Pelomyxa Research Articles

Identification of Pelomyxa palustris Endosymbionts

Pelomyxa palustris is a giant anaerobic/microaerobic amoeba, characterized by a number of exceptional cytological and physiological features, among them the presumed absence of energy producing organelles and the presence of endosymbiotic bacteria. These endosymbionts have been previously distinguished as: a large rectangular-shaped Gram-variable rod with a central cleft; a slender Gram-negative rod; and a slender Gram-positive rod. Using DNA extracted from P. palustris cysts, we have obtained three SSU rRNA gene sequences. We have determined that these sequences are affiliated to three different prokaryotic genera: Methanosaeta (a methanogenic archaea), Syntrophorhabdus (a syntrophic Gram-negative bacteria) and Rhodococcus (an aerobic chemoorganotrophic Gram-positive bacteria). To our knowledge, it is the first time that Syntrophorhabdus has been described as an endosymbiont in association with a methanogen. Strikingly, no traces of Methanobacterium formicicum could be detected, despite this methanogen had allegedly been isolated from trophozoites of P. palustris. It seems that the host and the endosymbionts have established a multipartite syntrophic consortium resembling to some extent those found in sewage treatment plants.

Evolution of Archamoebae: Morphological and Molecular Evidence for Pelobionts Including Rhizomastix, Entamoeba, Iodamoeba, and Endolimax

The archamoebae form a small clade of anaerobic/microaerophilic flagellates or amoebae, comprising the pelobionts (mastigamoebids and pelomyxids) and the entamoebae. It is a member of the eukaryotic supergroup Amoebozoa. We examined 22 strains of 13 species of Mastigamoeba, Pelomyxa and Rhizomastix by light-microscopy and determined their SSU rRNA gene sequences. The SSU rRNA gene sequences of Pelomyxa palustris and Mastigella commutans in GenBank are shown to belong to P. stagnalis and Mastigamoeba punctachora, respectively. Five new species of free-living archamoebae are described: Mastigamoeba abducta, M. errans, M. guttula, M. lenta, and Rhizomastix libera spp. nov. A species of Mastigamoeba possibly living endosymbiotically in Pelomyxa was identified. Rhizomastix libera, the first known free-living member of that genus, is shown to be an archamoeba. R. libera possesses an ultrastructure unique within archamoebae: a rhizostyle formed from a modified microtubular cone and a flagellum with vanes. While many nominal species of pelobionts are extremely hard to distinguish by light microscopy, transient pseudopodial characters are worthy of further investigation as taxonomic markers.

Draft Genome Sequence of Methanobacterium formicicum DSM 3637, an Archaebacterium Isolated from the Methane Producer Amoeba Pelomyxa palustris

Here is reported the draft genome sequence of Methanobacterium formicicum DSM 3637, which was isolated from the methane-producing amoeba Pelomyxa palustris. This bacterium was determined to be an endosymbiont living in the cytoplasm of P. palustris and the source of methane; however, the global characteristics of its genome suggest a free-living lifestyle rather than an endosymbiotic one.

Evolution: Revisiting the Root of the Eukaryote Tree

A recent phylogenomic investigation shows that the enigmatic flagellate Breviata is a distinct anaerobic lineage within the eukaryote super-group Amoebozoa and challenges the unikont-bikont rooting of the tree of eukaryotes.

