The Possible Involvement of Cyclic AMP and Volatile Substance (s) in the Development of a Macrocyst-Forming Strain of Dictyostelium mucoroides

A mutant MF1 previously isolated from Dictyostelium mucoroides -7 (Dm7) formed macrocysts with or without light when plated on agar at high cell dinsities. At lower cell densities, however, the MF1 cells formed only fruiting bodies. This failure to form macrocysts was shown to be due to the subthreshfold concentration of a volatile substance(s) required for macrocyst formation. Although ammonia is a volatile substance produced by both the Dm7 and MF1 cells, no evidence of its involvement in macrocyst formation was obtained. Mixing the Dm7 and MF1 in a one-to-one ratio resulted only in fruiting body formation suggesting that the Dm7 cells produced a factor which allowed MF1 cells to form fruiting bodies. This factor may be cyclic AMP (cAMP) since addition of cAMP to the medium directed development of MF1 cells to fruiting body formation. The effect of cAMP was exhibited most conspicuously when MF1 cells were exposed at the aggregation stage. Based on these results it is suggested that developmental pathway of the D. mucoroides macrocystforming strain Dm7 and its mutant MF1 may be determined by the relative concentrations of the volatile, macrocyst-inducing substance(s) and cAMP at the aggregation stage.     

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.     

Developmental aggregation of Myxococcus xanthus requires frgA, an frz-related gene

Myxococcus xanthus is a gram-negative bacterium which has a complex life cycle that includes multicellular fruiting body formation. Frizzy mutants are characterized by the formation of tangled filaments instead of hemispherical fruiting bodies on fruiting agar. Mutations in the frz genes have been shown to cause defects in directed motility, which is essential for both vegetative swarming and fruiting body formation. In this paper, we report the discovery of a new gene, called frgA (for frz-related gene), which confers a subset of the frizzy phenotype when mutated. The frgA null mutant showed reduced swarming and the formation of frizzy aggregates on fruiting agar. However, this mutant still displayed directed motility in a spatial chemotaxis assay, whereas the majority of frz mutants fail to show directed movements in this assay. Furthermore, the frizzy phenotype of the frgA mutant could be complemented extracellularly by wild-type cells or strains carrying non-frz mutations. The phenotype of the frgA mutant is similar to that of the abcA mutant and suggests that both of these mutants could be defective in the production or export of extracellular signals required for fruiting body formation rather than in the sensing of such extracellular signals. The frgA gene encodes a large protein of 883 amino acids which lacks homologues in the databases. The frgA gene is part of an operon which includes two additional genes, frgB and frgC. The frgB gene encodes a putative histidine protein kinase, and the frgC gene encodes a putative response regulator. The frgB and frgC null mutants, however, formed wild-type fruiting bodies.     

The effect of light on morphogenesis of Dictyostelium mucoroides

The effect of light on the production of macrocysts and sorocarps of Dictyostelium mucoroides, strain DM-7, has been studied with surface cultures grown on dilute lactose-peptone agar at 22 degrees C with Escherichia coli, strain B/r, as food bacteria. The production of sorocarps or macrocysts can be controlled by altering the light component of the environment. Far red light had no effect on macrocyst production, whereas visible light from 440 to 700 nm inhibited macrocyst production with production decreasing with increasing light intensity. Fluence response curves for macrocyst production were determined for twelve wavelengths of light between 400 and 700 nm. An action spectrum calculated from the fluence response curves shows a single major peak at about 425-430 nm.     

Morphogenetic effects of light and guanine derivatives on the fruiting myxobacterium Stigmatella aurantiaca.

When low cell densities of the myxobacterium Stigmatella aurantiaca were starved on an inorganic salts and agar medium, cell aggregation and fruiting body formation showed a striking dependency upon the presence of light. This dependency was not manifested when sufficient amounts of guanosine or guanine nucleotides were added to the medium. Light interacted cooperatively with suboptimal concentrations of guanine compounds to promote development. None of the other purine or pyrimidine derivatives, with the exception of adenine, stimulated development. However, aggregates that formed in the presence of adenine did not mature into fruiting bodies and instead disaggregated.     

