Vol. 122, No. 1 Printed in U.S.A.
JouRNAL oF BAcrioLoGY, Apr. 1975, p. 307-315 Copyright 0 1975 American Society for Microbiology
Flagellar Ultrastructure and Flagella-Associated Antigens of Campylobacter fetus E. C. McCOY,* D. DOYLE, H. WILTBERGER, K. BURDA, AND A. J. WINTER New York State Veterinary College, Cornell University, Ithaca, New York 14853 Received for publication 3 January 1975
Ultrastructural examinations of the flagellum of Campylobacter (Vibrio) fetus were performed throughout the growth cycle. Filament diameters, exceeding 17.6 nin during the exponential phase, were substantially greater than those reported for unsheathed flagella of other genera with the exception of Pseudomonas fluorescens. Filament diameters increased during growth, reaching a mean width of 21.2 nm in middle to late stationary phase. Internal flagellar structure, principally of the parallel lined variety, was observed during the later periods of growth but not during exponential or early stationary phase. Despite the unusually large filament sizes, no evidence of a flagellar sheath was observed after selected treatments (0.01 N HCl, 6 M urea, tris(hydroxymethyl) aminomethane-hydrochloride buffer, warm water) or examination of thin sections. To
determine whether alterations in filament size and variable ability to demonwere correlated with progressive changes in serological activity, agglutination and immobilization tests were conducted with antisera directed against intact flagella, the principal flagellar antigen, the 0 antigen, and a superficial glycoprotein which has been found in association with the flagellum and the cell envelope. Significant differences in the serological activity of cells at different growth intervals were not noted with any of the sera employed. strate filament fine structure
Campylobacter (Vibrio) fetus (30), a microaerophilic gram-negative rod, is a causative agent of infertility and abortion in cattle and of abortion in sheep. Although a number of reports have been published on the epidemiology and pathogenesis of vibrionic infections, the means whereby C. fetus survives host defenses and initiates infection are not precisely defined. Similarly, the basis for virulence in other gram-negative species is incompletely understood. Recent studies have emphasized the relationship of surface structures (8, 12, 13, 28), including flagella (3, 31), to the infectious process. The bacterial flagellum is composed of a least three antigenically distinct structural units: the filament, hook, and basal disks. The flagellar filament is a tubule varying in size from 12 to 15 nm, composed of identical protein subunits in one of two configurations (1, 15). Lowy and Hanson (16, 17) demonstrated that the arrangement of subunits in intact flagella gives rise to two different types of filament configuration: an A form, which is characterized by helically connected globules, and a thick-lined B form in which helical organization is not observed.
These authors suggest that with rare exception the predominant conformational type is a constant feature for a given bacterial species. The flagellar filament is continuous with a hook structure which extends into the outer envelope layers and is anchored to the cell wall and membrane layers by basal disks (29). In several species a filament accessory structure, a flagellar sheath, has been demonstrated (7, 9, 10, 24). Sheaths vary in size and structural organization, and the presence of sheath material may obscure the demonstration of filament ultrastructure. Although continuity between sheath and cell envelope layers, as observed in Bdellovibrio bacteriovorus (24), has not been demonstrated in Vibrio species, the finding that antibody produced against purified somatic antigen is functional in flagellar immobilization of V. cholera (31) is consistent with this possibility. The present report is part of an investigation of surface antigens of C. fetus. The inability to demonstrate filament ultrastructure during the exponential phase of growth and the recognition of unusually large filament diameter measurements suggested the presence of a flagellar
307
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McCOY ET AL.
