Induction of sporulation in developmental mutants of Bacillus subtilis.

Summary Eight alleles of spineless-aristapedia (ss ^a ) were analysed for penetrance and expressivity at 18° C and 25° C. All alleles are recessive and none exhibits a maternal effect. Both ss ^a40a and ss ^aB are temperature sensitive with a higher penetrance at 18° C than 25° C. ss ^aUCI , ss ^ak , ss ^a , ss ^ax and ss ^a are viable alleles with variable penetrance and expressivity whereas ss ^aCam is a lethal allele. All mutants were tested as hemizygotes using the Df(3R) bxd ¹⁰⁰ deletion. The viable alleles showed a more extreme penetrance and expressivity when hemizygous, several becoming lethal near eclosion. Each of the eight alleles was examined in heterozygous combinations with each of the others. The lethal allele ss ^aCam was viable in combination with all other alleles. The temperature sensitivity (t.s.) of ss ^aB and ss ^a40a when heterozygous with the non-t.s. alleles was variable; some combinations were t.s. with respect to penetrance and others with respect to expressivity, whilst some heterozygotes completely lost their sensitivity to temperature.It seems, therefore, that various aspects of the spineless-aristapedia phenotype, such as the temperature sensitivity, can be separated from the expressivity and penetrance in certain allelic combinations. This suggests a very complex gene function which is, however, experimentally separable into its components.     

Development of a new chemically defined sporulation medium for the yeasts in the genus Lipomyces

A chemically defined sporulation medium (AF medium) for the yeasts belonging to the genus Lipomyces was developed. The chemical composition was derived from chemical analyses of soybean extract. Some chemical modification of the AF medium indicated that the nitrogen sources (aspartic and glutamic acids) and zinc ion were essential for sporulation. The significance of medium pH was discussed.     

Induction of sporulation inBlastocladiella emersonii: Influence of nutritional variables

We have studied the induction of zoosporangial sporulation inBlastocladiella emersonii by determining the influence of nutritional variables on the formation of the basal septum. This is an easy-to-monitor structure that has proved to be a useful marker of initiation of this cellular differentiation process. At any time before formation of the septum, the cells returned to the growth phase when incubated in the presence of 2% casamino acids. However, after septum formation has occurred, the cells were committed to sporulation. Certain of the amino acid components of the defined growth medium, DM₂, either prevent (tyrosine, phenylalanine, tryptophan, histidine, and threonine) or delay (valine, serine, arginine, and methionine) formation of the septum when added to the cultures together with the sporulation solution (buffered 1 mM CaCl₂). Sugars do not prevent the formation of the septum, although they can interrupt sporulation at later developmental phases. Glucosamine and acetylglucosamine can prevent formation of the discharge papilla and glucose can block completion of zoospore production. Glutamine, adenosine, and guanosine also caused long delays in septum formation. Taken together, these data favor (a) the hypothesis that amino acid starvation is one of the most important variables with respect to the induction of sporulation, and (b) the use of certain environmental chemical conditions that block sporulation at different steps as developmental tools for the study of this cellular differentiation process.     

Induction of sporulation by inhibitory purines and related compounds.

Sporulation of Bacillus subtilis can be induced, in the presence of excess ammonia, glucose and phosphate, by many purine derivatives under conditions of partial growth inhibition. Some of the compounds are known inhibitors of purine nucleotide synthesis. For most compounds the effect is counteracted by adenine and guanine. Partial growth inhibition by amethopterin (methotrexate) causes sporulation in the absence of purines but not in their presence. Unable to induce sporulation at any concentration are inhibitors of DNA, RNA, and protein synthesis as well as base or amino acid analogs that are incorporated into these polymers.     

Factors which affect the frequency of sporulation and tetrad formation in Saccharomyces cerevisiae baker's yeasts.

To clarify the role that respiration, the mitochondrial genome, and interactions of mitochondria and nucleus play on sporulation and to improve the sporogenic ability of several baker's yeasts, an investigation of the effects of different media and culture conditions on baker's yeast sporulation was undertaken. When standard protocols were followed, the sporulation frequency varied between 20 and 60% and the frequency of four-spore asci varied between 1 and 6%. Different presporulation and sporulation media, the use of solid versus liquid media, and incubation at 22 versus 30 degrees C were checked, and the cells were collected from presporulation media in either exponential or stationary phase. Best results, yielding sporulation and four-spore ascus formation frequencies up to 97 and 60%, respectively, were obtained by collection of the cells in exponential phase from liquid presporulation medium with 10% glucose and transfer of them to sporulation medium with 0.5% potassium acetate at 22 degrees C. Under these conditions, the most important factor was the growth phase (exponential versus stationary) at which cells from presporulation medium were collected. Changes in sporulation frequencies were also measured after transfer of mitochondria from different sources to baker's yeasts. When mitochondria from laboratory, baker's, and wine yeasts were transferred to baker's and laboratory petite strains, sporulation and four-spore ascus formation frequencies dropped dramatically either to no sporulation at all or to less than 50% in both parameters. This transfer also resulted in an increase in the frequency of petite mutant formation but yielded similar growth and respiration rates in glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of microcycle sporulation in Bacillus brevis spp. AG4 by amethopterin

Addition of amethopterin to medium before inoculation inhibited DNA synthesis and induced microcycle sporogenesis in Bacillus brevis spp. AG4. Synthesis of RNA and proteins occurred at a considerably reduced rate.Abbreviations TVC total viable counts - HSC heat stable counts - CDGS chemically defined medium for growth and sporulation - TCA trichloro acetic acid     

Induction of autophagy by second-fermentation yeasts during elaboration of sparkling wines

Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed.     