Morphological study of cysts of Pelomyxa palustris Greeff, 1874

Pelomyxa palustris Greeff, 1874, is the only representative of pelomyxoid amoebae with rest cysts in its life cycle. The morphology of the P. palustris cysts was studied using light and electron microscopy. The encystation of P. palustris under the climatic conditions of northwestern Russia occurs in August through September. The rest of the cysts have complex, trilaminar walls. The most developed are the two inner layers, i.e., the electron-dense structureless endocyst and the laminated mesocyst; the thickness of each layer can reach 0.6–0.7 μm. The thickness of the superficial electron-dense ectocyst lamina usually does not exceed 0.1–0.2 μm. The encysted cell of P. palustris has a unique structure. About 60% of its cell volume is occupied by a giant central vacuole filled with prokaryotic cytobionts. This vacuole has also been found to contain vacuoles and vesicles of different natures, restricted by vacuole membranes, autophagosomes, and lipid droplets. The amoeba cytoplasm occupies the space between the endocyst inner surface and the central vacuole. It contains no inclusions, prokaryotic cytobionts and most of cell organelles. In the cytoplasm there are 4 large nuclei filled with relatively homogeneous karyoplasm. The nuclear envelope forms numerous long tubular outgrowths, piercing the cytoplasm and underlining the central vacuole membrane. In this state the encysted pelomyxoids survive until the beginning of excystation. The excystation of P. palustris in the studied region occurs in spring, during the second half of April through the beginning of May. The cysts undergo complex morphofunctional changes due to reorganization of the wall and formation of young multinucleate amoebae. Out of the three initial lamina of the wall, only one persists until the moment of encystation. The central vacuole is destroyed and its content penetrates into the cytoplasm. Pelomyxoid nuclei divide twice. Prokaryotic cytobionts are localized in the cytoplasm and in the perinuclear area. Young multinuclear P. palustris individuals exiting cysts do not differ from the adult forms by their organization.

The unusually long small subunit ribosomal RNA gene found in amitochondriate amoeboflagellate Pelomyxa palustris: its rRNA predicted secondary structure and phylogenetic implication

In order to ascertain a phylogenetic position of the freshwater amitochondriate amoeboflagellate Pelomyxa palustris its small subunit (SSU) rRNA gene was amplified and sequenced. It was shown to be 3502 bp long. The predicted secondary structure of its rRNA includes at least 16 separate expansion zones located in all the variable regions (V1–V9), as well as in some conservative gene regions. Most insertions are represented by sequences of low complexity that have presumably arisen by a slippage mechanism. Relatively conservative, uniformly positioned motifs contained in regions V4 and V7, as well as in some others, made it possible to perform folding. In maximum likelihood, maximum parsimony, and neighbor-joining trees, P. palustris tends to cluster with amitochondriate and secondary lost mitochondria amoebae and amoeboflagellates Entamoeba, Endolimax nana, and Phreatamoeba balamuthi, comprising together with them and aerobic lobose amoebae Vannella, Acanthamoeba, Balamuthia, and Hartmannella a monophyletic cluster. Another pelobiont, Mastigamoeba invertens, does not belong to this cluster. No specific similarity was discovered between the SSU rRNA of P. palustris and amitochondriate taxa of ‘Archezoa’: Diplomonada, Parabasalia, Microsporidia. Pelomyxa palustris SSU rRNA does not occupy a basal position in the phylogenetic trees and could be ascribed to the so-called eukaryotic ‘crown’ group if the composition of the latter were not so sensitive to the methods of tree building. Thus, molecular and morphological data suggest that P. palustris represents a secondarily modified eukaryotic lineage.

Reproduction of nuclei in Pelomyxa palustris

Light and electron micrographs were made of nuclei in Pelomyxa palustris, a unicellular, multinucleated giant amoeboid organism. We analyzed 1019 pelomyxae and classified their nuclei according to their location in the nuclear cycle. The majority of organisms (56.3%) had interphase nuclei, some of which contained spores of mostly 1-3 μm in diameter. The nuclei had disintegrated in 1.3 % of organisms that appeared to have no nuclei. The remainder (42.4%) had nuclei in the form of spores (1 to 10 μm spheroids) that were in various stages of development and growth. Mitotic figures were seen in some of them, with several chromosome pairs per nucleus. Interchromosomal fibers were seen at anaphase, and newly formed "young" interphase nuclei were observed.

Systematic and morphological diversity of endosymbiotic methanogens in anaerobic ciliates.

