JOURNAL OF BACTERIOLOGY, Jan. 1992, p. 263-268
Vol. 174, No. 1
0021-9193/92/010263-06$02.00/0 Copyright © 1992, American Society for Microbiology
Location of the Basal Disk and a Ringlike Cytoplasmic Structure, Two Additional Structures of the Flagellar Apparatus of Wolinella succinogenes STEPHAN C. SCHUSTER AND EDMUND BAEUERLEIN* Max-Planck-Institut fuer Biochemie, D-8033 Martinsried, Germany Received 30 July 1991/Accepted 25 October 1991 The basal body of Wolinella succinogenes consists of a central rod, a set of two rings (L and P rings), a basal disk front 70 to 200 nm in diameter, and a terminal knob. In negatively stained preparations of flagellar hook-basal body complexes, some disks remain fixed perpendicularly to the grid and show that such a disk is located on the distal side of the P ring. The basal disks have been isolated with and without the P ring; in both cases there is a hole in the center of the disk. The diameter of the disk is smaller in the presence of the P ring. The L-P ring complex is therefore assumed to be a bushing for the rod. Thin sections of whole bacteria and spheroplasts reveal that the disk is attached to the inner surface of the outer membrane. At the insertions of the flagellar hook-basal body-basal disk complexes, depressions are visible in negatively stained preparations of whole bacteria and spheroplasts. A new ringlike structure is connected to an elongation of the basal body into the cytoplasm in both preparations. Its diameter (60 nm) is larger than that of the M ring. A heavily stained compartment can be seen in between the new ringlike structure and the basal disk, which may be formed by the energy transducing units.
For a long time the entity called the intact flagellum (6-8) was thought to be the essential rotary device of the flagellar motor. In Escherichia coli and Salmonella typhimurium, this assembly consists of the flagellar filament, the hook, and the basal body. The basal body appears as a set of four rings coaxial with the rod. Two rings are very likely to be correlated to membranes: the L ring to the outer membrane and the M ring to the inner membrane. The P ring is in the periplasmic space and is assumed to interact with the peptidoglycan layer. The S ring is clearly visible in negatively stained electron micrographs in both bacteria, but from genetic, biochemical, and physiological studies no indications for its existence emerged until now (10, 11). In an extensive search for flagellar and motility mutants with the paralyzed or switch-defective phenotype, none mapped to the gene of the M ring (fliF) (10); all mapped to five genes encoding proteins that are not present on isolated basal bodies. Three of them (fiG, fliM, fliN) encode flagellar components that participate in both energy transduction and switching (15), whereas the other two (motA, motB) encode proteins that appear to function in energy transduction only (5, 18, 21, 22). It is therefore unlikely that the M ring is the active rotor of the bacterial flagellar apparatus. Furthermore, it was proposed about 13 years ago by Coulton and Murray (3, 4) that essential structures of the flagellar apparatus were lost during the procedures for isolation of intact flagella. They found circlets of about 15 particles around the rod in the cytoplasmic membrane and outer membraneassociated large disks, called concentric membrane rings, in Aquaspirillum serpens. From Wolinella succinogenes, an anaerobic, gram-negative bacterium with one polar flagellum, we recently isolated the flagellar hook-basal body complex, to which large disks were associated as additional structural elements of its flagellar apparatus (16). Here we describe (i) the location of *
these disks on the basal body and between the membranes, (ii) their isolation with and without the correlated rings, and (iii) new ringlike structures on extensions of the rod into the cytoplasm. MATERIALS AND METHODS
Bacterial strain. The W. succinogenes wild-type strain 1740 from Deutsche Sammlung Mikroorganismen was used for all experiments. E. coli 4200 HC (University of Konstanz) was also used. Chemicals and enzymes. All chemicals obtained from Merck Darmstadt were reagent grade; Triton X-100 was obtained from ROTH, Karlsruhe; hen egg white lysozyme and DNase I (grade II) were obtained from Boehringer Mannheim. Growth conditions. The standard Wolinella medium used consisted of the following: ammonium sulfate (4 mM), potassium phosphate (38 mM), fumaric acid (26 mM), and sodium formate (44 mM). After sterilization, magnesium chloride (120 FM) and iron (II) sulfate (7 ,uM) were added. Thioglycolic acid (0.38 RI/ml) was used together with a flux of nitrogen to maintain microaerobic conditions. Cells were grown at 37°C in stoppered Erlenmeyer flasks without shaking to the late-logarithmic phase. E. coli was grown in standard LB medium at 37°C to the early logarithmic phase. Preparation of spheroplasts. A 1-liter culture was harvested and resuspended in 100 ml of a cold solution of 0.5 M sucrose and 10 mM Tris (pH 8). After 30 min, lysozyme (0.02 mg/ml) and EDTA (final concentration, 10 mM) were added. The cells were then incubated overnight. The resulting spheroplasts were stable for several days. For negative staining a carbon-coated copper grid was laid on a 25-,lI drop of a spheroplast suspension. The grid was washed twice and then negatively stained. Isolation of intact flagella. A 100-ml suspension of spheroplasts, prepared as described above, was spun down (8,000
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FIG. 1. Basal disks standing upright perpendicular to the grid in more than one-half of their diameter. They are visible on then distal side of the P ring. In no case could the L ring be detected, suggesting that it may be an integral part of the basal disk. Negative staining. Bars, 100 tnm.
x g, 4°C, 20 min) and lysed in 10 ml of TET buffer (10 mM Tris, 5 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride [pH 8]). The DNA was digested with DNase in the presence of 10 mM MgSO4. After 1 h at room temperature the pH was raised to 11 with 5 N KOH. A low-spin centrifugation was applied (12,000 x g, 40C, 10 min) to remove cell debris, and the supernatant was centrifuged (60,000 x g, 4°C, 60 min) to pellet the intact flagella. The last two centrifugations were repeated three times. Purity could be estimated by a lack of color on the pellet. For E. coli the EDTA treatment at pH 11 was omitted; otherwise the procedure was like that used for W. succinogenes.