Induction by hypoxia of heterologous-protein production with the KlPDC1 promoter in yeasts

The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the KlPDC1 promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in Kluyveromyces lactis under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1beta was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The lacZ gene from Escherichia coli was cloned downstream of the KlPDC1 promoter in order to get beta-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in Saccharomyces cerevisiae and in Zygosaccharomyces bailii, and effects of hypoxic induction were measured and compared to those already observed in K. lactis cells. Interestingly, we found that the induction still worked in Z. bailii; thus, this promotor constitutes a possible inducible system for this new nonconventional host.     

Induction of petite mutation and inhibition of synthesis of respiratory enzymes in various yeasts

A systematic investigation covering a wide diversity of yeast species was made on the appearance of respiratory deficient (petite) mutants after treatment with acriflavine. Petite mutants were obtained from certain species only, but in these species all strains were found to have in common the property of giving rise to petite mutants; such species were designated as petite positive. Species failing to give rise to petite mutants were accordingly called petite negative. The primary action of acriflavine, namely the inhibition of the synthesis of the respiratory system, was shown to occur not only in petite positive yeasts, but also in petite negative ones. Some implications of the results are discussed.     

Heat resistance studies of yeasts; vegetative cells versus ascospores: erythromycin inhibition of sporulation in Kluyveromyces and Saccharomyces species

A comparative study of the heat resistance ( D ₆₀ values) of four Saccharomyces spp. and two Kluyveromyces spp. (21 strains) showed a 30–350-fold higher heat resistance of ascospores than of vegetative cells. It was also observed that small numbers of ascospores exhibiting a considerably higher heat resistance can easily be formed, even in a complete vegetative growth medium. This phenomenon may have led most other authors to report none or only slight differences between the heat resistance of yeast ascospores and their vegetative cells. Until more information has been collected about the ascospore load of acid (fruit) products and their heat resistance, accurate calculations of the minimum F values for heat preservation of these products may not be possible.     

Induction of citric acid cycle enzymes during initiation of sporulation by guanine nucleotide deprivation

In Bacillus subtilis, conditions causing partial deprivation of guanine nucleotides initiated sporulation and caused the synthesis of citrate synthase, aconitase, and alpha-ketoglutarate dehydrogenase. Alpha-ketoglutarate dehydrogenase could also be induced by acetate, and the specific activity of this enzyme was elevated in mutants that had high intracellular acetyl coenzyme A concentrations because they lacked citrate synthase activity. After deprivation of guanine nucleotides, the intracellular concentration of acetyl coenzyme A also increased, which explained the induction of alpha-ketoglutarate dehydrogenase. Furthermore, the decreases in alpha-ketoglutarate and L-malate concentrations observed during this deprivation accounted for the observed increases in citrate synthase activity (which was repressed by alpha-ketoglutarate and malate) and aconitase activity (which was repressed by alpha-ketoglutarate).     

Induction of Bacillus subtilis sporulation by decoyinine and the concomitant disappearance of ppGpp in vegetative cells

Sporulation of Bacillus subtilis, growing exponentially in the presence of rapidly metabolizable nutrients, was induced by addition of decoyinine (an antibiotic inhibitor of GMP synthesis), and intracellular amounts of ppGpp were determined after 2 M formic acid extraction by polyethyleneimine (PEI)-cellulose thin-layer chromatography. Consequently, it was found that the ppGpp in vegetative cells abruptly disappeared after the addition of decoyinine. This indicates that the disappearance of ppGpp is closely correlated to the initiation of B. subtilis sporulation.     

Induction of Bacillus subtilis sporulation by nucleosides: inosine appears to be sporogen.

Inosine completely reversed the selective inhibition of sporulation in Bacillus subtilis 168 caused bym-aminobenzeneboronic acid; guanosine and adenosine, but not xanthosine, partially reversed inhibition, whereas pyrimidine nucleosides were slightly effective. In addition, 0.005 to 0.025 mM inosine caused a four- to fivefold stimulation of sporulation of B. subtilis grown in minimal salts medium. Ultraviolet and infrared spectra and other physical and chemical properties of inosine were markedly similar to those of "sporogen," a previously described endogenous sporogenesis factor present in sporulating Bacillus species.     

Induction of sporulation of fungi isolated from Dactylis glomerata seed by exposure to near-ultraviolet radiation*

Pure cultures of fifty-two species of plant pathogenic and saprophytic fungi isolated from orchard (cock's-foot) grass seed (Dactylis glomerata L.) were incubated either in total darkness or exposed to a diurnal cycle of near-ultraviolet (NUV) radiation (12 h NUV/12 h darkness). Twenty-four species sporulated only after exposure to NUV including seven species of Drechslera, five species of Fusarium, as well as species olAscochyta, Photna, Septoria, Pyrenochaeta, Rhynchophoma and Stagonospora; six species sporulated moderately in darkness but more profusely following exposure to NUV; twenty species sporulated whether they were irradiated or not; and only two species failed to sporulate. To assess the pathogenic fungal microflora of orchard grass seed accurately, seeds should be incubated under a daily regime that includes NUV to induce sporulation.