sheath in C. fetus. The consistent association of a glycoprotein antigen with both cell envelope and purified flagellar preparations prompted an attempt to establish the identity of this antigen as a sheath structure. MATERIALS AND METHODS Bacterial strains. C. fetus subsp. fetus (intestinalis) strain 23, maintained in lyophilized form after its isolation in 1962 from an aborted 5-month bovine fetus and characterized previously (32), was used throughout this study. The host-dependent Bdellovibrio bacteriovorus 109J was obtained from S. C. Rittenberg (Department of Bacteriology, University of California at Los Angeles), and Bacillus subtilis C U 180 was supplied by Stanley Zahler (Department of Genetics, Cornell University). Media and growth conditions. Albimi agar and broth (Difco, Detroit, Mich.) supplemented with 12.5 mg of glutathione, 37.5 mg of sodium thioglycollate, and 200 mg of cysteine hydrochloride per liter of medium were used for the cultivation of C. fetus. Cultures maintained in semisolid Albimi agar (0.16%) were used for broth overlays in Blake bottles. Cultures were incubated at 37 C in vacuum desiccator jars in an atmosphere of 87.5% N2, 10% CO2, and 2.5% 02. For growth curve studies samples from 10- to 12-h overlay cultures were introduced into flasks containing Albimi broth to give a final absorbance of 0.05 at 525 nm. Flasks were incubated in a shaker incubator (New Brunswick) at 175 rpm and continuously gassed at a rate of 1.0 cubic foot per hour with 95% N2 and 5% 02. Samples were removed at selected time intervals and processed for viable counts (18), optical density measurements and electron microscopy. Batch cultivation of C. fetus in liquid medium was conducted in the shaker incubator in two ways. In earlier studies cells were cultivated as above in Albimi broth, collected after 16 h, and used for the extraction of soluble antigens. Later, cells were grown for 3 days in a modified Stuart medium with ascending concentrations of oxygen in the gas mixture as described by Clark et al. (2). This technique was employed since it increased cell yields approximately fivefold. These organisms were used for the isolation of flagella. The cultivation method used for B. bacteriovorus was that described by Rittenberg and Shilo (23). Broth samples from 24-h cultures were used for negative staining. B. subtilis was grown in Trypticase soy broth (Difco) for 4 to 6 h prior to preparation of negative stains. Extraction of antigens from whole cells of C. fetus. Cells were washed twice with distilled water and suspended in 100 ml of 0.2 M glycine-hydrochloride buffer (pH 2.2) per 4 g of packed cells. After stirring for 15 min at ambient temperature the mixture was centrifuged for 15 min at 11,000 x g. The supernatant fluid was neutralized, dialyzed exhaustively at 5 C against deionized H20, concentrated about 20-fold by ultrafiltration, and frozen. Acrylamide gel electrophoresis was performed by the method of Davis (5). Proteins were stained by Buffalo Black and carbohydrate with the periodic acid Schiff stain.
J. BACTERIOL.
To correlate stained bands with precipitin reactions, gel rods were sliced longitudinally; one-half was stained, and the other was incubated in agar parallel to a trough containing antiserum. 0 antigen was extracted from whole cells with tri-
chloroacetic acid by the procedure described by Staub
(27).
Purification of flagella. Freshly grown cells were separated from growth medium, washed twice in saline, and resuspended in borate buffer (0.05 M, pH 8.4) at a concentration of 1 g of packed cells per 4 ml of buffer solution. Flagella were sheared in an Omnimixer (Ivan Sorvall, Inc.) for 2 min at a predetermined speed sufficient to remove flagella without excessive fragmentation. Flagella were separated from cells by five cycles of low and high speed centrifugation, essentially as performed by Miwatani et al. (19). The final product, referred to as partially purified flagella, was devoid of cells but contained numerous globular bodies. It was stored at 5 C with toluene or NaN3 (0.02%) as preservative. Flagella were further purified by isopycnic gradient contrifugation in CsCl by the method of Shapiro and Maizel (25). Centrifugation at 25,000 rpm in a SW27 rotor (Beckman) was performed at 22 C for 2 days. Fractions were obtained by collecting drops either through a puncture in the bottom of the tube or by upward displacement, using an automatic system (ISCO Gradient Fractionator, model 182). CsCl was removed by dialysis. Acid solubilization of flagella. Aqueous suspensions of flagella were adjusted to a pH of 2.0 with HCl and held at ambient temperature with stirring for 1 h followed by centrifugation for 90 min at 70,000 x g. The pH of the supernatant fluid was adjusted to 7.0. Production of antisera. Male New Zealand white rabbits were immunized with