Archives of
Arch. Microbiol. I15, 207-213 (1977)
Hicrebialogy 9 by Springer-Verlag 1977
Electron Microscopic Investigation of the Hydrogen-Oxidizing Acetate-Forming Anaerobic Bacterium Acetobacterium woodii F R A N K MAYER, RUDI LURZ, and SIEGFRIED SCHOBERTH* Institut ffir Mikrobiologie der Universit~it G6ttingen, Grisebachstr. 8, D-3400 G6ttingen, Federal Republic of Germany
Abstract. Acetobacterium woodii is a Gram-positive anaerobic nonsporeforming bacterium able to grow on H2 and CO2 as sole sources of energy. The product of fermentation is acetic acid. Fine structural analysis showed rod-shaped flagellated cells, and coccoid cells without flagella arranged predominantly in pairs and chains. The cell wall was found to be composed of three layers. The cell surface exhibited a periodic array of particles consisting of subunits. The cytoplasmic membrane showed particles either in random distribution or in a hexagonal pattern. Intracytoplasmic membranes were rarely observed, whereas inclusion bodies of varying shapes, predominantly in an uncommon disc-shape, could frequently be observed. Their content was dissolved in ultrathin sections indicating hydrophobic nature. Key words: Acetobacterium woodii - Hydrogenoxidizing acetate-forming anaerobe - Fine structure - Electron microscopy.
Acetobacterium woodii, a new genus and species of strictly anaerobic nonsporeforming bacteria, was isolated recently in the laboratory of R. S. Wolfe (Schoberth and Balch, 1975; Balch et al., 1977). Like methane bacteria (Wolfe, 1971), A. woodii can grow o n ~7{2 and CO2 as sole sources of energy. The product of the fermentation carried out by this microorganism, however, is acetic acid and not methane. A. woodii also shows a homoacetic fermentation of fructose, and other compounds used as substrates are glucose, lactic, glyceric and formic acids. A. woodii seems to be abundant in nature and may play a considerable role * Present address: Institut ffir Biotechnologie der Kernforscbungsanlage Jiilich GmbH, D-5170 J/ilich, Federal Republic of Germany
in anaerobic methanogenic systems (Schoberth, 1977). The present paper describes the fine structure of A. woodii as seen in negatively stained, ultrathin sectioned and freeze-etched preparations.
MATERIALS AND METHODS Organism and Growth Conditions Acetobacterium woodii, type strain WB 1, isolated from a marine
estuary (Schoberth and Balch, 1975; Balch et al., 1977), kindly provided by W. E. Balch, was maintained in medium AM 6 in 5 ml volumes in Hungate tubes at 28~ Medium AM 6 contained in g/1 of glass distilled water (unless otherwise indicated) : fructose, 10.0; resazurine, 0.001; NH4CI, 1.0; 1 M K-phosphate buffer, pH 7.0, 5 ml; MgSOa - 7H20, 0./; yeast extract, 2.0; NaHCO3, 10.0; cysteine. HCI-HaO, 0.5; Na2S-9H20, 0.25; vitamin and mineral solutions (Wolin et al., 1963) were each added in a 20 ml amount; NaHCO3 was autoclaved separately as a 2 ~ solution; the gas phase was 80~o N 2 - 2 0 ~ CO2 (v/v). For stock cultures, ceils of A. woodii grown in medium AM 6 were transferred every four weeks into fresh medium9 Cells from stock cultures were also used for electron microscopic investigations by inoculating active cultures into medium AM 6. The amounts of cultures transferred were 0.03 and 0.5 ml. After overnight incubation at 28~ the cultures had reached optical densities of 0918 (A) and 0.75 (B) at 600 nm, corresponding to early and late logarithmic phase. Cultures A and B were used as indicated in the results section. For freeze-etching, cells grown in fermentors under a gas phase of H2 and CO, in a medium without fructose were used. The medium already described (Schoberth, 1977) was modified as follows: it contained in g/10l of glass distilled water (unless otherwise indicated): resazurine, 0.01; K2HPOr KHzPO4 and (NH)zSOr 2.5 each; NaC1, 0.5; MgSO4.7H20, 2.75; CaCla. 2H20, 0.63; FeSO4-7H20, 0.25; Na2WO4 - 2 H20, NazMoO4.2 HaO, and Na2SeO3 9 5 H20, 0,00025 each; yeast extract and tryptone, 20.0 each; Na~acetate- 3HzO, 1.63 ; Na-formate, 33.0; NaHCO3, 100.0; antifoam (polypropylene glycol P 2000, 2 ml in 100 ml ethanol), 2 ml; NaOH, 3.25; Na2S 9 9H20 and cysteine - HC1. H20, 5.0 each; vitamin and mineral solutions (s. above) were each added in a 125 ml amount. The fermentor, containing 10 1 medium, was inoculated with 5 ml culture from medium AM 6, incubated under 6 7 ~ H 2 - 3 3 ~ CO2 (v/v) at 36~ and harvested as previously described (Schoberth, 1977). The cell paste was kept frozen a* - 2 0 ~ until used. Cells from these cultures will be designated (C) in the results section.
