Autocides produced by Myxococcus xanthus.

Ethanol extracts of Myxococcus xanthus contained several substances, referred to as autocides, which were bactericidal to the producing strain but showed no activity against other bacteria. The autocides were produced by growing cells and remained largely cell bound throughout the growth cycle; ca. 5% of the autocidal activity was found in the supernatant fluid at the time cell lysis began. The autocides were separated by sequential-column and thin-layer chromatography into five active fractions (AM I through AM V). Each of the fractions was at least 20 times more active against M. xanthus than against the other gram-negative or gram-positive bacteria tested. AM I, AM IV, and AM V were inactive against yeasts, whereas a mixture of fractions AM II and AM III was active against Rhodotorula sp. At low concentrations, AM I reversibly inhibited the growth of M. xanthus; at higher concentrations of AM I, the cells lysed within 1 h. The lowest concentration of AM IV that showed any activity caused rapid cell death and lysis. The mode of action of the major autocide, AM V, was different from that of AM I and AM IV. During the initial 2 h of treatment, the viable count of M. xanthus cells remained constant; during the next few hours killing occurred without lysis; within 24 h lysis was complete. The autocidal activity of each of the fractions was expressed when the cells were suspended in buffer, as well as in growth medium. The possible role of autocides in developmental lysis of M. xanthus is discussed.     

Developmentally induced autolysis during fruiting body formation by Myxococcus xanthus.

The developmental events during fruiting body construction by the myxobacterium M. xanthus is an orderly process characterized by several sequential stages: growth leads to aggregation leads to formation of raised, darkened mounds of cells leads to autolysis leads to myxospore induction. The temporal sequence of autolysis followed by myxospore induction is consistent with the interpretation that developmental autolysis provides essential requirements for the surviving cells to induce to myxospores. At intermediate developmental times on agar plates a fraction of the cell population is irreversibly committed to lyse; i.e., lysis continues in liquid growth medium or in magnesium-phosphate buffer. Lysis is cell concentration independent and is therefore likely to be by an autolytic mechanism. The lysis sequence can be preliminarily characterized as having an early stage during which deoxyribonucleic acid synthesis continues and a later irreversible stage during which deoxyribonucleic acid synthesis does not occur. Irreversible lysis in liquid growth medium or in magnesium-phosphate buffer is initiated on agar plates during nutrient deprivation and such lysis results in the induction of a fraction of the population to myxospores. This induction is dependent upon the concentration of lysis products, thus providing evidence that developmentally induced autolysis is required for myxospore induction.     

Cell-cell interactions in developmental lysis of Myxococcus xanthus

The developmental events of sporulation and fruiting body formation in the prokaryote Myxococcus xanthus are preceded by a stage of massive cell death. Two phenotypically complementable strains of M. xanthus defective in developmental lysis were identified from a group of conditional sporulation mutants. Mixture of the two lysis groups resulted in full complementation of lysis, sporulation, and fruiting body formation; efficient sporulation was observed only in strain mixtures where lysis was complemented. We have identified a cell-free extract from developing cells that phenotypically complemented lysis, sporulation, and fruiting body formation in one group of mutants; the active component of this extract appeared to be tightly cell associated. The effect of the cell-free extract could be replaced by exogenously supplied glucosamine or mannosamine.     