The identities and taxonomic diversity of the endosymbiotic methanogens from the anaerobic protozoa Metopus contortus, Metopus striatus, Metopus palaeformis, Trimyema sp. and Pelomyxa palustris were determined by comparative analysis of their 16S ribosomal RNA sequences. Fluorescent oligonucleotide probes were designed to bind to the symbiont rRNA sequences and to provide direct visual evidence of their origins from methanogenic archaea contained within the host cells. Confocal microscopy was used to analyze the morphology of the endosymbionts in whole cells of Metopus palaeformis, Metopus contortus, Trimyema sp, and Cyclidium porcatum. The endosymbionts are taxonomically diverse and are drawn from three different genera; Methanobacterium, Methanocorpusculum and Methanoplanus. In every case the symbionts are closely related to, but different from, free-living methanogens for which sequences are available. It is thus apparent that symbioses have been formed repeatedly and independently. Ciliates which are unrelated to each other (Trimyema sp. and Metopus contortus) may contain symbionts which are closely related, and congeneric ciliates (Metopus palaeformis and M. contortus) may contain symbionts which are distantly related to each other. This suggests that some of the symbiotic associations must be relatively recent. For example, at least one of the symbioses in Metopus must postdate the speciation of M. palaeformis and M. contortus. Despite this, Metopus contortus, Trimyema sp., Cyclidium porcatum and their respective endosymbionts show sophisticated morphological interactions which probably facilitate the exchange of materials between the partners.

On the abundance and distribution of protozoa and their food in a productive freshwater pond

We have examined and quantified the protozoa living in a productive freshwater pond during a 2-day period in June 1987. Over 90 species were recognised. The planktonic and benthic communities were dominated by ciliates and heterotrophic flagellates although the large amoeba Pelomyxa palustris was abundant (102 ml(-1)) in anaerobic sediments. Picoplankton averaged 1.4 × 10(7) ml(-1), phototrophic nanoplankton 0.8 × 10(5) ml(-1), heterotrophic nanoplankton 0.9 × 10(5) ml(-1) and planktonic ciliates 1.3 × 10(2) ml(-1). Numbers were about two orders of magnitude higher in the sediment. Protozoan biomass ranged from 3% to 61% of the total plankton biomass. Heterotrophic flagellates were the principal grazers of the picoplankton. Planktonic ciliates fed mainly on phototrophic nanoplankton but they probably also ingested heterotrophic flagellates. Benthic ciliates were predominantly bactivorous. Competition between ciliate species was minimised by both spatial and food niche separation. Ten species of planktonic ciliates appeared to contain algal symbionts: one species (Strombidium viride) contained structures resembling sequestered chloroplasts. These findings concerning the diversity and abundance of protozoa in a freshwater pond are consistent with the consensus opinion expressed in the marine literature that protozoa play a fundamental role in microbial food webs within aquatic ecosystems.

Fine structure and taxonomic position of the giant amoeboid flagellate Pelomyxa palustris.

Specimens of Pelomyxa palustris from five collecting sites had numerous nonmotile flagella. The structures are called flagella because of morphological similarities to flagella and because P. palustris has affinities with amoeboid flagellates. Flagella were photographed on living cells and studied by transmission and scanning electron microscopy. From 64 to 742 flagella per cell were estimated from scanning electron microscopy of ten cells 204 to 1269 micron in length. The nonmotile flagella arise from basal granules which were, in one strain, surrounded by radiating electron-dense microtubules. This strain also had excess axonemal microtubules. Abundant cytoplasmic microtubules were arranged in several different patterns. In about half of the P. palustris cells in which nuclei were studied, microtubules were either apposed to the nuclear membrane in a parallel alignment (with some also radiating) or radiating from the nuclear membrane (with none parallel). Bacteria associated with nuclei were of three characteristic types: Gram-negative rods, Gram-positive rods, and large rods. All nuclei within a given trophozoite had similar perinuclear features. Recent proposals for separation of Pelomyxa to its own phylum (based on its proposed primitive, unique nature) can not be justified. Pelomyxa is a complex, highly specialized organism adapted to live in a specific fresh-water environment. Mastigamoebid amoeboid flagellates of the genera Mastigamoeba, Mastigella, Mastigina, and possibly Dinamoeba are placed with Pelomyxa within the order Pelobiontida Page, 1976, emend., containing two families. Pelomyxidae Schulze, 1877, and Mastigamoebidae Goldschmidt, 1907.

