Synchronous Growth and Sporulation of Bacillus megaterium

Filtration of late log-phase cultures of Bacillus megaterium ATCC 19213 grown on defined sucrose salts medium (SS) or SS plus glutamate medium (SSG) through nine layers of Whatman no. 40 filter paper in a fritted-glass disc Büchner funnel resulted in filtrates containing cells which showed synchronous growth and proceeded to sporulation. SS cells completed one synchronous division after filtration; sporulation ensued after the cessation of growth. SSG cells completed two synchronous divisions and sporulation occurred during the second division. A high degree of synchrony of vegetative growth of SSG cells was evident by the stepwise pattern of growth, by the doubling of cell numbers at each division, the high division index, and by the rapid formation of sporulation cell types and homogeneity of cell types in the filtered cultures when compared with asynchronous cultures. Because the described system gives both good growth and sporulation synchrony, the method should be useful in delineating early events in sporulation and their regulation.     

Nature and timing of some sporulation-specific protein changes in Saccharomyces cerevisiae.

During meiosis and spore formulation in Saccharomyces cerevisiae, changes that occur in a/alpha diploids, but not in isogenic nonsporulating a/a diploids, have been detected in cellular polypeptides. These were found by the technique of prelabeling growing cells with 35SO4(2-) and suspending them in sulfur-free sporulation medium. Under the conditions used, about 400 polypeptides were detected by two-dimensional gel electrophoresis, and 45 were altered during sporulation; of these, 21 changes were specific to a/alpha strains. These alterations were mainly due to the appearance of new polypeptides or to marked increases in the concentrations of a few polypeptides produced during vegetative growth. They could have been due either to modifications of existing polypeptides present in growing cells or to de novo synthesis of new gene products. They occurred at characteristic times during sporulation; whereas the majority of changes took place early (within the first 6 h in sporulation conditions), there were several changes characterizing the later stages of sporulation. Ten of the 35SO4(2-)-labeled polypeptides were also labeled with 32P in the presence of [32P]orthophosphate; of these, three were previously found to be sporulation specific. One of these was phosphorylated at all stages of sporulation and was labeled when [32P]orthophosphate was added either during growth of the culture of 1 h after transfer to sporulation medium. Another was labeled in the same way by adding 32P at either time, so that by 7 h in sporulation medium it was phosphorylated, but was dephosphorylated by 24 h. The third sporulation-specific peptide was labeled in extracts prepared at 7 h in sporulation medium (but not at 24 h) when [32P]-orthophosphate was added during presporulation growth, but not when [32P]-orthophosphate was added 1 h after transfer of the culture to sporulation medium. This polypeptide appeared early during sporulation; it is probably phosphorylated as it appears and is dephosphorylated at some time between 7 h and 24 h of sporulation.     

Requirement for peptidoglycan synthesis during sporulation of Bacillus subtilis.

Cultures of Bacillus subtilis were treated during sporulation with antibiotics (bacitracin and vancomycin) that affect peptidoglycan synthesis. The cells were resistant to the effects of the antibiotics only when the drugs were added about 2 h after the beginning of sporulation. This was about 1 h later than the escape time of a temperature-sensitive sporulation mutant that is unable to complete prespore septation. Similar experiments were done with a mutant temperature sensitive for peptidoglycan synthesis. This showed an escape curve similar to that shown by the antibiotics. When sporulating cells were treated with antibiotics, they produced alkaline phosphatase earlier than normal. Enzyme production was unaffected by inhibition of deoxyribonucleic acid synthesis but was inhibited by chloramphenicol. Sporulation mutants that are unable to make alkaline phosphatase under normal conditions were able to make it in the presence of bacitracin. The alkaline phosphatase made under these conditions was under "sporulation-type" control since its synthesis was repressible by casein hydrolysate and unaffected by inorganic phosphate. When cells were treated with bacitracin in the growth medium as well as in the sporulation medium, alkaline phosphatase synthesis was at the same level as in an untreated control. A number of other antibiotics and surfactants were tested for the ability to cause premature production of the phosphatase of those tested, only taurodeoxycholate whowed this behavior. Moreover, incubation of cells with taurodeoxycholate in the growth medium as well as in the sporulation medium prevented premature enzyme production.     

