Characteristics of exogenous dormancy of Aspergillus niger conidia]

Aspergillus niger conidia are characterized by exogenous dormancy: the first stage of their germination is accomplished in twice distilled water. However, germ tube formation requires the availability of carbon and nitrogen sources. Exogenous dormancy in A. niger conidia exhibits the following peculiar features: (i) nitrogen-containing substances are active stimulators of germination; (ii) temperature-dependent changes in the lipid bilayer and in the neutral lipid composition of conidia are virtually identical to those occurring in growing mycelium under temperature stress; and (iii) the spore viability threshold does not exceed 45 degrees C; i.e., the spores are more heat-resistant than the mycelium, but they are less heat-resistant than the spores that are in the state of endogenous dormancy. According to the current classification of the types of cell metabolism arrest, the exogenous dormancy of A. niger conidia resembles the pattern of metabolism characteristic of vegetative cells during the idiophase.     

On the nature of sporogenesis in some aerobic bacteria

Washed vegetative cells of various species of aerobic spore-forming bacteria sporulate abundantly when shaken in distilled water in air. The spores thus formed possess the same heat resistance as spores formed in a complete growth medium. Various factors influencing sporogenesis in water are described. Glucose in low concentration completely suppresses sporogenesis under these conditions and the suppression is relieved by the presence of ammonia as an exogenous source of nitrogen. Various amino acid and purine antimetabolite analogues inhibit sporogenesis and their inhibitory effects are completely reversed by much smaller amounts of the corresponding metabolites. Sporogenesis is thus regarded as a de novo synthesis of spore proteins from preexisting endogenous (enzyme) proteins. Cells low in protein fail to sporulate and the capacity of the cell to adaptively attack maltose and trehalose is strongly interfered with after the cell is irreversibly committed to sporulation, but not before that. Evidence is advanced supporting the hypothesis that sporogenesis is an endogenous process which commences when the supply of exogenous energy and carbon is depleted. It utilizes low molecular weight nitrogenous substances liberated by the degradation of preexisting enzyme proteins of the vegetative cell. Sporogenesis and adaptive enzyme formation are regarded as competitive synthetic processes, both utilizing endogenous enzyme proteins. The events of sporogenesis suggest that this process may be an adaptive protein synthesis, analogous to adaptive enzyme synthesis.     

Peculiarities of Exogenous Dormancy of Aspergillus nigerConidia

Aspergillus nigerconidia are characterized by exogenous dormancy: the first stage of their germination is accomplished in twice-distilled water. However, germ tube formation requires the availability of carbon and nitrogen sources. Exogenous dormancy in A. nigerconidia exhibits the following peculiar features: (i) nitrogen-containing substances are active stimulators of germination; (ii) temperature-dependent changes in the lipid bilayer and in the neutral lipid composition of conidia are virtually identical to those occurring in growing mycelium under temperature stress; and (iii) the spore viability threshold does not exceed 45°C; i.e., the spores are more heat-resistant than the mycelium, but they are less heat-resistant than the spores that are in the state of endogenous dormancy. According to the current classification of the types of cell metabolism arrest, the exogenous dormancy of A. nigerconidia resembles the pattern of metabolism characteristic of vegetative cells during the idiophase.     

Glycine Betaine Biosynthesized from Glycine Provides an Osmolyte for Cell Growth and Spore Germination during Osmotic Stress in Myxococcus xanthus

Glycine sarcosine methyltransferase (Gsm) and sarcosine dimethylglycine methyltransferase (Sdm) catalyze glycine betaine synthesis from glycine. Disruption of the M. xanthus gsmA (MXAN 7068) or sdmA (MXAN 3190) gene, encoding Gsm or Sdm homologue proteins, respectively, generated mutants that exhibited a longer lag period of growth and delayed spore germination under osmostress.Myxococcus xanthus is a Gram-negative bacterium that exhibits a complex multicellular developmental cycle (6, 7). These bacteria live in soil, where they prey on other microbes for food. In response to nutritional stress, hundreds of thousands of vegetative cells aggregate to form multicellular fruiting bodies containing differentiated myxospores. Once conditions become favorable for growth, the desiccation- and heat-resistant spores can germinate and initiate vegetative growth.It was reported previously that the receptor-type adenylyl cyclases CyaA and CyaB of M. xanthus act as osmosensors during spore germination and growth, respectively (8, 9). Glycine betaine is a very efficient osmolyte found in a wide range of prokaryotic and eukaryotic organisms, where it is accumulated at high cytoplasmic concentrations in response to osmotic stress (4, 16). In this study, it is reported that in M. xanthus glycine betaine can be biosynthesized from glycine and mainly functions as an osmoprotectant for cell growth and spore germination under osmotic stress conditions.     

