Hydrogen-oxidizing Methane Bacteria

JOURNAL OF BACTERIOLOGY, Mar. 1968, p. 1124-1129 Vol. 95, No. 3 Copyright 1968 American Society for Microbiology Printed in U.S.A. Hydrogen-oxidizing Methane Bacteria II. Electron Microscopy K. F. LANGENBERG, M. P. BRYANT, AND R. S. WOLFE Departmenits of Microbiology and Dairy Science, University of Illiniois, Urbana, Illin2ois 61801 Received for publication 29 December 1967 Comparison of the fine structure of the methanogenic organism from the culture known as Methanobacillus omelianskii with that of Methanobacterium formicicum revealed a great similarity. Both organisms exhibited a large number of intracytoplasmic membranous elements when stained with phosphotungstic acid. In contrast, no such elements were observed in Methanobacterium ruminantium. The taxonomic position of Methanobacillus omelianskii (Methanobacterium omelianskii) (2) has been uncertain for many years, and the organism has been regarded as a species incertae sedis of the family Bacillaceae (Bergey's Manual, 7th ed.). It has now been shown (6a) that the culture known as Methanobacillus omelianskii consists of an association of two distinct organisms: a motile rod which ferments ethyl alcohol to acetate and hydrogen (S organism), and a methanogenic, nonmotile rod which oxidizes hydrogen with the reduction of CO2 to CH4 (Methanobacterium strain M.o.H.). A study of the taxonomic position of these organisms is being pursued by one of us (M. P. Bryant). In this communication, the fine structure of Methanobacterium strain M.o.H. is compared with that of two other methane bacteria, which use H2 as the energy source and which reduce CO2. We present evidence that the fine structure of Methanobacterium strain M.o.H. is similar to that of M. formicicum (10), but dissimilar to that of M. ruminantium (11). MATERIALS AND METHODS Organisms and culture conditions. M. formicicum was kindly provided by Paul H. Smith. M. ruminiantium (strain Ml) and Methanobacterium strain M.o.H. were from our collection. Each organism was maintained and cultivated by the techniques described in the accompanying communication (6). Electron microscopy. A Jeolco, model JEM-T6S, electron microscope (Japan Electron Optics Laboratory Co., Ltd., Tokyo, Japan) was used for this study. It was fitted with self-cleaning thin-metal objective apertures (30, 50, and 70, in diameter) manufactured by C. W. French, Weston, Mass. Electron micrographs were made on Kodak projector slide plates, contrast grade. All specimens were mounted on carbon-coated, Formvar-filmed, 200-mesh perforated copper grids. Formvar films were cast on glass from 0.3% (w/v) chloroform solutions, as outlined by Bradlay (5), and were lightly coated with vacuum-evaporated carbon. Whole cells were negatively stained with a 0.02% (w/v) aqueous solution of phosphotungstic acid (PTA), which was adjusted to ph 7.0 with potassium hydroxide. Washed cells were added to PTA solution to produce a faintly turbid suspension. With a Pasteur pipette, a drop of this suspension was transferred to a specimen support grid which was held by a clamped pair of forceps. The drop of cell suspension was allowed to dry slowly and undisturbed so that cells would settle evenly on the membrane. Higher concentrations (up to 2%, w/v) of PTA and also of ammonium molybdate occasionally were used to stain cells negatively. In such cases, a drop of water suspension of cells was placed on a support grid and was allowed to evaporate slowly. Just before the drop had completely dried, a drop of negative stain was applied and allowed to stand for about 30 sec. Excess stain was removed by blotting with filter paper touched to the edge of the grid. Aqueous suspensions of washed whole cells to be metal-shadowed were dried slowly on support grids, and were shadowed at an angle of 15 to 200 with chromium or platinum. Preshadowed carbon replicas were made by the technique of Dalitz, as modified by deboer and Spit (7). A small drop of washed-cell suspension was placed on a warm glass slide; a second glass slide was used to make a smear from the drop. After drying, the smear was shadowed with platinum at an angle of 200 and then was coated with a layer of vacuum-evaporated carbon approximately 200 A thick. The carbon film was scored with a razor blade into 2-mm squares, and then the whole slide was immersed in 5%/C hydrofluoric acid for 1 min. The squares were floated free onto the surface of dichromate-sulfuric acid cleaning solution. After 1 hr, during which time the bacterial cells were dissolved from the carbon replica, the squares were transferred serially by means of a glass spoon through three beakers of 1124