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All dimensions are given in gm Figs. 1 - 5. Preparations negatively stained with uranyl acetate Figs. 6 - 1 2 . Ultrathin sectioned preparations Figs. 1 3 - 25. Freeze-etching preparations Figs. 1 and 2. The culture of Acetobacterium woodii prepared for electron microscopy contained flagellated cells and cells without flagella. The cells with flagella were rodshaped and showed, in most cases, one growing or completed cross wall. The cells without flagella formed chains consisting of short individual bacteria each having, at least, one or two "generations" of cross walls (Fig. 1, large and small
arrowheads) Fig. 3. Fig. 4.
Subpolar flagellation Proximal end of isolated flagellum showing the hook region (small arrowheads) and part of the filament with fine structure
(large arrowhead) Fig. 5.
Flagellated cells often showed flexible pill of diameter 6 - 7 nm
Electron Microscopy Negative Staining Procedure. Cells were negatively stained as described previously (Valentine et aL, 1968; Beuscher et al., 1974) using uranyt acetate ( 4 ~ , pH 5) dissolved in water.
Ultrathin Sectioning Technique. Cells suspended in agar were fixed by~ouble fixation with glutaraldehyde and osmium tetroxide. After dehydration, including a block staining with 2 ~ uranyl acetate, 4~'a graded series of acetone, fixed cells were embedded in Spurr's low viscosity medium (Spurr, 1969). Ultrathin sections were cut
with a LKB Ultrotome UM lII ultramicrotome with glass knifes. Sections were collected on carbon-coated formvar-copper grids and poststained with uranyl acetate (Frasca and Parks, :[965) and lead citrate (Venable and Coggeshall, 1965).
Freeze-Etching Technique. Ceils collected by centrifugation were frozen without glycerol in melting nitrogen. The freeze-etching preparation was performed in a BioEtch 2005 Automatic apparatus (Leybold-Heraeus, K61n, Federal Republic of Germany) at a vacuum of 2 x 10 .7 torr, a temperature of --100~ and an etching time of 90 s. Replica were produced by conventional platinum-
F~ Mayer et al. : Fine Structure of Acetobacterium woodii
209
Fig. 6. Cellswith growingor completedcross walls (largearrowheads)and disc-shaped electron transparent regions (smallarrowheads)most probably indicating that disc-shaped material had been extracted during preparation (see Figs.21 and 22) Fig.7. Cross-sectionedcell showing cytoplasmic membrane (1) and three wall layers (2,3,4). Layer 4 has periodic fine structure (P). IM intracytoplasmicmembranes Figs.8 and 9. Cytoplasmicmembrane and wall layers. 1 cytoplasmicmembrane, 2, 3, 4 wall layers carbon evaporation; they were stabilized by subsequent carbon evaporation, cleaned with chromic acid and washed several times with glass distilled water.
Electron Micrography. Electron micrographs were taken with a Philips EM 301 electron microscope. Magnifications were calibrated with a cross-lined grating replica.
RESULTS
Cell shape, Flagellation and Piliation Cultures of Acetobacterium woodii prepared for electron microscopy from early or late logarithmic growth phase contained two different cell types (Fig. 1): one type was rod-shaped and showed flagella and pili, the other type formed chains consisting of short cells without flagella and without pill (Fig.2). Both cell types usually contained complete (Figs. 1, 6 and 11) and growing (Figs. 1, 2, 6, 10, 22 and 25) cross walls. Flagellation was subpolar, with one or two flagella of diameter 1 6 n m (Figs. l, 3 and 4). The pili were flexible and showed a diameter of 6 - 7 nm (Fig. 5).
Cell Wall According to ultrathin sections, the walls of cells grown in medium with fructose (A and B, see "Materials and Methods") consisted of three layers differing in electron transparency (Figs.7,8,9 and 12). The outermost layer showed regularly arranged units with center-to-center distance of about 10 nm (Figs. 7, 10 and 1 3 - 16). In freeze-etching preparations of cells grown without fructose (C, see "Materials and Methods"), these units were seen to be composed of subunits (probably 4), with center-to-center distances of about 3 nm (Fig. 16). Figure 20 shows, most probably, an area of the inner wall surface with a periodic pattern with unit dimensions of 10--11 rim. Some indication for a periodic fine structure of this inner wall layer is also detectable in ultrathin sections (Fig. 12, layer 2).
Cytoplasmic Membrane In ultrathin sections the cytoplasmic membrane showed the usual triplelayered appearance and had a
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F. Mayer et al. : Fine Structure of Acetobacterium woodii
thickness of about 7 nm (Figs.7 and 9). Freezeetching preparations revealed membrane particles arranged in two different patterns: either in a more or less random distribution (Figs. 13-15 and 17), or in a hexagonal array with spacing of about 11 nm (Figs. 1 7 - 19). The units often showed central depressions indicating the existence of subunits (Fig. 18). Ribosomes and Nucleoplasma
Both ribosomes and nucleoplasma were visible in ultrathin sections (Figs. 6 - 8 and 10). They could not be identified as distinct structures or regions in freezeetching preparations. Ribosomes in ultrathin sections were randomly distributed over the whole cytoplasma (Figs. 6 and 10); an obvious concentration of nucleoplasma in the center of the cells could not be observed (Fig. 10). Intracytoplasmic Membranes and Inclusion Particles
Intracytoplasmic membranes were rarely seen and appeared as double membrane systems of low degree of complexity (Fig. 7). Intracytoplasmic inclusion particles were either globular (Figs. 13, 23 and 24), rodshaped (Fig. 24) or discshaped (Figs. 6, 10, 21 and 22). In ultrathin sections, the disc-shaped inclusions seemed to be dissolved (probably by organic solvents used during the preparation procedure; see "Materials and Methods"); only transparent disc-shaped areas were visible (Figs. 6 and 10). In freeze-etching preparations, however, the discs could easily be detected (Figs.21 and 22). Sometimes, the disc surface showed fracture lines (Fig. 21) indicating that very thin layers of the disc material had been broken away. The chemical nature and physiological role of the intracytoplasmic particles has not yet been investigated.