The effect of eight fungicides on germination of Conidiobolus obscurus resting spores

Conidiobolus obscurus resting spore germination was greater than 60% after 21 days incubation in 700 ppm concentrations of Rhodiasoufre, Pelt 44, Dithane M-45, Plantvax, and Saprol. No germ tubes were produced under the same treatment with Bavistine, Quin 20, or Benlate at the same dosage. Germination in distilled water was 85%. After only 24 hr contact with Quin 20 (700 ppm) followed by a distilled water wash, germination was not reduced. A higher ability to penetrate the spore wall may account for greater fungicide activity. Resting spores were found to be more resistant to certain fungicides than conidia; however, no general rule could be applied.     

Bacillus subtilis Cells Lacking Penicillin-Binding Protein 1 Require Increased Levels of Divalent Cations for Growth

Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg²⁺ or Ca²⁺ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of ponA cells in a medium low in Mg²⁺ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg²⁺ to growth media eliminated these phenotypes of a ponA mutant. In contrast to the effects of divalent cations, NaCl did not restore ponA cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg²⁺ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.     

Effects of pH shifts, bile salts, and glucose on sporulation of Clostridium perfringens NCTC 8798

The sporulation of Clostridium perfringens NCTC 8798 was studied after exposing vegetative cells to: pH values of 1.5 to 8.0 in fluid thioglycolate broth (for 2h) and then transferring them to Duncan-Strong (DS) sporulation medium; sodium cholate or sodium deoxycholate (0.3 to 6.5 mM) in DS medium; or Rhia-Solberg medium with 0.4% (wt/wt) starch, glucose, or both added at 0 to 55 mM. At pH 1.5, no culturable heat-resistant spores were formed. For cells exposed to pH 3.0, 4.0, 5.0, or 6.0, increases in heat-resistant spores were not seen until after a lag of 12 to 13 h, whereas the lag was only 2 to 3 h for cells exposed to pH 7.0 or 8.0. Maximal spore crops were produced after only 6 to 8 h for cells exposed to pH 7 or 8, but 16 to 18 h was required for production of maximal spore crops by cells exposed to the lower-pH media. The addition of sodium cholate (3.5 to 6.5 mM) to DS medium only slightly reduced the culturable heat-resistant spore count from 1.9 X 10(7) to 3 X 10(6)/ml. The addition of 1.8 mM or more sodium deoxycholate reduced the culturable heat-resistant spore count to less than 10/ ml. When either starch or glucose alone was added to Rhia-Solberg medium there was no production of culturable heat-resistant spores, but a combination of 0.4% (wt/wt) starch and 4.4 mM glucose yielded 6 X 10(5) spores/ml. The spore production remained at this level for glucose concentrations of 6 to 22 mM, but then declined to about 3 X 10(3) spores per ml at higher concentrations.     

Effects of pH shifts, bile salts, and glucose on sporulation of Clostridium perfringens NCTC 8798.

The sporulation of Clostridium perfringens NCTC 8798 was studied after exposing vegetative cells to: pH values of 1.5 to 8.0 in fluid thioglycolate broth (for 2h) and then transferring them to Duncan-Strong (DS) sporulation medium; sodium cholate or sodium deoxycholate (0.3 to 6.5 mM) in DS medium; or Rhia-Solberg medium with 0.4% (wt/wt) starch, glucose, or both added at 0 to 55 mM. At pH 1.5, no culturable heat-resistant spores were formed. For cells exposed to pH 3.0, 4.0, 5.0, or 6.0, increases in heat-resistant spores were not seen until after a lag of 12 to 13 h, whereas the lag was only 2 to 3 h for cells exposed to pH 7.0 or 8.0. Maximal spore crops were produced after only 6 to 8 h for cells exposed to pH 7 or 8, but 16 to 18 h was required for production of maximal spore crops by cells exposed to the lower-pH media. The addition of sodium cholate (3.5 to 6.5 mM) to DS medium only slightly reduced the culturable heat-resistant spore count from 1.9 X 10(7) to 3 X 10(6)/ml. The addition of 1.8 mM or more sodium deoxycholate reduced the culturable heat-resistant spore count to less than 10/ ml. When either starch or glucose alone was added to Rhia-Solberg medium there was no production of culturable heat-resistant spores, but a combination of 0.4% (wt/wt) starch and 4.4 mM glucose yielded 6 X 10(5) spores/ml. The spore production remained at this level for glucose concentrations of 6 to 22 mM, but then declined to about 3 X 10(3) spores per ml at higher concentrations.     

