CELLULAR SLIME MOLDS OF SOUTHEAST ASIA. I. DESCRIPTION OF NEW SPECIES

Sixteen species of cellular slime molds were isolated from Southeast Asian forest soils. Ten of these, Dictyostelium mucoroides Brefeld, D. purpureum Olive, D. polycephalum Raper, D. lacteum van Tieghem, D. rhizopodium Raper and Fennell, D. lavandulum Raper and Fennell, D. vinaceo-fuscum Raper and Fennell, D. coeruleo-stipes Raper and Fennell, Polysphondylium violaceum Brefeld, and P. pallidum Olive have been previously described and are well-recognized species occurring in other parts of the world. Two, one in the genus Dictyostelium and one belonging to the family Guttulinaceae, are considered species by the author but have not been formally described. Four are described in this paper as new: Dictyostelium intermedium, D. multi-stipes, D. bifurcatum, and Acytostelium subglobosum. A new variety papilloideum of D. lacteum is also described. One other discovery of special interest is an isolate of Polysphondylium violaceum which produces abundant macrocysts, now known to be the sexual stage in the life cycle of cellular slime molds.     

Cysteine proteinase changes during microcyst formation and germination inPolysphondylium pallidum

Microcyst formation inPolysphondylium pallidum WS320 was accompanied by a decrease in intracellular cysteine proteinase activity measured with the peptide nitroanilides Z-Arg-Arg-Nan and Bz-Pro-Phe-Arg-Nan. Some activity was released into the buffer, and secretion of that towards Z-Arg-Arg-Nan continued until encystment occurred. Cells grown in association withEscherichia coli had an electrophoretic proteinase pattern different from cells grown axenically. Microcysts formed from the two cell populations also had distinct proteinase patterns; those from bacterially grown cells retained significant quantities of proteinase ppCP22, whereas those derived from axenic cells were devoid of detectable proteinases. No significant changes in cysteine proteinase activities were observed during microcyst germination, although some changes in activity occurred subsequent to emergence. The results indicate that there is not a close correlation between particular cysteine proteinases and specific stages of microcyst formation. Intracellular proteinase loss and concomitant secretion are, however, processes typical of cellular slime molds developing in response to starvation.     

The regulation of cellular slime mold development: a factor causing development of Polysphondylium violaceum aggregation-defective mutants.

aggA mutants of Polysphondylium violaceum develop normally in synergistic mixtures with other aggregation-defective mutants. Cell to cell contact is not necessary for development. A small dialyzable factor(s) produced by wild-type and other aggregation-defective mutants triggers development of aggA mutants. This factor (D factor) is developmentally regulated, appearing early in development and then disappearing. Mutants require D factor until aggregation has just begun and then they can continue even in the absence of added factor. D factor is produced by many, but not all species of cellular slime molds and is developmentally regulated in Dictyostelium discoideum as well as P. violaceum.     

Regulation of cysteine proteinases during different pathways of differentiation in cellular slime molds

Cysteine proteinase activities have been determined using gelatin-SDS-PAGE analysis and assays based on peptide nitroanilides. Vegetative myxamoebae of all species examined contain high levels of cysteine proteinase activity present in multiple forms. In both Dictyostelium discoideum and Polysphondylium pallidum the proteinase content is dependent on whether the cells are grown axenically or in association with bacteria. In all instances development is accompanied by a decreased intracellular cysteine proteinase activity. This occurs during the formation of fruiting bodies in D. discoideum, microcysts in P. pallidum, and macrocysts in Dictyostelium mucoroides. Significant quantities of proteinase activity are always secreted by myxamoebae immediately on starvation. In D. mucoroides this leads to an almost total depletion of intracellular cysteine proteinases by the aggregation stage. As a consequence of this depletion it has been relatively easy to detect a developmentally regulated accumulation of cysteine proteinases at the enzyme activity level, something which has not yet proved possible with D. discoideum. Three cysteine proteinases are produced as D. mucoroides macrocysts develop and mature. In the case of microcyst formation in P. pallidum the proteinase contents of the developing cells and of the microcysts are dependent on how the myxamoebae are grown. In this developmental pathway at least, there is no absolute requirement for specific proteinases to be present (or absent) at a particular stage. The diversity of cysteine proteinases found in cellular slime molds and the variety of features apparent in their regulation suggest that they will prove to be very useful for investigating features of the structure/function relationships in this important group of enzymes.     