VOL. 95, 1968 HYDROGEN-UTILIZING METHANE BACTERIA. II distilled water to remove the acid and debris. Finally, the squares were picked up on specimen grids for examination by electron microscopy. For sectioning, cells were fixed with osmium tetroxide and were washed with 0.5% (w/v) uranyl acetate, by the method described by Kellenberger, Ryter, and Sechaud (9). Capsules were hardened ovemight at 40 C, and the next day and night, at 60 C. Ultrathin sections were cut with a glass knife in a MT-1 Porter-Blum Ultra-Microtome (Ivan Sorvall, Inc., Norwalk, Conn.). The general contrast of sections was increased by staining with heavy metals. Two methods were used for poststaining: (i) the high ph lead stain of Karnovsky (8) for 15 min, or (ii) 0.5% (w/v) aqueous uranyl acetate for 0.5 to 2 hr. The stain was washed away by dipping each grid repeatedly through the surface of each of three successive beakers of distilled water. RESULTS AND DISCUSSION Methanobacterium strain M.o.H. Negatively stained cells appeared turgid rather than rugose, indicating a gram-positive type of cell wall (12). Gram stains were variable, an observation which agrees with Barker's results (2). The dark patches distributed generally over the cells (Fig. 1 and 2) were the most conspicuous feature of negatively stained cells. These appear to be identical to the "intracytoplasmic membranous elements" observed by Bladen, Nylen, and Fitzgerald (4) in a Eubacterium from the rat cecum; by Bladen and Mergenhagen (3) in Veillonella; and by Zwillenberg (12) in Listeria monocytogenes. Large negatively stained intracytoplasmic membranous elements similar to the central inclusion(s) of the cells shown in Fig. 1 have been reported by Abram (1). The size and shape of these negatively stained internal elements varied from culture to culture. Several different types could be seen in a single cell (Fig. 1): doughnut shaped patches (p), clefts (c), and bag-like inclusions (s). Bladen et al. (4) have postulated a "pore" some 30 to 40 m,u in diameter in the cell wall surface. Various techniques were used in the present study in attempts to obtain a better picture of the postulated structures. Negatively stained cells, in which internal elements were clearly visible, were shadowed with metal from a small angle to show surface details. The lips of the presumed pores did not project perceptibly above the surface level of the cell (Fig. 2), nor was there any indication of an opening in the cell wall. Carbon replicas of the cell surface (Fig. 3) appeared smooth, corroborating the previous results. Round, membrane-bound intracytoplasmic bodies close to the cell membrane were seen in sections of osmium tetroxide-fixed cells (Fig. 4). These bodies (designated p) correspond in size 1125 to the negatively stained bodies previously observed, and are believed to be one and the same. No spores or sporelike bodies were observed in cells of strain M.o.H. M. formicicum. For comparison, another organism, M. formicicum, was studied. When examined electron microscopically, M. formicicum appeared similar to strain M.o.H. Almost all cells were found in clumps which could not be broken up by vigorous agitation in a vortex mixer. These clumps were bound by tightly meshed fibers approximately 7 m,u in diameter (Fig. 5). Very prominent negatively stained cytoplasmic membrane invaginations were observed, which generally appeared quite convoluted. The size and shape of cells observed were the same as for strain M.o.H. (Fig. 6 and 7). M. ruminantium. The appearance of M. ruminantium was as described by Smith and