Sporulation in Bacillus subtilis. Effect of medium on the form of chromosome replication and on initiation to sporulation in Bacillus subtilis

Thymine-requiring mutants of Bacillus subtilis and mutants that are temperature-sensitive for initiation of chromosome replication have been used to study the relationship between sporulation and chromosome formation. The DNA synthesis that normally occurs when cells are transferred to sporulation medium is essential for spore induction. This is shown by the fact that thymine-starved cells are unable to form spores and are unable to perform even the earlier steps of sporulation, such as septum formation or synthesis of alkaline phosphatase. The nature of the medium in which the cells are growing while the DNA is being completed is also important because it determines both the shape and the position of the daughter chromosomes. If the cells are in a rich medium, the newly synthesized chromosomes are discrete and compact bodies: the cells are primed for growth, and sporulation cannot be induced by transferring them at this stage to a spore-inducing medium. If DNA synthesis was completed with the cells in a poor medium the daughter chromosomes, by the time DNA synthesis has ceased, are spread in a single filamentous band and the cells are morphologically already in stage I of sporulation.     

Mutants ofBacillus megaterium with altered synthesis of an exocellular neutral proteinase

Germinated spores ofBacillus megaterium were mutagenized with ethyl methanesulphonate and spread on test agar with caseinate. Colonies with altered proteolytic zones or morphology were isolated and tested in liquid media. The mutants can be divided into four groups: A) those producing more proteinase in both growth and sporulation media, B) those producing the same amount of the enzyme in growth medium but higher amount in sporulation medium, C) those producing less proteinase in the growth medium and more in the sporulation one, D) those producing less or no enzyme. Clones of the first three groups were phenotypically asporogenic. All mutants producing more enzyme during growth retained their sensitivity to repression by amino acids. Isolation of mutants of types B) and C) supports the idea of differences in the control of proteinase synthesis during growth and during sporulation.     

Observations on the separation of Theileria sporozoites from ticks

Hyalomma anatolicum anatolicum ticks infected with Theileria annulata were partially fed on rabbits and then ground up with tissue culture medium. The ground up ticks were treated by centrifugation at 100 g, filtration through membranes of 8 μm pore diameter and centrifugation on a discontinuous density gradient of Percoll. Counts of sporozoites and tick debris were made from Giemsa stained slides of samples at each stage of the separation. Debris was removed during light centrifugation and filtration at a greater rate than sporozoites. After filtration approximately 41% of the original sporozoites remained in the suspension. After density gradient centrifugation most sporozoites were found in a distinct zone, at approx. 1·08 g/cm³ density, separate from most dense debris and light debris and soluble contaminants. After this final centrifugation approximately 24% of the original sporozoites remained in the recovered suspension.     

Sporulation of Saccharomyces cerevisiae in the Absence of a Functional Mitochondrial Genome

The role of the mitochondrial system during sporulation of Saccharomyces cerevisiae was studied. Addition of ethidium bromide (EthBr) to cells growing in acetate medium resulted in the quantitative (>98%) conversion of the culture to the petite genotype in one generation. The cells were respiratory active (derepressed) but contained no mitochondrial deoxyribonucleic acid (mtDNA) as demonstrated by analytical ultracentrifugation in CsCl. When transferred to acetate sporulation medium, the culture sporulated. Ascus production was only slightly below that of the control culture. Synthesis of mtDNA occurred during sporulation in the control but not in the EthBr-treated culture. Mitochondrial protein synthesis was virtually eliminated in the EthBr-treated culture. Therefore, completely derepressed cells can sporulate without a functional mitochondrial genetic system. When partially repressed cells were treated with EthBr, no ascus formation was observed after transfer to sporulation medium. Control cultures underwent respiratory adaptation in sporulation medium and then sporulated. Extensive derepression of the respiratory system is thus required for sporulation, and this adaptation is dependent on a functional mitochondrial system. Our results suggest that once the cells are fully derepressed no mitochondrial genetic information has to be expressed during meiosis and ascus formation.     

Chromosome age and segregation during sporulation of Bacillus megaterium.

The effect of chromosome age on segregation during sporulation was investigated. Vegetative cells of Bacillus megaterium were labeled with [Me-3H]thymine and then were grown at 30 degrees C in nonradioactive medium for various times before being allowed to sporulate. The ratio of the amount of label in sporal DNA to that in sporangial DNA, obtained after minor correction for the sporulation frequency, remained essentially constant as the postlabeling growth period was increased from one to seven generations. The spores were preferentially located at the older poles of sporangia, i.e. the poles formed by divisions occurring prior to those forming the sporangia. Therefore, it seems that old (labeled) chromosomes segregate randomly with respect to both the morphological and genealogical polarities of sporangia. Examination of total cell lysates by dye-buoyant density gradient centrifugation revealed the presence of covalently closed circular DNA from cells grown at 37 degrees C, but none was obtained from cells grown at 30 degrees C. Thus, possible interference by large amounts of extrachromosomal DNA in the determination of the chromosomal segregation pattern is unlikely.     

Thiomethylation of tyrosine transfer ribonucleic acid is associated with initiation of sporulation in Bacillus subtilis: effect of phosphate concentration.