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DISCUSSION Acetobacterium woodii is the only known nonsporeforming anaerobic bacterium able to ferment H2 and CO2 to acetic acid (Schoberth and Balch, 1975). Despite this fact, fine structural analysis did not reveal surprising results. A. woodii shares some morphological properties with Methanobacterium ruminantium (Smith and Hungate, 1958; Langenberg et al., 1968). Both bacteria are Gram-positive and show short coccoid rods having approximately the same cell size; they appear predominantly in pairs and chains. In both organisms spores were not detected, and both bacteria show states of multiple division, indicated by numerous partial septa. There are major physiological and chemical differences between these organisms, however, which show that the described overall cell shapes of A. woodii are not related to the specific physiological properties of this bacterium, and that they cannot be used for identification. The same can be concluded for the observed wall fine structure. Regular arrays of particles with subunits were detected on the surfaces of various Grain-negative and Grampositive bacteria (Glauert and Thornley, 1969; Hollaus and Sleytr, 1972; Sleytr, 1976). These surface layers consist of monolayers of macromolecules arranged in tetragonal or hexagonal arrays. The particles are composed mainly of protein or glycoprotein with molecular weights ranging from 70000 to 150000 (Sleytr, 1976). According to the measured dimensions of the particles in this layer in A. woodii, a molecular weight in this range can be estimated. The appearance of ribosomes and DNA was essentially identical with other bacteria. Intracytoplasmic membranes were rarely observed and normally occurred as double membrane systems of low complexity. As only few ultrathin sectioned cells showed these membranes, there is only a very low probability that they play an important role in the specific physiological properties
Fig. 10. Cell with cross wall formation (arrowhead), with disc-shaped transparent region (D, see also Fig. 6), periodic wall surface structure (P) and fibrillar nucleoplasma (circle) Fig. 11.
Dividing cell, cross wall completed
Fig. 12. Lysed cell showing cytoplasmic membrane (arrowhead) and three-layered cell wall (2, 3, 4; see Figs. 7 - 9). Layer 2 probably shows periodic fine structure, too Fig. 13, Two bacteria, both being in the state of cross wail formation as indicated by the ringshaped grooves (arrowheads). 4 surface layer with periodic fine structure, PF cytoplasmic membrane with more or less randomly arranged particles, G broken globular inclusion particle, AF fibrillar structures connecting cell wall and cytoplasmic membrane (see "Discussion") Fig. 14. Cell pole with cytoplasmic membrane showing particles. Large arrowheads point to different directions of wall surface particle orientations; arrows indicate a line where the two areas with these different particle orientations are in contact Figs. 15 and 16. Fine structure of periodic wall surface layer in face-on view. The particles (Fig. 16, encircled, center-to-center distances about 10 nm) sometimes show a tetrameric fine structure (Fig. 15) with nearest center-to-center distances of subunits of 3 nm (Fig. 16, arrows)
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Figs. 17 and 19. Broken cells with wall surface striation (arrowheadin Fig. 17) and cytoplasmic membrane showing particles with more or less random distribution (FF), and with strongly hexagonal arrangement (circle, PFR in Fig. 17, arrow in Fig. 19) Fig. 18. Hexagonally ordered particles of cytoplasmic membrane. Center-to-center distance is about 11 nm. Some particles show a central depression (arrowhead) Fig. 20. Area of inner wall surface (without randomly distributed particles), with striations (arrowhead,arrows)with periodicity of 10-11 nm (see Fig. 12, layer 2) Figs. 21 and 22. Disc-shaped inclusions seen in face-on view (Fig. 21, D) and in side-on view (Fig. 22, D). The inclusion particle in Figure 21 shows a fracture line (see "Discussion") (see Figs. 6 and 10). Arrowhead in Figure 22 points to a ringshaped zone at the inside of the cell wall indicating cross wall formation (see Fig. 25)
of A. woodii. Two structural features observed in A. woodii, however, are not common in other bacteria. These are the hexagonal arrangement of particles in the cytoplasmic membrane and the varying shapes of inclusion bodies, with the frequent presence of discshaped bodies. As no glycerol was used during the freezing procedure of the cells for freeze-etching, the hexagonal array of membrane particles, most probably, is not caused by artificial delocation and rearrangement of randomly distributed particles but represents their real distribution. Similar arrays were
described in freeze-etched yeast cells (Moor and Mtihlethaler, 1963). Membrane particles in other organisms and organells are believed to represent multienzyme complexes (Moor, 2966; Mfihlethaler et al., 1965), or possibly specialized sites associated with transport functions (Weinstein and Koo, 1968). Whether or not this is the case in A. woodii cannot be deduced from the data available. Fibrous elements (see Fig. 13) in contact with membrane particles and cell wall were observed in yeast and bacteria too (Bayer and Remsen, 1970; Moor and M/ihlethaler,
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F. Mayer et al. : Fine Structure of Acetobacterium woodii
Fig. 23.