bacterial cells, immunoprecipitates, or aqueous solutions emulsified in Freund complete adjuvant by methods previously employed (6). Two rabbits were immunized three times at 3-day intervals with 200 ,g of whole flagella by intravenous injection of aqueous suspensions. Rabbits were exsanguinated by cardiac puncture within 1 week after the last immunization. Serological reactions. Agglutination, immunoelectrophoresis, and immunodiffusion techniques were performed as previously described (6, 32). Immobilizing activity was tested by mixing bacteria with serum to obtain a final concentration of 1 x 108 cells/ml. A drop of this suspension was placed on a warmed slide and overlaid with a cover slip resting on a thin film of Vaseline. The preparation was examined by phase microscopy at a magnification of 500x. The percentage of cells retaining translational motility, either singly or clumped, was noted at intervals up to 3 min. In addition, mixtures of bacteria and antiserum were incubated at 37 C in a water bath and examined in similar fashion after 30 min. Preparation of samples and electron microscopy. Copper grids, 200 mesh, were coated with 0.18% Formvar and carbon stabilized. Samples from cultures standardized to an optical density of 0.5 at 525 nm were transferred to grids by flotation of the grid on a drop of cell suspension for 2 min. Grids were drained on filter paper and floated on distilled water for 10 to
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15 s, drained, transferred to a drop of negative stain for designated time intervals, and again drained. Staining procedures included: (i) uranyl acetate, 0.5% and 1.0% with no pH adjustment (pH 3.9) for 1 to 2 min; (ii) 1% uranyl acetate through a pH range of 3.4 to 6.8 for 2 min; (iii) 0.5 and 1.0% K and Na phosphotungstic acid (KPT or NaPT), pH 7.4, for 15S to 2 min. For short term exposure to chemicals (2 s to 2 min), grids with adfixed cells were floated on the chemical and washed prior to staining. When longer time intervals were used, the bacterial cells were mixed with the selected agent in test tubes prior to flotation of grids. Chemical agents employed included: 0.01 N HCl through a time interval of 2 s to 10 min; 6 M urea (pH 7.5) through a time interval of 2 s to 15 min; 40 C distilled water and tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (0.05 M, pH 7.5). External diameters were estimated by averaging at least three measurements along the length of negatively stained flagella, avoiding areas of curvature. Up to three individual filaments were measured per negative, and for statistical analysis a grand mean value of filament diameters obtained from one negative was taken as a single observation. Measurements were taken from negatives and converted to angstrom units using catalase crystals as an internal standard
(33). For thin-section preparation, bacteria from liquid cultures were centrifuged from the medium and enrobed in agar. The agar blocks were cut to 1-mm cubes and placed in 1% OSO4 in veronal acetate buffer, pH 6.8 (14) at room temperature for 1 h and refrigerated for an additional 45 min. After three washes in veronal acetate buffer samples were divided in half. One-half was placed on 0.5% uranyl acetate for 1 h, whereas the other was kept in buffer for the same period (26). Dehydration in graded series of alcohol and propylene oxide was followed by embedding in a modified Epon-Araldite plastic mixture (20). Sections were cut with a diamond knife on an LKB Ultratome III. Grey to silver sections were picked up on uncoated 200-mesh copper grids and stained with 2% uranyl acetate followed by lead citrate (21). Sections and negative stains were viewed in a Phillips E.M.-300 electron microscope at 80 kV.
RESULTS Extraction of bacteria in glycine-hydrochloride buffer. Immunodiffusion and immunoelectrophoresis with whole cell antiserum revealed the presence of 3 antigens in supernatant fluids, none of which corresponded to the 0 antigen (32). 0 antigen was present in some preparations as a faint line in immunodiffusion tests. The component present in the highest concentration has been termed [a], the anodal component adjacent to it [b], and the most cathodal one [c] (Fig la). Prior to treatment with acid buffer, C. fetus cells were inagglutinable in 0 antiserum, but after treatment agglutinability in 0 antiserum was complete,
309
