Utilization of trehalose during Dictyostelium discoideum spore germination

An analysis of metabolism by measurement of respiratory quotient values indicates that reduced substances, such as lipids and/or amino acids, are the primary respiratory substrates of dormant Dictyostelium discoideum spores. The spores appear to consume both reduced substances and carbohydrates during the swelling stage of germination. The respiration of emerged myxamoebae is again dominated by the consumption of reduced substances. The pool of trehalose remains largely intact during heat-induced activation and also during postactivation lag. The initiation of spore swelling is accompanied by a decrease in the trehalose pool; the majority of trehalose is consumed before late spore swelling. Upon placing heat-activated spores under restrictive environmental conditions, swelling and trehalose hydrolysis are both prevented. Release from these conditions results in rapid swelling and hydrolysis of trehalose. Trehalase, the enzyme responsible for trehalose breakdown, is present in dormant spores at basal levels. This preformed enzyme is responsible for the hydrolysis of trehalose even though there is a significant increase in trehalase activity with the emergence of myxamoebae. RNA and protein synthesis inhibitors do not prevent trehalose hydrolysis or spore swelling. It is concluded that oxidation of reduced substances occurs in dormant, activated, and swollen spores, as well as in emerged myxamoebae of D. discoideum. Carbohydrate utilization dominates over the oxidation of reduced substances only during the swelling stage of germination.     

Properties of Germinating Spores of Dictyostelium discoideum

The process of spore germination in Dictyostelium discoideum consists of three sequential stages: activation of dormant spores, swelling of activated spores, and emergence of myxamoebae from swollen spores. Dormant and activated spores are resistant to heating, freezing, or drying. Drying and freezing, moreover, may maintain the activated state until the spores are returned to normal conditions. Low temperature incubation after heat shock or the presence of an autoinhibitor will return activated spores to the dormant state. The entire spore germination process is aerobic, being inhibited at any point by oxygen deprivation or respiratory poisons. Each spore of this social organism appears to germinate at its own rate and independent of the other spores in the suspension.     

Activation and killing of Dictyostelium discoideum spores with urea.

The optimal conditions for activation of Dictyostellium discoideum spores are an 8 M urea treatment for 30 min. The lag between activation and swelling is 45 min. Lower concentrations of urea do not activate entire spore populations. Incubating spores in 8 M urea for 60 min or treatment with 10 M urea for 30 min results in a lengthening of the post-activation lag and a decrease in the final percentage of germination. Urea-activated spores can be deactivated by azide, cyanide, osmotic pressure, and low-temperature incubation. Activated spores do not germinate if incubated in 1 M urea for 24 h but will complete germination upon resuspension in urea-free buffer. Shocking spores at 45 degrees C in 8 M urea or incubating spores in 4-8 M urea for 10 h at 23.5 degrees C causes inactivation. When suspended in urea-free buffer, a larger percentage of these dead spores release spheroplasts through a longitudinal split in the spore case. Sequential enzyme treatment of spheroplasts with cellulase and pronase causes them to release lysable protoplasts. The data of these experiments suggest that shedding of the outer and middle wall layers during physiological spore swelling may be a physical process rather than an enzymatic one.     

RasG protein accumulation occurs just prior to amoebae emergence during spore germination in Dictyostelium discoideum

Abstract RasG protein levels in dormant and germinating spores of Dictyostelium discoideum strains JC1 and SG1 were estimated by Western blotting. Ras Glevels were very low in dormant spores and remained low during the lag period, regardless of whether spores were heat activated or treated with autoactivator during the early stages of spore germination. RasG levels increased late during spore swelling just prior to the emergence stage of germination. These data are consistent with a requirement for RasG during vegetative growth.     

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.     

Fine structure, physiology and biochemistry of arthrospore germination in Streptomyces antibioticus.