Inhibitory Effects of Turf Pesticides on Bacillus popilliae and the Prevalence of Milky Disease

Fourteen pesticides (fungicides, herbicides, and insecticides) were tested to determine whether they had deleterious effects on the bioinsecticide Bacillus popilliae, the causal agent of milky disease. All of these pesticides reduced levels of spore viability, spore germination, and/or vegetative cell growth when they were tested over a range of concentrations from 0 to 1,000 ppm of active ingredient, and the fungicides had the greatest detrimental effects. As determined by tests in water, the level of spore viability was significantly reduced by chlorothalonil, iprodione, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and 2-[(4-chloro-o-tolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid. In tests performed with iprodione, loss of viability was evident at concentrations less than the concentration calculated to result from recommended use. Tests performed in soil demonstrated that triadimefon, chlorothalonil, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and pendimethalin at concentrations resulting from recommended rates of application reduced spore titers. Spore germination did not occur in the continued presence of 2-[(4-chloro-otolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid, isofenphos, and chlordane, whereas exposure of spores to triadimefon or pendimethalin for 2 days stimulated germination. The tests to determine effects on spore germination were inconclusive for all other pesticides. Triadimefon, chlorothalonil, iprodione, pendimethalin, and chlorpyrifos at concentrations less than the concentrations recommended for use inhibited vegetative cell growth of B. popilliae, and chlordane at a concentration that was twice the concentration expected to result from the recommended rate of application repressed cell growth. My data support the hypothesis that use of synthetic pesticides can contribute to a low incidence of milky disease in white grubs.     

Ultraviolet Sensitivity of Bacillus subtilis Spores upon Germination and Outgrowth

A strain of Bacillus subtilis, UVSSP-42-1, which produces ultraviolet (UV)-sensitive spores and vegetative cells, was found to possess germinated spores 25 times more UV resistant than the resting spores. This relative resistance achieved upon germination was associated with the transition of the heat-resistant refractile spores to the heat-sensitive phase-dark forms. Several generations of outgrowth were required before the cells attained the level of UV sensitivity characteristic of the vegetative cell. The UV sensitivity of germinated spores was compared with other strains with various combinations of mutations affecting deoxyribonucleic acid repair capabilities. The presence of hcr and ssp mutations which are known to abolish the removal of photoproducts from deoxyribonucleic acid did not alter significantly the sensitivity of the germinated forms. However, the addition of the recA mutation and, to some extent, the pol mutation increased the UV sensitivity of the germinated spores. These results indicate that deoxyribonucleic acid repair mechanisms dependent on the recA gene are active in the germinated spores. The chemical nature of the damage repaired by the recA gene product is not known. This study indicates that the life cycle of sporulating bacilli consists of at least three photobiologically distinct forms: spore, germinated spore, and vegetative cell.     

Composition of the Cellular Envelopes of Anabaena cylindrica

Comparative chemical analyses were made of the walls of vegetative cells, heterocysts, and spores, and of the mucilage of Anabaena cylindrica. The wall of the vegetative cell is composed predominantly of amino compounds, with a mannose-rich carbohydrate component comprising only 18% of the dry weight. In contrast, 62% of the heterocyst wall and 41% of the spore wall is carbohydrate. The carbohydrate moieties of the heterocyst wall and spore wall are similar in that the ratio of glucose, mannose, galactose, and xylose is approximately 75:20:3:4 in both walls. It appears that, during the differentiation of a vegetative cell into either a spore or a heterocyst, a glucose-rich wall polysaccharide is produced that is different from the polysaccharide component of the wall of the vegetative cell and of the sheath. In the case of the heterocyst, the wall was estimated to account for approximately 52% of the dry weight of the whole cell.     