Nutritional induction and suppression of fruiting in Myxococcus xanthus FBa

A defined agar medium (A agar) containing 15 amino acids in concentrations between 0.5 and 2 mm was developed for studying the fruiting cycle of Myxococcus xanthus FBa. Cells grew only vegetatively in this medium unless the initial concentration of one of nine required or stimulatory amino acids was lowered about 50-fold. In the latter circumstance, fruiting bodies developed after several days of vegetative growth. The conclusion was that fruiting occurred when any amino acid required for normal growth became limiting in the environment. High concentrations (10 mm) of phenylalanine, tryptophan, or methionine prevented fruiting without affecting growth. Mutants requiring arginine, thymidine, or adenine could not be induced to fruit by limiting their unique requirement although they responded to the same deprivations which brought about fruiting of the wild type. A histidine auxotroph formed fruiting bodies when histidine was lowered to growth-limiting concentrations, provided that the medium was supplemented with purines. A uracil auxotroph was isolated that, perhaps secondarily, had lost some of the mechanisms which control the formation of fruiting bodies; if uracil was present, it formed fruits even when no amino acid was limiting. No concentration of uracil was sufficient to prevent fruiting. Fruiting bodies were formed when mixtures of the uracil auxotroph and wild-type cells were inoculated on A agar plus uracil, even when 75% of the cells were wild type. Microcysts of both strains were present in the fruiting bodies.     

Multicellular development in Myxococcus xanthus is stimulated by predator-prey interactions

Myxococcus xanthus is a predatory bacterium that exhibits complex social behavior. The most pronounced behavior is the aggregation of cells into raised fruiting body structures in which cells differentiate into stress-resistant spores. In the laboratory, monocultures of M. xanthus at a very high density will reproducibly induce hundreds of randomly localized fruiting bodies when exposed to low nutrient availability and a solid surface. In this report, we analyze how M. xanthus fruiting body development proceeds in a coculture with suitable prey. Our analysis indicates that when prey bacteria are provided as a nutrient source, fruiting body aggregation is more organized, such that fruiting bodies form specifically after a step-down or loss of prey availability, whereas a step-up in prey availability inhibits fruiting body formation. This localization of aggregates occurs independently of the basal nutrient levels tested, indicating that starvation is not required for this process. Analysis of early developmental signaling relA and asgD mutants indicates that they are capable of forming fruiting body aggregates in the presence of prey, demonstrating that the stringent response and A-signal production are surprisingly not required for the initiation of fruiting behavior. However, these strains are still defective in differentiating to spores. We conclude that fruiting body formation does not occur exclusively in response to starvation and propose an alternative model in which multicellular development is driven by the interactions between M. xanthus cells and their cognate prey.     

Territorial interactions between two Myxococcus Species.

It is unusual to find fruiting bodies of different myxobacteria occupying the same territory on natural samples. We were thus interested in determining whether myxobacteria establish territorial dominance and, if so, what the mechanism of that interaction is. We had previously observed that vegetative swarms of Myxococcus xanthus and Stigmatella aurantiaca placed close to each other on an agar surface initially merged but eventually separated. Further studies indicated that these two species also formed separate fruiting bodies when mixed together on developmental agar (unpublished observation). We examined the interactions between two more closely related myxobacteria, M. xanthus and M. virescens, in greater detail. When mixtures of a kanamycin-resistant strain of M. xanthus and a kanamycin-sensitive strain of M. virescens were placed together under developmental conditions, the cells sorted themselves out and established separate fruiting body territories. In addition, differential viable counts of a mixture of the two species during development indicated that each strain was producing an extracellular component that inhibited the growth and development of the other. Nevertheless, finally, M. virescens invariably outcompeted M. xanthus at all input ratios of M. xanthus/M. virescens tested. This is consistent with the observation that M. virescens is by far the more commonly encountered of the two species. The properties of the inhibitory substance from M. virescens are consistent with the possibility that it is a bacteriocin. Our working hypothesis is that the bacteriocin plays a role in the establishment of myxobacterial territoriality. If so, this is an example of an ecological function of bacteriocins.     

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.     

