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PubMed1968

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%.

Bacteria↗

PubMed1972

Physiological and biochemical changes associated with macroconidial germination in Microsporum gypseum.

A study was made of the metabolic processes associated with macroconidial germination in Microsporum gypseum. The optimum conditions for stimulation of endogenous respiration, changes in chemical composition as germination proceeds, and the uptake and synthetic fates of amino acids, glucose, and uracil were investigated. The assimilation and conversion of (14)C-glucose, (14)C-amino acids, and (14)C-uracil into the cell pool and into trichloroacetic acid-precipitable material were studied during the early stages of germination (i.e., prior to germ-tube emergence). The macroconidia were not metabolically inert for any significant period of time after exposure to germination conditions. Rather, the spores rapidly assimilated all metabolites and slowly converted them into macromolecules. Investigations of the effect of inhibitors of nucleic acid and protein synthesis prior to germ-tube emergence and during early germ-tube elongation suggested significant changes in metabolism and cell permeability may be correlated with the emergence of germ tubes. Radioactivity of incorporated glucose was found to be associated largely with the lipid fractions of the macroconidia early in germination.

Amino Acids↗

PubMed1971

Reduced germination of Clostridium botulinum type A spores in vitro by polymorphonuclear leukocytes from chronic granulomatous disease.

Leukocytes from two patients with chronic granulomatous disease of childhood (CGD) and from one female carrier were examined for their capacity to induce germination of Clostridium botulinum type A spores in vitro. Normal human leukocytes induced germination of C. botulinum spores to the same extent as guinea pig neutrophils. Germination was depressed by more than half when cells from CGD patients were used. A noticeable, but less severe, abnormality was present in leukocytes from a female carrier for this X-linked trait. CGD leukocytes are defective in cellular production of H(2)O(2), and the latter is known to be effective in inducing germination of clostridial spores. Lysozyme is also known to be effective in spore germination and is present in CGD leukocytes in normal amounts. The posibility that C. botulinum spores require a mechanism which sensitizes them to the action of H(2)O(2) and lysozyme is raised, and it is suggested that the methodology used in this work measures a defect of such a mechanism in CGD leukocytes.

Animals↗

PubMed1999

Association of a myosin immunoanalogue with cell envelopes of Aspergillus fumigatus conidia and its participation in swelling and germination.

A myosin immunoanalogue was identified in conidia of Aspergillus fumigatus by Western blotting, indirect immunofluorescence assay, and gold immunoelectron microscopy with two different antimyosin antibodies. The distribution pattern of this protein was followed during the early stages of germination. A single 180-kDa polypeptide, detected predominantly in a cell envelope extract, was found to cross-react with monoclonal and polyclonal antibodies raised against vertebrate muscle myosin. Immunoelectron microscopy permitted precise localization of this polypeptide, indicating that myosin analogue was mainly distributed along the plasma membrane of resting and swollen conidia. In germinating conidia, indirect immunofluorescence microscopy revealed myosin analogue at the periphery of germ tubes, whereas actin appeared as dispersed punctate structures in the cytoplasm that were more concentrated at the site of germ tube emergence. A myosin ATPase inhibitor, butanedione monoxime, greatly reduced swelling and blocked germination. In contrast, when conidia were treated with cytochalasin B, an inhibitor of actin polymerization, swelling was not affected and germination was only partially reduced. Butanedione monoxime-treated conidia showed accumulation of cytoplasmic vesicles and did not achieve cell wall reorganization, unlike swollen conidia. Collectively, these results suggest an essential role for this myosin analogue in the deposition of cell wall components during germination of A. fumigatus conidia and therefore in host tissue colonization.

Actins↗

PubMed2006

Expression of genes coding for GerA and GerK spore germination receptors is dependent on the protein phosphatase PrpE.

The ability of Bacillus subtilis to form spores is a strategy for survival under unfavorable environmental conditions. It is equally crucial to break spore dormancy and return to vegetative growth at the appropriate time. Here we present data showing that the PrpE phosphatase is involved in the control of expression of genes coding for GerA receptors, which are necessary for L-alanine-induced spore germination. Moreover, PrpE is also involved in aspartic acid, glucose, fructose, and potassium (AGFK)-induced spore germination by controlling expression of genes coding for GerK receptors. In the absence of PrpE, the production of spores was essentially normal. However, L-alanine-induced spore germination and, to a lesser extent, the AGFK-induced pathway were abolished. In contrast, the germination pathway dependent on Ca2+-dipicolinate or dodecylamine remained intact. A protein phosphatase PrpE-green fluorescent protein fusion was localized to the prespore and to the dormant spore, consistent with a role in controlling expression of genes coding for GerA receptors. We propose that PrpE is an important element in a signal transduction pathway in Bacillus subtilis that controls the expression of genes coding for germination receptors.