Globular inclusion of unknown composition (see Fig. 13)
Fig. 24.
Inclusions of various shape, unknown composition
Fig. 25.
Cell with cross wall formation (arrowhead)
1963; Sleytr, 1970), but can be assumed to be artificial plastic deformations caused by the fracturing process during the freeze-etching procedure. Disc-shaped inclusion bodies in bacteria are not common. The solubility of their contents in organic solvents and their behaviour during the fracturing process in freezeetching [fracture lines similar to those observed in yeast cells (Moor and Mfihlethaler, 1963)] indicate that they are composed of hydrophobic material as it is the case for some storage material (e.g. polyhydroxybutyrate, PHB) in other bacteria. A relationship between the described two uncommon morphological features of A. woodii and its unique physiological properties cannot be postulated unless other bacteria with similar physiological characteristics are investigated. Acknowledgements. This work was supported in part by a grant of the Stiftung Volkswagenwerk. The freeze-etching preparations were made possible by the kindness of Leybold-Heraeus, K61n. We thank Miss Kirsten Christensen and Miss Claudia Vogeley for skilful technical assistance.
REFERENCES Balch, W. E., Schoberth, S., Tanner, R.S., Wolfe, R. S.: Acetobacterium, a new genus of hydrogen-oxidizing carbon dioxidereducing anaerobes, and characterization of Acetobacterium woodii sp. nov. Int. J. Syst. Bacteriol. (1977, in press) Bayer, M. E., Remsen, C. C.: Structure of Escherichia coli after freeze-etching. J. Bacteriol. 101, 304-313 (1970) Beuscher, N., Mayer, F., Gottschalk, G. : Citrate lyase from Rhodopseudomonas gelatinosa: Purification, electron microscopy and subunit structure. Arch. Microbiol. 100, 307-328 (1974) Frasca, J. M., Parks, V. R.: A routine technique for doublestaining ultrathin sections using uranyl and lead Salts. J. Cell. Biol. 25, 157-161 (1965) Glauert, A. M., Thornley, M. J. : The topography of the bacterial cell wall. Ann. Rev. Microbiol. 23, 159-198 (1969)
Hollaus, F., Sleytr, U. : On the taxonomy and fine structure of some hyperthermophilic saccharolytic Clostridia. Arch. Mikrobiol. 86, 129-146 (1972) Langenberg, K. F., Bryant, M. P., Wolfe, R. S. : Hydrogen-oxidizing methane bacteria. II. Electron microscopy. J. Bacteriol. 95, 1124-1129 (1968) Moor, H.: Use of freeze-etching in the study of biological ultrastructure. Int. Rev. Exp. Pathol. 5, 179-216 (1966) Moor, H , Mfihlethaler, K,: Fine structure in frozen-etched yeast cells. J. Cell Biol. 17, 609-628 (1963) M~hlethaler, K., Moor, H., Szarkowski, J. W. : The ultrastructure of chloroplast lamellae. Planta (Berl.) 67, 305- 323 (1965) Schoberth, S. : Acetic acid from Ha and CO2 : formation of acetate by cell extracts of Acetobacterium woodii. Arch. Microbiol. 114, 143 - 148 (1977) Schoberth, S., Balch, W. E.: A new hydrogen-oxidizing acetateforming anaerobe. Am. Soc. Microbiol., Abstr. Ann. meeting, I 90 (1975) Sleytr, U. : Fracture faces in intact cells and protoplasts of Bacillus stearothermophilus. A study by conventional freeze-etching and freeze-etching of corresponding fracture moieties. Protoplasma (Wien) 71,295-312 (1970) Sleytr, U. B.: Self-assembly of the hexagonally and tetragonally arranged subunits of bacterial surface layers and their reattachment to cell walls. J. Ultrastruct. Res. 55, 360-377 (1976) Smith, P. H., Hungate, R. E.: Isolation and characterization of Methanobaeterium ruminantium n. sp. J. Bacteriol. 75, 713- 718 (1958) Spurr, A. R. : A low-viscosity epoxy resin embedding medium for electron microscopy, J. Ultrastruct. Res. 26, 31-43 (1969) Valentine, R. C., Shapiro, B. M., Stadtman, E. R.: Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from E. coll. Biochemistry 7, 2143- 2152 (1968) Venable, J. H., Coggeshall, R. : A simplified lead citrate stain for use in electron microscopy. J. Cell. Biol. 25, 407-408 (1965) Weinstein, R. S., Koo, V. M.: Penetration of red cell membranes by some membrane-associated particles. Proc. Soc. Exp. Biol. Med. 128, 353-357 (1968) Wolfe, R. S.: Microbial formation of methane. Adv. Microbiol. Physiol. 8, 107-146 (1971) Wolin, E. A., Wolin, M, J., Wolfe, R. S.: Formation of methane by bacterial extracts. J. Biol. Chem. 238, 2882-2886 (1963) Received August 19, 1977
Arch. Microbiol. I15, 207-213 (1977)
Hicrebialogy 9 by Springer-Verlag 1977
Electron Microscopic Investigation of the Hydrogen-Oxidizing Acetate-Forming Anaerobic Bacterium Acetobacterium woodii F R A N K MAYER, RUDI LURZ, and SIEGFRIED SCHOBERTH* Institut ffir Mikrobiologie der Universit~it G6ttingen, Grisebachstr. 8, D-3400 G6ttingen, Federal Republic of Germany
Abstract. Acetobacterium woodii is a Gram-positive anaerobic nonsporeforming bacterium able to grow on H2 and CO2 as sole sources of energy. The product of fermentation is acetic acid. Fine structural analysis showed rod-shaped flagellated cells, and coccoid cells without flagella arranged predominantly in pairs and chains. The cell wall was found to be composed of three layers. The cell surface exhibited a periodic array of particles consisting of subunits. The cytoplasmic membrane showed particles either in random distribution or in a hexagonal pattern. Intracytoplasmic membranes were rarely observed, whereas inclusion bodies of varying shapes, predominantly in an uncommon disc-shape, could frequently be observed. Their content was dissolved in ultrathin sections indicating hydrophobic nature. Key words: Acetobacterium woodii - Hydrogenoxidizing acetate-forming anaerobe - Fine structure - Electron microscopy.