Sporulation timing in Myxococcus xanthus is controlled by the espAB locus

The fruiting body development of Myxococcus xanthus consists of two separate but interacting pathways: one for aggregation of many cells to form raised mounds and the other for sporulation of individual cells into myxospores. Sporulation of individual cells normally occurs after mound formation, and is delayed at least 30 h after starvation under our laboratory conditions. This suggests that M. xanthus has a mechanism that monitors progress towards aggregation prior to triggering sporulation. A null mutation in a newly identified gene, espA (early sporulation), causes sporulation to occur much earlier compared with the wild type (16 h earlier). In contrast, a null mutation in an adjacent gene, espB, delays sporulation by about 16 h compared with the wild type. Interestingly, it appears that the espA mutant does not require raised mounds for sporulation. Many mutant cells sporulate outside the fruiting bodies. In addition, the mutant can sporulate, without aggregation into raised mounds, under some conditions in which cells normally do not form fruiting bodies. Based on these observations, it is hypothesized that EspA functions as an inhibitor of sporulation during early fruiting body development while cells are aggregating into raised mounds. The aggregation-independent sporulation of the espA mutant still requires starvation and high cell density. The espA and espB genes are expressed as an operon and their translations appear to be coupled. Expression occurs only under developmental conditions and does not occur during vegetative growth or during glycerol-induced sporulation. Sequence analysis of EspA indicates that it is a histidine protein kinase with a fork head-associated (FHA) domain at the N-terminus and a receiver domain at the C-terminus. This suggests that EspA is part of a two-component signal transduction system that regulates the timing of sporulation initiation.     

Sporulation of Bacillus subtilis in Continuous Culture

Sporulation of Bacillus subtilis 168 was studied in chemostat cultures. Sporulation occurred at high frequency under limitation of growth by glucose or the nitrogen source in minimal medium, whereas rates of sporulation were low for Mg(2+), phosphate, citrate, or tryptophan limitation. Sporulation was found at all growth rates tested, and the incidence of spores increased with decrease in growth rate of the culture. Within the range of growth rates up to the maximum obtainable with the defined medium, no threshold effect of growth rate on sporulation was observed. By studying transient states, it was possible to determine the time taken for the appearance of a refractile spore after initiation of a cell to sporulation. Under conditions of glucose limitation, cells were found to be committed to sporulation as soon as they were initiated. In nitrogen-limited cultures, however, a partial relief of nitrogen limitation prevented the development of spores during the first hour after initiation. The results of experiments with multistep changes in dilution rate of a chemostat culture indicate that initiation to sporulation is probably restricted to a particular point in the cell division cycle.     

Effects of glucosamine on lysis, glycerol formation, and sporulation in Myxococcus xanthus.

Glucosamine (GlcN), which has previously been shown to rescue fruiting body formation, lysis, and sporulation in a developmental mutant (G. Janssen and M. Dworkin, Dev. Biol. 112:194-202, 1985), induced lysis in vegetative and developing wild-type cells and inhibited fruiting body formation. It also resulted in a transient, intracellular increase in the concentration of glycerol, a known sporulation inducer, and sporulation of the surviving cells. Phospholipase activity, which was shown to be normally developmentally regulated, increased 7.6-fold after treatment of vegetative cells with 50 mM GlcN. Likewise, autocidal activity, which normally increased 18 to 24 h after the initiation of development, increased 20% when vegetative or developing cells were exposed to GlcN. Two mutants resistant to GlcN-induced lysis (MD1021 and MD1022) were isolated and showed neither an increase in autocide production nor an increase in phospholipase activity in response to added GlcN. MD1021 was developmentally deficient, and GlcN rescued fruiting body formation as well as phospholipase activity and autocide production. We propose that GlcN exerts its lytic effect by regulating the activity of phospholipase enzymes that release autocides, compounds that are believed to be responsible for developmental autolysis. GlcN-induced sporulation was found to depend on several factors: the initial cell density, the amount of lysis induced by GlcN, and the presence of tan-phase variants. An initial cell density of greater than 2 x 10(5) cells per ml was required to support GlcN-induced sporulation, and sporulation did not occur unless 50 to 75% of these cells had lysed. Mutants that were resistant to GlcN-induced lysis also did not sporulate in the presence of GlcN. The effects of GlcN on developing cells depended on the concentration of GlcN added; the addition of low concentrations of GlcN resulted in enhancement of sporulation, while higher concentrations resulted in the inhibition of sporulation. The ultrastructure of GlcN-induced spores resembled that of spores induced by the exogenous addition of glycerol, in contrast to spores isolated from mature fruiting bodies. A model by which GlcN may regulate both lysis and sporulation is presented.     

Cell-density-dependent killing of Myxococcus xanthus by autocide AMV.

