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FEMS MicrobiologyEcology31 (1985) 187-192 Published by Elsevier 187 FEC 00023 Endosymbiofic meth ogenic bacteria of the sapropelic amoeba Mastigella (Flagellated amoebae; Pelomyxa palustris," anaerobic protozoa) Johan J.A. van Bruggen, Claudius K. Stumm, Kor B. Zwart and Godfried D. Vogels Departmentof Microbiology,Facultyof Science, Universityof Nijmegen, Toernooivela~NL-6525 El) Nijmegen, The Netherlands Received11 April 1985 Revisionreceivedand accepted26 April 1985 1. SUMMARY Large numbers of Gram-positive methanogenic bacteria, which morphologically resembled Methanobacterium formicicum, were found in the flagellated amoeba Mastigella from fresh water sapropel. A non-methanogenic Gram-positive bacterium with a characteristic axial cleft was found to live in close association with the amoebal nucleus. The similarity between the symbiotic system of Mastigella and that of the giant amoeba Pelomyxa is discussed, as well as the contribution of sapropelic amoebae to the methanogenesis of the sapropel ecosystem. 2. INTRODUCTION Intracellular bacteria in sapropelic protozoa have been known to exist for many years [1-4]. Recently, it was shown in 8 sapropelic protozoa investigated, that the majority of these .endosymbionts were methanogenic bacteria [5]. The identification of the methanogens was documented by fluorescence microscopy [6], demonstration of coenzyme F420 and 7-methylpterin, and methane production by an isolated cell of the giant amoeba Pelomyxa palustris. Moreover, Methanobacterium formicicum was isolated from the ciliate Metopus striatus [7]. P. palustris was found to contain 2 types of methanogens [5] and a non-methanogenic Gram-positive rod with a characteristic axial cleft [8,9], whereas several organdies normally present in eukaryotic cells were absent. In this paper, we describe the endosymbionts of another sapropelic amoeba, Mastigella. Striking similarities were observed with the endosymbionts of the (taxonomically unrelated) amoeba P. palustris. 3. MATERIALS AND METHODS 3.1. Organisms The amoeba was studied in sapropel samples taken from several eutrophic ponds near Nijmegen and--as a constant source of the organism--from a mud-filled 200-1 aquarium in the laboratory. The organism was identified according to Goldschmidt [1] and Lemmermann [10]. 3.2. Microscopical procedures For a rapid identification of methanogenic bacteria, Leitz epifluorescence microscopy, according to Doddema and Vogets [6], was used. Differential interference contrast (DIC) microscopy 0168-6496/85/$03.30 © 1985 Federation of European MicrobiologicalSocieties 188 was performed with the Leitz T-device on an Ortholux microscope. Electron microscope preparations were made as reported previously [7] and were studied in a Philips EM300. 3.3. Quantification of amoebae Using a pipette, 27.7 ml water containing a small quantity of sapropel was sampled from the muddy surface of the aquarium. In a second experiment, 6 sapropel samples were taken from different places in the aquarium and pooled (total volume approx. 50 ml) and 4 samples (2.2 ml) were studied. The material was transferred to a petri dish and examined under a dissection microscope. Using a small pipette, virtually all the Mastigella and P. palustris cells present were collected and counted. The dry weight of the sapropel was determined after collection by membrane filtration and subsequent drying for 24 h at 75°C in vacuo. 3.4. Quantification of endosymbionts 200 Mastigella t,ells were subjected to 3 pipette transfers of 10 mM NazHPOa/KH2PO4 buffer, pH 7.0. The mean volume of the endoplasm of the cells was estimated by measuring the diameter of the cells, which had rounded after pipetting. Subsequently, the endosymbionts were released from the Mastigella cells by homogenisation in a defined buffer volume, and counted in a Bi~rker-T'urk counting chamber (W. Schreck, Hofheim/Ts, F.R.G.) by means of phase-contrast microscopy. 