Acetobacterium woodii, a new genus and species of strictly anaerobic nonsporeforming bacteria, was isolated recently in the laboratory of R. S. Wolfe (Schoberth and Balch, 1975; Balch et al., 1977). Like methane bacteria (Wolfe, 1971), A. woodii can grow o n ~7{2 and CO2 as sole sources of energy. The product of the fermentation carried out by this microorganism, however, is acetic acid and not methane. A. woodii also shows a homoacetic fermentation of fructose, and other compounds used as substrates are glucose, lactic, glyceric and formic acids. A. woodii seems to be abundant in nature and may play a considerable role * Present address: Institut ffir Biotechnologie der Kernforscbungsanlage Jiilich GmbH, D-5170 J/ilich, Federal Republic of Germany
in anaerobic methanogenic systems (Schoberth, 1977). The present paper describes the fine structure of A. woodii as seen in negatively stained, ultrathin sectioned and freeze-etched preparations.
MATERIALS AND METHODS Organism and Growth Conditions Acetobacterium woodii, type strain WB 1, isolated from a marine
estuary (Schoberth and Balch, 1975; Balch et al., 1977), kindly provided by W. E. Balch, was maintained in medium AM 6 in 5 ml volumes in Hungate tubes at 28~ Medium AM 6 contained in g/1 of glass distilled water (unless otherwise indicated) : fructose, 10.0; resazurine, 0.001; NH4CI, 1.0; 1 M K-phosphate buffer, pH 7.0, 5 ml; MgSOa - 7H20, 0./; yeast extract, 2.0; NaHCO3, 10.0; cysteine. HCI-HaO, 0.5; Na2S-9H20, 0.25; vitamin and mineral solutions (Wolin et al., 1963) were each added in a 20 ml amount; NaHCO3 was autoclaved separately as a 2 ~ solution; the gas phase was 80~o N 2 - 2 0 ~ CO2 (v/v). For stock cultures, ceils of A. woodii grown in medium AM 6 were transferred every four weeks into fresh medium9 Cells from stock cultures were also used for electron microscopic investigations by inoculating active cultures into medium AM 6. The amounts of cultures transferred were 0.03 and 0.5 ml. After overnight incubation at 28~ the cultures had reached optical densities of 0918 (A) and 0.75 (B) at 600 nm, corresponding to early and late logarithmic phase. Cultures A and B were used as indicated in the results section. For freeze-etching, cells grown in fermentors under a gas phase of H2 and CO, in a medium without fructose were used. The medium already described (Schoberth, 1977) was modified as follows: it contained in g/10l of glass distilled water (unless otherwise indicated): resazurine, 0.01; K2HPOr KHzPO4 and (NH)zSOr 2.5 each; NaC1, 0.5; MgSO4.7H20, 2.75; CaCla. 2H20, 0.63; FeSO4-7H20, 0.25; Na2WO4 - 2 H20, NazMoO4.2 HaO, and Na2SeO3 9 5 H20, 0,00025 each; yeast extract and tryptone, 20.0 each; Na~acetate- 3HzO, 1.63 ; Na-formate, 33.0; NaHCO3, 100.0; antifoam (polypropylene glycol P 2000, 2 ml in 100 ml ethanol), 2 ml; NaOH, 3.25; Na2S 9 9H20 and cysteine - HC1. H20, 5.0 each; vitamin and mineral solutions (s. above) were each added in a 125 ml amount. The fermentor, containing 10 1 medium, was inoculated with 5 ml culture from medium AM 6, incubated under 6 7 ~ H 2 - 3 3 ~ CO2 (v/v) at 36~ and harvested as previously described (Schoberth, 1977). The cell paste was kept frozen a* - 2 0 ~ until used. Cells from these cultures will be designated (C) in the results section.