Signalling and sex in the social amoebozoans

The social amoebozoans have a life tricycle consisting of asexual multicellular development leading to fruiting bodies, sexual multicellular development resulting in macrocysts, and unicellular development generating microcysts. This review covers the events of sexual development in the best‐studied heterothallic (Dictyostelium discoideum) and homothallic (D. mucoroides) mating systems. Sexual development begins with pheromonal interactions that produce fusion‐competent cells (gametes) which undergo cell and pronuclear fusion. Calcium‐ and calmodulin‐mediated signalling mediates these early events. As they initiate chemotactic signalling, each zygote increases in size becoming a zygote giant cell. Using cyclic AMP (cAMP), the zygote chemotactically lures in amoebae and engulfs them in an act of cannibalistic phagocytosis. Chemotaxis proceeds more quickly than endocytosis because the breakdown products of cAMP (5‐AMP, adenosine) bind to a presumptive adenosine receptor to inhibit sexual phagocytosis. This slowing of phagocytosis allows amoebae to accumulate around the zygote to form a precyst aggregate. Zygote giant cells also produce several other signalling molecules that feed back to regulate early events. The amoebae surrounding the zygote seal their fate as zygotic foodstuff by secreting a primary cellulose wall, the extracellular sheath, around the zygote and aggregated amoebae, which prevents their escape. Phagocytosis within this precyst continues until all peripheral amoebae are internalized as endocytes and the final macrocyst wall is formed. Endocyte digestion results in a mature macrocyst with a uniform cytoplasm containing a diploid nucleus. After detailing the morphological events of heterothallic and homothallic mating, we review the various intercellular signalling events and other mechanisms involved in each stage. This complete and comprehensive review sets the stage for future research on the unique events that characterize sex in the social amoebozoans.     

Cystein proteinase changes during macrocyst formation in Dictyostelium mucoroides

Cysteine proteinases were detected in vegetative myxamoebae of Dictyostelium mucoroides DM7 using chromogenic substrates and by electrophoretic analysis (gelatin-SDS-PAGE) which revealed three enzymes, dmCP30, dmCP35 and dmCP46 (a minor form). During the initial stages of macrocyst formation the cysteine proteinaes were secreted and disappeared almost completely from the cells. High extracellular levels of activity towards N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine 4-nitroanilide and of dmCP30 persisted throughout macrocyst development. Three new intracellular proteinases, dmCP31, dmCP36 and dmCP40, were produced as macrocysts formed but their activity was only detected by gelatin-SDS-PAGE. Their appearance was specific to the developmental pathway leading to macrocyst formation. This is the first direct evidence for the accumulation of cysteine proteinases during a developmental process in a cellular slime mould.     

Scanning electron microscopy of fruiting body formation by myxobacteria.

Scanning electron microscopy was used to follow fruiting body formation by pure cultures of Chondromyces crocatus M38 and Stigmatella aurantica. Vegetative cells were grown on SP agar and then transferred to Bonner salts agar for fructification. Fruiting in both species commences with the formation of aggregation centers which resemble a fried egg in appearance. In Chondromyces the elevated center or "yolk" region of the aggregation enlarges into a bulbous structure under which the stalk forms and lengthens. At maximum stalk height the bulb extends laterally as bud-like swellings appear. These are immature sporangia and are arranged in a distintive radial pattern around the top of the stalk. This symmetry is lost as more sporangia are formed. Stigmatella does not form a bulb; rather the yolk region of the aggregation center projects upward to form a column-like stalk which is nearly uniform in diameter throughout its length. At maximum stalk height, the terminus of the stalk develops an irregular pattern of bud-like swellings. These differentiate into sporangia. Stalks of 2-week-old mature fruiting bodies of both species appear to be cellular in composition. Stereomicrographs suggest orientation of these cells parallel to the long axis of the stalk. Stalks of 8-week-old fruiting bodies of Chondromyces were acellular and consisted of empty tubules, suggesting that the cells undergo degeneration with aging of the fruiting body.     

Aggregation during fruiting body formation in Myxococcus xanthus is driven by reducing cell movement

When starved, Myxococcus xanthus cells assemble themselves into aggregates of about 10(5) cells that grow into complex structures called fruiting bodies, where they later sporulate. Here we present new observations on the velocities of the cells, their orientations, and reversal rates during the early stages of fruiting body formation. Most strikingly, we find that during aggregation, cell velocities slow dramatically and cells orient themselves in parallel inside the aggregates, while later cell orientations are circumferential to the periphery. The slowing of cell velocity, rather than changes in reversal frequency, can account for the accumulation of cells into aggregates. These observations are mimicked by a continuous agent-based computational model that reproduces the early stages of fruiting body formation. We also show, both experimentally and computationally, how changes in reversal frequency controlled by the Frz system mutants affect the shape of these early fruiting bodies.     