Conservation and variation of ribosomal proteins in several species of the cellular slime molds Dictyostelium and Polysphondylium

Genus- and species-specific composition of ribosomal proteins was investigated in four species of the genus Dictyostelium (D. discoideum, D. purpureum, D. murcoroides and D. giganteum) and two species of the genus Polysphondylium (P. pallidum and P. violaceum). Ribosomal proteins were resolved by a high-resolution, two-dimensional gel method. In general, the numbers and distributions for the majority of ribosomal proteins were similar within the species of each genus, although some differences were detected. More differences were observed between Dictyostelium and Polysphondylium than among the individual species within each genus. Stage-specific ribosomal proteins previously demonstrated in D. discoideum were found to be developmentally regulated in other Dictyostelium species, and in both Polysphondylium species. The study shows that ribosomal proteins may be a potentially useful new biochemical parameter for the molecular taxonomy of the cellular slime molds.     

Contact sites in aggregating cells of Polysphondylium pallidum

Aggregating cells of the cellular slime mold Polysphondylium pallidum are completely dissociated by univalent antibody fragments (Fab) directed against membrane antigens. The blocking effect on cell adhesion is species specific: Fab against P. pallidum has little effect on cells of Dictyostelium discoideum, and vice versa. Suspended cells of these species agglutinate together, but within the agglutinates they sort out into separate areas.Absorption of the Fab with growth phase cells removes only part of its blocking activity. This indicates the expression of a new class of target sites of adhesion blocking Fab during cell differentiation from the growth phase to the aggregation competent stage. Another class of target sites is already present on the surface of growth phase cells. In both developmental stages cell adhesion is largely resistant to EDTA.The major target sites of adhesion blocking Fab appear to differ from carbohydrate-binding proteins known as pallidin. Removal of the adhesion blocking activity by absorption of Fab with intact cells does not deplete for anti-pallidin Fab. Cell adhesion is only weakly affected by Fab specific for pallidin I and II.     

Folic Acid as Second Chemotactic Substance in the Cellular Slime Moulds

IT has been known for some time that in certain species of cellular slime moulds acrasin, the substance which attracts the amoebae to central collection points during the aggregation phase, is cyclic AMP^1–4. We were also able to show that E. coli gave off another substance besides cyclic AMP (henceforth referred to as bacterial factor, or BF) which attracted the vegetative amoebae of Dictyostelium discoideum⁵. Here we demonstrate that this second attractant has the properties of folic acid or one of its derivatives. We also show that folic acid and related compounds not only attract the vegetative amoebae of D. discoideum (No. NC-4H) but also the amoebae of six other species (Dictyostelium rosarium No. CC-7; D. mucoroides No. 11; D. purpureum No. 2; D. minutum No. V-3; Polysphondylium violaceum No. 1; P. pallidum No. 2). For the latter three species cyclic AMP is not the aggregative attractant (ref. 6 and J. T. B., E. M. H., S. Noller, F. B. Oleson and A. B. Roberts, in preparation) which raises the interesting question of whether their acrasin might be related to the folates.     

Dictyostelids from Mediterranean forests of the south of Europe

The first results of a study of dictyostelid diversity in soils of Mediterranean vegetation are presented. Surface soil samples were collected during 2003–2004 from different Mediterranean forests in Spain and Portugal and were plated out for cellular slime moulds. Isolated during this study were 14 species of dictyostelids. One species, Dictyostelium firmibasis is reported for the first time from Europe, and nine (Dictyostelium aureo-stipes, Dictyostelium sp. 1, Dictyostelium fasciculatum, Dictyostelium giganteum, Dictyostelium implicatum, Dictyostelium leptosomum, Dictyostelium sphaerocephalum, Polysphondylium candidum, Polysphondylium pallidum) are new records for the Mediterranean region. Members of the three genera Dictyostelium (ten species), Polysphondylium (three species) and Acytostelium (one species) are present in this region of the world. Comments on their diversity and distribution are included.     

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.     

Novel chlorinated dibenzofurans isolated from the cellular slime mold,Polysphondylium filamentosum,and their biological activities

Cellular slime molds are expected to have the huge potential for producing secondary metabolites including polyketides, and we have studied the diversity of secondary metabolites of cellular slime molds for their potential utilization as new biological resources for natural product chemistry. From the methanol extract of fruiting bodies of Polysphondylium filamentosum, we obtained new chlorinated benzofurans Pf-1 (4) and Pf-2 (5) which display multiple biological activities; these include stalk cell differentiation-inducing activity in the well-studied cellular slime mold, Dictyostelium discoideum, and inhibitory activities on cell proliferation in mammalian cells and gene expression in Drosophila melanogaster.     

