Morphogenetic movements and multicellular development in the fruiting Myxobacterium, Stigmatella aurantiaca.

Stigmatella aurantiaca, strain DW-4, is a bacterium that grows as single cells in liquid culture but will synchronously aggregate and construct multicellular fruiting bodies when starved on an agar surface. The fruiting body consists of a stalk and several sporangia housing differentiated myxospores. Fruiting body development is stimulated by exposure of the aggregating cells to incandescent light.     

Effects of specific cations on aggregation and fruiting body morphology in the myxobacterium Stigmatella aurantiaca.

The cation requirements for fruiting body formation in the myxobacterium Stigmatella aurantiaca on agarose were determined. Calcium alone caused the cells to aggregate into interconnecting ridges. Under these conditions, stalk formation was severely depressed but sporangia frequently formed. The combination of magnesium and manganese was necessary for optimal formation of discrete aggregates (rather than ridges) and stalks. Manganese inhibited sporangium development. The inclusion of calcium into the magnesium-manganese medium overcame the inhibition by manganese and stimulated the production of multiple sporangia.     

Analysis of fruiting body development in the aggregative ciliate Sorogena stoianovitchae (Ciliophora, Colpodea)

The fruiting body-forming ciliate Sorogena stoianovitchae is a protist that is multicellular in one stage of its life cycle. When nutrient levels are depleted, a number of Sorogena cells aggregate beneath the water surface to form an aerial fruiting body. Based on morphologies and the inhibition of protein synthesis, fruiting body development is divided into five distinct stages: (1) aggregation before sunrise, (2) compact aggregation after sunrise, (3) secretion of mucous matrix, (4) stalk-elongation, and (5) completion of the fruiting-body. In the aggregation stage, the cells were trapped in a matrix material that stained orange with 4',6-diamino-2-phenylindole (DAPI), but differed from the mucous matrix in the later stage. A short interruption of the dark period, at 6-8 h after the onset of dark, inhibited fruiting body development. Irrespective of the length of the dark period (10-16 h), the cells remained in the aggregation stage until the beginning of the light period. Therefore, an uninterrupted dark period of more than 8 h is critical for the initial aggregation of cells, but subsequent development is triggered by light.     

Germination of myxospores from the fruiting bodies of Myxococcus xanthus.

Germination of myxospores from fruiting bodies of Myxococcus xanthus was examined under a light microscope as well as by analyzing the incorporation of [3H]uracil into the RNA fraction. Efficient germination was observed in 0.2% Casitone containing 8 mM MgSO4 and 1 mM CaCl2 at 30 degrees C. Under this condition, spherical myxospores were converted into rod-shaped vegetative cells within 5 to 6 h. The germination was severely inhibited in the presence of 1 mM phenylmethylsulfonyl fluoride, a protease inhibitor, indicating that a serine protease(s) is required for the myxospore germination. EGTA (1 mM) also completely blocked germination, indicating that Ca2+ plays an important role in myxospore germination. In 1% Casitone without added Mg2+ and Ca2+ or 0.2% Casamino Acids with 8 mM MgSO4 and 1 mM CaCl2, myxospores lost their refractility under a phase microscope, while no RNA synthesis took place within 6 h, as judged by the incorporation of [3H]uracil. A group of proteins were found to be specifically synthesized during an early stage of germination. In addition, a new major spore-associated protein with a size of 41.5 kDa became detectable in the spore shell fraction 3 h after germination. The present results demonstrate that myxospore germination occurs in at least two steps: the loss of myxospore refractility, followed by an outburst of metabolic activities. The first step can occur even in the absence of energy metabolism, while the second step was blocked by rifampin, EGTA, and protease inhibitors.     