The thiomethylation of Bacillus subtilis tyrosine transfer ribonucleic acid (tRNATyr) (i6A) has been shown to occur during the slowing-down of growth. The extent of this modification in stationary-phase cells grown in defined medium has been determined in parallel with the sporulation frequency. We observed that the presence of phosphate repressed sporulation and also inhibited the thiomethylation of tRNATyr (i6A) of B. subtilis W168. These effects were partially eliminated by decreasing the glucose concentration until it was growth limiting. In the case of strain W23S, in which sporulation is insensitive to glucose repression, sporulation and tRNATyr thiomethylation were not inhibited by nonlimiting concentrations of phosphate. These results suggest that both sporulation and tRNATyr hyper-modification share some common regulatory process.     

The Effect of Ochre Suppression on Meiosis and Ascospore Formation in Saccharomyces

The effect of altered tyrosyl-tRNAs on the developmental process of sporulation was examined. Mutations in eight independent loci resulting in tyrosine-inserting nonsense suppressor were tested for their effects on sporulation. Different levels of inhibition were found ranging from SUP3-omicron, which caused the greatest reduction of sporulation (7-17% of wild type), to SUP11-omicron which caused no reduction in sporulation. Since the SUP3-omicron mutation exhibited the greatest effect, it was studied in detail. Although SUP3-omicron is a dominant nonsense suppressor, its effect on sporulation is recessive. Expression of the sporulation deficiency is dependent upon the stage of transfer from glucose growth medium (i.e., log, early stationary, etc.) to sporulation medium. SUP3-omicron/SUP3-omicron diploid cells transferred from log or early stationary phase are capable of sporulation, whereas cells transferred after early stationary phase (i.e., after adaptation to respiration) exhibit poor sporulative ability. Sporulation events were examined under restrictive conditions to observe those events completed by SUP3-omicron/SUP3-omicron diploids. The early events of sporulation occur in these cells. Later events are completed by progressively fewer cells. Premeiotic DNA synthesis occurred in approximately 40% of the cells, nuclear segregation occurred in 20%, and finally, only 2% formed asci. The fact that fewer late-sporulation events occur under restrictive conditions can be explained by increased efficiency of suppression.     

Membrane protein alterations during the early stages of sporulation of bacillus subtilis

Membrane protein alterations during the early stages of sporuloation were examined by polyacrylamide gel electrophoresis. Solubilized samples of the vegetative cell membrane (VCM), sporulation membrane fraction (SMF), and inner forespore membranes (IFM) were compared with respect to their protein compositions. The VCM contained 39 protein components, distinguishable as separate bands on gel electrophoresis, and these ranged in molecular weight from 16,000 to greater than 100,000. During the first 5 hr of sporulation, 6 of these 39 protein bands disappeared, 8 increased and 12 decreased in concentration, and 13 showed no discernible change. In addition, 15 new protein components were identified in the SMF during the fireist 5 hr. The new components consisted of 7 protein bands that were transiently associated with the SMF, and 8 proteins that persisted in the SMF from their time of appearance until at least T5 of sporulation. Comparison of the protein composition of the IFM with those of the VCM and SMF revealed that membrane protein alterations occur during sporulation. The turnover of H³-tryptophan-labeleld membrane protein was followed during growth and sporulation. During the 30 min of growth following a simple chase with excess unlabeled tryptophan, membrane protein appeared stable, whereas 5–10% of the nonmembrane protein turned over to acid-soluble material. However, manipulation of the cells by dilution ito fresh medium, or centrifugation, as part of the chase procedure, resulted in elution of membrane protein to the cytoplasm. In contrast, proteins labeled during vegetative growth were always eluted to the cytoplasm during the first 2 hr of sporulation, and this was followed by a period of reassociation with the membrane fraction. The results are discussed with respect to membrane differentiation as it relates to spore development.     

Protein degradation and protease activity during the late cycle of Blastocladiella emersonii

Analysis of protein degradation during the life cycle of Blastocladiella emersonii showed that (i) protein degradation is especially high during two phases of differentiation (sporulation, 12%/h and germination, 5%/h) in contrast with a much smaller degradation rate in the other phases (growth and zoospores, less than 1%/hr); (ii) protein degradation during germination in growth medium, as well as most of the germination process, is quantitatively unaffected by cycloheximide; (iii) a caseinolytic protease (pH optimum 5.5, apparent molecular weight 55,000 to 60,000) is present in extracts of zoospores and germinating cells; (iv) this protease activity is very low (perhaps absent) in extracts of late growth phase cells, but reappears during induced sporulation; (v) a different class of caseinolytic protease activity (pH optima 7 and 10; apparent molecular weight 25,000 to 30,000) is found in cellular extracts of late growth phase and early phases of sporulation; (vi) the latter class of enzyme activity is released into the medium during later phases of sporulation and is replaced in the cells by the former class. Speculations as to the roles of protein degradation in cell differentiation are discussed.     

