Effect of Auxin and Gibberellin on Sporulation in Yeast

Effect of auxin and gibberellic acid on sporulation of a yeast, Saccharomyces ellipsoideus, was studied. When added to the sporulation media, gibberellic acid promoted sporulation. The sporulation rate was higher in the medium SGV with vitamins than in the vitamin-free SG, but the effect of gibberellic acid was more pronounced in the latter. Auxin (IAA, 2,4-D, and NAA) inhibited sporulation in SGV, but promoted it in SG. This sporulation-promoting effect of IAA was reversed by an antiauxin, 2,4,6-T. Preculturing in the presence of added IAA increased sporulation. Added to the preculture medium, gibberellic acid alone showed little effect on sporulation, but in combination with IAA it enhanced sporulation conspicuously. IAA and gibberellic acid were effective in sporulation promotion only when added before the nuclear enlargement occurred in sporulation culture.     

Correlation among turnover of nucleic acids,ribonuclease activity and sporulation ability of Saccharomyces cerevisiae

The turnover of nucleic acids and changes in ribonuclease activity during sporulation of Saccharomyces cerevisiae were studied. In the sporulating strains, 37–58% of vegetatively synthesized RNA were degraded during the sporulation process. The degree of degradation of vegetative RNA was proportional to the sporulation ability. In the non-sporulating strains, the degradation of vegetative RNA was less than 28% in the sporulation medium. Accompanied by the degradation of vegetative RNA, a ribonuclease activity increased several times during sporulation. We have found a close relation among the sporulation rate, the degree of the degradation of vegetative RNA and the increase in ribonuclease activity in the sporulation medium, using cells of which sporulation ability was repressed by changing the age or carbon source in various degrees.     

Differential amino acid requirements for sporulation in Bacillus subtilis

The amino acid requirements for sporulation were studied by use of auxotrophic mutants of Bacillus subtilis 168. Cells were grown to T(0) in medium containing the test amino acid and were then transferred to a minimal medium lacking that amino acid. Omission of leucine caused no reduction in sporulation. Omission of methionine, lysine, and phenylalanine appeared to cause reduced levels of sporulation, and sporulation was completely inhibited when isoleucine, tryptophan, and threonine were omitted. The amino acids in this third class showed a sequence of requirements, with tryptophan required earlier than isoleucine, which in turn was required earlier in the sporulation process than threonine. Isoleucine omission did not affect the early sporulation functions of extracellular protease formation or septum formation, but prevented the increased levels of protein synthesis and oxygen consumption that normally accompany early sporulation stages. Isoleucine did not appear to be metabolized to other compounds in significant amounts during sporulation. The role of isoleucine in the sporulation process remains unclear.     

SPORULATION OF THE YEAST, HANSENULA SATURNUS: I. EFFECT OF MAGNESIUM AND CALCIUM IONS

Sporulation in a strain of the wild yeast, Hansenula saturnus,was investigated. The yeast was found to form spores even indistilled water. The sporulation rate (percentage of ascus-bearingindividuals) in this case was found to be markedly affectedby the cell concentration adopted in the test. The addition of inorganic nutrients to the sporulation mediumstimulates sporulation. The yeast requires either magnesiumor calcium for growth and sporulation. Higher concentrationsof these ions are required for sporulation than for growth.In both cases magnesium is effective at more dilute concentrationsthan calcium. Under the conditions of the experiments, in which the yeastforms a pellicle, the sporulation rate in the pellicle far exceedsthat in the sediment. The effects of environmental factors on the sporulation wasconsidered in relation to growth. It was found that, under theconditions of poor growth in the sporulation culture, no exogenousmagnesium and calcium are required for sporulation. In suchcases, the yeast cells are inferred to have an endogenous stockof magnesium and calcium enough for the sporulation. ¹ Present address: Laboratory of Microbiology, Department ofAgriculture, Tôhoku University, Sendai. (Received May 4, 1961; )     

Biochemical studies on sporulation in blue-green algae. II. Factors affecting glycogen accumulation

Inorganic nitrogens sources like nitrate, nitrite enhanced sporulation and glycogen accumulation in Anabaena sp. but ammonium chloride neither influenced sporulation nor glycogen accumulation. Acetate and citrate also stimulated early sporulation and glycogen level was higher over nitrogen free control. Nitrogen and carbon sources in combination proved to be useful in inducing early sporulation and increased content of glycogen. Phosphate and calcium also affected glycogen accumulation significantly, although, the sporulation was found to be of the same order as in nitrogen free medium. Sulphate initiated early sporulation, the mechanism of which is not known.     