The control of meiosis progression in the fungus Coprinus cinereus by light/dark cycles

Meiosis progression in Coprinus cinereus is controlled by light/dark cycles. Light is essential to propel basidia into karyogamy and light intensity determines the timing of meiotic events. The higher the light intensities, the faster the fruiting bodies enter karyogamy. The critical period when light has this influence is between 16 and 6 h before karyogamy. The control is highly stage specific. A 3-h dark period is essential for a Java dikaryon and the Japanese A(mut)B(mut) homokaryon to enter meiotic metaphase; without it the fruit body is permanently arrested at diffused diplotene. This arrest is light intensity-dependent (>20 hlx) and temperature-dependent (e.g., 27 degrees C). The placement of the dark period is very stage specific; it has no effect when placed before karyogamy stage. A dikaryon of London origin is light blind and able to complete meiosis under continuous high light regime. Fruiting bodies arrested under a continuous high light can be rescued by a 3-h dark treatment, but there is always an 8-h lag time to enter meiotic metaphase. It is possible that the dark effect signals cellular processes leading to division events. Cytological studies of arrested fruiting bodies showed that chromosomes are normal in meiotic prophase through pachytene and diplotene, but are unable to undergo chromosome condensation. Genetic crosses between a monokaryon of Java stock J6;5.4 and a monokaryon BL55 or H5 of London stock showed that light-blindness is dominant, and is controlled by a single Mendelian gene.     

Pheromone produced by the myxobacterium Stigmatella aurantiaca.

An extracellular, diffusible signaling molecule (pheromone) was produced by Stigmatella aurantiaca during fruiting body formation. The pheromone decreased the aggregation period in both the light and the dark and substituted for light in stimulating the maturation of aggregates into fruiting bodies. The cells were more sensitive to lower concentrations of pheromone in the light than in the dark, possibly explaining the stimulation of aggregation and fruiting body formation by light. The pheromone also interacted cooperatively with GMP to shorten the aggregation period. The pheromone behaved chemically as a low-molecular-weight lipid.     

Synergism between morphogenetic mutants of Myxococcus xanthus.

Myxococcus xanthus, a social procaryotic microorganism, forms fruiting bodies and myxospores. We have isolated a collection of mutants of M. xanthus that are defective in fruiting morphogenesis and have studied synergistic interaction in pairwise mixtures of these mutants. Certain pairs of these fruiting-defective mutants can fruit when mixed together. Similarly, certain mutants that cannot sporulate under standard fruiting conditions can form myxospores in the presence of wildtype or other nonsporulating mutants. The pattern of synergism between pairs of conditional nonsporulating mutants defines at least three and probably four groups of mutants, such that members of a group cannot synergize with each other but can synergize with members of other groups.     

Morphogenesis in Myxococcus xanthus and Myxococcus virescens (Myxobacterales)

1. Myxococcus xanthus B and M. virescens V2 were compared with a view to establishing the control of their morphogenetic cycles. Both organisms are typical myxococci and on solid media with low concentrations of nutrient they form fruiting bodies, within which vegetative cells convert to myxospores. Ultrathin sections of vegetative M. virescens resembled those of M. xanthus and contained prominent heavily stained bodies, presumed to be polyphosphate granules. Shadowed preparations showed fimbriae associated with M. xanthus but not with M. virescens. 2. M. xanthus B converted to myxospores in liquid medium in response to certain alcohols. M. virescens V2 produced phase-refractile spheres, which were not viable and had an unusual ultrastructure. 3. The distributions of fruiting bodies on solid media containing 0.02% Casitone were recorded for the two species and were compared with a Poisson distribution. Cells responded to differences in cell density in a manner suggestive of a response to a chemotactic attractant. Cells growing vegetatively and also cells forming fruiting bodies produced 3',5'-cyclic adenosine monophosphate (cAMP) as measured by the incorporation of exogeneous [3H] adenosine into cAMP. 4. The significance of these findings for theories of fruiting body formation are discussed.     