Preparation of basal disks. Basal disks could be obtained by sonification of spheroplasts for 10 min in a Branson Sonifier 450. The procedure described above resulted in a preparation good enough for electron microscopic investigation. Electron microscopy. Electron microscopy was done on a Philips CM 10 electron microscope at 80 kV. For negative staining procedures in E. coli and W. succinogenes, we used carbon-coated copper grids and a 2% uranyl acetate stain. Cells studied in thin sections were fixed directly in culture medium with 2% glutaraldehyde for 30 min at 370C. After
centrifugation the cells were taken up into agar blocks (1% agar; Sigma). The agar blocks were incubated for 1 h in 2% glutaraldehyde in Soerensen buffer and then washed for 10 min in Soerensen buffer. Postfixation was done for 1 h in 1% osmium peroxide in Kellenberger buffer. Embedding was in Epon. Sections were picked up on naked 200-mesh copper grids and stained with 1% uranyl acetate.
RESULTS Location of the basal disk on the rod. Flagellar hook-basal body (HBB) complexes of W. succinogenes, to which the basal disks are associated (16), were isolated by solubilization of spheroplasts by mild detergents. In electron micrographs of negatively stained preparations, some of the disks remained standing upright perpendicularly to the grid in more than one half of the diameter, whereas the remaining part was lying on the grid under its HBB complex. It was therefore possible to see the basal disk, located on the distal side of the P ring, probably between the P and L rings (Fig. 1). Though in no case could the L ring be observed, this may be taken as an indication that the L ring is an integral part of the basal disk. If the basal disk is completely attached to the grid, both rings of the distal ring complex, the L and P rings,
FIG. 2. Preparation of basal disks with (A) and without (B) P rings by ultrasonic treatment of spheroplasts and flagellar HBB-basal disk complexes. Diameters: inner ring, 10 nm; outer ring, 24 nm; central hole, 13 nm. Bar, 100 nm.
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FIG. 3. Thin sections of whole bacteria (A), spheroplasts (B), and vesicles (C) arising spontaneously from spheroplasts. In all three preparations high electron density is closely related to the inner surface of the outer membrane. The diameter of this area corresponds to the average diameter of the basal disks. Bars, 100 nm.
are detected always in the center of the basal disk (16). It is therefore very probable that the basal disk is coaxial with the L-P ring complex. In a new procedure the basal disks alone could be obtained by ultrasonic treatment of spheroplasts and of flagellar HBB complexes. In negatively stained preparations, most of these disks are found together with a correlated basal ring in their center; only a few were found without it (Fig. 2). It is probable that this ring corresponds to the P ring (Fig. 1), since its outer diameter is about 23 nm. The diameter of the
central hole is about 13 nm without a ring and about 10 nm with a ring. From all this we assume that the P-L ring basal disk complex serves as bushing for the rod. The basal disk is attached to the inner surface of the outer membrane. To study a possible function of the basal disk, we examined the association of the disk with the membranes. Thin sections of whole bacteria and of spheroplasts were obtained. In electron micrographs of whole cells (Fig. 3A), the depression formed by the basal disk could be seen in the inner and outer membranes. In thin sections of spheroplasts (Fig. 3B and C) a high electron density was found at the insertion of the flagellum on the inner side of the outer membrane. The diameter of this density corresponds with that of an average basal disk. An electron density much higher than that in any other part of the cytoplasmic membrane was found underneath the basal disk. In many cases an extension of this high density into the cytoplasm was detected (Fig. 3B). On rarer occasions two flagellar motors were found in close proximity in one vesicle. Most likely these vesicles resulted from a dividing cell with one motor on each pole; in no case were more than two motors found in one vesicle (Fig. 3C). A new ringlike structure in the cytoplasm, probably connected to the basal body. Just below the insertion of the HBB complex a ringlike structure is visible in negatively stained preparations of whole cells (Fig. 4A) and of spheroplasts (Fig. 4B). Its diameter of 57 to 60 nm is larger than that of the M ring (37 to 39 nm). A more detailed structure is seen in spheroplasts, suggesting that globular particles are fixed at the border of this ringlike structure (Fig. 4B, arrows). Within the area between the basal disk and the ringlike cytoplasmic
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FIG. 4. Ringlike structures coaxial with an extension of the rod into the cytoplasm. (A) Negatively stained whole bacterium. The ringlike structure below the cytoplasmic membrane has a diameter of about 57 to 60 nm and a thickness of about 10 nm. From its outer diameter a compartment of high electron density extends to the basal disk. (B) Negatively stained spheroplasts, the size of which is probably determined by the diameter of the basal disk (diameter, 120 nm). Here the ringlike structure appears to be composed of or occupied by globular particles. The compartment of high electron density is visible also. Bar, 100 nm.
structure is a heavily stained compartment; it appears to be divided into two halves by the motor axis (Fig. 4). Its dimensions are smaller than the diameter of the basal disk but comparable to that of the ringlike cytoplasmic structure. Both of these findings are supported by an electron micrograph of a small spheroplast (Fig. 4B), the size of which is apparently determined by the diameter of the basal disk (120 nm). In this case the depression is more localized to the insertion of the HBB complex, and the outer and inner membranes are no longer distinguishable. The ringlike structure is found also in this more artificial preparation, and even the elongation of the basal body to it may be detected. Additional basal body-associated structures of E. coli: elongation of the rod and M ring-associated arm and caps. The flagellar HBB complexes of E. coli were prepared in a mild way, with low concentrations of EDTA (5 mM) at pH 7.