indicating the removal of superficial antigens which prevent such agglutination of fresh cells. As expected, examination of negative stains disclosed that acid treatment had also caused complete removal of flagella. The presence of only very low concentrations of 0 antigen in the extracts indicated that the extraction process had not disrupted cells and that antigens [a], [b], and [c] were therefore located superficially. However, their identification as somatic or flagellar components could not be resolved. Antigen [a] has been identified in acrylamide gel electrophoresis. It migrated with a retardation factor of 0.41 and stained with protein and carbohydrate stains. CsCl separation of sheared flagella. Partially purified flagella was composed of flagellar filaments, some of which had retained hooks, and a large number of globular bodies (Fig. 2a) which resembled structures located on the bacterial cell surface (Fig. 2b). Similar structures have been reported in prior studies with C. fetus (22) and V. parahemolyticus (19). After centrifugation in CsCl gradients, two discrete but very closely spaced bands developed (Fig. lc). The upper of these, located at a specific gravity of 1.29, contained flagella almost free of globular bodies (Fig. 2c). The lower band contained flagella with somewhat greater contamination. However, the reason for the distinct separation of these bands was not established. A third broad band beneath the others contained globular bodies. Intact flagella from the topmost band produced no reactions in immunodiffusion tests. Solubilization of flagella with acid caused the release of three antigens (Fig. lb). One of these was [a]; and the other two are referred to as [d] and [e]. Antigen [d] has not been identified in immunoelectrophoresis. Antigen [e] was present in the greatest abundance, was electrophoretically heterogeneous, and migrated on each side of the origin well (Fig. la). Growth kinetics. The growth phases of C. fetus cells as determined by optical density measurements and viable counts were as follows; exponential phase, 6 to 12 h; early stationary phase 12 to 15 h; mid-stationary phase 18 to 24 h, and late stationary phase 48 h. Flagella measurements. Diameter measurements of intact flagella from overlay cultures of C. fetus in the stationary phase of growth (24 to 48 h) and of control cultures of B. bacteriovorus and B. subtilis, negatively stained for 1 min with 0.5% uranyl acetate (pH 3.9) or 0.5% NaPT (pH 6.8), are recorded in Table 1. Filament diameter did not appear to be affected by the staining method employed, although staining
310
McCOY ET AL.
J. BACTERIOL.
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05
b
FIG. 1. (a) Immunoelectrophoresis patterns produced by extracts of Campylobacter fetus strain 23 cells. Anode is to the right. Top well: acid solubilized flagella; middle well: acid extract of whole cells; bottom well: trichloroacetic acid extract of whole cells (O antigen). Troughs contain a whole cell antiserum. Bands produced by antigens [a], [b], [c], [e], and 0 are labeled. (b) Immunodiffusion patterns produced with extracts of Campylobacter fetus. Well 1: acid-solubilized flagella; well 2: antigen [a] separated from an acid extract by pevikon block electrophoresis; wells 3, 4, and 5: antiserum prepared with acid solubilized flagella. Bands produced by antigens [a], [d], and [e] are labeled. (c) Cesium chloride gradient separation of sheared flagella. The two closely spaced white bands contained flagella; the diffuse band beneath these contained globular bodies.
with 0.5% uranyl acetate proved superior for demonstrating sheath-core delineation in B. bacteriovorus (Fig. 3c), as well as filament fine structure in C. fetus (Fig. 3a). Internal structure in C. fetus flagella was predominantly of the B type in which six or infrequently seven equidistant lines of stain, including the outer edges, were present. In many filaments regions of flagellar A structure were also apparent. No evidence of a sheath was observed. B. subtilis
flagella possessed B structure (Fig. 3b) as has been observed by Ichiki and Martinez (11). No internal filament fine structure was discernible in B. bacteriovorus flagella. Initial flagellar diameter measurements in C. fetus (48 h) approached in size the published values for flagella with the large sheath type (7). Further attempts were made to demonstrate a flagellar sheath in this organism despite its apparent absence in negatively stained prepara-
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CAMPYLOBACTER FETUS FLAGELLA
31
uu .2um b FIG. 2. (a) and (c) Electron micrographs of sheared flagella preparations of Campylobacter fetus, negatively stained with uranyl acetate, before (a) and after (c) fractionation in a CsCI gradient. (b) C. fetus cell from a 2-day broth overlay culture, negatively stained with uranyl acetate, showing surface structures resembling the globular bodies found. TABLE 1. Diameter measurements of negatively stained flagella Organism
Campylobacter fetus (24 h)b Camplyobacter fetus (48 h)b Bdellovibrio bacteriovorus Bacillus subtilis
No. of obser-
vationsa
11 9 9 9
Filament diameter (nm)
18.2 21.2 28.3 13.6
± ± ± ±
2.0 2.6 4.7 1.0
a The number of observations refers to the number of individual preparations examined. " Broth overlay cultures.