During germination, Streptomyces antibioticus arthrospores passed through stages: darkening, swelling and germ tube emergence. The first stage, darkening, whose main features were a decrease in absorbance and a loss of refractility, only required exogenous divalent cations (Ca2+, Mg2+ or Fe2+) and energy that can be obtained from the spore reserves. This stage was blocked by agents that inhibit ATP formation but not by antibiotics that inhibit macromolecular synthesis. The second stage, swelling, needed an exogenous carbon source and was not blocked by mitomycin C. In this stage, the spores exhibited the highest cytochrome oxidase and catalase activities and respiratory quotient. The last stage, germ tube emergence, required additional carbon and nitrogen sources. Ammonium compounds were superior to nitrate. Dry weight remained constant during the stages of darkening and swelling, with a rapid increase from the moment of germ tube emergence. Optimum pH and temperature for germination were 8.0 and 45 degrees C, respectively. Heat treatment (55 degrees C for 10 min) had no effect on germination. The fine structure of the spore underwent important changes during germination. The wall of the swollen spore became stratified and the inner layer was continuous with the germ tube wall. Macromolecular synthesis occurred in the sequence RNA, protein and then DNA. Rifampicin, streptomycin and mitomycin C prevented synthesis when added at the start of incubation. The same effect was obtained if the addition was made during germination, except with mitomycin C which inhibited DNA, but not RNA and protein synthesis.     

The identification of a self-inhibitor from Syncephalastrum racemosum and its effect upon sporangiospore germination

High concentrations of Syncephalastrum racemosum spores germinated less readily than low concentrations. Extensive washing of spores alleviated this inhibition of germination. Analysis of the spore washings revealed the main constituent to be nonanoic acid. Exogenously added nonanoic acid was found to mimic the self-inhibition, in that it delayed the time of germ tube emergence and increased the lag before spore swelling commenced.     

Germination of spores of Micromonospora chalcea: physiological and biochemical changes

Germinating spores of Micromonospora chalcea pass through three morphological stages: darkening, swelling and germ tube emergence. The process of germination has pH and temperature optima of 8.0 and 40 degrees C, respectively, and is not affected by activation treatments. Darkening, accompanied by a loss of heat resistance and refractility and a decrease in absorbance of the dormant spores, needs only energy, which can be obtained from endogenous sources, and exogenous cations. Agents that inhibit ATP formation block darkening, but inhibitors of macromolecular synthesis do not affect it. Swelling requires exogenous carbon but not nitrogen sources and is characterized by a 30 to 40% increase in spore diameter. RNA synthesis is necessary for swelling and inhibitors of protein synthesis delay this process. During this stage, maximum respiratory, cytochrome oxidase and catalase activities are reached. DNA synthesis starts at the beginning of germ tube emergence. This final stage requires both exogenous carbon and nitrogen sources and the sequence of macromolecular synthesis is RNA, protein and, finally, DNA. Rifampicin, streptomycin and mitomycin C prevent protein and DNA synthesis regardless of when added during germination. Rifampicin inhibits [3H]uridine incorporation immediately but there is a delay of about 160 min in the case of streptomycin or mitomycin C.     

Spore swelling in Dictyostelium is a dynamic process mediated by calmodulin

Abstract Spore swelling is a necessary prelude to the emergence of amoebae during spore germination in Dictyostelium discoideum . Previous work has shown that the initiation of this event requires the activity of the calcium-dependent regulatory protein calmodulin. In this study, the use of trifluoperazine, an inhibitor of calmodulin function, has shown that calmodulin activity is required throughout the swelling phase. When fully swollen spores were treated with trifluoperazine they rapidly returned to the same size and shape observed prior to swelling. These data suggest that spore swelling in D. discoideum is a dynamic process which is mediated by calmodulin.     

