Ultrastructure of the plasmodial slime moldPerichaena vermicularis

Summary The development of the peridium ofPerichaena vermicularis has been examined using light and electron microscopy and acid phosphatase localization. A newly formed fruiting body consists of undifferentiated protoplasm which is enveloped by a slime coat. Almost immediately after formation of the plasmodiocarp, the protoplasm differentiates into autolytic and fruiting regions. The autolytic region is located at irregular intervals between the slime coat and the fruiting region and separated from both of them by membranes.Soon after the autolytic region has formed, additional signs of degeneration appear in the autolytic region including unusual appearance of nuclei, increase in autophagic vacuoles, and the presence of clear areas in the ground substance. The plasma membrane, which once completely separated the slime coat from the autolytic region, is no longer continuous. Electron micrographs of the autolytic region from later developmental stages show formation of extensive channels which contain protoplasm in various stages of degradation. Acid phosphatase is present in the channels of the autolytic region. The morphological evidence and the presence of hydrolytic enzyme suggest the region is being digested and re-adsorbed.After the autolytic region has been digested, an even layer of peridial wall material is laid down, and at regular intervals additional wall material is produced. The additional wall material forms the reticulation on the inside of the peridial wall.This work was supported by National Sciences Foundation grants (GB-5883 and GB-8537) to Dr.Ian K.Ross and an NSF Traineeship (GZ 445 and 796) to I.Charvat.This constitutes a portion of a thesis presented to the Regents of the University of California by the first author in partial fulfillment of the requirements for the Ph. D. degree.     

Microcyst formation in the plasmodial slime moldDidymium iridis

Summary Myxamoebae ofDidymium iridis were removed from the bacterial food source and induced to encyst by transfer to 10 mM phosphate buffer. After 24 hours of induction approximately 90% of the myxamoebae had differentiated into microcysts. The kinetics of encystment were not significantly affected by pH or osmolarity of the encystment medium. Early stages of encystment were distinguished by the appearance of autophagic vacuoles and an extracellular slime-like sheath. The outer wall layer, consisting of dense fibrils, was unevenly deposited after 4 hours. An electron-lucent, second wall layer appeared between 5–10 hours followed by a densely packed, third wall layer adjacent to the plasma membrane. Wall formation appeared to involve smooth-membraned vesicles of possible Golgi origin. The vesicle contents and outer wall layer reacted with the periodic acid-silver methenamine stain for polysaccharide. The density of intramembrane particles of the protoplasmic fracture face increased during encystment with a gradual formation of aggregates of particles.Florida Agricultural Experiment Station Journal Series No. 3473.     

Species-specific cell mobility of bacteria-feeding myxamoebae in plasmodial slime molds

On decaying wood or litter in forests, plasmodial slime molds (myxomycetes) represent a large fraction of eukaryotic protists that feed on bacteria. In his seminal book Experimental Physiology of Plants (1865), Julius Sachs referred to the multinucleate plasmodium of myxomycetes, which were considered at that time as primitive plants (or fungi). Today it is well established that myxomycetes are members of the Amoebozoa (Protista). In this study we compare the mobility of myxamoebae of 3 European species, Lycogala epidendrum (order Liceales), Tubulifera arachnoidea, and Trichia decipiens (order Trichiales). Using agar plates, on which 3 separate bacterial species were cultivated as prey organisms (Methylobacterium mesophilicum, Escherichia coli, Agrobacterium tumefaciens), we document large differences in cell motility between the myxomycetes investigated. In addition, we show that the 3 species of myxamoebae can be distinguished based on their average cell size. These data shed light on the mode of co-occurrence via differential substrate utilization in these members of the Amoebozoa.     

Evolution of hierarchical cytoplasmic inheritance in the plasmodial slime mold Physarum polycephalum

A striking linear dominance relationship for uniparental mitochondrial transmission is known between many mating types of plasmodial slime mold Physarum polycephalum. We herein examine how such hierarchical cytoplasmic inheritance evolves in isogamous organisms with many self-incompatible mating types. We assume that a nuclear locus determines the mating type of gametes and that another nuclear locus controls the digestion of mitochondria DNAs (mtDNAs) of the recipient gamete after fusion. We then examine the coupled genetic dynamics for the evolution of self-incompatible mating types and biased mitochondrial transmission between them. In Physarum, a multiallelic nuclear locus matA controls both the mating type of the gametes and the selective elimination of the mtDNA in the zygotes. We theoretically examine two potential mechanisms that might be responsible for the preferential digestion of mitochondria in the zygote. In the first model, the preferential digestion of mitochondria is assumed to be the outcome of differential expression levels of a suppressor gene carried by each gamete (suppression-power model). In the second model (site-specific nuclease model), the digestion of mtDNAs is assumed to be due to their cleavage by a site-specific nuclease that cuts the mtDNA at unmethylated recognition sites. Also assumed is that the mtDNAs are methylated at the same recognition site prior to the fusion, thereby being protected against the nuclease of the same gamete, and that the suppressor alleles convey information for the recognition sequences of nuclease and methylase. In both models, we found that a linear dominance hierarchy evolves as a consequence of the buildup of a strong linkage disequilibrium between the mating-type locus and the suppressor locus, though it fails to evolve if the recombination rate between the two loci is larger than a threshold. This threshold recombination rate depends on the number of mating types and the degree of fitness reduction in the heteroplasmic zygotes. If the recombination rate is above the threshold, suppressor alleles are equally distributed in each mating type at evolutionary equilibrium. Based on the theoretical results of the site-specific nuclease model, we propose that a nested subsequence structure in the recognition sequence should underlie the linear dominance hierarchy of mitochondrial transmission.     