tions. To this end, cells were examined in earlier phases of growth, treatments were employed which enhance the differentiation of sheath and core components, and thin sections were examined. Minimal flagellar diameter measurements were obtained from cells harvested in the exponential and early stationary phase of growth (Table 2). Examination of young cultures grown in Albimi medium with and without supplements or in brain heart infusion broth (Difco) failed to reveal any evidence of internal filament structure (Fig 4). Antigen [a] was present on the cell surface at all stages of growth, as revealed by agglutination tests with specific antiserum. Immunodiffusion tests with broth supernatants indicated that the release of some
antigen [a] had also occurred into the growth medium by mid-stationary phase. Treatment effects. Representative samples of negatively stained preparations of C. fetus and B. bacteriovorus flagella after exposure to 0.01 N HCl are included in Fig. 5. Rapid removal of sheath material was noted when B. bacteriovorus flagella were exposed to acid. However, core filaments were intact after 1 min of treatment (Fig 5d). Differential effects of a comparable nature were never observed in C. fetus. Brief exposure (2 s) of C. fetus cells to acid resulted in a loss of rigidity of the entire flagellum (Fig. 5a); upon longer treatment (10 to 15 s) complete depolymerization was observed (Figs. 5b and c). Results were the same regardless of the phase of growth examined. Treatment of the respective organisms with 6 M urea produced final results similar to those obtained with HCI. However, the initial effect noted in C. fetus cells exposed to urea for 10 to 15 s was breakage of the flagellum at the proximal end. Extraction of C. fetus cells with warm water and Tris-hydrochloride buffer (pH 7.5) resulted in an altered appearance of filaments from cells in the later phases of growth, as well as release of antigen [a] into the supernatant fluid. Internal structure could not be demonstrated in filaments after treatment. Similarly, washing stationary phase cells with distilled water prior
312
McCOY ET AL.
J. BACTERIOL.
1. ...
1
.
FIG. 3. Electron micrographs of uranyl acetate-stained flagella. (a) Campylobacter fetus filament from a 24-h broth overlay culture. Note filament substructure of the lined variety. (b) Bacillus subtilis filament from a 4-h broth culture showing filament substructure of the lined variety. (c) Bdellovibrio bacteriovorus filament from a 24-h broth culture. Note the presence of a flagellar sheath structure. TABLE 2. Characteristics of Campylobacter fetus flagella during the growth cycle No. of
Phase of grwh observagrowth tions'
Exponential (9 h) Early stationary (12 h) Stationary (15 to 24 h)
Filament diameter
(nm)
4
17.6 4 7.6
2
17.2 ± 7.6
5
18.6 ± 16.4
Itra
InternalI
stuur
None demonstrable None demonstrable Lined form
a The number of observations refers to the number of individual preparations examined.
to flotation on grids adversely affected the demonstration of filament ultrastructure. Tris treatment of Bdellovibrio flagella resulted in an apparent loosening of the sheath at intervals along the filament length. Thin sections. No evidence of a flagellar sheath continuous with the cell envelope could be demonstrated in C. fetus or B. subtilis. The results obtained with B. bacteriovorus, in agreement with the findings of Seidler and Starr (24), clearly demonstrated the relationship of the
Bdellovibrio flagellar sheath with the outer cell envelope layer. Agglutination and immobilization studies. Since flagella from cells in different phases of growth having relatively different quantities of free and cell-associated [a] antigen appeared to be structurally different by electron microscope studies, attempts were made to determine whether differences in serological behavior could be demonstrated. Agglutination and immobilization reactions were performed with cells collected from broth cultures at 9, 12, 18, 24 and 48 h. Cells were tested with antisera prepared against antigens [a], [e], whole flagella, acid-solubilized flagella, and boiled cells. No appreciable differences were noted with cells of different ages in agglutination reactions. The following effects were observed in the 3-min immobilization reaction: no effect (single, motile cells); formation of small, motile clumps; and formation of large, nonmotile clumps. The incidence of large, nonmotile clumps was low (1 to 5%) and occurred with antisera against whole flagella, solubilized flagella, and antigen [a]. On longer incubation (30 min) a marked increase in the percentage of large nonmotile
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VOL. 122, 1975
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FIG. 4. Electron micrographs of uranyl acetate-stained flagella from Campylobacter fetus broth cultures sampled at 9 h (a) 12 h (b) 24 h (c) and 48 h (d) growth time intervals. Note increases in filament width and the development of internal structure at successive stages of growth. Oblique pattern in filament substructure may be seen in Fig. 5 (arrow).