Water potential,glycerol synthesis,and water content of germinating Phycomyces spores

During early germination, the sporangiospores of Phycomyces blakesleeanus synthesized large amounts of glycerol. Glycerol started leaking out of the spores after some 20 min germination. Simultaneously the water content of the spores greatly increased. Water uptake was accompanied by disapperance of the phase contrast halo and an increase in spore cross-sectional area which all occurred during the same period between 10 and 30 min germination. When spores were incubated in 0.5 or 1 M sucrose, glycerol accumulated in the spores to much higher concentrations and the increase in cellular water content was greatly reduced and retarded. Glycerol synthesis and the concomitant lowering of spore osmotic potential was not the only mediator of spore swelling since equally important glycerol concentrations loaded into dormant spores did not cause spore water uptake or swelling. Also the swelling of the spores was less affected than water uptake by decreases in ambient water potential. Apparently also cell wall loosening was involved in the swelling phenomenon which might have important implications for cellular metabolism.     

Expression of proteolytic enzymes during Dictyostelium discoideum spore germination

The specific activity of cathepsin B-like, cathepsin D-like, and leucine aminopeptidase enzymes was measured in dormant, aging, and germinating spores of wild-type and mutant Dictyostelium discoideum.The activity of leucine aminopeptidase was relatively constant during spore aging and spore germination. The level of cathepsin D-like activity was highest in young dormant spores but decreased during germination or aging.The level of cathepsin B-like activity remained constant in wild-type spores which were aged for 13 days. The dormant spores of spontaneous germination mutants initially contained low levels of cathepsin B-like activity which increased during aging. Thus, there was no correlation between the level of endogenous cathepsin B activity and the ability to be autoactivated or heat-activated. The level of cathepsin B-like activity does not have a role in the generation of energy for the swelling stage of germination. Finally, the combined level of endogenous and exogenous cathepsin B activity increased more than 20-fold during the emergence of myxamoebae suggesting that the enzyme(s) may play a role at this development stage of germination.     

Characterization of cDNA clones specific for sequences developmentally regulated during Dictyostelium discoideum spore germination.

Spore germination in the slime mold Dictyostelium discoideum was used as a model to study the developmental regulation of protein and mRNA synthesis. Changes in the synthesis of these macromolecules occur during the transition from dormant spore to amoebae. The study of the mechanisms which regulate the quantity and quality of protein synthesis can best be accomplished with cloned genes. cDNA clones which hybridized primarily with mRNAs from only spores or germinating spores and not with growing amoebae were collected. Three such clones, denoted pLK109, pLK229, and pRK270, were isolated and had inserts of approximately 500, 1,200, and 690 base pairs, respectively. Southern blot hybridization experiments suggested that each of the genes is present in multiple copies in the D. discoideum genome. RNA blot hybridizations were performed to determine the sizes of the respective mRNAs and their developmental regulation. The mRNA that hybridized to pLK109 DNA was present predominantly in spores and at 1 h after germination but was absent in growing amoebae. Its concentration dramatically dropped at 3 h. The mRNA present in spores is apparently larger (approximately 0.5 kilobase) than in the later stages of germination (0.4 kilobase), indicating processing of the RNA during germination. The mRNA that hybridized to pLK229 DNA was approximately 1.0 kilobase and was present in very low amounts during growth. Its concentration rose until 1 h after spore germination and decreased thereafter. pRK270-specific RNA was approximately 2.7 kilobases and was found predominantly at 1 h after germination. It was present in lower concentrations at 2 and 3 h after germination and was absent in spores and amoebae. In vitro translation of mRNA selected from 1-h polyadenylated RNA which was hybridized to pLK109 or pLK229 DNA gave proteins of molecular weights consistent with the sizes of the mRNAs as determined by the RNA blot analysis.     