Closed spindle nuclear division in the plasmodial phase of the acellular slime moldEchinostelium minutum

Summary Mitosis in the plasmodium ofEchinostelium minutum is intranuclear (closed spindle) and centrioles are not present at the spindle poles. The nuclear envelope remains essentially intact throughout mitosis with polar fenestrae appearing in anaphase and persisting through telophase. During anaphase there is a shortening in the distance of the chromosomes to the poles followed by a further separation of the poles. Zippering of microtubules may be the basis for these two anaphasic movements. During telophase the polar MTOCs are extruded into the cytoplasm through the polar fenestrae prior to reconstitution of the nuclear envelope. It is proposed that during sporulation such MTOCs are responsible for the differentiation of the centrioles which subsequently persist in the myxamoebal phase of this species.Based on the doctoral dissertation of the first author presented to the Department of Botany, University of Washington, Seattle, WA 98195, U.S.A.     

Fine structure of plasmodial nuclei in the slime moldPhysarum polycephalum I. Comparison of diploid and haploid vegetative mitosis

Summary The same basic ultrastructural features of interphase and mitotic nuclei were found for both the haploid Colonia and the diploid wild type strains of the myxomycete,Physarum polycephalum. Differences in nuclear size and chromocenter numbers were observed, but the nucleolar cycle and the intranuclear and acentriolar type of mitosis characteristic of the plasmodial stage of the diploid is present in haploid plasmodia, ruling out any relation between ploidy level and type of mitotic figure.     

Alterations in superoxide dismutase, glutathione, and peroxides in the plasmodial slime mold Physarum polycephalum during differentiation

Changes in the level of antioxidant defenses and the concentration of free radical by-products were examined in differentiating (M3cVII and LU897 X LU863), non-differentiating (LU887 X LU897), and heterokaryon microplasmodia of the slime mold Physarum polycephalum during spherulation in salts-only medium. As differentiation proceeded, superoxide dismutase activity increased by as much as 46 fold; glutathione concentration and the rate of oxygen consumption decreased; cyanide-resistant respiration, hydrogen peroxide, and organic peroxide concentrations increased. The non-differentiating culture failed to exhibit any of these changes. A heterokaryon obtained by the fusion of differentiating and non-differentiating strains was observed to differentiate at a very retarded rate and to exhibit the changes observed in the spherulating strains at a correspondingly slower rate. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.     

Regulation of DNA replication in the nuclei of the slime mold Physarum polycephalum. Transplantation of nuclei by plasmodial coalescence

Nuclei in G2 phase of the slime mold Physarum polycephalum, when transplanted, by plasmodial coalescence, into an S-phase plasmodium, failed to start another round of DNA synthesis. In the reciprocal combination, S-phase nuclei in a G2-phase host continued DNA synthesis for several hours without appreciable decrease in rate. It is suggested that the beginning of DNA replication is determined by an event, either during or shortly after mitosis, which renders the chromosomes structurally competent for DNA replication.     

In the shadow of Darwin: Anton de Bary’s origin of myxomycetology and a molecular phylogeny of the plasmodial slime molds

In his Origin of Species (John Murray, London, 1859), Charles Darwin described the theory of descent with modification by means of natural selection and postulated that all life may have evolved from one or a few simple kinds of organisms. However, Darwin’s concept of evolutionary change is entirely based on observations of populations of animals and plants. He briefly mentioned ‘lower algae’, but ignored amoebae, bacteria and other micro-organisms. In 1859, Anton de Bary, the founder of mycology and plant pathology, published a seminal paper on the biology and taxonomy of the plasmodial slime molds (myxomycetes). These heterotrophic protists are known primarily as a large composite mass, the plasmodium, in which single nuclei are suspended in a common ‘naked’ cytoplasm that is surrounded by a plasma membrane. Here we summarize the contents of de Bary’s 1859 publication and highlight the significance of this scientific classic with respect to the establishment of the kingdom Protoctista (protists such as amoebae), the development of the protoplasmic theory of the cell, the introduction of the concept of symbiosis and the rejection of the dogma of spontaneous generation. We describe the life cycle of the myxomycetes, present new observations on the myxamoebae and propose a higher-order phylogeny based on elongation factor-1 alpha gene sequences. Our results document the congruence between the morphology-based taxonomy of the myxomycetes and molecular data. In addition, we show that free-living amoebae, common protists in the soil, are among the closest living relatives of the myxomycetes and conclude that de Bary’s ‘Amoeba-hypothesis’ on the evolutionary origin of the plasmodial slime molds may have been correct.     