Ammonia Determines the Alternative Pathways of Sexual or Asexual Development in the Cellular Slime Mold Dictyostelium discoideum

The sexual development, macrocyst formation, of Dictyostelium discoideum is initiated by sexual fusion of cells. The sexual fusion is only taken place under the culture conditions of excess water and darkness. Under these conditions, cells acquire the fusion competence, but lose it when cell density is high. The loss of the fusion competence is caused by accumulation of ammonia excreted by cells in a culture. Ammonia suppresses the fusion competence of cells at a certain concentration, and consequently inhibits formation of macrocysts and induces fruiting-body formation. Thus, excess water induces the sexual development by diluting ammonia and lack of water induces the asexual development.     

Aggresomes, inclusion bodies and protein aggregation

Intracellular and extracellular accumulation of aggregated protein are linked to many diseases, including ageing-related neurodegeneration and systemic amyloidosis. Cells avoid accumulating potentially toxic aggregates by mechanisms including the suppression of aggregate formation by molecular chaperones and the degradation of misfolded proteins by proteasomes. Once formed, aggregates tend to be refractory to proteolysis and to accumulate in inclusion bodies. This accumulation has been assumed to be a diffusion-limited process, but recent studies suggest that, in animal cells, aggregated proteins are specifically delivered to inclusion bodies by dynein-dependent retrograde transport on microtubules. This microtubule-dependent inclusion body is called an aggresome.     

Short-Range Guiding Can Result in the Formation of Circular Aggregates in Myxobacteria Populations

Myxobacteria are social bacteria that upon starvation form multicellular fruiting bodies whose shape in different species can range from simple mounds to elaborate tree-like structures. The formation of fruiting bodies is a result of collective cell movement on a solid surface. In the course of development, groups of flexible rod-shaped cells form streams and move in circular or spiral patterns to form aggregation centers that can become sites of fruiting body formation. The mechanisms of such cell movement patterns are not well understood. It has been suggested that myxobacterial development depends on short-range contact-mediated interactions between individual cells, i.e. cell aggregation does not require long-range signaling in the population. In this study, by means of a computational mass-spring model, we investigate what types of short-range interactions between cells can result in the formation of streams and circular aggregates during myxobacterial development. We consider short-range head-to-tail guiding between individual cells, whereby movement direction of the head of one cell is affected by the nearby presence of the tail of another cell. We demonstrate that stable streams and circular aggregates can arise only when the trailing cell, in addition to being steered by the tail of the leading cell, is able to speed up to catch up with it. It is suggested that necessary head-to-tail interactions between cells can arise from physical adhesion, response to a diffusible substance or slime extruded by cells, or pulling by motility engine pili. Finally, we consider a case of long-range guiding between cells and show that circular aggregates are able to form without cells increasing speed. These findings present a possibility to discriminate between short-range and long-range guiding mechanisms in myxobacteria by experimentally measuring distribution of cell speeds in circular aggregates.     

Biological Activity of the Alternative Promoters of the Dictyostelium discoideum Adenylyl Cyclase A Gene

Amoebae of the Dictyostelium discoideum species form multicellular fruiting bodies upon starvation. Cyclic adenosine monophosphate (cAMP) is used as intercellular signalling molecule in cell-aggregation, cell differentiation and morphogenesis. This molecule is synthesized by three adenylyl cyclases, one of which, ACA, is required for cell aggregation. The gene coding for ACA (acaA) is transcribed from three different promoters that are active at different developmental stages. Promoter 1 is active during cell-aggregation, promoters 2 and 3 are active in prespore and prestalk tip cells at subsequent developmental stages. The biological relevance of acaA expression from each of the promoters has been studied in this article. The acaA gene was expressed in acaA-mutant cells, that do not aggregate, under control of each of the three acaA promoters. acaA expression under promoter 1 control induced cell aggregation although subsequent development was delayed, very small fruiting bodies were formed and cell differentiation genes were expressed at very low levels. Promoter 2-driven acaA expression induced the formation of small aggregates and small fruiting bodies were formed at the same time as in wild-type strains and differentiation genes were also expressed at lower levels. Expression of acaA from promoter 3 induced aggregates and fruiting bodies formation and their size and the expression of differentiation genes were more similar to that of wild-type cells. Expression of acaA from promoters 1 and 2 in AX4 cells also produced smaller structures. In conclusion, the expression of acaA under control of the aggregation-specific Promoter 1 is able to induce cell aggregation in acaA-mutant strains. Expression from promoters 2 and 3 also recovered aggregation and development although promoter 3 induced a more complete recovery of fruiting body formation.     