Properties and developmental roles of the lysyl- and tryptophanyl-transfer ribonucleic acid synthetases of Bacillus subtilis: common genetic origin of the corresponding spore and vegetative enzymes

The lysyl-transfer ribonucleic acid synthetase (LRS) and tryptophanyl-transfer ribonucleic acid synthetases (TRS) (l-lysine:tRNA ligase [AMP], EC 6.1.1.6; and l-tryptophan:tRNA ligase [AMP], EC 6.1.1.2) have been purified 60- and 100-fold, respectively, from vegetative cells and spores of Bacillus subtilis. There are no significant differences between the corresponding spore and vegetative enzymes with respect to their elution characteristics from columns of phosphocellulose or hydroxylapatite, their molecular weight (~130,000 for LRS and ~87,000 for TRS as determined by gel filtration), their kinetic constants for substrates (in the amino acid-dependent adenosine triphosphate-pyrophosphate exchange reaction), and the kinetics of inactivation by heat and by antibody. The Mg(2+) requirement for optimal enzyme activity of the corresponding spore and vegetative enzyme differ slightly. Mutants having defective (temperature sensitive) vegetative LRS or TRS activities produce spores in which these enzymes are also defective. The mutant spores are more heat sensitive than the parental type, but contain normal levels of dipicolinic acid. They germinate normally at the restrictive temperature (43 C), but are blocked at specific developmental stages in outgrowth. No modification in temperature sensitivity phenotype occurs during outgrowth, nor is there a change in molecular weight of the two enzymes. The implication is that the LRS and TRS activities of the vegetative and spore stages are each coded (at least in part) by the same structural gene. The temperature sensitivity of mutant spores is discussed with respect to those factors which are involved in the formation of the heat-resistant state.     

Electron microscopy of spore germination and cell wall formation in Mucor rouxii

Summary The fine structure of ungerminated and aerobically germinated sporangiospores of Mucor rouxii was compared. The germination process may be divided into two stages: I, spherical growth; II, emergence of a germ tube. In both stages, germination is growth in its strictest sense with overall increases in cell organelles; e.g., the increase in mitochondria is commensurate with the overall increase in protoplasmic mass. Noticeable changes occurring during germination are the disappearance of electron-dense lipoid bodies, formation of a large central vacuole and, most strikingly, formation of a new cell wall. Unlike many other fungi, M. rouxii does not germinate by converting the spore wall into a vegetative wall. Instead, as in other Mucorales, a vegetative wall is formed de novo under the spore wall during germination stage I. This new wall grows out, rupturing the spore wall, to become the germ tube wall. Associated with the apical wall of the germ tube is an apical corpuscle previously described. The vegetative wall exhibits a nonlayered, uniformly microfibrillar appearance in marked distinction to the spore wall which is triple-layered, with two thin electron dense outer layers, and a thick transparent inner stratum. The lack of continuity between the spore and vegetative walls is correlated with marked differences in wall chemistry previously reported. A separate new wall is also formed under the spore wall during anaerobic germination leading to yeast cell formation. On the other hand, in the development of one vegetative cell from another, such as in the formation of hyphae from yeast cells, the cell wall is structurally continuous. This continuity is correlated with a similarity in chemical composition of the cell wall reported earlier.     

Facile Method for Monitoring Inhibition of Anaerobic Spore Outgrowth

A device is presented for the laboratory monitoring of spore outgrowth under controlled temperature and anaerobic conditions. Alterations in pH, redox potential, headspace composition, and optical density are followed as the activated spores grow out into vegetative cells. An interlock system allows the addition of test solutions or the removal of medium under anaerobic conditions. The device may also be used for rapid (<4 h) chemical inhibition studies or adapted for temperature injury studies of aerobic or anaerobic cells. Data on outgrowth of Clostridium sporogenes and inhibition by nitrite solutions are presented.     