Bacillus subtilis↗

PubMed1970

Stable messenger ribonucleic acid and germination of Myxococcus xanthus microcysts.

We have examined germination, protein synthesis and ribonucleic acid (RNA) synthesis by microcysts of the fruiting myxobacterium Myxococcus xanthus. The morphological aspects of microcyst formation were completed at about 2 hr after induction had begun. In such microcysts, germination, RNA synthesis, and protein synthesis were inhibited by actinomycin D (Act D). At 6 hr after induction, germination and protein synthesis had become relatively resistant to Act D, whereas RNA synthesis was inhibited by about 95%. Experiments with (3)H-Act D indicated that the deoxyribonucleic acids of both young and old microcysts bind Act D equally. Resistance of germination to Act D was acquired 4 to 5 hr after induction of microcyst formation, and was due to an Act D-sensitive synthesis at that time. Vegetative cells and microcysts were pulsed with uridine-5-(3)H and chased for 60 min; the RNA was extracted and analyzed by means of sucrose density gradient centrifugation and gel electrophoresis. Both microcysts and vegetative cells were found to contain grossly the same types of RNA in the same proportions. RNA pulse-labeled in microcysts was more stable than that in vegetative cells. No particular portions of the microcyst pulse-labeled RNA were selectively stabilized. These data indicate that a stable messenger RNA required for synthesis of germination proteins was synthesized during microcyst formation. This may be the same as the RNA synthesized 4 to 5 hr after initiation of microcyst formation. We suggest that the existence of such stable messenger RNA in microcysts is consistent with the limited biosynthetic activities of such cells.

Bacteria↗

PubMed1974

Phosphate-mediated alteration of the Microsporum gypseum germination protease specificity for substrate: enhanced keratinase activity.

Inorganic phosphate was found to decrease the caseinolytic and ethyl-esterase activities of the Microsporum gypseum germination protease. The germination protease possessed exokeratinase (beta-keratinase) activity immediately after release from the fungal spore. After phosphate treatment of the enzyme, the germination protease also possessed endo-keratinase (alpha-keratinase) activity. Phosphate altered the protease's pH optimum from 9.0 to 7.0 and decreased the molecular weight from 33,000 to 16,000. These values were identical to those found for the keratinase. Alpha- and beta-keratinase activities were stimulated in excess of 200-fold by disulfide reducing agents. Natural and suspected keratin degradation products also enhanced keratinase activity. Cell fractionation and in vitro conversion of the alkaline germination protease into a functional keratinase suggested that the subunits comprising the germination protease and the keratinase were of a common origin.

Animals↗

PubMed1975

Neurospora crassa conidial germination: role of endogenous amino acid pools.

The levels of the endogenous amino acid pools in conidia, germinating conidia, and mycelia of wild-type Neurospora crassa were measured. Three different chromatographic procedures employing the amino acid analyzer were used to identify and quantitatively measure 28 different ninhydrin-positive compounds. All of the common amino acids were detected in conidial extracts except proline, methionine, and cystine. The levels of these three amino acid pools were also very low in mycelia. During the first hour of germination in minimal medium, the levels of most of the free amino acid pools decreased. The pool of glutamic acid, the predominant free amino acid in conidia, decreased 70% during the first hour. Very little glutamic acid or any other amino acid was excreted into the medium. During the first 20 min of germination, the decrease in the glutamic acid pool was nearly equivalent to the increase in the aspartic acid pool. The aspartic acid and lambda-aminobutyric acid pools were the only amino acid pools that increased to maximum levels within the first 20 min of germination and then decreased. It is proposed that an important metabolic event that occurs during the early stages of conidial germination is the production of reduced pyridine nucleotides. The degradation of the large glutamic acid pool existing in the conidia (2.5% of the conidial dry weight) could produce these reduced coenzymes.

Amino Acids↗

PubMed1975

Mutants of Dictyostelium discoideum defective in spore germination.