Isolation of a Methanogenic Endosymbiont of the Sapropelic Amoeba Pelomyxa palustris Greeff

ABSTRACT. One of the two methanogenic endosymbionts of the giant sapropelic amoeba Pelomyxa palustris was isolated in pure culture. The cells were slender non‐motile rods (3 × 0.4 μm), sometimes occurring in chains of 3–4 cells. Ultrathin sections revealed a Gram‐positive cell wall and conically pointed ends with mesosome‐like structures in the cytoplasm. The isolate had a generation time of 10 and 12 h during growth on H2/CO2 and formate, respectively. The optimum growth temperature was 40°C and the optimum pH was 7.8. The G+C content of its DNA was found to be 37.7% mole percent. The isolate was identified as Methanobacterium formicicum.

The Extent of Algal and Bacterial Endosymbioses in Protozoa1,2

Long neglected has been the extensive and more or less intimate association of protozoa with a wide variety of other cells, either prokaryotic or eukaryotic in nature. Yet study of such relationships can provide important information concerning certain basic aspects of cellular evolution in general. A survey is offered here of the whole range of such symbiotic associations (i.e. with species of protozoa serving as hosts) with the purposes of drawing attention to the exciting possibilities of such research and of reviewing significant findings made to date. Because of the vastness of the overall field, examples and discussion are primarily limited to consideration of the following major studies: methanogenic bacteria in certain ciliates, bacterial endosymbionts of the large freshwater amoeba Pelomyxa palustris (itself an amazing organism from an evolutionary/phylogenetic point of view), the rod‐shaped bacteria found in Amoeba proteus, the “Greek‐letter” prokaryotes of Paramecium species, the xenosomes (sensu stricto) of the marine scuticociliate Parauronema acutum, and the diverse algal endosymbionts of similarly diverse protozoan taxa–ciliates, flagellates, radiolarians, acantharians, and foraminifera.

Symbiosis of methanogenic bacteria and sapropelic protozoa

Fluorescent bacteria were demonstrated to be abundantly spread as single cells throughout the cytoplasm of the giant amoeba Pelomyxa palustris, the sapropelic ciliate Metopus striatus and six other anaerobic protozoa examined. The endosymbionts of P. palustris and M. striatus were identified as methanogenic bacteria on the basis of the presence of the deazaflavin coenzyme F420 and the pterin compound F342. Moreover individuals of P. palustris produced methane over a long period of incubation. The number of methanogenic bacteria was above 1010 cells/ml protozoal cytoplasm. Two types of methanogenic bacteria together with unidentified thick bacteria were found in P. palustris. The physiological background of this endosymbiosis and its functioning in degradation processes in the anoxic environment are discussed.

Respiratory Studies on the Giant Amoeba Pelomyxa palustris1

ABSTRACT. The large fresh‐water microaerobic amoeba Pelomyxa palustris does not contain mitochondria, but three types of bacterial endosymbionts are always present. Thus, it is of interest in the discussion of the possible origin of mitochondria from primitive prokaryotes. Gas exchanges (O2, CO2) and concentration of endosymbionts were determined in individual amoebae, in which the life cycle stage was noted. Grey type (stationary phase) amoebae had a lower O2 uptake and lower endosymbiont concentration than light type (growth phase) amoebae, and highest O2 uptake was found in centrifugal pieces of light type Pelomyxa, centrifuged in vivo, which contained nearly all of the endosymbionts. In light type amoebae, the respiratory activity was independent of O2 concentration between 1 and 21%, and, when compared on the basis of dry weight and protein, of the same order as that of other free‐living protozoa. The R.Q. was slightly higher than 1, indicating that glycolysis does not play a significant role in energy metabolism. It is concluded that P. palustris is fully aerobic, and suggestions are presented as to the role of the endosymbionts in its respiratory metabolism.

The establishment of mitochondria: Paracoccus and Rhodopseudomonas.