Additional basal body-associated structures are visible in negatively stained preparations without preceding fixation (Fig. 5). An essential feature of this preparation is that the rod, the motor axis, is elongated on the other side of the M ring (Fig. 5A), suggesting its extension into the cytoplasma. A caplike structure (Fig. 5B) obviously wraps the extended rod and is sometimes partially lost, forming symmetric arms on both sides of the M ring (Fig. 5A). DISCUSSION The basal body-associated disk of W. succinogenes is located on the distal site of the P ring, as visible in electron micrographs of perpendicularly standing basal disks (Fig. 1). The P ring is clearly visible on the proximal side of the basal disk, whereas the L ring cannot be observed on either side.
FIG. 5. Additional basal body-associated structures of E. coli in negatively stained, unfixed preparations of its flagellar HBB complex. (A) Extension of the rod and symmetric arms on both sides of the M ring. (B) A caplike structure wrapping the extension of the rod. Bars, 100 nm.
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FIG. 6. Thin section of W. recta (reprinted from reference 17 with permission).
In earlier electron micrographs where the basal disks lay completely on the grid, either the terminal knob, the S-M ring complex, or two rings (the L-P ring complex) are visible (Fig. 2D, E, and F and Fig. 3A in reference 16). In all cases the L-P ring complex was located in the center of the basal disk. It is therefore very probable that L and P rings form a cylinderlike complex that serves as bushing for the rod. This hypothesis is supported by the fact that the P ring is surrounded by stain in most of negatively stained flagellar HBB preparations with upright standing disks (Fig. 1). In addition, subcomplexes termed staples were obtained by acidic degradation of S. typhimurium HBB complexes, which were thought to be L-P ring complexes (1). Two types of images were found in electron micrographs: a rectangle with one edge missing (staple) and an annulus. Both were correlated to the subcomplex from its side and axial views. Because axial sections of staples and L-P rings on intact basal bodies have showed a striking resemblance in image reconstruction, it has been concluded that staples are L-P ring complexes (23). Additional support for this has come from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of staples, in which only three protein bands have been found, a 27-kDa protein (L ring), a 38-kDa protein (P ring), and a 65-kDa protein (M ring) (1, 12). When spheroplasts or flagellar HBB-basal disk complexes were sonificated, predominantly basal disks were obtained; in the center of the basal disks a ring could be seen. Only a
few of the disks were without a ring (Fig. 2). It is therefore probable that the observed ring or the L-P ring complex interacts more strongly with the basal disk than with the rod, a conclusion that is consistent with the postulated function of the L-P ring complex as a bushing for the rod. The basal disk itself is attached to the inner surface of the outer membrane, providing a rigid area into which one bushing of the flagellar apparatus, the L-P ring complex, is inserted. It is probable that the basal disk is involved in the pole formation of W. succinogenes and the correct arrangement of the energy-transducing units that are assumed to be the intramembrane particle ring structures in the cytoplasmic membranes. These particles were found first in A. serpens (4) and then in Streptococcus sp. and E. coli (13). When a nonfunctional mocha operon was present in E. coli, flagellated but immobile cells that lacked the particle rings were obtained. Both the motility and the ring structure were recovered when motA and motB were introduced simultaneously (13). A common feature of the monoflagellated poles in gramnegative bacteria appears to be a depression formed around the insertion of the flagellum in, for example, W. succinogenes (Fig. 4A). Similar depressions have been found in thin sections of Campylobacter jejuni (2), Campylobacter fetus (formerly Vibrio fetus) (19), and Wolinella recta (17), suggesting that outer membrane, basal disk, and peptidoglycan layer are close together, at least in their central part around the flagellum. This is supported by electron micrographs of spheroplasts from W. succinogenes (Fig. 4B). It is unknown even today whether the basal disk and cytoplasmic membrane are close together within the area of the flagellar apparatus. In negatively stained spheroplasts of W. succinogenes the spatial arrangement of the cytoplasmic membrane could not be distinguished relative to the flagellar apparatus
(Fig. 4B).
Although no basal disks have been isolated from gramnegative bacteria with peritrichous flagella, L ring-associated structures of variable (8) and symmetrical (24) appearance have been found on intact flagellum preparations of S. typhimurium. New ringlike cytoplasmic structures visible in negatively stained whole bacteria (Fig. 4A) of W. succinogenes are probably on an elongation of the basal body. In the spheroplasts, globulelike units appear to be attached around this
Filament
Hook
L-ring
/ P-ring
::*,i]: I
outer membrane
petidoglycan layer
Heavily stained compartment cytoplasmic structure
terminal knob
FIG. 7. Schematic diagram of the HBB-basal disk complex with the additional cytoplasmic structures.