4. RESULTS 4.1. Description of the amoeba Under the light microscope, the uninucleate amoeba, with a diameter of 133 + 14 #m, showed a granular endoplasm with many ingested plant particles. The ectoplasm was hyaline with numerous pointed or blunt, erupting pseudopodia. The cell possessed a short flagellum (Fig. 1) which showed sweeping and curling movements; no contribution to the mobility of the amoeba by this organelle was observed. The prominent nucleus, with a diameter of about 35 /~m, showed periph- f Fig. 1. Living,migratingMastigellacellwithpointedand blunt pseudopodia, clear ecto- and granular endoplasm,and flagellum. ( --, ) Differentialinterferencecontrast (DIC), electronic flash. Bar represents20/~m. eral nucleoli (dispersed type, Fig. 2); no connection Was observed between the nucleus and the flagellum origin. Division stages of the nucleus were not observed. However, ceils with 2 nuclei were sometimes found, presumably representing a stage shortly prior to cytokinesis (Fig. 3). On the basis of these characteristics, the organism was identified as a member of the genus Mastigella, a group of flagellated amoebae. (Order, Pantostomatinae; family, Rhizomastigaceae. Lemmermann [10]). Its morphological characteristics were compared with those of some other Mastigella species (Table 1). Except for the diameter of the nucleus, the present organism most resembled M. vitrea. The presence of endosymbiotic bacteria in this organism had already been reported in 1907 by Goldschmidt [1], who also noticed the similarity of these endosymbionts with those found in P. palustris [8,9]. 4.2. Endosymbionts Throughout the endoplasm of the Mastigella cells, numerous slender rods (3.4 x 0.3 /~m) were 189 ? i 1 !i!~ ii~!iii~/!~ !i~.... Fig. 2. Optical transverse section of the nucleus of a riving Mastigella cell. The round bodies inside the nucleus are nucleolar granules. The surface of the nucleus is covered with thick rods, As a result of the rapid streaming of the cytoplasm the thickness of the bacterial layer is not uniform. DIC, electronic flash. Bar represents 10/~m. always observed. Flattening of the Mastigella cells by a slight pressure on the cover glass facilitated the visibility of these endosymbionts, which showed the characteristic bluish fluorescence of methanogenic bacteria (Fig. 4) and stained Gram-positively. Fig. 3. Binucleate Mastigella cell. The lower nucleus is slightly out of focus in this Giemsa-stained preparation. Both nuclei are surrounded by masses of thick rods. Bar represents 20/~m. Another endosymbiont always present was a thick rod (4.5 × 0.8 #m) surrounding the nucleus in a uni- or multicellular layer (Figs. 2 and 3). Aggregates of these bacteria were sometimes found separated from the nucleus in the endoplasm. This organism also stained Gram-positively but did not show fluorescence. The mean number of slender and thick rods per Table 1 Morphological properties of some Mastigella species Organism Cell diameter (~tm) Nucleus diameter (~m) Endosymbionts Flagellum length Mastigella sp. Mastigella vitrea Mastigella nitens Mastigellapolymastix 120-150 125-150 80,90 32-80 35 10-15 _a 18-20 present present _a absent 40-70 120-250 45 > 80 b * This paper Not mentioned. b 1-4 Flagella present. a References (~tm) [*] [1,10] [10] [11] 190 Table 2 N u m b e r s of amoebae and amoebae-derived methanogenic bacteria in the aquarium sapropel Mastigella Pelomyxa A m o e b a e / m l sapropel A m o e b a e / m g dried sapropel Methanogens/cell M e t h a n o g e n s / m g dried sapropel M e t h a n o g e n s / m l sapropel 52 _+29 a 106 22 x 103 2.3 x 106 1.1 x 106 9 +_7 a 16 50 x 103 b 0.8 >( 106 0.4 x 106 Mean + S D of 5 samples. b The mean diameter of Pelomyxa cells present was considered as 200 lam. The concentration of methanogenic bacteria is 1.2 x 101° per ml cytoplasm [5]. a Fig. 4. Epifluorescence micrograph of a single unfixed and slightly flattened Mastigella cell. The methanogenic bacteria are visible as thin rods. Thick rods are non-fluorescent and not visible in this micrograph. Bar represents 10/xm. Fig. 5. Ultra-thin section of Mastigella demonstrating the 2 endosymbiotic bacteria. The thick rod-shaped bacteria contain a typical axial cleft ( ---, ) and are separated from the nucleus only by a thin layer of vesicles. The thin rods are highly osmiophilic and, as shown in the inset, contain mesosome-like structures ( ~ ). Bar represents 1 #m. N = nucleus. 