Hungate (11); cells occurred in chains of 2 to 25 cells. A closer look at the gross appearance of M. ruminantium was obtained by negative staining with low concentrations (0.01 to 0.02%) of PTA at ph 7; electron micrographs are presented in Fig. 8 and 9. The most striking features of these cells were the number of dividing cells and the complete absence of the intracytoplasmic membranous elements found in M.o.H. and M. formicicum by negative staining. Nearly all cells in actively growing cultures were in a state of cell division or multiple cell division, as indicated by one or more cross walls which appear as dark bands when negatively stained. Negatively stained cells appeared turgid, the cell surface appeared to be smooth, and the cell membrane did not look convoluted; these are characteristics of negatively stained gram-positive cells (12). Ultrathin sections revealed a sharply defined, smooth, and rigid cell wall as well as a granular cytoplasm which contained a prominent nuclear region (Fig. 10). Cell walls of M. ruminantium were triple-layered, with a total thickness of about 15 m,u. The innermost layer was very electron-dense and 1.5 to 2.0 m,u thick. The middle layer was less electron-dense and 7 to 10 m,u thick. The outer layer was slightly less electron-dense than the inner layer and also was 1.5 to 2.0 m,u thick. Cytoplasmic membranous bodies such as (CI) in Fig. 10 were observed often in sections of M. ruminantium, especially in older cells. The membranes enclosing these bodies appear to be identical to the cytoplasmic membrane, all being unit membranes of the same dimensions and electron density. These were not identified with any function of the cell, and no surface openings,

0 cw o :. S.f. ' X : ' e$ S,E, X* N s '...X2 eg. w.. / w... X. b. i.s8'... :. _ o f } e tw s * F,$! b tt > g., 4 E+' n ';*.' *.. E.:.,... ^ '-@ it. + ss tz ' si # WEe'.: w F " ([ '! ws t J S st4: 4':... @ fz$.r " g SE _' ;: L r t t E :. t.s is fi.. ;E t:... i,.e i R ; w 3r. he.,, - * >E'S '' i! * 4 rtzt t,,.t, >--s @ x.*t -.. vlb ;....; jo... L :-. i sr,u D :<ii 'lifo:.. S;,,,rS *e, +..:.sf w f is $r s: ^ff t... t: i,s W.. ^ w:slr.e: & vw,,,..x.e,,,.s. :,WW.. Sa^ v i.t.>.*..:.<#. +.. So, 9 :' {s.. :: _ )<ifs E; N X a,. F W.' 's 5,,. st. m Fe,.,. FIG. 1. Methanobacterium strain M.o.H., negatively stained with 0.02% phosphotungstate, at ph 7.0. Numerous internal structures are penetrated by stain and appear as patches (P), clefts (C) or pits, and sac-like inclusions (S). Surrounding the cells is material (X) which may originate from the internal structures. The cell wall is not visible. X 33,000. FIG. 2. Internal and external structure of Mo.H. The cell was stained with 0.01% phosphotungstate to reveal internal elements (i) and then was shadowed with chromium metal from an angle of 15 to show surface irregularities. No depressions or protrusions were observed in association with internal structures. X 33,000. FIG. 3. Carbon replica of the surface of Methanobacterium strain M.o.H. No surface structure was fowud associated with internal structure. A small bit of debris (D) is visible. X 33,000. FIG. 4. Section of Methanobacterium strain M.o.H. Cells are bounded by a triple-layered cell wall (CW). Pocket (P) is postulated to be an infolding of the cytoplasmic membrane and to correspond to the internal elements stained by phosphotungstate. The cytoplasm (Cyt) appears as a matrix of light and dark granules. Cells usually contained two shtarply defined nuclear regions (N). Also commonly found were centrally or terminally located cytoplasmic regions or granules (G) of medium electron density, thought to be polysaccharide storage material. X 60,000. 1126

VOL. 95, 1968 HYDROGEN-UTILIZING METHANE BACTERIA. II 1127 Downloaded from http://jb.asm.org/ 0 FIG. 5. Detail of fibrillar meshwork (F) which binds clumps of cells of Methanobacterium formicicum. Negatively stained with 2% phosphotungstate. X 72,000. FIG. 6. Clump of cells of Methanobacterium formicicum. Convoluted internal structures (i) like those fountd in Methanobacterium strain M.o.H. are found in all cells. X 26,000. FIG. 7. Isolated cell of Methanobacterium formicicum, free of fibrillar material. This cell is identical in size and appearance to M.o.H. Internal structures near the top (T.i.) and near the bottom (B.i.) surfaces of the cell may be seen. X 33,000. on October 21, 2018 by guest