Requirement of Deoxyribonucleic Acid Synthesis for Microcycle Sporulation in Bacillus megaterium

Bacillus megaterium cells have been examined during outgrowth for their macromolecular content, ability to undergo microcycle sporulation, the time of their growth division, the time of deoxyribonucleic acid (DNA) replication initiation, and their ability to synthesize DNA after transfer to sporulation medium. The increase in total DNA content of the cells increased discontinuously beginning at 90 min. Thymidine incorporation became insensitive to chloramphenicol between 90 and 105 min of outgrowth. At 90 min the cells acquired the ability to undergo microcycle sporulation and the degree of sporulation depended on the time spent in outgrowth, with maximal sporulation occurring at 180 min. During outgrowth, cells underwent one synchronous growth division beginning at 225 min and ending at 270 min. Outgrowing cells were not able to continue DNA synthesis after transfer to sporulation medium. The data suggest that DNA replication starts before cells are able to undergo microcycle sporulation; however, the initiation of replication may not be the only requirement for microcycle sporulation.     

Inactivation of maltose permease and maltase in sporulating Saccharomyces cerevisiae

Maltose transport and maltase activities were inactivated during sporulation of a MAL constitutive yeast strain harboring different MAL loci. Both activities were reduced to almost zero after 5 h of incubation in sporulation medium. The inactivation of maltase and maltose permease seems to be related to optimal sporulation conditions such as a suitable supply of oxygen and cell concentration in the sporulating cultures, and occurs in the fully derepressed conditions of incubation in the sporulation acetate medium. The inactivation of maltase and maltose permease under sporulation conditions in MAL constitutive strains suggests an alternative mechanism for the regulation of the MAL gene expression during the sporulation process.     

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 Metarhizium anisopliae and Beauveria bassiana on Coptotermes formosanus and in vitro

The sporulation of 22 total isolates of Metarhizium anisopliae and Beauveria bassiana was quantified on cadavers of the Formosan subterranean termite, Coptotermes formosanus. Conidial production increased significantly over 11 days post-death. Effects of isolates of M. anisopliae and B. bassiana on in vivo sporulation were significant. Although the overall effects of fungal species on in vivo sporulation were not significant, the interactions between fungal species and certain times post-death were significant, indicating different sporulation patterns between the two fungal species. B. bassiana isolates could be categorized into a group with high total sporulation (day 11) and low quick sporulation (on days 2 and 3), while M. anisopliae isolates fell into another group with high quick sporulation and low total sporulation. This could give M. anisopliae an advantage over B. bassiana in termite microbial control due to termite defensive social behaviors. Conidial production was significantly higher in vitro than in vivo. In vitro and in vivo sporulation differed by as much as 89x and 232x among the selected isolates of B. bassiana and M. anisopliae, respectively. Correlation between in vivo and in vitro conidial production was positive and significant. This may allow preliminary in vitro screening of a large number of isolates for high in vivo sporulation.     

Effect of cyclic 3',5'-adenosine monophosphate on the sporulation of Saccharomyces cerevisiae

Cyclic 3',5'-adenosine monophosphate and sodium dibutyryl cyclic3',5'-adenosine monophosphate had no effect on sporulation ofSaccharomyces cerevisiae, when added to a sporulation mediumnot enriched with glucose. They did, however, reverse the repressionof sporulation by glucose, when added to the sporulation mediumtogether with glucose. 5'-AMP, 5'-ADP and 5'-ATP did not reversethe repression of sporulation by glucose. (Received February 24, 1972; )     

Isolation and characterization of sporulation-initiation mutation in the Bacillus subtilis prfB gene

A Bacillus subtilis prfB45 mutant grew at 42 degrees C, but its sporulation was severely defective at 37 degrees C. Sporulation-specific induction of kinA, spo0A, and spo0H genes was inhibited in the mutant. The effects of temperature up-shift and down-shift on sporulation of the prfB45 mutant was observed at an early stage of sporulation. UGA readthrough frequency at non-permissive temperatures for sporulation was higher in the mutant than in the wild-type strain. Temperature-sensitive sporulation of the prfB45 mutant was suppressed by mutations in rpsL coding for S12 of ribosomes, required for accurate termination of translation. Additionally, spontaneous second-site mutations that suppressed the sporulation phenotype of the prfB45 strain were found in the rpoB gene. These results suggest that accurate termination of translation is required for proper initiation of sporulation.     

Intergenic suppression of stage II sporulation defects by a mutation in the major vegetative sigma factor gene (rpoD) of Bacillus subtilis.

