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.     

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 .     

Pelomyxa palustris Greeff

Summary 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.Mitochondria, Golgi bodies, contactile vacuoles, and crystal vacuoles were definitely absent in P. palustris. The cytoplasm contained many food vacuoles and clear vacuoles of various sizes. Vacuole-like aggregations, probably containing glycogen, were present.Work supported by the U. S. Atomic Energy Commission.     

Fine Structure and Taxonomic Position of the Giant Amoeboid Flagellate Pelomyxa palustris1,2

ABSTRACT 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 μm 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.     

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.     

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.     

The Free Amino Acid Levels of Pelomyxa carolinensis,Amoeba dubia and Proteus*

SYNOPSIS. The free amino acid extracts of 3 species of the free-living amoebae, Pelomyxa carolinensis, Amoeba dubia and A. proteus, were studied. Both the free amino acid patterns and levels in each species were different. Altho it appears that the free amino acid patterns were not altered by culture routines or starvation, the free amino acid levels were altered by these factors. The possibility of using free amino acid levels as a criterion for recognizing genetic differences is discussed.     

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 morphological study of the cysts Pelomyxa palustris Greeff, 1874

Pelomyxa palustris Greeff, 1874, is the only species of pelomixoid amoebas with the rest cysts in its life cycle. The morphology of the P. palustris has been studied by the light and electronic microscopy. Encystation of P. palustris under climatic conditions of North-West of Russia occurs within August-September. Rest cysts have a complex, trilaminar wall. Two inner lamina are the dense endocyst and the laminated mesocyst, thickness of each layer runs up to 0.6-0.7 microm. Thickness of the electron-dense ectocyst usually does not exceed 0.1-0.2 microm. The encystated cell of P. palustris has the unique structure. About 60 % of the cell volume are occupied by a huge vacuole placed in the center and filled up with the prokaryotic cytobionts. Different vacuoles, small vesicles of various nature, autophagosomes and lipid drops could be found inside that huge vacuole. The amoebae cytoplasm occupies the space in between endocyst's inner surface and the central vacuole. No any inclusions, prokaryotic cytobionts and most of cell organelles are absent in the cytoplasm. There are 4 large nuclei filled with relatively homogeneous karyoplasm lying in the cytoplasm. Nuclear envelope forms a lot of long tubular channels, running through the cytoplasm and lining the membrane of the central vacuole. Encysted pelomixoid stay in this state up until the beginning of excystation. Excystation of P. palustris in the studied region occurs in spring, during the latter half of April and the beginning of May. Cysts undergo complex morphofunctional changes, related to the reorganization of the wall and formation of young multinucleate amoebas. Only one wall lamina of the 3 initial ones is left up to the moment of excystation. The central vacuole endures ruination and its content penetrates into the cytoplasm. Pelomixoid nuclei divide twice. Prokaryotic cytobionts are localized in cytoplasm and in the perinuclear area. Young multinuclear species of P. palustris coming out of the cysts do not differ in their structure from the adult forms.     

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.     

Pelomyxa palustris Greeff. I. Cultivation and general observations

SUMMARY. A rich source of supply of Pelomyxa palustris has recently been found. The findings and views of previous investigators on the geographical and seasonal distribution, longevity in the laboratory and reproduction of this organism are reviewed and discussed. Clone and mass cultures were successfully obtained in Carrel flasks with Spirogyra as food. Feeding is an extremely slow process so that this Pelomyxa is incapable of capturing and ingesting actively motile organisms under ordinary circumstances. Multiplication is solely by simple or multiple plasmotomy.     

The Protein Amino Acid Compositions of Amoeba proteus,A. discoides,A. dubia and Pelomyxa carolinensis*

SYNOPSIS. A comparison has been made of the protein amino acid compositions of Amoeba proteus, A. discoides, A. dubia and Pelomyxa carolinensis. The protein amino acid compositions of each of these species differed in 1–3 of the following amino acids: arginine, aspartic acid-asparagine, threonine and glycine. The possibility of using these characteristics as acceptable genetic markers is discussed.     

A Lecithinase from the Amoeba Hartmannella rhysodes*

SYNOPSIS A lipogenic toxin produced by the amoeba Hartmannella rhysodes, Fernald strain, made mammalian cells in culture round up and fill with fat droplets. From this toxin an enzyme was obtained with lecithinase and lysolecithinase activities. This enzyme is different from any isolated elsewhere. When purified enzyme was added to strain L mouse fibroblasts in culture, the cells rounded up and became fatty. Much of the lipogenic activity of the original toxin can be ascribed to this enzyme.     

Contractility of Glycerinated Amoeba proteus and Chaos-chaos*

Immediate contact with large volumes of cold 50% (v/v) buffered glycerol preserved typical ameboid shape of Chaos chaos and Amoeba proteus with no visible distortions. These technics allowed determination of the contraction sites in these glycerinated models upon application of ATP-Ca-Mg-solutions. The ectoplasmic tube was the main site of contraction. Preliminary EM investigations revealed thick and thin filaments, associated with the ectoplasmic tube near the plasmalemma, which appeared to be the basis for the contractility of the ectoplasmic tube. There was no predominant contraction of the pseudopodial tips or the endoplasm in these models. The changes of volume were as much as 50%, and in some cases were not accompanied by any change in the length of the ameba; however, lengthwise contractions of the ectoplasmic tube in some amebae occurred to as much as 25%. The data substantiate a basic requirement of the ectoplasmic tube contraction theory of ameboid locomotion.