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ring (Fig. 4B). The diameter of the whole structure is about 57 to 60 nm, and it differs clearly from the M ring (diameter of 37 to 39 nm). A compartment of high electron density that was found in whole bacteria and spheroplasts between the basal disk and this new ringlike structure (Fig. 4) apparently determines its diameter. A cylinderlike structure can be seen in thin sections of W. recta (17) (Fig. 6) and Spirillum volutans (25). In several electron micrographs of various bacteria, similar but not so differentiated cytoplasmic areas of high electron density may be observed, for example, in C. jejuni (2) and C. fetus (19). From the elements described above, we propose a hypothetical model of the HBB complex with respect to the cell wall (Fig. 7). In E. coli, additional basal body-associated structures have been found in unfixed, negatively stained preparations (Fig. 5); these structures are very similar to those in fixed, negatively stained preparations of S. typhimurium (9). Recently the structures described above were found in unfixed cells in S. typhimurium (9a). In both, an extension of the motor axis into the cytoplasmic side of the M ring is observed (Fig. 5A). In other preparations a caplike structure covers it; this caplike structure is either a part of the M ring or associated with it (Fig. SB). This cap apparently may be removed or partially destroyed, so that on both sides of the M ring two symmetrical arms are formed (Fig. 5A). In thin sections of S. volutans, the cylinderlike structures appear to be cut through (25). It is possible that these walls become arms if the cylinder is destroyed or parts of it are released. On the other hand, these caplike and armlike structures of S. typhimurium (9) and E. coli (Fig. 5) may be parts of a more irregular, bell-shaped extension of the flagellar base into the cytoplasm as found with rapid-freeze electron microscopy in S. typhimurium (14). In a hypothetical view the cylinderlike or bell-shaped cytoplasmic structures of the flagellar apparatus appear to be part of it. The new ringlike structure may be a switch or a rotor or a combination of both. The basal disk is probably responsible for the mechanical stability of the rotary device, at least in bacteria with polar flagella.
J. BACTERIOL. 6. DePamphilis, M. L., and J. Adler. 1971. Purification of intact flagella from Escherichia coli and Bacillus subtilis. J. Bacteriol.
105:376-383.
7. DePamphilis, M. L., and J. Adler. 1971. Fine structureture and isolation of the hook-basal body complex of flagella from Escherichia coli and Bacillus subtilis. J. Bacteriol. 105:384-395. 8. DePamphilis, M. L., and J. Adler. 1971. Attachment of flagellar basal bodies to the cell envelope: specific attachment to the outer, lipopolysaccharide membrane and the cytoplasmic membrane. J. Bacteriol. 105:396407. 9. Driks, A., and D. J. DeRosier. 1990. Additional structures associated with bacterial flagellar basal body. J. Mol. Biol. 211:669-672. 9a.Driks, A., and D. J. DeRosier. 1991. Personal communication. 10. Homma, M., S. I. Aizawa, G. E. Dean, and R. M. Macnab. 1987. Identification of the M-ring protein of the flagella motor of Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 84:74837487. 11. Jones, C. J., and R. M. Macnab. 1990. Flagella assembly in
12. 13. 14.
15.
16.
17.
ACKNOWLEDGMENTS
18.
We greatly appreciate stimulating discussions with R. M. Macnab, H. Engelhardt, and S. Khan. We also thank R. M. Macnab for critical reading of the manuscript and valuable comments.
19. 20.
1.
2. 3. 4.
5.
REFERENCES Aizawa, S.-I., G. E. Dean, C. J. Jones, R. M. Macnab, and S. Yamaguchi. 1985. Purification and characterization of the flagellar hook-basal body complex of Salmonella typhimurium. J. Bacteriol. 161:836-849. Brock, F. M., and R. G. E. Murray. 1988. The ultrastructure and ATPase nature of polar membrane in Campylobacter jejuni. Can. J. Microbiol. 34:594-604. Coulton, J. W., and R. G. E. Murray. 1977. Membrane-associated components of the bacterial flagellar apparatus. Biochim. Biophys. Acta 465:290-310. Coulton, J. W., and R. G. E. Murray. 1978. Cell envelope associations of Aquaspirillum serpens flagella. J. Bacteriol. 136:1037-1049. Dean, G. E., R. M. Macnab, J. Stader, P. Matsumura, and C. Buks. 1984. Gene sequence and predicted amino acid sequence of MotA protein, a membrane associated protein required for flagellar rotation in Escherichia coli. J. Bacteriol. 159:991-999.