A genetic melanotic neoplasm of Drosophila melanogaster

The construction of mature fruiting bodies occurs during the culmination stage of development of Dictyostelium discoideum. These contain at least two different cell types, spores and stalks, which originate from an initially homogenous population of vegetative amoebas. As an attempt to identify proteins whose synthesis is regulated in each cell type during differentiation, we have analyzed the two-dimensional profiles of proteins synthesized by spore and stalk cells during the culmination stage. We have identified 5 major polypeptides which are specifically synthesized by spore cells during culmination and 9 which are only made by stalk cells. Furthermore, synthesis of about 20 polypeptides appears to be enriched either in the spore or in the stalk cells. We also show that synthesis of actin, a major protein synthesized during Dictyostelium development, is specifically inhibited in the spore cells during culmination. Synthesis of most of the cell type-specific proteins initiates at 19–20 hr, during culmination. Moreover, the proteins whose synthesis is induced after formation of tight aggregates, the time when the major change in gene expression occurs, are not specifically incorporated into spores or stalk cells, and appear to be synthesized by both cell types. We conclude that a new class of genes is expressed during the culmination stage in Dictyostelium, giving rise to specific patterns of protein synthesis in spore and stalk cells.     

Induction of fruiting bodies in a macrocyst-forming mutant of the cellular slime mold, Dictyostelium mucomides

Some wild-type strains of Dictyostelium mucoroides exhibit dimorphism in development depending on culture conditions: on agar, fruiting bodies containing stalk and spore cells are formed, whereas under water, a thick-walled structure lacking spore and stalk cells (the macro-cyst) is formed. The mutant, MF-1, was derived from one of these wild-type strains. It forms macrocysts on an agar surfxe as well as under water. It was found that MF-1 could be induced to form fruiting bodies in two ways. First, when an aggregation center from the wild-type strain was grafted to an MF-1 aggregation center. MF-1 cells migrated to the center and formed a large aggregate that gave rise to many slugs that became fruiting bodies. This result, along with the observation that MF-1 aggregates have no tip, suggests that MF-1 normally produces an aggregation center that is unable to organize the aggregate to form a slug. Second, when MF-1 cells were allowed to develop on 1.2 mM ethionine (an analog of methionine), they formed aggregates with tips and developed into fruiting bodies with thick stalks instead of macrocysts. The effect of ethionine was blocked by the presence of 2.4 mM methio-nine. Two other methionine analogs were also tested, i.e., α-methylmethionine and norleucine. When cultured on the former at concentrations ranging from 1.2 to 9.6 mM, MF-1 cells still produced macrocysts; when cultured on norleucine at concentrations ranging from 2.4 to 9.6 mM, MF-1 cells aggregated into large clumps that formed numerous slugs, but these failed to continue development to fruiting bodies. In vertebrates, it is known that a major biochemical effect of ethionine is the inhibition of the methylation of nucleic acids, proteins, and phospholipids. Norleucine and a-methylmethionine inhibit methylation to a lesser extent. Thus, it can be speculated that the biological effects of ethionine on MF-1 cells may result from its interference with methylation reactions, suggesting that macrocyst formation may involve excess methylation as compared with the situation during fruiting-body development.     

Morphologies and phylogenetic classification of cellulolytic myxobacteria

The evolutionary distances of the 16S rDNA sequences in cellulolytic myxobacteria are less than 3%, which units all the strains into a single genus, Sorangium. The size of myxospores and the shape of sporangioles, rather than fruiting body colors or swarm morphologies are consistent with the changes of the 16S rDNA sequences. It is suggested that there are at least two species in the genus Sorangium: one includes strains with small myxospores and spherical sporangioles, and the color of the fruiting bodies is normally orange or brown, though sometimes yellow or black. The second species has large myxospores, polyhedral sporangioles with many inter-cystic substrates, and normally deep brown to black color.     