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All dimensions are given in gm Figs. 1 - 5. Preparations negatively stained with uranyl acetate Figs. 6 - 1 2 . Ultrathin sectioned preparations Figs. 1 3 - 25. Freeze-etching preparations Figs. 1 and 2. The culture of Acetobacterium woodii prepared for electron microscopy contained flagellated cells and cells without flagella. The cells with flagella were rodshaped and showed, in most cases, one growing or completed cross wall. The cells without flagella formed chains consisting of short individual bacteria each having, at least, one or two "generations" of cross walls (Fig. 1, large and small
arrowheads) Fig. 3. Fig. 4.
Subpolar flagellation Proximal end of isolated flagellum showing the hook region (small arrowheads) and part of the filament with fine structure
(large arrowhead) Fig. 5.
Flagellated cells often showed flexible pill of diameter 6 - 7 nm
Electron Microscopy Negative Staining Procedure. Cells were negatively stained as described previously (Valentine et aL, 1968; Beuscher et al., 1974) using uranyt acetate ( 4 ~ , pH 5) dissolved in water.
Ultrathin Sectioning Technique. Cells suspended in agar were fixed by~ouble fixation with glutaraldehyde and osmium tetroxide. After dehydration, including a block staining with 2 ~ uranyl acetate, 4~'a graded series of acetone, fixed cells were embedded in Spurr's low viscosity medium (Spurr, 1969). Ultrathin sections were cut
with a LKB Ultrotome UM lII ultramicrotome with glass knifes. Sections were collected on carbon-coated formvar-copper grids and poststained with uranyl acetate (Frasca and Parks, :[965) and lead citrate (Venable and Coggeshall, 1965).
Freeze-Etching Technique. Ceils collected by centrifugation were frozen without glycerol in melting nitrogen. The freeze-etching preparation was performed in a BioEtch 2005 Automatic apparatus (Leybold-Heraeus, K61n, Federal Republic of Germany) at a vacuum of 2 x 10 .7 torr, a temperature of --100~ and an etching time of 90 s. Replica were produced by conventional platinum-
F~ Mayer et al. : Fine Structure of Acetobacterium woodii
209
Fig. 6. Cellswith growingor completedcross walls (largearrowheads)and disc-shaped electron transparent regions (smallarrowheads)most probably indicating that disc-shaped material had been extracted during preparation (see Figs.21 and 22) Fig.7. Cross-sectionedcell showing cytoplasmic membrane (1) and three wall layers (2,3,4). Layer 4 has periodic fine structure (P). IM intracytoplasmicmembranes Figs.8 and 9. Cytoplasmicmembrane and wall layers. 1 cytoplasmicmembrane, 2, 3, 4 wall layers carbon evaporation; they were stabilized by subsequent carbon evaporation, cleaned with chromic acid and washed several times with glass distilled water.
Electron Micrography. Electron micrographs were taken with a Philips EM 301 electron microscope. Magnifications were calibrated with a cross-lined grating replica.
RESULTS
Cell shape, Flagellation and Piliation Cultures of Acetobacterium woodii prepared for electron microscopy from early or late logarithmic growth phase contained two different cell types (Fig. 1): one type was rod-shaped and showed flagella and pili, the other type formed chains consisting of short cells without flagella and without pill (Fig.2). Both cell types usually contained complete (Figs. 1, 6 and 11) and growing (Figs. 1, 2, 6, 10, 22 and 25) cross walls. Flagellation was subpolar, with one or two flagella of diameter 1 6 n m (Figs. l, 3 and 4). The pili were flexible and showed a diameter of 6 - 7 nm (Fig. 5).
Cell Wall According to ultrathin sections, the walls of cells grown in medium with fructose (A and B, see "Materials and Methods") consisted of three layers differing in electron transparency (Figs.7,8,9 and 12). The outermost layer showed regularly arranged units with center-to-center distance of about 10 nm (Figs. 7, 10 and 1 3 - 16). In freeze-etching preparations of cells grown without fructose (C, see "Materials and Methods"), these units were seen to be composed of subunits (probably 4), with center-to-center distances of about 3 nm (Fig. 16). Figure 20 shows, most probably, an area of the inner wall surface with a periodic pattern with unit dimensions of 10--11 rim. Some indication for a periodic fine structure of this inner wall layer is also detectable in ultrathin sections (Fig. 12, layer 2).
Cytoplasmic Membrane In ultrathin sections the cytoplasmic membrane showed the usual triplelayered appearance and had a
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F. Mayer et al. : Fine Structure of Acetobacterium woodii
thickness of about 7 nm (Figs.7 and 9). Freezeetching preparations revealed membrane particles arranged in two different patterns: either in a more or less random distribution (Figs. 13-15 and 17), or in a hexagonal array with spacing of about 11 nm (Figs. 1 7 - 19). The units often showed central depressions indicating the existence of subunits (Fig. 18). Ribosomes and Nucleoplasma
Both ribosomes and nucleoplasma were visible in ultrathin sections (Figs. 6 - 8 and 10). They could not be identified as distinct structures or regions in freezeetching preparations. Ribosomes in ultrathin sections were randomly distributed over the whole cytoplasma (Figs. 6 and 10); an obvious concentration of nucleoplasma in the center of the cells could not be observed (Fig. 10). Intracytoplasmic Membranes and Inclusion Particles
Intracytoplasmic membranes were rarely seen and appeared as double membrane systems of low degree of complexity (Fig. 7). Intracytoplasmic inclusion particles were either globular (Figs. 13, 23 and 24), rodshaped (Fig. 24) or discshaped (Figs. 6, 10, 21 and 22). In ultrathin sections, the disc-shaped inclusions seemed to be dissolved (probably by organic solvents used during the preparation procedure; see "Materials and Methods"); only transparent disc-shaped areas were visible (Figs. 6 and 10). In freeze-etching preparations, however, the discs could easily be detected (Figs.21 and 22). Sometimes, the disc surface showed fracture lines (Fig. 21) indicating that very thin layers of the disc material had been broken away. The chemical nature and physiological role of the intracytoplasmic particles has not yet been investigated.