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Salmonella typhimurium: analysis with temperature-sensitive mutants. J. Bacteriol. 172:1327-1339. Jones, C. J., R. M. Macnab, H. Okino, and S.-I. Aizawa. 1990. Stoichiometric analysis of the flagellar hook-basal body complex of Salmonella typhimurium. J. Mol. Biol. 212:377-387. Khan, S., M. Dapice, and T. Reese. 1988. Effects of mot gene expression on the structure of the flagellar motor. J. Mol. Biol. 202:575-585. Khan, S., I. H. Khan, and T. Reese. 1991. New structural features of the flagellar base in Salmonella thyphimurium revealed by rapid freeze electron microscopy. J. Bacteriol. 173: 2888-2896. Kihara, M., M. Homma, K. Kutsukake, and R. M. Macnab. 1989. Flagella switch of Salmonella typhimurium: gene sequences and deduced protein sequences. J. Bacteriol. 171:32473257. Kupper, J., I. Wildhaber, Z. Gao, and E. Baeuerlein. 1989. Basal-body-associated disks are additional structural elements of the flagellar apparatus isolated from Wolinella succinogenes. J. Bacteriol. 171:2803-2810. Lai, C. H., M. A. Listgarden, A. C. R. Tanner, and S. S. Soeransky. 1981. Ultrastructures of Bacteroides gracilis, Campylobacter concisus, Wolinella recta, and Eikenella corrodeus, all from human with periodontal disease. Int. J. Syst. Bacteriol. 31:465475. Macnab, R. M., and D. J. DeRosier. 1988. Bacterial flagellar structure and function. Can. J. Microbiol. 34:442-451. Ritchie, A. E., R. F. Keeler, and J. M. Bryner. 1966. Anatomical features of Vibrio fetus: electron microscopy survey. J. Gen. Microbiol. 43:427438. Schuster, S., and E. Baeuerlein. 1989. Basal body associated disks as new structural elements of the flagella apparatus of Wolinella succinogenes. Biol. Chem. Hoppe-Seyler 370:958. Silverman, M., P. Matsumura, and M. Simon. 1976. The identification of the mot gene product with Escherichia coli-lambda hybrids. Proc. Natl. Acad. Sci. USA 73:3126-3130. Stader, J., P. Matsumura, D. Vacante, G. E. Dean, and R. M. Macnab. 1986. Nucleotide sequence of the Escherichia coli motB gene and site-limited incorporation of its product into the cytoplasmic membrane. J. Bacteriol. 166:244-252. Stallmeyer, M. J. B., S.-I. Aizawa, R. M. Macnab,, and D. J. DeRosier. 1989. Image reconstruction of the flagellar basal body of Salmonella typhimurium. J. Mol. Biol. 205:519-528. Suzuki, T., T. lino, T. Horiguchi, and S. Yamaguchi. 1978. Incomplete flagellar structures in nonflagellate mutants of Salmonella typhimurium. J. Bacteriol. 133:904-915. Swan, M. A. 1985. Electron microscopic observations of structures associated with the flagella of Spirillum volutans. J. Bacteriol. 161:1137-1145.
Vol. 174, No. 1
0021-9193/92/010263-06$02.00/0 Copyright © 1992, American Society for Microbiology
Location of the Basal Disk and a Ringlike Cytoplasmic Structure, Two Additional Structures of the Flagellar Apparatus of Wolinella succinogenes STEPHAN C. SCHUSTER AND EDMUND BAEUERLEIN* Max-Planck-Institut fuer Biochemie, D-8033 Martinsried, Germany Received 30 July 1991/Accepted 25 October 1991 The basal body of Wolinella succinogenes consists of a central rod, a set of two rings (L and P rings), a basal disk front 70 to 200 nm in diameter, and a terminal knob. In negatively stained preparations of flagellar hook-basal body complexes, some disks remain fixed perpendicularly to the grid and show that such a disk is located on the distal side of the P ring. The basal disks have been isolated with and without the P ring; in both cases there is a hole in the center of the disk. The diameter of the disk is smaller in the presence of the P ring. The L-P ring complex is therefore assumed to be a bushing for the rod. Thin sections of whole bacteria and spheroplasts reveal that the disk is attached to the inner surface of the outer membrane. At the insertions of the flagellar hook-basal body-basal disk complexes, depressions are visible in negatively stained preparations of whole bacteria and spheroplasts. A new ringlike structure is connected to an elongation of the basal body into the cytoplasm in both preparations. Its diameter (60 nm) is larger than that of the M ring. A heavily stained compartment can be seen in between the new ringlike structure and the basal disk, which may be formed by the energy transducing units.
For a long time the entity called the intact flagellum (6-8) was thought to be the essential rotary device of the flagellar motor. In Escherichia coli and Salmonella typhimurium, this assembly consists of the flagellar filament, the hook, and the basal body. The basal body appears as a set of four rings coaxial with the rod. Two rings are very likely to be correlated to membranes: the L ring to the outer membrane and the M ring to the inner membrane. The P ring is in the periplasmic space and is assumed to interact with the peptidoglycan layer. The S ring is clearly visible in negatively stained electron micrographs in both bacteria, but from genetic, biochemical, and physiological studies no indications for its existence emerged until now (10, 11). In an extensive search for flagellar and motility mutants with the paralyzed or switch-defective phenotype, none mapped to the gene of the M ring (fliF) (10); all mapped to five genes encoding proteins that are not present on isolated basal bodies. Three of them (fiG, fliM, fliN) encode flagellar components that participate in both energy transduction and switching (15), whereas the other two (motA, motB) encode proteins that appear to function in energy transduction only (5, 18, 21, 22). It is therefore unlikely that the M ring is the active rotor of the bacterial flagellar apparatus. Furthermore, it was proposed about 13 years ago by Coulton and Murray (3, 4) that essential structures of the flagellar apparatus were lost during the procedures for isolation of intact flagella. They found circlets of about 15 particles around the rod in the cytoplasmic membrane and outer membraneassociated large disks, called concentric membrane rings, in Aquaspirillum serpens. From Wolinella succinogenes, an anaerobic, gram-negative bacterium with one polar flagellum, we recently isolated the flagellar hook-basal body complex, to which large disks were associated as additional structural elements of its flagellar apparatus (16). Here we describe (i) the location of *