Cell patterning in branched and unbranched fruiting bodies of the cellular slime mold Polysphondylium pallidum

Cell patterning, the percentage of spores and stalk cells, was measured in branched and unbranched asexual fruiting bodies of Polysphondylium pallidum. Unlike D. discoideum, where small and large fruiting bodies are more stalky than average-sized fruiting bodies, the overall cell patterning was the same in branched and unbranched fruiting bodies of all sizes in P. pallidum. Light greatly increased the numbers of fruiting bodies in P. pallidum per unit area (or decreased aggregation territory size) so that most fruiting bodies formed in the light were small and unbranched. By contrast, light had little effect on the cell patterning of P. pallidum, although there was a slight increase in the percentage of stalk cells in the light compared to the dark. This indicates that the mechanisms governing light sensitivity of aggregation territory size and cell patterning have different components in P. pallidum. The accuracy of cell patterning of individual branches of branched fruiting bodies was so imprecise as to leave doubt that patterning is occurring at the branch level. Individual whorls of branched fruiting bodies had a greater percentage spores (90%) than whole fruiting bodies (78%) and the cell patterning was relatively imprecise. Only in whole fruiting bodies was the spore:stalk ratio highly correlated. These findings are consistent with cell pattern determination operating at the whole aggregate level, rather than at the individual whorl or branch level in P. pallidum.     

Sporulation of Myxococcus xanthus in liquid shake flask cultures.

When suspended in a liquid starvation medium, exponentially growing Myxococcus xanthus sporulated within 3 days. These myxospores were similar to spores developed within fruiting bodies, as determined by electron microscopy and the production of spore-specific protein S. This liquid sporulation system may be useful as a means of preparing large quantities of myxospores and extracellular fluid for biochemical studies, including isolation of chemical signals produced during the sporulation process.     

Fruiting-body formation and myxospore differentiation and germination in Mxyococcus xanthus viewed by scanning electron microscopy.

Streaming cells, fruiting bodies, and single cells undergoing myxospore differentiation and germination were examined in the FB strain of Myxococcus xanthus by scanning electron microscopy. Myxospores differentiated in fruiting bodies differed in size, in kinetics of germination, in the fate of the myxospore capsule, and in the external structure of the walls of newly emerged cells when compared with myxospores differentiated in liquid medium after glycerol induction. Vegetative cells outgrowing from glycerol-induced myxospores were regularly pleomorphic, a condition that persisted through the first cell division.     

Alternative Developmental Pathways Determined by Environmental Conditions in the Cellular Slime Mold Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum grows in the soil as a population of independent, uninucleate amoebae. Upon entrance to the stationary phase, the amoebae collect in multicellular aggregates to form organized fruiting bodies composed of spores and stalk cells. Depending upon environmental conditions, the developing aggregate either constructs the fruiting body at the site of aggregation or transforms into a structure that can migrate to a more favorable location. Environmental conditions that favor migration are (i) the accumulation of metabolite(s) produced by the aggregate and (ii) a low ionic strength in the substratum. Conditions that prevent migration or that stop a migrating slug are (i) the presence of buffer and (ii) illumination by overhead light.     

Loss of the beta-catenin homologue aardvark causes ectopic stalk formation in Dictyostelium

Aardvark (Aar) is a Dictyostelium beta-catenin homologue with both cytoskeletal and signal transduction roles during development. Here, we show that loss of aar causes a novel phenotype where multiple stalks appear during late development. Ectopic stalks are preceded by misexpression of the stalk marker ST-lacZ in the surrounding tissue. This process does not involve the kinase GSK-3. Mixing experiments show that ectopic ST-lacZ expression and stalk formation are cell non-autonomous. The protein-cellulose matrix surrounding the stalk of aar mutant fruiting bodies is defective, and damage to the stalk of wild-type fruiting bodies leads to ectopic ST-lacZ expression. We postulate that poor synthesis of the stalk tube matrix allows diffusion of a stalk cell-inducing factor into the surrounding tissue.     