Electron Microscopy of Solitary and Aggregated Slime Mould Cells

Polysphondylium violaceum and Dictyostelium discoideum myxamoebae have simple double-layered nuclear membranes, a cytoplasmic reticulum of particle-covered membranes, and small mitochondria consisting of convoluted tubules tightly packed in double membranes. In addition to objects still recognisable as bacteria, their food vacuoles contain concentric (or spiral) membranes, apparently formed secondarily from undigested material; these are ultimately ejected. Where the triple-layered plasma membranes (~70 A wide) of cells in the early aggregates are apposed to one another, they run parallel but separated by a layer of rather constant thickness (~200 A), as in many unspecialised metazoan tissues. Thus studies on slime moulds may well increase our understanding of cell adhesion and tissue formation in metazoa.     

Distinct lectin activities from six species of cellular slime molds.

Extracts of cohesive cells of four species of cellular slime mold, D. mucoroides, D. purpureum, D. rosarium and P. violaceum agglutinate erythrocytes in a manner that is similar to that previously observed with extracts of D. discoideum and P. pallidum. We determined inhibitory activity of a series of sugars on the agglutination activity of each of these extracts, using both semiquantitative and quantitative agglutination assays. The inhibitory potency of this series of sugars was distinct for each extract, although only slight differences were found between several species, especially D. discoideum and P. violaceum. A possible role of these agglutinins in species-specific cell cohesion is considered.     

The effect of monoterpenoids on growth of a cellular slime mold,<Emphasis Type="Italic">Polysphondylium pallidum</Emphasis>

A cellular slime mold,Polysphondylium pallidum was isolated from the forest floor of Mountain Muhak. The effect of 11 selected monoterpenoids on the growth ofP. pallidum was studied. We tested four different concentrations (1,0.1, 0.01, and 0.001 μg/μl) for each compound by using a disk volatilization technique. Each compound was treated after germination of spores ofP. pallidum. The growth ofP. pallidum was inhibited by the treatment of the monoterpenoids at all concentrations tested. The microscopic analysis further supported these results. Most of the inhibitory effects of the compounds were represented by changes in the shapes of the fruiting bodies, such as very short sorophores, smaller sized sori and sori without spores. Especially, the monotepenoids changed the shape of whorls of side branches. These results suggested that selected monoterpenoids inhibit the growth ofP. pallidum.     

Multiple acid proteinases in the cellular slime mould Dictyostelium discoideum.

Proteinase activity in the cellular slime mould Dictyostelium discoideum has been analyzed by electrophoresis on polyacrylamide gels containing denatured hemoglobin. At least eight bands due to acid proteinases have been defined using extracts of myxamoebae, four bands A-D which move faster than the fifth and major band E, a minor band E' which moves just behind E and two slow bands G and H. Fruiting body formation was accompanied by the appearance of one new proteinase band F. The proteinases were present in extracts of both axenically-grown and bacterially-grown cells. Differences between the pH dependence and stability of the individual proteinases were detected. Inhibitor studies suggested that the faster proteinases A-D may be cathepsin B-like, whilst the slower enzymes E, E' and F do not fit readily into any known group of proteinases since they were sensitive to HgCl2 but not to other inhibitors of cathepsin B and not to inhibitors of cathepsin D-like proteinases under standard conditions. None of the proteinases was apparently formed during or after preparation of extracts and the proteinases could be re-run on polyacrylamide gels to give only the band expected from the first run. The bands are believed to reflect multiple proteinase activities within the cell.     

THE DISTRIBUTION OF DICTYOSTELID CELLULAR SLIME MOLDS IN SOUTHERN CALIFORNIA WITH TAXONOMIC NOTES ON SELECTED SPECIES

The occurrence and distribution of Dictyostelid cellular slime molds in southern California were investigated by means of a clonal isolation technique that permitted quantitative sampling of populations. Thirteen species were isolated. These include (in approximate order of decreasing frequency): Dictyostelium rosarium Raper and Cavender, D. mucoroides Brefeld, D. sphaerocephalum (Oud.) Sacc. and March., D. aureum Olive, Polysphondylium pallidum Olive, D. minutum Raper, D. giganteum Singh, D. purpureum Olive, P. violaceum Brefeld, D. mucoroides variant, Acytostelium leptosomum Raper and Quinlan, D. polycephalum Raper, and a new species of Dictyostelium. D. rosarium and D. aureum, both rarely reported in the literature, were common and widespread. Trends in habitat preference of the Dictyostelids encountered are discussed. Selected taxonomic details are provided for D. rosarium, D. aureum and members of the “D. mucoroides complex“—D. mucoroides, D. sphaerocephalum, D. giganteum, and a D. mucoroides variant.     