these disks on the basal body and between the membranes, (ii) their isolation with and without the correlated rings, and (iii) new ringlike structures on extensions of the rod into the cytoplasm. MATERIALS AND METHODS
Bacterial strain. The W. succinogenes wild-type strain 1740 from Deutsche Sammlung Mikroorganismen was used for all experiments. E. coli 4200 HC (University of Konstanz) was also used. Chemicals and enzymes. All chemicals obtained from Merck Darmstadt were reagent grade; Triton X-100 was obtained from ROTH, Karlsruhe; hen egg white lysozyme and DNase I (grade II) were obtained from Boehringer Mannheim. Growth conditions. The standard Wolinella medium used consisted of the following: ammonium sulfate (4 mM), potassium phosphate (38 mM), fumaric acid (26 mM), and sodium formate (44 mM). After sterilization, magnesium chloride (120 FM) and iron (II) sulfate (7 ,uM) were added. Thioglycolic acid (0.38 RI/ml) was used together with a flux of nitrogen to maintain microaerobic conditions. Cells were grown at 37°C in stoppered Erlenmeyer flasks without shaking to the late-logarithmic phase. E. coli was grown in standard LB medium at 37°C to the early logarithmic phase. Preparation of spheroplasts. A 1-liter culture was harvested and resuspended in 100 ml of a cold solution of 0.5 M sucrose and 10 mM Tris (pH 8). After 30 min, lysozyme (0.02 mg/ml) and EDTA (final concentration, 10 mM) were added. The cells were then incubated overnight. The resulting spheroplasts were stable for several days. For negative staining a carbon-coated copper grid was laid on a 25-,lI drop of a spheroplast suspension. The grid was washed twice and then negatively stained. Isolation of intact flagella. A 100-ml suspension of spheroplasts, prepared as described above, was spun down (8,000
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FIG. 1. Basal disks standing upright perpendicular to the grid in more than one-half of their diameter. They are visible on then distal side of the P ring. In no case could the L ring be detected, suggesting that it may be an integral part of the basal disk. Negative staining. Bars, 100 tnm.
x g, 4°C, 20 min) and lysed in 10 ml of TET buffer (10 mM Tris, 5 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride [pH 8]). The DNA was digested with DNase in the presence of 10 mM MgSO4. After 1 h at room temperature the pH was raised to 11 with 5 N KOH. A low-spin centrifugation was applied (12,000 x g, 40C, 10 min) to remove cell debris, and the supernatant was centrifuged (60,000 x g, 4°C, 60 min) to pellet the intact flagella. The last two centrifugations were repeated three times. Purity could be estimated by a lack of color on the pellet. For E. coli the EDTA treatment at pH 11 was omitted; otherwise the procedure was like that used for W. succinogenes.
Preparation of basal disks. Basal disks could be obtained by sonification of spheroplasts for 10 min in a Branson Sonifier 450. The procedure described above resulted in a preparation good enough for electron microscopic investigation. Electron microscopy. Electron microscopy was done on a Philips CM 10 electron microscope at 80 kV. For negative staining procedures in E. coli and W. succinogenes, we used carbon-coated copper grids and a 2% uranyl acetate stain. Cells studied in thin sections were fixed directly in culture medium with 2% glutaraldehyde for 30 min at 370C. After
centrifugation the cells were taken up into agar blocks (1% agar; Sigma). The agar blocks were incubated for 1 h in 2% glutaraldehyde in Soerensen buffer and then washed for 10 min in Soerensen buffer. Postfixation was done for 1 h in 1% osmium peroxide in Kellenberger buffer. Embedding was in Epon. Sections were picked up on naked 200-mesh copper grids and stained with 1% uranyl acetate.
RESULTS Location of the basal disk on the rod. Flagellar hook-basal body (HBB) complexes of W. succinogenes, to which the basal disks are associated (16), were isolated by solubilization of spheroplasts by mild detergents. In electron micrographs of negatively stained preparations, some of the disks remained standing upright perpendicularly to the grid in more than one half of the diameter, whereas the remaining part was lying on the grid under its HBB complex. It was therefore possible to see the basal disk, located on the distal side of the P ring, probably between the P and L rings (Fig. 1). Though in no case could the L ring be observed, this may be taken as an indication that the L ring is an integral part of the basal disk. If the basal disk is completely attached to the grid, both rings of the distal ring complex, the L and P rings,
FIG. 2. Preparation of basal disks with (A) and without (B) P rings by ultrasonic treatment of spheroplasts and flagellar HBB-basal disk complexes. Diameters: inner ring, 10 nm; outer ring, 24 nm; central hole, 13 nm. Bar, 100 nm.
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FIG. 3. Thin sections of whole bacteria (A), spheroplasts (B), and vesicles (C) arising spontaneously from spheroplasts. In all three preparations high electron density is closely related to the inner surface of the outer membrane. The diameter of this area corresponds to the average diameter of the basal disks. Bars, 100 nm.