191 cell were estimated at 22000 and 6500, respectively. Since the amoeba endoplasm had a mean volume of 0.7 x 10 -6 ml, the numbers of bacteria correspond to a concentration per ml endoplasm of 3 × 101° and 0.9 × 101° for slender and thick rods, respectively. 4.3. Ultrastructure of Mastigella Electron microscopy revealed that Mastigella cells lack mitochondria, Golgi apparatus and microbodies. The ultrastructure of the nucleus is similar to that of P. palustris [8,9], but inclusions like sand and paraglycogen grains, which are typical for the giant amoeba P. palustris, were not found in Mastigella. 2 Types of endosymbionts were observed in the cytoplasm. Slender, osmiophilic, Gram-positive rods, with conically pointed ends, containing mesosome-like structures, were encountered throughout the cytoplasm. These slender endosymbionts, considered to be methanogenic bacteria, strongly resembled Methano- bacterium formicicum. The second endosymbiont, a thick Gram-positive rod, had a rectangular shape and a deep, incision-like cleft along the whole length of the cell (Fig. 5). Many thick rods were observed in close association with the nucleus, separated from it only by a thin layer of vesicles. This bacterium is probably identical to the thick rod found in P. palustris [8,9]. Both endosymbionts were separated from the amoeba cytoplasm by a membrane and were actively growing, as was apparent from the frequent observation of dividing bacteria. 4.4. Numbers of amoebae in the sapropel Both Mastigella and P. palustris cells were present in great numbers in the upper layer of the aquarium sapropel (Table 2). Mastigella cells were fairly constant in size, whereas the P. palustris cell diameter ranged from 100-1000 #m. The mean concentration of amoeba-derived methanogens in the upper layer of the sapropel was estimated to be 1.5 × 106/ml. This is a minimum concentration, since it is likely that some amoebae escaped collection from the mud samples. 5. DISCUSSION 5.1. Characterization of Mastigella The amoeba described here and found ubiquitously in the upper layers of the sapropel was identified as a Mastigella species and resembled M. vitrea. The cells contained large numbers of methanogenic and non-methanogenic endosymbiotic bacteria. The methanogens resembled morphologically the Gram-positive methanogens occurring in P. palustris (manuscript in preparation), another sapropel amoeba. The non-methanogenic thick rods are most probably identical to the thick rods of P. palustris [9]. Apart from the endosymbionts, the ultrastructures of both anaerobic amoebae share many features. Mitochondria, Golgi apparatus and microbodies are absent. In contrast to the multinucleated Pelomyxa, Mastigella has a single nucleus and contains no paraglycogen. The presence of methanogenic endosymbionts is not unique to anaerobic amoebae, but seems to be a common feature among sapropel protozoa [5]. 5.2. Function of the endosymbionts No attempts have yet been made to isolate and characterise the methanogenic endosymbionts of Mastigella. Therefore, no information is available on the substrate specificity of this organism. However, hydrogen or formate are common substrates for methanogenic rods, and probably also for the methanogenic rods of Mastigella. As a result of methane production by these endosymbionts, the intracellular hydrogen levels may remain very low and, as a consequence, the eukaryote can easily reoxidize its reduced electron acceptors and profit from an energetically favourable situation. The question arises as to where the reduction equivalents are produced, as hydrogen or formate, in the Mastigella cell. Except for P. palustris and the now described Mastigella species, all anaerobic sapropel protozoa so far investigated ([7] and unpublished results) contain hydrogenosome-like microbodies which are usually closely associated with the methanogenic bacteria in the cells. It is not likely that hydrogen has a cytoplasmic origin in both anaerobic amoebae, since hydrogenase appears to be