Ultrastructural Changes During Germination of Dictyostelium discoideum Spores

Spores of Dictyostelium discoideum undergo significant changes in fine structure during germination. The mitochondria progressively become less dense and lose their peripherally attached ribosomes, and the tubuli become more pronounced as germination proceeds. During this period, the three-layered spore wall breaks down in two stages: first, the outer and middle layers are ruptured as a unit, and, second, the inner wall is breached. Crystals and dark (lipid) bodies disappear shortly before or during emergence of the myxamoebae. Autophagic vacuoles are found in dormant spores and throughout the entire germination process. The addition of cycloheximide to germinating spores inhibited the loss of the crystals and dark (lipid) bodies. In addition, the drug inhibited the breakdown of the inner wall layer. Cycloheximide did not prevent the formation of the water expulsion vesicle or the apparent function of the autophagic vacuoles.     

The physiological effects of restrictive environmental conditions on Dictyostelium discoideum spore germination.

Spores may be reversibly activated by the application of heat, dimethyl sulfoxide, urea, or ethylene glucol. Severe changes in four environmental variables (high osmotic pressure, low oxygen tension, low or high pH, and low or high temperature) interfere with the germination process. Spores at the end of the postactivation lag phase of germination were usually deactivated if exposed to severe environmental conditions and thus did not swell; spores in the swelling and oxygen uptake which began during spore activation was primarily attributable to a cyanide-sensitive pathway and secondarily to a salicylhydroxamic acid (SHAM) sensitive pathway. Inhibition of the SHAM-sensitive pathway did not cause spore deactivation while the addition of cyanide resulted in rapid spore deactivation. Treatment of activated spores with azide or environmental shifts also resulted in inhibition of oxygen uptake and spore deactivation. Deactivating spores did not demonstrate the amino acid incorporation, uridine incorporation, and expression of trehalase activity which is found in the later stages of germinating control spores. Protein synthesis inhibitors did not cause spore deactivation or a decrease in oxygen uptake but they inhibited amino acid incorporation and the expression trehalase activity in swollen spores. It is concluded that control of respiratory activity is involved in regulation of reversible activation.     

Respiratory competence of Dictyostelium discoideum spores.

Analysis of the respiratory chain of spores of Dictyostelium discoideum, which lack a cyanide-sensitive respiration, indicated that cytochromes a-a3, b, and c-c1 are present at levels identical to those found in the vegetative amoebae. The specific activities of enzymes of both the respiratory chain and the citric acid cycle in the 600 x g supernatant fraction of sonically treated spores were at least as high as in similar preparations of amoebae. The activities of glutamic dehydrogenase and oligomycin-sensitive adenosine triphosphatase were reduced in the spores 30 and 56%, respectively. Intact spores appeared to lack a cyanide-sensitive respiration as a result of inadequate quantities of respiratory substrate and, more importantly, as a result of a lack of the cofactor nicotinamide adenine dinucleotide. The emergence phase of spore germination was sensitive to the antibiotic chloramphenicol, which is a specific inhibitor of mitochondrial protein synthesis. It is concluded that germination requires the early synthesis of oxidized nicotinamide adenine dinucleotide and generation of respiratory substrates and one or more mitochondrially synthesized proteins.     

Unequal accumulation of 26S and 17S RNAs in ribosomes during spore germination in Dictyostelium discoideum

Ribosome synthesis was studied in spores at the swelling stage and compared with freshly emerged and logarithmically growing vegetative amoebae. During the swelling stage of spore germination, ribosome synthesis was abnormal. Newly made ribosomes accumulated unequal amounts of 26S and 17S rRNAs. The stoichiometric ratio 26S:17S was 0.5 in swelling spores, compared with 0.9 in amoebae. The relative level of pre-rRNA persisting in the nucleus was apparently 2- to 3-fold higher in swelling spores than in amoebae. All of the known ribosomal proteins, except for a few, were made during the swelling stage and were associated with the newly made ribosomes in expected amounts. Analysis of the 2'-O-methyl ribose content in the newly made rRNAs suggest that methylation was defective in swelling spores. Compared with growing amoebae, the methyl content was 30 and 64% less in 26S and 17S RNAs from the swelling stage, respectively. It is suggested that undermethylation could be partly responsible for the differential accumulation of newly made 26S and 17S RNAs during the early stages of spore germination in Dictyostelium discoideum.     