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VOL. 95, 1968 HYDROGEN-UTILIZING METHANE BACTERIA.I1129 such as postulated in M.o.H., were seen in M. ruminantium. Most membranous bodies appeared to be located centrally near one side, thus placing them close to the site of new cell wall formation. Connection to the site of new wall formation was observed in a few cases. The initial stage of cell division in M. ruminantium was distinguished by a slight indentation of the cytoplasmic membrane accompanied by a break in the innermost layer of the cell wall and by a thickening of the middle layer of the cell wall. It was this thickening which was stained by PTA, producing the banded appearance of growing cells (Fig. 8 and 9). The similarities observed in the fine structure of strain M.o.H. and of M. formicicwn suggest that they are very closely related organisms. This is supported by our unpublished observations that M. formicicum is gram-variable, rather than gram-negative as originally indicated by Schnellen (10), and that colonies of the two organisms are practically identical. The only differences known are the fibrillar meshwork in the one strain of M. formicicum studied and the inability of strain M.o.H. to produce methane from formate. The relationship of mesosomes to the intracytoplasmic membranous elements in these two strains is not clear. ACKNOWLEDGMENTS We thank Janet Jeffery and Norman Ryckman for assistance, and Stanley C. Holt for suggestions on the sectioning of M. ruminantium. This investigation was supported by grant GB- 4481X from the National Science Foundation and by Public Health Service grant UI-SW00045-01. LITERATURE CrrED 1. ABRAM, D. 1965. Electron microscope observations on intact cells, protoplasts, and the cytoplasmic membrane of Bacillus stearothermophilus. J. Bacteriol. 89:855-873. 2. BARKER, H. A. 1940. Studies upon the methane fermentation. IV. The isolation and culture of Methanobacterium omelianskii. Antonie van Leeuwenhoek J. Microbiol. Serol. 6:201-220. 3. BLADEN, H. A., AND S. E. MERGENHAGEN. 1964. Ultrastructure of Veillonella and morphological correlation of an outer membrane with particles associated with endotoxic activity. J. Bacteriol. 88:1482-1492. 4. BLADEN, H. A., M. U. NYLEN, AND R. J. FITZ- GERALD. 1964. Internal structures of a Eubacterium sp. demonstrated by the negative staining technique. J. Bacteriol. 88:763-770. 5. BRADLEY, D. E. 1965. The preparation of specimen support films. In D. Kay [ed.], Electron microscopy, 2nd ed. F. A. Davis Co., Philadelphia. 6. BRYANT, M. P., B. C. MCBRIDE, AND R. S. WOLFE. 1968. Hydrogen-oxidizing methane bacteria. I. Cultivation and methanogenesis. J. Bacteriol. 95:1118-1123. 6a. BRYANT, M. P., E. A. WOLIN, M. J. WOLIN, AND R. S. WOLFE. 1967. Methanobacillus omelianskii, a symbiotic association of two species of bacteria. Arch. Mikrobiol. 59:20-31. 7. DEBOER, W. E., AND B. J. SPIT. 1964. A new type of bacterial cell wall structure revealed by replica technique. Antonie van Leeuwenhoek J. Microbiol. Serol. 30:239-248. 8. KARNOVSKY, M. J. 1961. Simple methods for "staining with lead" at high ph in electron microscopy. J. Cell Biol. 11:729-732. 9. KELLENBERGER, E., A. RYTER, AND J. SECHAUD. 1958. Electron microscope study of DNAcontaining plasms. II. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in different physiological states. J. Cell Biol. 4:671-678. 10. SCHNELLEN, C. G. T. P. 1947. Onderzoekingen over de methaangisting (Dissertation, Technical University, Delft). De Maasstad, Rotterdam. 11. SMITH, P. H., AND R. E. HUNGATE. 1958. Isolation and characterization of Methanobacterium ruminantium n. sp. J. Bacteriol. 75:713-718. 12. ZWILLENBERG, L. 0. 1964. Electron microscopic features of Gram-negative and Gram-positive bacteria embedded in phosphotungstate. Antonie van Leeuwenhoek J. Microbiol. Serol. 30 :154-162.