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DISCUSSION Acetobacterium woodii is the only known nonsporeforming anaerobic bacterium able to ferment H2 and CO2 to acetic acid (Schoberth and Balch, 1975). Despite this fact, fine structural analysis did not reveal surprising results. A. woodii shares some morphological properties with Methanobacterium ruminantium (Smith and Hungate, 1958; Langenberg et al., 1968). Both bacteria are Gram-positive and show short coccoid rods having approximately the same cell size; they appear predominantly in pairs and chains. In both organisms spores were not detected, and both bacteria show states of multiple division, indicated by numerous partial septa. There are major physiological and chemical differences between these organisms, however, which show that the described overall cell shapes of A. woodii are not related to the specific physiological properties of this bacterium, and that they cannot be used for identification. The same can be concluded for the observed wall fine structure. Regular arrays of particles with subunits were detected on the surfaces of various Grain-negative and Grampositive bacteria (Glauert and Thornley, 1969; Hollaus and Sleytr, 1972; Sleytr, 1976). These surface layers consist of monolayers of macromolecules arranged in tetragonal or hexagonal arrays. The particles are composed mainly of protein or glycoprotein with molecular weights ranging from 70000 to 150000 (Sleytr, 1976). According to the measured dimensions of the particles in this layer in A. woodii, a molecular weight in this range can be estimated. The appearance of ribosomes and DNA was essentially identical with other bacteria. Intracytoplasmic membranes were rarely observed and normally occurred as double membrane systems of low complexity. As only few ultrathin sectioned cells showed these membranes, there is only a very low probability that they play an important role in the specific physiological properties
Fig. 10. Cell with cross wall formation (arrowhead), with disc-shaped transparent region (D, see also Fig. 6), periodic wall surface structure (P) and fibrillar nucleoplasma (circle) Fig. 11.
Dividing cell, cross wall completed
Fig. 12. Lysed cell showing cytoplasmic membrane (arrowhead) and three-layered cell wall (2, 3, 4; see Figs. 7 - 9). Layer 2 probably shows periodic fine structure, too Fig. 13, Two bacteria, both being in the state of cross wail formation as indicated by the ringshaped grooves (arrowheads). 4 surface layer with periodic fine structure, PF cytoplasmic membrane with more or less randomly arranged particles, G broken globular inclusion particle, AF fibrillar structures connecting cell wall and cytoplasmic membrane (see "Discussion") Fig. 14. Cell pole with cytoplasmic membrane showing particles. Large arrowheads point to different directions of wall surface particle orientations; arrows indicate a line where the two areas with these different particle orientations are in contact Figs. 15 and 16. Fine structure of periodic wall surface layer in face-on view. The particles (Fig. 16, encircled, center-to-center distances about 10 nm) sometimes show a tetrameric fine structure (Fig. 15) with nearest center-to-center distances of subunits of 3 nm (Fig. 16, arrows)
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Figs. 17 and 19. Broken cells with wall surface striation (arrowheadin Fig. 17) and cytoplasmic membrane showing particles with more or less random distribution (FF), and with strongly hexagonal arrangement (circle, PFR in Fig. 17, arrow in Fig. 19) Fig. 18. Hexagonally ordered particles of cytoplasmic membrane. Center-to-center distance is about 11 nm. Some particles show a central depression (arrowhead) Fig. 20. Area of inner wall surface (without randomly distributed particles), with striations (arrowhead,arrows)with periodicity of 10-11 nm (see Fig. 12, layer 2) Figs. 21 and 22. Disc-shaped inclusions seen in face-on view (Fig. 21, D) and in side-on view (Fig. 22, D). The inclusion particle in Figure 21 shows a fracture line (see "Discussion") (see Figs. 6 and 10). Arrowhead in Figure 22 points to a ringshaped zone at the inside of the cell wall indicating cross wall formation (see Fig. 25)
of A. woodii. Two structural features observed in A. woodii, however, are not common in other bacteria. These are the hexagonal arrangement of particles in the cytoplasmic membrane and the varying shapes of inclusion bodies, with the frequent presence of discshaped bodies. As no glycerol was used during the freezing procedure of the cells for freeze-etching, the hexagonal array of membrane particles, most probably, is not caused by artificial delocation and rearrangement of randomly distributed particles but represents their real distribution. Similar arrays were
described in freeze-etched yeast cells (Moor and Mtihlethaler, 1963). Membrane particles in other organisms and organells are believed to represent multienzyme complexes (Moor, 2966; Mfihlethaler et al., 1965), or possibly specialized sites associated with transport functions (Weinstein and Koo, 1968). Whether or not this is the case in A. woodii cannot be deduced from the data available. Fibrous elements (see Fig. 13) in contact with membrane particles and cell wall were observed in yeast and bacteria too (Bayer and Remsen, 1970; Moor and M/ihlethaler,
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F. Mayer et al. : Fine Structure of Acetobacterium woodii
Fig. 23.