Photoperiodic response of Coprinus congregatus: Effects of light breaks on fruiting

Light initiated fruit-body primordia of Coprinus congregatus Bull, ex Fr. were subjected to different dark periods (4 h to 24 h) and exposed to short blue light pulses at different times. The light break inhibited the development of primordia as did continuous light. The time of maximum sensitivity to a light break was dependent on the duration of the dark period. A short light break imposed two-thirds of the way through the dark period produced strong inhibition of fruiting. The higher the temperature during the dark period, the lower the irradiance required for 50% inhibition of fruiting at the time of maximal sensitivity. During a very long dark period (48 h) a light break was no longer inhibitory. The light break fulfilled the light requirement for normal morphogenesis and defined the time the fruit-bodies sporulated.     

Morphogenesis of Stigmatella aurantiaca fruiting bodies.

Scanning electron micrographs of intermediate stages of fruiting body formation in the myxobacterium Stigmatella aurantiaca suggest that fruiting body formation can be divided into several stages distinguishable on the basis of the motile behavior of the cells. Aggregates formed at sites where cells glide as groups in circles or spirals. Thus, each aggregate was surrounded by a wide band of cells. Several streams of cells were pointed toward and connected to the wide band of cells at the base of the aggregate, suggesting directed cell movement toward the aggregate. The pattern of cells at the base of taller, more mature aggregates suggested that groups of cells enter the aggregate from the surrounding band of cells by changing the pitch of their movement, thus creating an ascending spiral. Stalk formation was characterized by a distinctly different pattern, which suggested that single cells emerge from the band of cells and move toward the aggregate, under it, and then vertically to create the stalk. At this stage, the aggregate appeared to be torn from the substrate as it was lifted off the surface. The cells in the completed stalks were well separated, and most had their long axes pointed in a vertical direction. A great deal of the stalk material appeared to be slime in which the cells were embedded and through which they were presumably moving in the live material. Some suggestions regarding factors that may direct the observed morphogenetic movements are discussed.     

Quantifying aggregation dynamics during Myxococcus xanthus development

Under starvation conditions, a swarm of Myxococcus xanthus cells will undergo development, a multicellular process culminating in the formation of many aggregates called fruiting bodies, each of which contains up to 100,000 spores. The mechanics of symmetry breaking and the self-organization of cells into fruiting bodies is an active area of research. Here we use microcinematography and automated image processing to quantify several transient features of developmental dynamics. An analysis of experimental data indicates that aggregation reaches its steady state in a highly nonmonotonic fashion. The number of aggregates rapidly peaks at a value 2- to 3-fold higher than the final value and then decreases before reaching a steady state. The time dependence of aggregate size is also nonmonotonic, but to a lesser extent: average aggregate size increases from the onset of aggregation to between 10 and 15 h and then gradually decreases thereafter. During this process, the distribution of aggregates transitions from a nearly random state early in development to a more ordered state later in development. A comparison of experimental results to a mathematical model based on the traffic jam hypothesis indicates that the model fails to reproduce these dynamic features of aggregation, even though it accurately describes its final outcome. The dynamic features of M. xanthus aggregation uncovered in this study impose severe constraints on its underlying mechanisms.     

Mutants of Myxococcus xanthus insensitive to glycerol-induced myxospore formation.

Mutants of Myxococcus xanthus FBt unable to form myxospores in response to 0.5 M glycerol arise spontaneously with a frequency of 1--3 X 10(-5). These mutants are designated glc. Ultraviolet mutagenesis increases the frequency to a maximum of 7% of the survivors. The reversion frequency following ultraviolet irradiation of spontaneous glc mutants is less than 10(-3). Of four glc mutants examined, none form myxospores in response to the alternative inducers, ethylene glycol and dimethyl sulphoxide. One glc mutant is induced by 1.5 M glycerol; strain FBt responds to this glycerol concentration with low efficiency myxospore formation. Strain FBt and glc mutants all produce myxospores with low efficiency in response to phenyl ethanol. Of 117 glc mutants tested, 109 form fruiting bodies containing mature myxospores; thus, mutations to the glc phenotype do not normally block myxospore formation within the fruiting cycle of the organism.