clumps was observed only with antisera against solubilized flagella and antigen [a]. Notable differences in immobilization attributable to phase of growth were not observed. DISCUSSION Sheathed flagella have been demonstrated in few bacterial genera which include Bdellovibrio, Vibrio, Pseudomonas, and Proteus. The flagellar sheath may contribute significantly to filament diameter (7, 9, 10, 24) or produce a minimal increment in flagellar width (17). Examinations of negatively stained and thin-sectioned preparations of C. fetus during the stationary phase of growth provided no indication of the presence of a sheath of either type. Fuerst and Hayward (9) reported that flagellar sheathing appears to be a stable characteristic and stressed the importance of the sheath as a taxonomic criterion. In this respect our results are consistent with the generic separation of C. fetus from the genus Vibrio (30). Comparisons of C. fetus flagella from different growth phases revealed several points of interest. The diameters of filaments from exponential phase cultures of C. fetus were smaller than those from stationary phase but exceeded diameters reported for unsheathed species with
the exception of P. fluorescens, which has a mean width of 17.3 nm (17). Demonstration of filament fine structure was not achieved in C. fetus during expontial phase and could not be enhanced by manipulation of the growth environment or suspending milieu. However, in flagella from stationary phase cells filament fine structure, resembling that in P. fluorescens with respect to the number of internal lines in the B filament, was obscured by distilled water washing or suspension in Tris buffer. Czajkowski et al. (4) reported the failure to demonstrate internal structure in intact flagella of P. mirabilis and B. subtilis which were grown overnight and water washed by centrifugation. They suggested that linear patterns, associated with an increase in filament diameter, could be ascribed to filament degradation. Our findings with B. subtilis, in which young cells were not washed prior to grid preparation, disagree with those of Czajkowski et al., and experiments with C. fetus cells in stationary phase revealed that washing prior to grid preparation caused the loss of filament substructure. The cellular localization and function of antigens [a], [b], and [c] are not fully clarified, and the identification of flagellar components [d] and [e] with specific flagellar structures re-
314
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McCOY ET AL.
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FIG. 5. Electron micrographs of uranyl acetate-stained preparations of flagella following exposure to 0.01 N HCI. (a) Campylobacter fetus flagellum exposed to acid for 2 s. (b) C. fetus flagellum after a 5-s exposure to acid. (c) C. fetus flagellum exposed to acid for 15 s. (d) Bdellovibrio bacteriovorus flagellum after a 5-s exposure to acid. Note the delineation of sheath and core components.
mains to be established. The location of component [a] on the cell body has recently been confirmed (E. C. McCoy et al., submitted for publication). Its detection in flagellar prepara-
tions may have been the result of minute contamination of those products with globular bodies, although the possibility that this antigen represents a superficial constituent of both
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CAMPYLOBACTER FETUS FLAGELLA
the cell body and flagellum cannot be excluded. The latter possibility is supported by the observation that antiserum prepared against intact flagella is unable to effect significant immobilization of cells possessing [a] antigen. However, the same antiserum is extremely effective in immobilizing a mutant of strain 23 which lacks [a] antigen (E. C. McCoy, unpublished data). The possibility that [a] antigen covers a site on the flagellum critical for immobilization is under study. ACKNOWLEDGMENTS We would like to acknowledge the advice and assistance of John Telford in electron microscopy and Rafael Martinez for critical review of the manuscript. This study was supported by Public Health Service grant AI 11160 from the National Institute of Allergy and Infectious Diseases.