After activation, wild-type Dictyostelium discoideum spores germinate rapidly and synchronously in phosphate buffer as well as in complex medium. Mutants defective in spore germination were isolated and characterized. These mutants (called grm) did not germinate normally in buffer but did germinate in complex medium in the presence of bacteria. One mutant (grm B) swelled normally, but amoebae were not formed. Another mutant (grm F) swelled and germinated poorly in buffer. The members of the third group of mutants (A, C, D, and E) did not swell or give rise to amoebae in buffer.

Buffers↗

PubMed1977

Initiation of Bacillus spore germination by hydrostatic pressure: effect of temperature.

Suspensions of Bacillus cereus T, B. subtilis, and B. pumilus spores in water or potassium phosphate buffer were germinated by hydrostatic pressures of between 325 and 975 atm. Kinetics of germination at temperatures within the range of 25 to 44 degrees C were determined, and thermodynamic parameters were calculated. The optimum temperature for germination was dependent on pressure, species, suspending medium, and storage time after heat activation. Germination rates increased significantly with small increments of pressure, as indicated by high negative deltaV values of -230 +/- 5 cm3/mol for buffered B. subtilis (500 to 700 atm) and B. pumilus (500 atm) spores and -254 +/- 18 cm3/mol for aqueous B. subtilis (400 to 550 atm) spores at 40 degrees C and -612 +/- 41 cm3/mol for B. cereus (500 to 700 atm) spores at 25 degrees C. The ranges of thermodynamic constants calculated at 40 degrees C for buffered B. pumilus and B. subtilis spores at 500 and 600 atm and for aqueous B. subtilis spores at 500 atm were: Ea = 181,000 to 267,000 J/mol; deltaH = 178,000 to 264,000 J/mol; deltaG = 94,000 to 98,300 J/mol; deltaS = 264 to 544 J/mol per degree K. These values are consistent with the concept that the transformation of a dormant to a germinating spore induced by hydrostatic pressure involves either hydration or a reduction in the visocosity of the spore core and a conformational change of an enzyme.

Bacillus↗

PubMed1977

Initiation of deoxyribonucleic acid replication in germinating spores of Bacillus subtilis 168 carrying the dnaB (Ts)134 mutation.

The nature of the deoxyribonucleic acid synthesis reported by others to occur at 45 degrees C in germinating spores of the temperature-sensitive deoxyribonucleic acid initiation mutant of Bacillus subtilis 168, TsB134, has been investigated. Density transfer experiments, using 5-bromouracil, show that a normal round of replication can occur in a significant fraction of the spore population under such conditions. No repair synthesis is detectable. The possibility raised by this finding, that initiation of the first round of replication during spore outgrowth is unique in that its initiation is determined prior to germination, has been investigated by comparing the behavior of germinating spores of isogenic strains of B. subtilis 168, one carrying and the other without the dnaB (Ts)134 mutation. It is shown that deoxyribonucleic acid synthesis in the Ts strain is very sensitive to temperature in the vicinity of 45 degrees C. At a slightly higher temperature, 49 degrees C, initiation of the first round of replication in the Ts strain is completely (greater than 96%) blocked, but it proceeds normally in the Ts(+) strain. Thus, it is concluded that, after the germination of a spore, the action of the dnaB134 gene product is an obligatory requirement for initiation of the first round of replication. The initiation of replication that can occur in spores of the original TsB134 strain germinating at 45 degrees C is presumably due to incomplete inactivation of the dnaB134 gene product under such conditions.

Bacillus subtilis↗

PubMed1977

Function of S-adenosylmethionine in germinating yeast ascospores.

Germination and outgrowth of ascospores of Saccharomyces cerevisiae 4579 require both methionine and adenine, whereas leucine is only required for outgrowth. The methionine requirement may be satisfied by S-adenosylmethionine, but this sulfonium compound will not substitute for adenine. Between 30 and 70 min of protein synthesis is initially required for the completion of germination in strain 4579. The inhibition of S-adenosylmethionine synthetase by trifluoromethionine prevents both germination and protein synthesis. During the initial stages of germination, the S-adenosylmethionine synthetase, S-adenosylmethionine decarboxylase, and transfer ribonucleic acid methyltransferases increased significantly, indicating that polyamines and/or the methylation of transfer ribonucleic acid are required for the initiation of germination.

Adenine↗

PubMed1977

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.

Acetates↗

PubMed1979

Glucose-triggered germination of Bacillus megaterium spores.