A mutant strain of Polysphondylium pallidum deficient in production of cyclic AMP.

A mutant strain (PN507) of the cellular slime mold Polysphondylium pallidum is described which: (a) is morphogenetically abnormal in stalk formation; (b) secretes unusually low quantities of cyclic AMP; (c) responds to exogenous cyclic AMP in the same manner as wild type, by differentiating stalk cells and synthesizing several specific proteins; (d) complements with other morphogenetic mutants secreting normal amounts of cyclic AMP to produce fruiting structures resembling wild type. The tentative conclusion is that the critical defect of PN507 is low production of cyclic AMP.     

Proteinase Activity during Tracheary Element Differentiation in Zinnia Mesophyll Cultures

The zinnia (Zinnia elegans) mesophyll cell culture tracheary element (TE) system was used to study proteinases active during developmentally programmed cell death. Substrate-impregnated gels and single-cell assays revealed high levels of proteinase activity in differentiating TEs compared with undifferentiated cultured cells and expanding leaves. Three proteinases (145, 28, and 24 kD) were exclusive to differentiating TEs. A fourth proteinase (59 kD), although detected in extracts from all tissues examined, was most active in differentiating TEs. The 28- and 24-kD proteinases were inhibited by thiol proteinase inhibitors, leupeptin, and N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine (E-64). The 145- and 59-kD proteinases were inhibited by the serine proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF). Extracts from the TE cultures contained sodium dodecyl sulfate-stimulated proteolytic activity not detected in control cultures. Sodium dodecyl sulfate-stimulated proteolysis was inhibited by leupeptin or E-64, but not by PMSF. Other tissues, sucrose-starved cells and cotyledons, that contain high levels of proteolytic activity did not contain TE-specific proteinases, but did contain higher levels of E-64-sensitive activities migrating as 36- to 31-kD enzymes and as a PMSF-sensitive 66-kD proteinase.     

The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas

Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts, pathogenic amoebas resist antibiotics, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA, with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates Amoebozoan encystation and that components of the cAMP signalling pathway could be effective targets for therapeutic intervention with encystation.     

Partial purification and characterization of endoproteinases from senescing barley leaves

Two major endoproteinases were purified from senescing primary barley leaves. The major enzyme (EP₁) appeared to be a thiol proteinase and accounted for about 85% of the total proteolytic activity measured in vitro. This proteinase was purified 5,800-fold and had a molecular weight of 28,300. It was highly unstable in the absence of dithiothreitol or at a pH greater than 7.5. Leupeptin, at a concentration of 10 micromolar, inhibited this enzyme 100%. A second proteinase (EP₂) was purified approximately 50-fold and had a molecular weight of 67,000. It was inhibited 20% by 1 millimolar dithiothreitol and 50% by 1 millimolar phenylmethyl sulfonylfluoride. EP₂ contributed about 15% of the total proteolytic activity measured in vitro. Both proteinases hydrolyzed a variety of artificial and protein substrates, and both had pH optima of 5.5 to 5.7 when either azocasein or [¹⁴C]ribulose-1,5-bisphosphate carboxylase ([¹⁴C]RuBPCase) was the substrate. The thiol endoproteinase hydrolyzed azocasein linearly but hydrolyzed [¹⁴C]RuBPCase biphasically. A third endoproteinase (EP₃), not detected by standard proteolytic assays, was observed when [¹⁴C]RuBPCase was the substrate.     

Induction of cell fusion by a factor released by the cellular slime mold Polysphondylium pallidum

Heterokaryons and hybrid cells, which are extremely useful for research in cell biology, can be produced artificially by treating cells with either polyethylene glycol or certain inactivated viruses that alter the plasma membrane. We report here a novel cell-fusion inducing factor secreted by CK-8 strain cells of cellular slime mold Polysphondylium pallidum. Treatment of other strains or other species of cellular slime molds, such as NC-4 of Dictyostelium discoideum with the diluted fraction, containing molecules larger than 50 kDa, of the conditioned medium of CK-8 cell culture induces cell fusion at high frequency and produces multinucleated large cells. This cell fusion is inducible between cells of either a single strain or of two different strains of cellular slime molds.Abbreviations BSS Bonner's salt solution - CM conditioned medium - EDTA ethylenediaminetetraacetic acid - F2 fraction containing cell-fusion induction factor - Mr molecular mass