Autocide AMV of Myxococcus xanthus was purified and identified as phosphatidylethanolamine. Alkaline hydrolysis of AMV yielded a high proportion of mono- and diunsaturated fatty acids. The bactericidal activity of AMV on M. xanthus depended upon the density of target cells: the greater the cell density, the greater the killing by AMV. For example, at 2 U of AMV per ml, 0, 50, and 99% killing was measured with 2 X 10(4), 2 X 10(5), and 2 X 10(7) target cells per ml, respectively. The cell-density-dependent activity of AMV was also observed on solid medium. Studies with model lipid compounds suggest that the inhibitory activity of AMV is due to the fatty acid moiety, released from phosphatidylethanolamine by the concerted (enzymatic) activity of many cells. Mutants of M. xanthus selected for resistance to AMI (a mixture of fatty acids) were also resistant to AMV. The possible role of AMV in developmental lysis is discussed.     

Characterization of a new sporulation factor in Bacillus subtilis.

We report the existence and partial purification of sporulation factor, which stimulates sporulation of Bacillus subtilis at low cell density. Proline or arginine is required for stimulation under the conditions of our assay. Sporulation factor is a small heat-stable substance produced by the cells during exponential growth phase. It is required in small amounts and is resistant to various proteolytic agents. Several spo mutants were tested for the ability to produce functional sporulation factor. All of these mutants produce factor and do not sporulate in the presence of factor from wild-type cells. Sporulation factor is not involved in the induction of alpha-amylase synthesis at the initiation of sporulation.     

Control of sporulation in the filamentous cyanobacteriumAnabaena torulosa

In the cyanobacteriumAnabaena torulosa, sporulation occurred even during the logarithmic growth phase. Sporulation was initiated by differentiation of the vegetative cell on one side, adjoining the heterocyst followed by differentiation of the vegetative cell on the other side. Subsequently, spores were differentiated alternately on either side to form spore strings. The sequence of sporulation supports the previous notion that a gradient of spore maturation exists in cyanobacteria and also indicates that the gradient is manifested unequally on either side of heterocysts. Sporulation was absent or negligible in a minerally enriched medium but ocurred readily in a minimal medium. The extent of sporulation was inversely related to phosphate concentration. Sporulation was enhanced at higher temperature. Incandescent light, but not fluorescent light, greatly stimulated sporulation suggesting possible involvement of red light in spore differentiation. Addition of filtrate, from 5 to 8 day old cultures, to freshly inoculatedA. torulosa greatly enhanced sporulation indicating the influence of extracellular products in spore formation.     

The rate of development of a microsporidan in moth cell culture.

A microsporidan parasite of the forest tent caterpillar Malacosoma disstria infected cells and replicated in vitro in a line from the moth Heliothis zea. After spore germination, the incidence of infected cells increased with time until leveling off with sporulation. During the first 24 hr, there was a static number of parasites, followed by a 2-day logarithmic growth phase during which the population doubled five to six times. The growth rate was 9 to 11 hr per population doubling. Sporulation commenced on day 3, and 40 to 50 spores were recovered from each infected cell. The life cycle was completed within 6 days, culminating in spores that were infectious for cultured cells. The antibiotic fumagillin at a dose of 1 ppm in the culture medium was microsporida-static.     

Cell density-dependent growth of Myxococcus xanthus on casein.

When Myxococcus xanthus FB was grown on 0.2% casein it exhibited a phenomenon we call cooperative growth. That is, above 104 cells per ml, both strains that were studied exhibited increasing growth rates as a function of increasing cell numbers. Between 104 and 107 cells per ml, the mean doubling times of strains YS and TNS decreased from 15.2 to 8 h and 26 to 8.5 h, respectively. The extracellular proteinase activity of the two strains was equivalent and directly proportional to cell number. Cooperative growth was correlated with increased concentration of hydrolyzed casein in the medium, suggesting cooperative hydrolysis of casein. At low cell densities neither strain was capable of measurable growth on casein in liquid media, and we have calculated that the average concentration of hydrolyzed casein in the medium was indeed too low to support growth. At low cell densities, growth on hydrolyzed casein (Casitone) was normal and independent of cell concentration. Demonstration of cooperative growth at higher cell densities supports the suggestion that the communal behavior of myxobacteria results in more efficient feeding.     