Glucose-induced pathways for actin tyrosine dephosphorylation during Dictyostelium spore germination

In the presence of germination signals, dormant spores of Dictyostelium discoideum rapidly germinate to start a new life cycle. Previously we have shown that half of the actin molecules in spores are maintained in a tyrosine-phosphorylated state, and a decline of the actin phosphorylation levels is a prerequisite for spore swelling. In this study, we have established d-glucose as a trigger molecule for the actin dephosphorylation. Present in a nutrient germination medium, d-glucose both may act as a trigger molecule and/or may serve as a substrate within a pathway for actin dephosphorylation depending upon spore age. However, the glucose-induced actin dephosphorylation was insufficient for spores to swell. Other factors in the nutrient medium were required for complete germination of young spores aged 1 to 5 days. In contrast, dispersion in nonnutrient buffer was necessary and sufficient for a decline of actin phosphorylation levels and even the emergence of amoebae in older spores (6 days and beyond). Moreover, the dephosphorylation pathway in the older spores was independent of energy production. We propose that the diversification of the actin dephosphorylation pathway may enable spores to increase their probability of germination upon spore aging.     

Dictyostelium discoideum mRNAs developmentally regulated during spore germination have short half-lives.

mRNA decay was studied during spore germination in Dictyoselium discoideum by the use of three previously isolated cDNA clones, pLK109, pLK229, and pRK270, which are specific for mRNAs developmentally regulated during spore germination. The half-life of a constitutive mRNA, pLK125, which is present throughout germination, growth, and development, as also determined. Nogalamycin, a DNA-intercalating compound, was used to inhibit RNA synthesis. Total RNA was isolated at intervals after addition of the drug, and the decay of mRNAs specific for the cDNA clones was determined by both Northern blot and RNA dot hybridization. If nogalamycin was added immediately after activation of dormant spores, neither pLK229 nor pLK109 mRNA decayed, but pLK125 mRNA did decay. Although pLK109 mRNA did not decay under these conditions, the RNA was smaller 1 h after activation than in dormant spores, indicating that it was processed normally. At 1 h after activation, pLK229-, pLK125-specific mRNAs decayed exponentially, with half-lives of 24, 39, and 165 min, respectively. Under the same conditions, decay of pLK109-specific mRNA was biphasic. Thirty-eight percent of the mRNA decayed with a half-life of 5.5 min, and the remainder decayed with a half-life of 115 min. It seems likely that nogalamycin inhibits the synthesis of an unstable component of the mRNA degradative pathway which is needed continuously for the decay of pLK109 mRNA. By extrapolating the curve representing the rapidly decaying component, a half-life of 18 min was calculated for pLK109-specific mRNA. The mRNAs developmentally regulated during spore germination have half-lives shorter than that of the constitutive messenger and shorter than the average half-life of 3 to 4 h previously determined for total Dicyostelium polyadenylated mRNA.     

Spore Germination in Strains of Dictyostelium discoideum and Other Members of the Dictyosteliaceae

Spores of all strains of Dictyostelium discoideum tested in this study germinated after a heat shock of 45 C for 30 min. Whereas the strains differed in their rates of germination, the rate for each strain was constant. A correlation existed between the rate of germination and the rate of vegetative growth when spores were inoculated into bacterial streaks. Heat shock clearly increased spore germination in D. purpureum, but the response was less dramatic than in D. discoideum. Enhancement also occurred in D. rosarium, but only in media containing peptone. Strains of D. mucoroides gave varied responses, and these could be divided into those which required mutrients for spore germination and those which did not. The spores of Polysphondylium pallidum were resistant to mild heat (45 C), but were not activated; peptone was required for germination. In contrast, the microcysts of this species were heat-labile and required no added nutrients for excystment.