Triggering of germination in Bacillus megaterium QM B1551 spores with D-glucose was studied. First, the interaction of glucose with spores for less than 1 min resulted in triggering almost 90% of the spores after the glucose was removed by dilution. Therefore only a brief time is needed for glucose to trigger germination, and then the continuous presence of glucose is not necessary. Detectable uptake of glucose began 2 to 3 min after absorbance loss started, and a non-metabolizable glucose analog, methyl-alpha-D-glucopyranoside, triggered germination in the absence of detectable uptake. Several inhibitors that reduced or eliminated glucose uptake did not block triggering of germination. Therefore, glucose uptake may be a relatively late event and not a prerequisite for triggering of germination.

Bacillus megaterium↗

PubMed1980

Expression of glycosidase activities during germination of Dictyostelium discoideum spores.

Several lysosomal glycosidase activities were examined in vitro during heat-induced germination of Dictyostelium discoideum spores and were found not to be coordinately controlled. The level of beta-glucosidase activity increased significantly during the emergence stage of germination. Both alpha-glucosidase and N-acetyl-beta-glucosaminidase activities remained relatively constant until postemergence, when they increased slightly; alpha-mannosidase activity decreased during all stages of germination. The activity of beta-galactosidase increased slightly during spore swelling, fell below the level initially found in spores at zero time, and increased slightly during postemergence. The expression of all of these enzyme activities, except the increase in beta-galactosidase, appeared to require protein synthesis. Spores in the lag phase of germination which were exposed to severe environmental stress were deactivated and exhibited reduced levels of alpha-glucosidase, beta-glucosidase, and N-acetyl-beta-glucosaminidase activities. Prolonged heat activation treatment reduced the levels of lysosomal glycosidase activities in postactivated spores but did not change the subsequent enzyme patterns during the spore-swelling and emergence stages of germination.

Acetylglucosaminidase↗

PubMed1980

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.

Arsenazo III↗

PubMed1981

Levels of H+ and other monovalent cations in dormant and germinating spores of Bacillus megaterium.

Previous investigators using the extent of uptake of the weak base methylamine to measure internal pH have shown that the pH in the core region of dormant spores of Bacillus megaterium is 6.3 to 6.5. Elevation of the internal pH of spores by 1.6 U had no significant effect on their degree of dormancy or their heat or ultraviolet light resistance. Surprisingly, the rate of methylamine uptake into dormant spores was slow (time for half-maximal uptake, 2.5 h at 24 degrees C). Most of the methylamine taken up by dormant spores was rapidly (time for half-maximal uptake, less than 3 min) released during spore germination as the internal pH of spores rose to approximately 7.5. This rise in internal spore pH took place before dipicolinic acid release, was not abolished by inhibition of energy metabolism, and during germination at pH 8.0 was accompanied by a decrease in the pH of the germination medium. Also accompanying the rise in internal spore pH during germination was the release of greater than 80% of the spores K+ and Na+. The K+ was subsequently reabsorbed in an energy-dependent process. These data indicate (i) that between pH 6.2 and 7.8 internal spore pH has little effect on dormant spore properties, (ii) that there is a strong permeability barrier in dormant spores to movement of charged molecules and small uncharged molecules, and (iii) that extremely early in spore germination this permeability barrier is breached, allowing rapid release of internal monovalent cations (H+, Na+, and K+).

Bacillus megaterium↗

PubMed1984

Differential gene expression during aerobic germination of Mucor racemosus sporangiospores.

Evidence is provided suggesting that several modes of differential gene expression operate concomitantly during the first 60 min of germination of Mucor racemosus sporangiospores under air. Protein synthesis was initiated immediately upon exposure of the spores to nutrient medium and accelerated exponentially throughout the period of observation. All translation during the first 30 min of germination occurred using only preformed stable mRNAs as a template. Analysis of the protein products synthesized in vivo was performed by L-[35S]methionine labeling, one- and two-dimensional polyacrylamide gel electrophoresis, and autoradiography. The population of proteins accumulated during spore formation and present at the time of harvest differed significantly from those proteins synthesized during spore germination. Autoradiographs displayed several proteins synthesized during the former but not the latter morphogenetic process. Conversely, other proteins were synthesized during the first 30 min of germination but not during spore formation, even though the mRNA specifying these proteins must have been synthesized and stored in the dormant spore. A posttranscriptional regulatory mechanism that directs selective translation thus appears to exist in the developing spore. In addition, autoradiographs showed that many proteins, although made throughout the intervals examined, displayed significant changes in their relative rates of synthesis. One gene product exemplified a possible case of post-translational modification during the first hour of sporangiospore germination.

Aerobiosis↗