Sporulation Synchrony of Saccharomyces cerevisiae Grown in Various Carbon Sources

Sporulation of several strains of Saccharomyces cerevisiae grown in a variety of carbon sources that do not repress the tricarboxylic acid cycle enzymes was more synchronous than the sporulation of cells grown in medium containing dextrose which does repress those enzymes. Dextrose-grown cells showed optimal sporulation synchrony when inoculated into sporulation medium from early stationary phase when the dextrose in the medium is exhausted. Logarithmic-phase cells grown in either non-fermentable carbon sources (acetate and glycerol) or a fermentable carbon source that does not repress tricarboxylic acid cycle enzymes (galactose) sporulated more synchronously than the early stationary-phase dextrose cells. Attempts were made to sporulate cells taken from both complex and semidefined media. The semidefined acetate medium failed to support the growth of a number of strains. However, cells grown in the complex acetate medium, as well as both complex and semidefined glycerol and galactose media, sporulated with better synchrony than did the dextrose-grown cells.     

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 during Growth in a Gut Isolate of Bacillus subtilis

Sporulation by Bacillus subtilis is a cell density-dependent response to nutrient deprivation. Central to the decision of entering sporulation is a phosphorelay, through which sensor kinases promote phosphorylation of Spo0A. The phosphorelay integrates both positive and negative signals, ensuring that sporulation, a time- and energy-consuming process that may bring an ecological cost, is only triggered should other adaptations fail. Here we report that a gastrointestinal isolate of B. subtilis sporulates with high efficiency during growth, bypassing the cell density, nutritional, and other signals that normally make sporulation a post-exponential-phase response. Sporulation during growth occurs because Spo0A is more active per cell and in a higher fraction of the population than in a laboratory strain. This in turn, is primarily caused by the absence from the gut strain of the genes rapE and rapK, coding for two aspartyl phosphatases that negatively modulate the flow of phosphoryl groups to Spo0A. We show, in line with recent results, that activation of Spo0A through the phosphorelay is the limiting step for sporulation initiation in the gut strain. Our results further suggest that the phosphorelay is tuned to favor sporulation during growth in gastrointestinal B. subtilis isolates, presumably as a form of survival and/or propagation in the gut environment.     

Characteristics of pulse-labeled ribonucleic acid in relation to medium pH and acetate concentration during meiosis of yeast

Summary Cellular impermeability associated with sporulating cells of Saccharomyces cerevisiae is caused by a rapid increase in the medium pH. Three factors have been identified as being important in regulating the rise in medium pH: 1) the cell density, 2) the potassium acetate concentration of the sporulation medium, and 3) and initial pH below 6.0. Sporulation conditions were established for strain 4579 which resulted in optimum uptake of ³H-adenine at T₇, a period when the cells would be normally impermeable. Pulse-labeled polysomal RNA was characterized at T₄ in naturally permeable cells of strain SK-1 and impermeable cells which required manipulation of the medium pH to facilitate uptake. Transfer ribonucleic acid (RNA), poly A-containing RNA and ribosomal RNA were synthesized in both cultures during the 20 min pulse. Furthermore, the rate of ribosomal RNA synthesis and processing into functional ribosomes approached the rate reported for vegetative cells. Initial sporulation conditions which caused a prolonged delay in the rise in medium pH adversely affected the kinetics of appearance and number of ascospores. The affect was shown to be on meiotic events since a reduction of sporulation was always accompanied by a reduction in the amount of intragenic recombination.     