Genes involved in Dictyostelium discoideum sexual reproduction

Macrocyst formation in the cellular slime moulds is a sexual process induced under dark and humid conditions. Normal development life cycle in these organisms involves proliferation by cell division and, upon starvation, formation of multicellular aggregates and fruiting bodies, consisting of spores and stalk cells. Macrocyst formation, cell division by binary fission and spore formation are thus three alternative modes of reproduction, for which it is of interest to understand how a choice is made. The genetic basis of asexual development and fruiting body formation is well known, by contrast information on the genetic control of sexual reproduction during macrocyst formation is scarce. In Dictyostelium discoideum, the most widely used species, several cell-surface proteins relevant to sexual cell fusion have been identified using cell fusion-blocking antibodies, but isolation of the relevant genes has been unsuccessful. Analysis of sexually deficient mutants, some of which are normal for asexual development, has shown that sexual reproduction is regulated by both specific genes and genes that are also involved in asexual development. Reverse genetic analysis of 24 genes highly enriched in a gamete-specific subtraction library has revealed four genes involved in the regulation of sexual cell interactions. One of them was found to be a novel regulator of the cAMP signalling pathway specific to sexual development. Studies on the molecular genetic control of the sexual cycle will be reviewed and their contribution to our understanding of the organization and function of the D. discoideum genome as a whole discussed.     

Genes involved in Dictyostelium discoideum sexual reproduction

Macrocyst formation in the cellular slime moulds is a sexual process induced under dark and humid conditions. Normal development life cycle in these organisms involves proliferation by cell division and, upon starvation, formation of multicellular aggregates and fruiting bodies, consisting of spores and stalk cells. Macrocyst formation, cell division by binary fission and spore formation are thus three alternative modes of reproduction, for which it is of interest to understand how a choice is made. The genetic basis of asexual development and fruiting body formation is well known, by contrast information on the genetic control of sexual reproduction during macrocyst formation is scarce. In Dictyostelium discoideum, the most widely used species, several cell-surface proteins relevant to sexual cell fusion have been identified using cell fusion-blocking antibodies, but isolation of the relevant genes has been unsuccessful. Analysis of sexually deficient mutants, some of which are normal for asexual development, has shown that sexual reproduction is regulated by both specific genes and genes that are also involved in asexual development. Reverse genetic analysis of 24 genes highly enriched in a gamete-specific subtraction library has revealed four genes involved in the regulation of sexual cell interactions. One of them was found to be a novel regulator of the cAMP signalling pathway specific to sexual development. Studies on the molecular genetic control of the sexual cycle will be reviewed and their contribution to our understanding of the organization and function of the D. discoideum genome as a whole discussed.     

Quantification of transformation efficiency using a new method for clonal growth and selection of axenic Dictyostelium cells

A new method for clonal growth of Dictyostelium axenic amoebae has been developed. Cells are plated in growth medium containing 1% ultra-low gelling temperature agarose. Cells grow normally in the agarose and form colonies up to several millimeters in diameter. When the colonies have grown to a sufficient size, they begin multicellular development. Pseudoplasmodia are formed, migrate to the surface of the agar, and then undergo fruiting body formation. Cells can be removed from the soft agarose colonies with a toothpick or by picking spores from the fruiting bodies. This method should be useful for drug, auxotrophic, and temperature selections where clonal maintenance of axenic colonies is important. This method has been used in combination with a selection for resistance to G418 to isolate independent colonies following DNA-mediated transformation. Several parameters in the calcium phosphate and electroporation transformation protocols have been optimized and the transformation frequency quantified. Independent transformed colonies are obtained at a frequency of 1 in 10(4) to 1 in 10(5) cells when integrating plasmids are introduced using calcium phosphate coprecipitation. The frequency is about tenfold higher when extrachromosomal shuttle vectors are introduced into cells.