organelle-bound in all eukaryotic cells 192 studied. For that reason, it is tempting to suggest that the non-methanogenic, thick rod serves as a hydrogen source for the methanogens. Such a metabolic interaction could be a remarkable example of inter-endosymbiotic hydrogen transfer, avoiding the disadvantage of mass transfer resistance. Unfortunately, all attempts so far to grow the thick rod in pure or mixed cultures have failed, and its substrate spectrum remains unknown. The close association of these bacteria with the nucleus strongly suggests a mutual metabolic relationship. In spite of the presence of a flagellum, Mastigella and Pelomyxa must be considered as descendants of early stages in eukaryotic evolution, since organelles (except for the nucleus) are absent and endosymbiotic bacteria seem to be essential for growth of these primitive eukaryotes. This view differs strongly from that of Bovee and Jahn [12], who suggested that Mastigella might have evolved from mitochondria-containing amoebae. 5.3. Contribution of amoeba-derived methanogens to total methane production The numbers of methanogenic bacteria reported in the sapropel vary with the methods applied and with the kind of sediment analysed. Approx. 1.6 × 105 methanogens per g wet sediment were found by applying a most-probablenumber method [13], whereas 5 × 104 methanogens per ml wet sediment were found by determining the number of colony-forming units [14] and 105-109 methanogens per g dry sediment were estimated by means of a direct fluorescent-antibody technique [15]. The presence of at least 1.5 × 106 amoeba-derived endosymbiotic methanogenic bacteria per ml of the upper layer of the sapropel clearly indicates that such methanogens contribute considerably to the methane production in this part of the sapropel. ACKNOWLEDGEMENTS We thank Dr. C. Chapman-Andresen, Dr. F.C. Page and Mr. F.J. Siemensma for their valuable suggestions with regard to the identification of the organism. REFERENCES [1] Goldschmidt, R. (1907) Lebensgeschichte der MastigamiSben Mastigella vitrea n. sp. und Mastigina setosa n. sp. Arch. Protistenk. 1 (Suppl.), 83-168. [2] Gould-Veley, L.J. (1905) A further contribution to the study of Pelomyxa palustris (Greeff). J. Linn. Soc. 29, 374-395. [3] Kahl, A. (1930-1935) Wimpertiere oder Ciliata (Infusoria), in Die Tierwelt Deutschlands (Dahl, Ed.), parts 18, 21, 25 and 30. Fischer, Jena. [4] Liebmann, H. (1937) Bakteriensymbiose bei Faulschlammciliaten. Biol. Zbl. 57, 442-445. [5] Van Bruggen, J.J.A., Stumm, C.K. and Vogels, G.D. (1983) Symbiosis of methanogenic bacteria and sapropelic protozoa. Arch. Microbiol. 136, 89-95. [6] Doddema, H.J. and Vogels, G.D. (1978) Improved identification of methanogenic bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 36, 752-754. [7] Van Bruggen, J.J.A., Zwart, K.B., Van Assema, R.M., Stumm, C.K. and Vogels, G.D. (1984) Methanobacteriurn formicicum, an endosymbiont of the anaerobic ciliate Metopus striatus McMurrich. Arch. Microbiol. 139, 1-7. [8] Chapman-Andresen, C. (1971) Biology of the large amoebae. Ann. Rev. Microbiol. 25, 27-48. [9] Whatley, J.M. (1976) Bacteria and nuclei in Pelomyxa palustris: comments on the theory of serial endosymbiosis. New Phytol. 76, 111-120. [10] Lemmermann, E. (1914) Flaggellatae, I, in Die Stlsswasserflora Deutschlands, Osterreichs and der Schweiz (Pascher, A., Ed.), pp. 43-46. Fischer, Jena. [11] Frenzel, J. (1892) Untersuchungen ilber die mikroskopische Fauna Argentiniens, I. Die Protozoen. Bibliotheca Zoologica 12, 38-40. [12] Bovee, E.C. and Jahn, T.L. (1973) Taxonomy and phylogeny, in The Biology of Amoebae (Jeon, K.W., Ed.), pp. 37-82. Academic Press, New York. [13] Zaiss, U. (1981) Seasonal studies of methanogenesis and desulfurication in sediments of the River Saar. Zbl. Bakt. Hyg., I. Abt. Orig. C2, 76-89. [14] Cappenberg, Th.E. and Verdouw, H. (1982) Sedimentation and breakdown kinetics of organic matter in the anaerobic zone of Lake Vechten. Hydrobiologia 95, 165-179. [15] Ward, T.E. and Frea, J.I. (1980) Sediment distribution of methanogenic bacteria in Lake Erie and Cleveland Harbor. Appl. Environ. Microbiol. 39, 597-603.