Globular inclusion of unknown composition (see Fig. 13)
Fig. 24.
Inclusions of various shape, unknown composition
Fig. 25.
Cell with cross wall formation (arrowhead)
1963; Sleytr, 1970), but can be assumed to be artificial plastic deformations caused by the fracturing process during the freeze-etching procedure. Disc-shaped inclusion bodies in bacteria are not common. The solubility of their contents in organic solvents and their behaviour during the fracturing process in freezeetching [fracture lines similar to those observed in yeast cells (Moor and Mfihlethaler, 1963)] indicate that they are composed of hydrophobic material as it is the case for some storage material (e.g. polyhydroxybutyrate, PHB) in other bacteria. A relationship between the described two uncommon morphological features of A. woodii and its unique physiological properties cannot be postulated unless other bacteria with similar physiological characteristics are investigated. Acknowledgements. This work was supported in part by a grant of the Stiftung Volkswagenwerk. The freeze-etching preparations were made possible by the kindness of Leybold-Heraeus, K61n. We thank Miss Kirsten Christensen and Miss Claudia Vogeley for skilful technical assistance.
REFERENCES Balch, W. E., Schoberth, S., Tanner, R.S., Wolfe, R. S.: Acetobacterium, a new genus of hydrogen-oxidizing carbon dioxidereducing anaerobes, and characterization of Acetobacterium woodii sp. nov. Int. J. Syst. Bacteriol. (1977, in press) Bayer, M. E., Remsen, C. C.: Structure of Escherichia coli after freeze-etching. J. Bacteriol. 101, 304-313 (1970) Beuscher, N., Mayer, F., Gottschalk, G. : Citrate lyase from Rhodopseudomonas gelatinosa: Purification, electron microscopy and subunit structure. Arch. Microbiol. 100, 307-328 (1974) Frasca, J. M., Parks, V. R.: A routine technique for doublestaining ultrathin sections using uranyl and lead Salts. J. Cell. Biol. 25, 157-161 (1965) Glauert, A. M., Thornley, M. J. : The topography of the bacterial cell wall. Ann. Rev. Microbiol. 23, 159-198 (1969)
Hollaus, F., Sleytr, U. : On the taxonomy and fine structure of some hyperthermophilic saccharolytic Clostridia. Arch. Mikrobiol. 86, 129-146 (1972) Langenberg, K. F., Bryant, M. P., Wolfe, R. S. : Hydrogen-oxidizing methane bacteria. II. Electron microscopy. J. Bacteriol. 95, 1124-1129 (1968) Moor, H.: Use of freeze-etching in the study of biological ultrastructure. Int. Rev. Exp. Pathol. 5, 179-216 (1966) Moor, H , Mfihlethaler, K,: Fine structure in frozen-etched yeast cells. J. Cell Biol. 17, 609-628 (1963) M~hlethaler, K., Moor, H., Szarkowski, J. W. : The ultrastructure of chloroplast lamellae. Planta (Berl.) 67, 305- 323 (1965) Schoberth, S. : Acetic acid from Ha and CO2 : formation of acetate by cell extracts of Acetobacterium woodii. Arch. Microbiol. 114, 143 - 148 (1977) Schoberth, S., Balch, W. E.: A new hydrogen-oxidizing acetateforming anaerobe. Am. Soc. Microbiol., Abstr. Ann. meeting, I 90 (1975) Sleytr, U. : Fracture faces in intact cells and protoplasts of Bacillus stearothermophilus. A study by conventional freeze-etching and freeze-etching of corresponding fracture moieties. Protoplasma (Wien) 71,295-312 (1970) Sleytr, U. B.: Self-assembly of the hexagonally and tetragonally arranged subunits of bacterial surface layers and their reattachment to cell walls. J. Ultrastruct. Res. 55, 360-377 (1976) Smith, P. H., Hungate, R. E.: Isolation and characterization of Methanobaeterium ruminantium n. sp. J. Bacteriol. 75, 713- 718 (1958) Spurr, A. R. : A low-viscosity epoxy resin embedding medium for electron microscopy, J. Ultrastruct. Res. 26, 31-43 (1969) Valentine, R. C., Shapiro, B. M., Stadtman, E. R.: Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from E. coll. Biochemistry 7, 2143- 2152 (1968) Venable, J. H., Coggeshall, R. : A simplified lead citrate stain for use in electron microscopy. J. Cell. Biol. 25, 407-408 (1965) Weinstein, R. S., Koo, V. M.: Penetration of red cell membranes by some membrane-associated particles. Proc. Soc. Exp. Biol. Med. 128, 353-357 (1968) Wolfe, R. S.: Microbial formation of methane. Adv. Microbiol. Physiol. 8, 107-146 (1971) Wolin, E. A., Wolin, M, J., Wolfe, R. S.: Formation of methane by bacterial extracts. J. Biol. Chem. 238, 2882-2886 (1963) Received August 19, 1977