Many aerobic bacteria (both facultative and obligate) possess a number of those biochemical features of mitochondria which are concerned with energy metabolism. However, only restricted number, notably Paracoccus denitrificans and Rhodopseudomonas spheroides, have the majority of these features. The theory of endosymbiosis proposes that a primitive eukaryote took up bacteria to yield mitochondria. The present-day Paracoccus then resembles the ancestral bacterium in many respects the primitive amoeba, Pelomyxa palustris, which lacks mitochondria but contains a permanent population of unique symbiotic bacteria, has many of the characteristics of a present-day transitional form. The evolution of mitochondria from endosymbiotic bacteria would involve their integration with the host cell both biochemically and structurally: a number of the intermediate steps are discussed. Attention is drawn to the existence in some ciliates of hydrogenosomes, which function as anaerobic mitochondria.

A numerical taxonomic study of six species of the free-living Amoeba

A study using six species of the free-living amoeba was carried out using multivariate analysis of ninety-seven morphological, physiological and ecological characters. The derived taxonomy indicates that 1. Amoeba discoides and Amoeba proteus are intraspecific instead of interspecific, 2. Chaos carolinensis and Chaos illinoisensis belong in the same genus, 3. the above two pairs are more closely related to each other than they are to Amoeba dubia and 4. all four species are distantly related to Pelomyxa palustris . Although the numerical taxonomix scheme is essentially identical to the natural scheme by Bovee and Jahn (1973) it differs from that postulated by Kudo (1966). This study is also in agreement with the limited serological studies that have so far been carried out. (Taxonomy multivariate analysis, Amoeba discoides, Amoeba proteus, Amoeba dubia, Chaos carolinensis, Chaos illinoisensis, Pelomyxa palustris .)

BACTERIA AND NUCLEI IN PELOMYXA PALUSTRIS: COMMENTS ON THE THEORY OF SERIAL ENDOSYMBIOSIS

SUMMARYThe primitive giant amoeba, Pelomyxa palustris, contains a population of endosymbiotic bacteria. The distinctive ultrastructure of one of the species of bacteria is described in detail. A particularly close relationship exists between these bacteria and the nuclei of Pelomyxa. The significance of some aspects of this relationship is discussed with reference to the endosymbiotic theory of the origin of mitochondria from bacterial precursors.

Membrane activity in freshwater amebae.

SYNOPSIS. The constantly changing shape and surface area/volume ratio of Chaos and Amoeba provide sufficient evidence for continuous membrane activity during locomotion, endocytosis, and cell division. Factors influencing membrane intake and renewal are discussed on the basis of data from tracer experiments and from studies on fine structure. The concept of permanent pinocytosis as an essential part of locomotion is discussed, together with the differences between induced and permanent pinocytosis and the factors which might be responsible for the “induction” of permanent pinocytosis. The large saprobiotic ameba, Pelomyxa palustris differs from Chaos and Amoeba in membrane distribution. P. palustris is generally monopseudopodial, and changes in its shape are more limited. The mucous coat, less pronounced in this species, is most prominent at the very active uroid, the site of endocytosis. The membrane renewal cycle in Pelomyxa has not been studied to date.

The Effects of Weightlessness on Pelomyxa II. Nuclear and Cellular Division

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Pelomyxa palustris Greeff

The Pelomyxa palustris amoebae used in this study were multinucleate, herbivorous protozoans. All nuclei within a single organism were similar, but several types of nuclei were seen in different amoeba. These nuclei might represent various stages of mitosis although metaphase and anaphase stages were never seen. Rod-shaped bacteria within vesicles characteristically surrounded the nuclei. Bacterial rods of this as well as another type also occurred within vesicles in the cytoplasm. The nuclear envelope contained annuli and it was covered externally by minute vesicles. Nucleoli and micronucleoli were most frequently located along the inner surface of the nuclear envelope. Clusters of electron-opaque spheroids were found within the nucleoli; sometimes, they existed free in the nucleoplasm. Intranuclear globules of lipidlike material were often seen.