Ultrastructure of the capsule of Klebsiella pneumoniae and slime of Enterobacter aerogenes revealed by freeze etching

Summary The capsule of Klebsiella pneumoniae type I and slime of Enterobacter aerogenes strain A3 (SL) was examined by electron microscopy using the freeze etch technique. The capsules of K. pneumoniae were found to be composed of several layers of polysaccharide 10 nm thick; while the polysaccharide slime of E. aerogenes strain A3 (SL) was found to be composed of a diffuse network of fibrils. This work represents the first effort to visualize the replica of the unfixed, partially hydrated bacterial capsule or slime in the electron microscope. The slime of E. aerogenes strain A3 (SL) which was purified, and then freeze etched, resembled the layered structure of the capsule of K. pneumoniae. It is suggested that the charge or dielectric constant of the slime polysaccharide polymers was altered during purification, thereby permitting the layering to occur.Paper presented at the Annual Meeting of the American Society for Microbiology, Philadelphia, Pa. (U.S.A.), 1972.     

higher-order phylogeny of plasmodial slime molds (Myxogastria) based on elongation factor 1-A and small subunit rRNA gene sequences

The Myxogastria are common soil microorganisms with a life cycle comprised of a plasmodial trophic stage and large fruiting bodies generally visible with the unaided eye. Until now, their classification has been based exclusively on a combination of morphological, ultrastructural, and developmental characters. Our study is the first attempt to examine phylogenetic relationships among these taxa using molecular data. Partial small-subunit ribosomal RNA and/or elongation factor 1-alpha gene sequences were obtained from eleven, mostly field-collected species representing the five orders of Myxogastria. Nineteen sequences were obtained and subjected to phylogenetic analysis together with 10 sequences available from GenBank. Separate and combined analyses of the two data sets support the division of Myxogastria into three distinct groups. The most basal clade consists of the Echinosteliales, an order considered to have affinities with Protostelia. The three species examined possess unpigmented or slightly pigmented spores. The second group consists of Liceales and Trichiales, taxa characterized by the presence of clear, but pigmented, spores. The third group consists of the two remaining orders, Physarales and Stemonitales, both possessing dark spores. This suggests that spore pigmentation is an evolutionarily conservative character in myxogastrians, and that the simple morphology of echinostelids is not a derived feature.     

Ultrastructure of the thread cells in the slime gland of Japanese hagfishes,Paramyxine atami and Eptatretus burgeri

The thread cells in the slime gland of Japanese hagfishes, Paramyxine atami and Eptatretus burgeri were studied by light and electron microscopy. The mature thread cells are large elements (180 times 80 mu) filled with an intricately coiled thread, approximately 2 mu in diameter. The protein nature of the thread has been confirmed by histochemical examination. In the initial stage of growth, the thread consists of a bundle of distinctly parallel filaments approximately 90-120 A in diameter and a centrally located tubular component approximately 230-260 A in diameter which occurs singly or occasionally as a double and triple structure. The developing thread displays thin filaments, approximately 30-60 A in diameter. The thin filaments are composed of fine fibrous structures, subfilaments, approximately 10-30 A in diameter. On the outer surface of the thread a coating is apparent, giving it a fluffy appearance. Polysomal clusters consisting of five or six ribosomes are predominant. Fine fibrous structures are also found among the threads; they seem to have a spatial relationship with the polysomes and resemble the subfilament constituents of the thin filaments. From these results, it may be suggested that the fine fibrous structures synthesized by polysomes, twist together and coalesce into a thread. The problem of the polysome size and the molecular weight of the fibrous protein synthesized is discussed.     

Intraerythrocytic plasmodial calcium metabolism

Both prokaryotes and eukaryotes require Ca⁺⁺ for a variety of cellular functions. Intraerythrocytic plasmodia, however, exist within a cell ordinarily impermeable to external Ca⁺⁺. Our investigations of Ca⁺⁺ homeostasis in murine Plasmodium berghei reveal that (1) infected erythrocytes contain 10 – 15 times as much Ca⁺⁺ as do uninfected red cells, (2) these large amounts of Ca⁺⁺ are located almost exclusively within the parasite, and (3) the parasite obtains at least a portion of this Ca⁺⁺ through causing increased permeability of the host cell membrane to external Ca⁺⁺.