are detected always in the center of the basal disk (16). It is therefore very probable that the basal disk is coaxial with the L-P ring complex. In a new procedure the basal disks alone could be obtained by ultrasonic treatment of spheroplasts and of flagellar HBB complexes. In negatively stained preparations, most of these disks are found together with a correlated basal ring in their center; only a few were found without it (Fig. 2). It is probable that this ring corresponds to the P ring (Fig. 1), since its outer diameter is about 23 nm. The diameter of the
central hole is about 13 nm without a ring and about 10 nm with a ring. From all this we assume that the P-L ring basal disk complex serves as bushing for the rod. The basal disk is attached to the inner surface of the outer membrane. To study a possible function of the basal disk, we examined the association of the disk with the membranes. Thin sections of whole bacteria and of spheroplasts were obtained. In electron micrographs of whole cells (Fig. 3A), the depression formed by the basal disk could be seen in the inner and outer membranes. In thin sections of spheroplasts (Fig. 3B and C) a high electron density was found at the insertion of the flagellum on the inner side of the outer membrane. The diameter of this density corresponds with that of an average basal disk. An electron density much higher than that in any other part of the cytoplasmic membrane was found underneath the basal disk. In many cases an extension of this high density into the cytoplasm was detected (Fig. 3B). On rarer occasions two flagellar motors were found in close proximity in one vesicle. Most likely these vesicles resulted from a dividing cell with one motor on each pole; in no case were more than two motors found in one vesicle (Fig. 3C). A new ringlike structure in the cytoplasm, probably connected to the basal body. Just below the insertion of the HBB complex a ringlike structure is visible in negatively stained preparations of whole cells (Fig. 4A) and of spheroplasts (Fig. 4B). Its diameter of 57 to 60 nm is larger than that of the M ring (37 to 39 nm). A more detailed structure is seen in spheroplasts, suggesting that globular particles are fixed at the border of this ringlike structure (Fig. 4B, arrows). Within the area between the basal disk and the ringlike cytoplasmic
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FIG. 4. Ringlike structures coaxial with an extension of the rod into the cytoplasm. (A) Negatively stained whole bacterium. The ringlike structure below the cytoplasmic membrane has a diameter of about 57 to 60 nm and a thickness of about 10 nm. From its outer diameter a compartment of high electron density extends to the basal disk. (B) Negatively stained spheroplasts, the size of which is probably determined by the diameter of the basal disk (diameter, 120 nm). Here the ringlike structure appears to be composed of or occupied by globular particles. The compartment of high electron density is visible also. Bar, 100 nm.
structure is a heavily stained compartment; it appears to be divided into two halves by the motor axis (Fig. 4). Its dimensions are smaller than the diameter of the basal disk but comparable to that of the ringlike cytoplasmic structure. Both of these findings are supported by an electron micrograph of a small spheroplast (Fig. 4B), the size of which is apparently determined by the diameter of the basal disk (120 nm). In this case the depression is more localized to the insertion of the HBB complex, and the outer and inner membranes are no longer distinguishable. The ringlike structure is found also in this more artificial preparation, and even the elongation of the basal body to it may be detected. Additional basal body-associated structures of E. coli: elongation of the rod and M ring-associated arm and caps. The flagellar HBB complexes of E. coli were prepared in a mild way, with low concentrations of EDTA (5 mM) at pH 7.
Additional basal body-associated structures are visible in negatively stained preparations without preceding fixation (Fig. 5). An essential feature of this preparation is that the rod, the motor axis, is elongated on the other side of the M ring (Fig. 5A), suggesting its extension into the cytoplasma. A caplike structure (Fig. 5B) obviously wraps the extended rod and is sometimes partially lost, forming symmetric arms on both sides of the M ring (Fig. 5A). DISCUSSION The basal body-associated disk of W. succinogenes is located on the distal site of the P ring, as visible in electron micrographs of perpendicularly standing basal disks (Fig. 1). The P ring is clearly visible on the proximal side of the basal disk, whereas the L ring cannot be observed on either side.
FIG. 5. Additional basal body-associated structures of E. coli in negatively stained, unfixed preparations of its flagellar HBB complex. (A) Extension of the rod and symmetric arms on both sides of the M ring. (B) A caplike structure wrapping the extension of the rod. Bars, 100 nm.
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FIG. 6. Thin section of W. recta (reprinted from reference 17 with permission).
In earlier electron micrographs where the basal disks lay completely on the grid, either the terminal knob, the S-M ring complex, or two rings (the L-P ring complex) are visible (Fig. 2D, E, and F and Fig. 3A in reference 16). In all cases the L-P ring complex was located in the center of the basal disk. It is therefore very probable that L and P rings form a cylinderlike complex that serves as bushing for the rod. This hypothesis is supported by the fact that the P ring is surrounded by stain in most of negatively stained flagellar HBB preparations with upright standing disks (Fig. 1). In addition, subcomplexes termed staples were obtained by acidic degradation of S. typhimurium HBB complexes, which were thought to be L-P ring complexes (1). Two types of images were found in electron micrographs: a rectangle with one edge missing (staple) and an annulus. Both were correlated to the subcomplex from its side and axial views. Because axial sections of staples and L-P rings on intact basal bodies have showed a striking resemblance in image reconstruction, it has been concluded that staples are L-P ring complexes (23). Additional support for this has come from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of staples, in which only three protein bands have been found, a 27-kDa protein (L ring), a 38-kDa protein (P ring), and a 65-kDa protein (M ring) (1, 12). When spheroplasts or flagellar HBB-basal disk complexes were sonificated, predominantly basal disks were obtained; in the center of the basal disks a ring could be seen. Only a
few of the disks were without a ring (Fig. 2). It is therefore probable that the observed ring or the L-P ring complex interacts more strongly with the basal disk than with the rod, a conclusion that is consistent with the postulated function of the L-P ring complex as a bushing for the rod. The basal disk itself is attached to the inner surface of the outer membrane, providing a rigid area into which one bushing of the flagellar apparatus, the L-P ring complex, is inserted. It is probable that the basal disk is involved in the pole formation of W. succinogenes and the correct arrangement of the energy-transducing units that are assumed to be the intramembrane particle ring structures in the cytoplasmic membranes. These particles were found first in A. serpens (4) and then in Streptococcus sp. and E. coli (13). When a nonfunctional mocha operon was present in E. coli, flagellated but immobile cells that lacked the particle rings were obtained. Both the motility and the ring structure were recovered when motA and motB were introduced simultaneously (13). A common feature of the monoflagellated poles in gramnegative bacteria appears to be a depression formed around the insertion of the flagellum in, for example, W. succinogenes (Fig. 4A). Similar depressions have been found in thin sections of Campylobacter jejuni (2), Campylobacter fetus (formerly Vibrio fetus) (19), and Wolinella recta (17), suggesting that outer membrane, basal disk, and peptidoglycan layer are close together, at least in their central part around the flagellum. This is supported by electron micrographs of spheroplasts from W. succinogenes (Fig. 4B). It is unknown even today whether the basal disk and cytoplasmic membrane are close together within the area of the flagellar apparatus. In negatively stained spheroplasts of W. succinogenes the spatial arrangement of the cytoplasmic membrane could not be distinguished relative to the flagellar apparatus
(Fig. 4B).
