-labeled 20 min at pH 6.0 incorporated 40 times more ¹⁴C-adenine into RNA than sporulating cells pulse-labeled at pH 8.0. This increased incorporation can be attributed to a 100-fold increase in labeled adenosine triphosphate in cells pulse-labeled at pH 6.0 where maximum uptake occurs.     

Genome-Wide Transcriptional Profiling of Clostridium perfringens SM101 during Sporulation Extends the Core of Putative Sporulation Genes and Genes Determining Spore Properties and Germination Characteristics

The formation of bacterial spores is a highly regulated process and the ultimate properties of the spores are determined during sporulation and subsequent maturation. A wide variety of genes that are expressed during sporulation determine spore properties such as resistance to heat and other adverse environmental conditions, dormancy and germination responses. In this study we characterized the sporulation phases of C. perfringens enterotoxic strain SM101 based on morphological characteristics, biomass accumulation (OD₆₀₀), the total viable counts of cells plus spores, the viable count of heat resistant spores alone, the pH of the supernatant, enterotoxin production and dipicolinic acid accumulation. Subsequently, whole-genome expression profiling during key phases of the sporulation process was performed using DNA microarrays, and genes were clustered based on their time-course expression profiles during sporulation. The majority of previously characterized C. perfringens germination genes showed upregulated expression profiles in time during sporulation and belonged to two main clusters of genes. These clusters with up-regulated genes contained a large number of C. perfringens genes which are homologs of Bacillus genes with roles in sporulation and germination; this study therefore suggests that those homologs are functional in C. perfringens. A comprehensive homology search revealed that approximately half of the upregulated genes in the two clusters are conserved within a broad range of sporeforming Firmicutes. Another 30% of upregulated genes in the two clusters were found only in Clostridium species, while the remaining 20% appeared to be specific for C. perfringens. These newly identified genes may add to the repertoire of genes with roles in sporulation and determining spore properties including germination behavior. Their exact roles remain to be elucidated in future studies.     

Protein degradation during sporulation ofBacillus megaterium: Effect of actinomycin D

The first acceleration of protein degradation in cells ofBacillus megaterium was found at the stage 0–I of sporulation, the second one at the stage II–III, where the sporulation process became irreversible. These accelerations were reduced by actinomycin D inhibiting RNA and protein syntheses by more than 95%. In the presence of the antibiotic, only 8% of prelabeled proteins were degraded. Actinomycin D did not lower either the concentration of ATP or the proteolytic activity in the homogenate prepared from sporulating cells. This indicates that the inhibition of protein catabolism by actinomycin D was not owing to the absence of ATP or proteolytic enzymes. Actinomycin probably inhibited an unknown step preceding the proteolytic attack of the protein molecules during sporulation, because it had no significant effect on proteolysis during vegetative growth.     

Endocellular fatty acid composition during batch growth and sporulation of Bacillus thuringiensis kurstaki

Bacillus thuringiensis kurstaki (HD-1) was grown on two different complex media to study its fatty acid composition during vegetative growth and sporulation. In contrast to literature results, iso-even branched-chain fatty acids were found to predominate after early vegetative growth and throughout sporulation.     

Sporulation in Hansenula wingei Is Induced by Nitrogen Starvation in Maltose-Containing Media

The sexually agglutinative yeast Hansenula wingei lives in association with bark beetles that inhabit coniferous trees. This yeast was induced to sporulate by malt extract, which contains a high percentage of maltose (50%) and a low percentage of nitrogen (0.5%). A solution of 1.5% maltose without any growth factors also induced ascosporogenesis in H. wingei. Thus, only a carbon source is required for sporulation as in Saccharomyces. However, potassium acetate did not induce sporulation in H. wingei as it does in S. cerevisiae. Instead, disaccharides (such as maltose, sucrose, or cellobiose) promote sporulation better than either monosaccharides (such as dextrose, fructose, or mannose) or respiratory substrates (such as ethanol or glycerol). The specificity of disaccharides in promoting sporulation in H. wingei may be considered an adaptation since these disaccharides are present in the natural environment of this yeast. In addition, the specificity of disaccharides may be related to the induction of the disaccharidase because cells precultured on dextrose sporulate well on maltose, but cells precultured on maltose sporulate poorly on maltose. When (NH₄)₂SO₄ was added at a low concentration (3 mM) to synthetic sporulation medium (1.5% maltose solution), sporulation was abolished, whereas other salts and nitrogen sources inhibited to a lesser extent and vitamins and trace elements had no effect. Oxygen was required for sporulation, as expected for an obligate aerobe. Maximal sporulation was achieved in 2% malt extract broth at high cell density (10⁹ cells per ml), pH 5, and 25°C. By using these optimal physiological conditions and hybrid strains selected from an extensive genetic breeding program, about 30% asci (10% tetrads) were obtained routinely. Thus, the genetics of cell recognition in this yeast can now be studied.     

Chemically defined media for growth and sporulation of Entomophthora virulenta

Amino acids, salts, and vitamins were combined with dextrose to test their effect on growth and sporulation of Entomophthora virulenta in liquid shake culture. The addition of a vitamin solution to the tested media did not enhance growth or sporulation. MgSO₄·7H₂O was the only salt individually tested that allowed for good growth and sporulation. MgSO₄·7H₂O concentrations exceeding 250 mg/liter in media lacking other salts inhibited sporulation. A simple medium of l-arginine, l-leucine, glycine, and mineral salts allowed high growth and sporulation.