THE PARASPORAL BODY OF BACILLUS LATEROSPORUS LAUBACH

On sporulation the slender vegetative rods swell and form larger spindle-shaped cells in which the spores are formed. When the spores mature they lie in a lateral position cradled in canoe-shaped parasporal bodies which are highly basophilic and can be differentiated from the surrounding vegetative cell cytoplasm with dilute basic dyes. On completion of sporulation the vegetative cell protoplasm and the cell wall lyse, leaving the spore cradled in its parasporal body. This attachment continues indefinitely on the usual culture medium and even persists after the spores have germinated. In thin sections of sporing cells the bodies are differentiated from the cell protoplasm by differences in structure. Whereas the protoplasm has a granular appearance, in both longitudinal and cross-sections the parasporal body comprises electron-dense lamellae running parallel with the membranes of the spore coat and less electron-dense material in the interstices of the lamellae. The inner surface of the body is contiguous with that of the spore coat as if it were part of the spore, rather than a separate body attached to the spore. The staining reactions of the parasporal body are not consistent with those of any substance described in bacteria. With Giemsa the bodies stain like chromatin, but the Feulgen reaction indicates that they do not contain the requisite nucleic acid. With an aqueous solution of toluidine blue they stain metachromatically, but with an acidified solution the results are variable. Neisser's stain for polyphosphate is negative. The basophilic substance is removed from the body with some organic solvents. This basophilic substance has not been specifically identified with any material seen in ultrathin sections, but it is suggested that it might be the less electron-dense material in the interstices of the lamellar structure. In contrast to the spore coat of B. laterosporus, those of its two relatives B. brevis and B. circulans take up basic stain like the parasporal body. Thin spore sections of these species have shown that the walls are thicker than those surrounding the spores of B. laterosporus, and it is suggested that the outer stainable layer of brevis and circulans spores is an accessory coat which in laterosporus may have been deformed to give a parasporal body.     

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.     

Biochemical Studies of Bacterial Sporulation and Germination XIV. Phospholipids in Bacillus megaterium

The principal phospholipids of Bacillus megaterium throughout the cycle of growth and sporulation were found to be phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and a hitherto unidentified isomer of glycosaminyl-phosphatidylglycerol. Phosphatidylglycerol predominated during vegetative cell growth and then declined as spores developed, whereas diphosphatidylglycerol became more prominent during spore maturation. The latter phosphatide was relatively inaccessible in the vegetative cell and was more accessible in the spore, as judged by solvent extraction under various conditions.     

Resting spore formation ofLeptocylindrus danicus (Bacillariophyceae) during short time-scale upwelling and its significance as predicted by a simple model

Resting spore formation during short time-scale upwelling and its significance were investigated in the field and by a simple theoretical model. Field observations of spore formation ofLeptocylindrus danicus were made off Izu Peninsula, Japan. A rapid increase in ratio of resting spore to vegetative cell numbers indicated thatL. danicus formed resting spores quickly as a response to nutrient depletion in the upwelled water, although only a very low number of resting spores was found in the upwelling. A simple model was constructed to investigate the possible advantages of spore formation during short time-scale upwelling. This showed that there is a critical time-scale for resting spore formation to be advantageous. The nutrient depletion period of the upwelling off Izu was shorter than the critical time-scale determined by the model. Rapid-sinking of resting spores may increase further the critical time-scale, unless spores return with upwelling water. For short time-scale upwelling, the vegetative cell may be better suited than the resting spore for enduring a short period of nutrient depletion. Contribution from Shimoda Marine Research Center, University of Tsukuba, No. 475.     

Peptide Synthesis by Extracts from Bacillus subtilis Spores

Cell-free peptide synthesis by extracts from vegetative cells and spores of Bacillus subtilis was analyzed and compared. The initial rate of phenylalanine incorporation in a polyuridylate-directed system was found to be in a similar range for the two extracts. However, spore extracts frequently incorporated less total phenylalanine as did the vegetative cell system. Optimal conditions for amino acid incorporation by spore extracts were found to be similar to those of vegetative cell extracts. Polyphenylalanine synthesis was stimulated by preincubation of both extracts prior to the addition of polyuridylic acid (poly U) and labeled phenylalanine. Both systems showed a dependence on an energy-generating system and were inhibited by chloramphenicol and puromycin. Ribonuclease, but not deoxyribonuclease, inhibited the reaction significantly. The presence of methionine transfer ribonucleic acid (tRNA(F)) and methionyl-tRNA(F) transformylase was demonstrated in spore extracts. An analysis of several aminoacyl-tRNAs in spores revealed that the relative amounts of these tRNAs were similar to those found in vegetative cells. Only lysine tRNA was found to be present in relatively greater amounts in spores. These results indicate that dormant spores of B. subtilis contain the machinery for the translation of genetic information.