Although no basal disks have been isolated from gramnegative bacteria with peritrichous flagella, L ring-associated structures of variable (8) and symmetrical (24) appearance have been found on intact flagellum preparations of S. typhimurium. New ringlike cytoplasmic structures visible in negatively stained whole bacteria (Fig. 4A) of W. succinogenes are probably on an elongation of the basal body. In the spheroplasts, globulelike units appear to be attached around this
Filament
Hook
L-ring
/ P-ring
::*,i]: I
outer membrane
petidoglycan layer
Heavily stained compartment cytoplasmic structure
terminal knob
FIG. 7. Schematic diagram of the HBB-basal disk complex with the additional cytoplasmic structures.
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ring (Fig. 4B). The diameter of the whole structure is about 57 to 60 nm, and it differs clearly from the M ring (diameter of 37 to 39 nm). A compartment of high electron density that was found in whole bacteria and spheroplasts between the basal disk and this new ringlike structure (Fig. 4) apparently determines its diameter. A cylinderlike structure can be seen in thin sections of W. recta (17) (Fig. 6) and Spirillum volutans (25). In several electron micrographs of various bacteria, similar but not so differentiated cytoplasmic areas of high electron density may be observed, for example, in C. jejuni (2) and C. fetus (19). From the elements described above, we propose a hypothetical model of the HBB complex with respect to the cell wall (Fig. 7). In E. coli, additional basal body-associated structures have been found in unfixed, negatively stained preparations (Fig. 5); these structures are very similar to those in fixed, negatively stained preparations of S. typhimurium (9). Recently the structures described above were found in unfixed cells in S. typhimurium (9a). In both, an extension of the motor axis into the cytoplasmic side of the M ring is observed (Fig. 5A). In other preparations a caplike structure covers it; this caplike structure is either a part of the M ring or associated with it (Fig. SB). This cap apparently may be removed or partially destroyed, so that on both sides of the M ring two symmetrical arms are formed (Fig. 5A). In thin sections of S. volutans, the cylinderlike structures appear to be cut through (25). It is possible that these walls become arms if the cylinder is destroyed or parts of it are released. On the other hand, these caplike and armlike structures of S. typhimurium (9) and E. coli (Fig. 5) may be parts of a more irregular, bell-shaped extension of the flagellar base into the cytoplasm as found with rapid-freeze electron microscopy in S. typhimurium (14). In a hypothetical view the cylinderlike or bell-shaped cytoplasmic structures of the flagellar apparatus appear to be part of it. The new ringlike structure may be a switch or a rotor or a combination of both. The basal disk is probably responsible for the mechanical stability of the rotary device, at least in bacteria with polar flagella.
J. BACTERIOL. 6. DePamphilis, M. L., and J. Adler. 1971. Purification of intact flagella from Escherichia coli and Bacillus subtilis. J. Bacteriol.
105:376-383.
7. DePamphilis, M. L., and J. Adler. 1971. Fine structureture and isolation of the hook-basal body complex of flagella from Escherichia coli and Bacillus subtilis. J. Bacteriol. 105:384-395. 8. DePamphilis, M. L., and J. Adler. 1971. Attachment of flagellar basal bodies to the cell envelope: specific attachment to the outer, lipopolysaccharide membrane and the cytoplasmic membrane. J. Bacteriol. 105:396407. 9. Driks, A., and D. J. DeRosier. 1990. Additional structures associated with bacterial flagellar basal body. J. Mol. Biol. 211:669-672. 9a.Driks, A., and D. J. DeRosier. 1991. Personal communication. 10. Homma, M., S. I. Aizawa, G. E. Dean, and R. M. Macnab. 1987. Identification of the M-ring protein of the flagella motor of Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 84:74837487. 11. Jones, C. J., and R. M. Macnab. 1990. Flagella assembly in
12. 13. 14.
15.
16.
17.
ACKNOWLEDGMENTS
18.
We greatly appreciate stimulating discussions with R. M. Macnab, H. Engelhardt, and S. Khan. We also thank R. M. Macnab for critical reading of the manuscript and valuable comments.
19. 20.
1.
2. 3. 4.
5.
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