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.     

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)     

Purification of mitochondria and secretory granules isolated from pancreatic beta cells using Percoll and Sephacryl S-1000 superfine

Mitochondria and secretory granules were isolated from beta-cell-rich pancreatic islets of ob/ob mice. Crude fractions obtained by differential centrifugation were subjected to centrifugation on isotonic Percoll. The gradient medium was removed from the resulting fractions by gel filtration on Sephacryl S-1000 Superfine. When compared to the crude fractions, the purified mitochondrial fraction exhibited a 4.5-fold increase in citrate synthase activity, a 70% reduction of lysosomal arylsulfatase, and a 40% decrease of contamination with granular insulin. In the purified secretory granule fraction, the insulin content was as high as 60% of the total protein (albumin standard) with arylsulfatase unchanged and no detectable citrate synthase activity.     

Requirement for Acetate and Glycine (or Serine) for Sporulation Without Growth of Bacillus subtilis

Cells of Bacillus subtilis sporulate when they are transferred, at any time of growth in nutrient sporulation medium, to a potassium-phosphate buffer containing slowly utilizable carbon sources such as l-aspartate, citrate, l-glutamate, or lactate. Transfer to buffer containing more rapidly utilizable carbon sources such as malate or glucose leads to sporulation only when the cells either had reached the end of growth or when the transfer medium also contains glycine. Acetate, which as a sole carbon source does not allow growth, also does not alone permit sporulation; however, the presence of both acetate (0.05 m) and glycine or l-serine (0.01 m) in the buffer medium allows sporulation if the cells are transferred to this medium after they have grown in the nutrient sporulation medium beyond the end of the exponential growth phase (T(0)). The development, required before transfer, does not seem to involve the end of a round of deoxyribonucleic acid duplication, as experiments with tryptophan-starved cells have indicated. Glycine or serine cannot be replaced by any of the known metabolites, which are partially derived from them. Amino acid analysis of nutrient sporulation medium showed that glycine (but not serine) is present at a concentration of 0.3 mm at the beginning of the developmental period, thus allowing, in combination with an acetyl-coenzyme A (CoA) precursor, sporulation but not growth. Acetyl-CoA is required not only for adenosine-triphosphate synthesis but also for some other reactions.     

Partial purine deprivation causes sporulation of Bacillus subtilis in the presence of excess ammonia, glucose and phosphate.

In strains of Bacillus subtilis able to synthesize purines de novo, massive sporulation is suppressed by the combination of excess ammonia, glucose and phosphate. Purine auxotrophs, blocked in the general or the guanine-specific portion of the branched purine pathway, sporulated in such a medium when the purine required for normal growth was removed from the medium. The resulting spore titre and the sporulation frequency increased with the residual growth rate in the purine-free medium, i.e. with the leakiness of the purine mutation. Sporulation was further increased by allowing residual growth in growth-limiting amounts of guanosine. None-leaky purine mutants blocked before 5'-phosphoribosyl-5-amino-4-imidazole carboxamide also sporulated well when supplied with 5-amino-4-imidazole carboxamide at concentrations (2 mM) that supported growth at a suboptimal rate.     

Inhibiton by sulfanilamide of sporulation in Saccharomyces cerevisiae.

The antimetabolite sulfanilamide inhibits sporulation in Saccharomyces cerevisiae strain AP1. Cells exposed to sulfanilamide at various times during the sporulation process become progressively insensitive to the drug, although accumulation of sulfanilamide by the cells increases with time. Vegetative growth of AP1 is practically unaffected by sulfanilamide; pregrowth of the cells in the presence of the drug does not prevent sporulation. Thus, inhibition is confined to the meiotic phase of the cell cycle. Sensitivity to sulfanilamide is independent of pH. Increasing the time cells are exposed to sulfanilamide results in a progressive reduction of ascus formation; however, the inhibition is reversible since sporulation can occur in cells exposed to the drug for greater than 24 h. The drug arrests the cells at a point before commitment to sporulation, since yeast cells exposed to sulfanilamide for 12 h do not complete the sporulation process when returnedto vegetative medium, but resume mitotic growth instead. Meiotic nuclear division is largely prevented by sulfanilamide, and synthesis of RNA and protein is severely retarded. DNA synthesis is inhibited up to 50%; glycogen synthesis is approximately 90% inhibited. Other yeast strains showed varying sensitivity to sulfanilamide; homothallic strains were generally less affected.