Adenosine 5′-Triphosphate Release and Membrane Collapse in Glycerol-Requiring Mutants of Bacillus subtilis

Glycerol-requiring mutants of Bacillus subtilis could not sporulate in nutrient sporulation medium even when additional glycerol was added from the beginning of growth. Sporulation could be partially restored either by the frequent addition of small amounts of glycerol during the developmental period or by the single addition of both 10 mM glycerol and 10 mM malate. But sporulation could be completely restored by the addition of 50 mM glycerol-phosphate from the beginning. At the end of growth of the glycerol mutants in nutrient sporulation medium, the cell membrane collapsed and separated from the cell wall, and much of the cellular adenosine 5'-triphosphate was released into the medium. These observations were made in two glycerol mutants, one derived from strain 168 containing glycerol-teichoic acid in the cell wall and the other derived from strain W23 containing ribitol-teichoic acid.     

Stimulation of Yeast Sporulation by Glycerol

S ummary : Glycerol stimulated sporulation of Saccharomyces cerevisiae Hanson, especially when the cells were precultured in a complex growth medium instead of a chemically defined medium. Optimum spore yields occurred with 1–4% of glycerol but some were produced in 16% glycerol. Sporulation in glycerol was much less sensitive to ammonium sulphate inhibition than it was in acetate. Growth occurred with glycerol as sole carbon source and glutamic acid as sole nitrogen source, but not with ammonium sulphate as the sole nitrogen source.     

Murein components rescue developmental sporulation of Myxococcus xanthus

Murein (peptidoglycan) components are able to rescue sporulation in certain sporulation-defective mutants of Myxococcus xanthus. N-Acetylglucosamine, N-acetylmuramic acid, diaminopimelic acid, and D-alanine each increase the number of spores produced by SpoC mutants. When all four components are included they have a synergistic effect, raising the number of spores produced by SpoC mutants to the wild-type level. Murein-rescued spores are resistant to heat and sonic oscillation and germinate when plated on a nutrient-rich medium. They appear to be identical to fruiting body spores in their ultrastructure, in their protein composition, and in their resistance to boiling sodium dodecyl sulfate. Murein rescue of sporulation, like fruiting body sporulation, requires high cell density, a low nutrient level, and a solid surface.     

Comparison of the Wheat Brown and Yellow Rusts for Monocyclic Sporulation and Infection Processes, and their Polycyclic Consequences

Sporulation capacity and infection efficiency of wheat brown and yellow rusts were measured daily in favourable controlled conditions. Monocyclic sporulation capacity for a single lesion of yellow rust was 9 times greater than for an isolated lesion of brown rust, and 40 times greater than for a lesion of brown rust at medium infection density. Infection efficiency fluctuated and reached about 40 % for brown rust but remained under 5% for yellow rust. For both fungi, sporulation capacity and infection efficiency compensated for each other, but their product, the daily multiplication factor, was greater for yellow rust than for brown rust. Progeny/parent ratio was 3 times greater for yellow rust. Effect of daily multiplication factor variations on epidemic progress was simulated using a simple matrix model. Increase in number of lesions was faster in brown rust than in yellow rust because of a latent period shorter by 2 days. Semi-systemic growth of yellow rust fungus reduced, however, the difference between both fungi when sporulating surface was calculated.     

Effect of growth rate and hydrophobicity on bacteria surviving protozoan grazing

Measurements were made of the predation by Tetrahymena thermophila on several bacterial species in media containing heat-killed Escherichia coli cells to serve as an alternative prey. If grazing pressure was initially not intense on a mixture of bacterial species, the species that survived protozoan feeding at greater densities were those that grew quickly before the onset of active predation. If members of several species were incubated individually at similar initial densities with actively grazing T. thermophila, some species survived at ca. 10(4)/ml, some survived at ca. 10(2)/ml, and others were eliminated. Members of the first two groups but not the third group were able to multiply in the medium in the absence of the protozoan, but the growth rates in the protozoan-free medium did not correlate with the number of survivors. However, the species that persisted at the higher densities possessed highly hydrophobic cell surfaces. The size of the surviving population of four bacterial species whose growth was prevented by chloramphenicol correlated with the initial cell density that was incubated with T. thermophila. It is concluded that the individual species surviving predation on a mixture of species is related to the capacity of the bacterium to grow, the hydrophobicity of its cell surface, and the population density of the species before the onset of intense grazing.