The Bacillus subtilis intergenic suppressor mutations crsA and rvtA, previously shown to restore sporulation competence to a variety of strains containing stage 0 sporulation defects, also suppress lesions in the stage II sporulation genes spoIIF, spoIIN and spoIIJ. They do not rescue sporulation in other stage II through stage V sporulation mutations. Cells containing spoIIN, spoIIF96 and spoIIJ::Tn917 mutations fail to transcribe spoIID, a late stage II gene. Introduction of crsA47 into spoIINts279, spoIIF96, or spoIIJ::Tn917 mutant backgrounds circumvents the need for the spoIIF, IIN, and IIJ products, restoring both expression of spoIID, and sporulation competence.     

Calcium and malate are sporulation-promoting factors of Physarum polycephalum

Fruiting body formation (sporulation) is a distinctive, irreversible differentiation process in the life cycle of the slime mold Physarum polycephalum. The most important requirement for sporulation of Physarum is a period of starvation, and normally sporulation proceeds in the light. It is shown here that by omitting the liquid sporulation medium and elevating the temperature from 21 to 25 degrees C, sporulation can occur routinely in the dark. It is further shown that this autocrine signaling in the dark requires calcium ions and malate. A putative sporulation control factor was detected in conditioned media derived from plasmodia starved in the dark, which was then identified as polymalate. As an additional role for this previously detected polyanion, specific for the plasmodial state of Physarum, it is suggested that the secreted compound serves as a source for both malate and calcium ions and thus promotes sporulation without light signaling.     

Biochemical changes during sporulation of Bacillus stearothermophilus.

The process of sporulation was studied in Bacillus stearothermophilus. A medium is described that supports good growth and sporulation of the organism. In this medium, which contains glucose, salts, and amino acids, acetate starts to accumulate before any of the glucose is catabolized. Enzymes of the tricarboxylic acid cycle are present at all times during growth and sporulation and are found in dormant spores. As the glucose in the culture is consumed, acetate rapidly increases and the pH of the medium drops. The acetate rapidly disappears during sporulation and the pH rises. Dipicolinic acid appears during sporulation and several key-enzyme activities fluctuate in a characteristic pattern.     

Cell-density-dependent lysis and sporulation of Myxococcus xanthus in agarose microbeads.

Vegetative cells of Myxococcus xanthus were immobilized in 25-microns-diameter agarose microbeads and incubated in either growth medium or sporulation buffer. In growth medium, the cells multiplied, glided to the periphery, and then filled the beads. In sporulation buffer, up to 90% of the cells lysed and ca. 50% of the surviving cells formed resistant spores. A strong correlation between sporulation and cell lysis was observed; both phenomena were cell density dependent. Sporulation proficiency was a function of the average number of cells within the bead at the time that sporulation conditions were imposed. A minimum of ca. 4 cells per microbead was necessary for efficient lysis and sporulation to proceed. Increasing this number accelerated the lysis and sporulation process. No lysis occurred when an average of 0.4 cell was entrapped per bead. Entrapping an average of 1.7 cells per bead resulted in 46% lysis and 3% sporulation of survivors, whereas entrapping an average of 4.2 cells per bead yielded 82% lysis and 44% sporulation of the surviving cells. Sporulation and lysis also depended upon the cell density in the culture as a whole. The existence of these two independent cell density parameters (cells per bead and cells per milliliter) suggests that at least two separate cell density signals play a role in controlling sporulation in M. xanthus.     

Dielectric monitoring of growth and sporulation of <Emphasis Type="BoldItalic">Bacillus thuringiensis</Emphasis>

On-line permittivity and optical density measurements have been used to monitor biomass concentration and sporulation status during growth of a spore-forming bacterium, Bacillus thuringiensis, in fed-batch culture. The correlation between permittivity, optical density and other observations showed three distinct phases of growth: growth itself, transition and sporulation. The permittivity variations during the transition and sporulation phases could be related to the sporulation development: the evolution pattern of the ratio of optical density to permittivity was representative of the culture state, and during the sporulation phase, a permittivity index could be build to measure the extend of spore liberation.     

Hydrolytic enzymes and spore formation in Bacillus intermedius

The investigation of the activity of extracellular hydrolytic enzymes and sporulation in the bacterium Bacillus intermedius 3-19 showed that the activity of ribonuclease is maximal in the glucose-containing growth medium, in which sporulation is suppressed. At the sporulation stages II-IV, the synthesis of phosphatase was not regulated by the factors that influence this synthesis in the phase of growth retardation. Caseinolytic activity exhibited two peaks. The first peak was observed when thiol-dependent proteinase began accumulating in the medium. The second peak corresponded to the late stages of sporulation, i.e., the stages of spore maturation and the autolysis of sporangium. The regulatory relationship between proteinase synthesis and sporulation and the possible role of extracellular phosphatases and proteinases in the sporulation are discussed.