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flagellar antigens from Salmonella adelaide. Aust. J. Exp. Biol. Med. Sci. 42:267-282. Clark, B. L., J. H. Dufty, and M. J. Monsbourgh. 1972. Immunization against bovine vibriosis. 1. Comparison of the protective properties of bacterins prepared by two methods. Aust. Vet. J. 48:376-381. Corbeil, L. B., G. D. Schurig, J. R. Duncan, R. R. Corbeil, and A. J. Winter. 1974. Immunoglobulin classes and biological functions of Campylobacter (Vibrio) fetus antibodies in serum and cervicovaginal mucus. Infect. Immun. 10:422-429. Czajkowski, J., V. Soltesz, and C. Weibull. 1974. Absence of an electron microscopic substructure in intact flagella of Proteus mirabilis and Bacillus subtilis. J. Ultrastruct. Res. 46:79-86. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-436. Duncan, J. R., B. N. Wilkie, F. Hiestand, and A. J. Winter. 1972. The serum and secretory immunoglobulins of cattle: characterization and quantitation. J. Immunol. 128:965-976. Follett, E. A. C., and J. Gordon. 1963. An electron microscope study of Vibrio flagella. J. Gen. Microbiol. 32:235-239. Frasch, C. E., and E. C. Gotschlich. 1974. An outer membrane protein of Neisseria meningitidis group B responsible for serotype specificity. J. Exp. Med. 140:87-104. Fuerst, J. A., and A. C. Hayward. 1969. The sheathed flagellum of Pseudomonas stizolobii. J. Gen. Microbiol. 58:239-245. Glauert, A. M., D. Kerridge, and R. W. Horne. 1963. The fine structure and mode of attachment of sheathed flagellum of Vibrio metchnikovii. J. Cell Biol. 18:327-336. Ichicki, A. J., and R. J. Martinez. 1969. Antigenic heterology between flagellin and flagella of Bacillus subtilis. J. Bacteriol. 98:481-485. Jones, G. W., and J. M. Rutter. 1972. Role of K88 antigen in the pathogenesis of neonatal disease caused by Escherichia coli in piglets. Infect. Immun. 6:918-297. Kaijser, B. 1973. Immunology of Escherichia coli: K antigen and its relation to urinary tract infection. J.
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Infect. Dis. 127:670-677. 14. Kellenberger, E., A. Ryter, and J. Sechaud. 1958. Electron microscope study of DNA-containing plasm. II. Vegetative and mature phage DNA as compared with
normal bacterial nucleoids in different physiological states. J. Biophys. Biochem. Cytol. 4:671-678. 15. Koffler, H. and R. W. Smith. 1972. Nature of flagellar antigens, p. 31-66. In A. Nowotny (ed.), Cellular antigens. Springer-Verlag, New York. 16. Lowy, J., and J. Hanson. 1964. Structure of bacterial flagella. Nature (London) 202:538-540. 17. Lowy, J., and J. Hanson. 1965. Electron microscopic studies of bacterial flagella. J. Mol. Biol. 11:293-313. 17a. McCoy, E. C., D. Doyle, K. Burda, L. B. Corbeil, and A. J. Winter. 1975. Superficial antigens of Campylobacter (Vibrio) fetus: characterization of an antiphagocytic component E. Infect. Immun. 11:517-525. 18. Miles, A. A., and S. S. Misra. 1938. The bacteriocidal power of the blood. Hygiene 38:732-748. 19. Miwatani, T., S. Shinoda, and T. Fujino. 1970. Purification of monotrichous flagella of Vibrio parahemolyticus. Biken J. 13:149-155. 20. Mollenhauer, H. H. 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39:111-114. 21. Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17:208-213. 22. Ritchie, A. E,, R. F. Keeler, and J. H. Bryner. 1966. Anatomical features of Vibrio fetus: electron microscope survey. J. Gen. Microbiol. 43:427-438. 23. Rittenberg, S. C., and M. Shilo. 1970. Early host damage in the infection cycle of Bdellovibrio bacterivorus. J. Bacteriol. 102:149-160. 24. Seidler, R. J., and M. P. Starr. 1968. Structure of the flagellum of Bdellovibrio bacteriovorus. J. Bacteriol. 95:1952-1955. 25. Shapiro, L., and J. V. Maizel. 1973. Synthesis and structure of Caulobacter crescentus flagella. J. Bacteriol. 113:478-485. 26. Silva, M. T., and J. C. F. Sousa. 1973. Ultrastructure of the cell wall and cytoplasmic membrane of gram-negative bacteria with different fixation techniques. J. Bacteriol. 113:953-962. 27. Staub, A. M. 1967. Preparation of cell wall antigens from gram negative bacteria, p 29-30. In C. A. Williams and M. W. Chase (ed.), Methods in immunology and immunochemistry, vol. I. Academic Press, New York. 28. Swanson, J., S. J. Kraus, and E. C. Gotschlich. 1971. Studies on gonococcus infection. I. Pili and zones of adhesion: their relation to gonococcal growth patterns. J. Exp. Med. 134:886-905. 29. Vaitazis, Z., and R. N. Doetsch. 1969. Relationship between cell wall, cytoplasmic membrane, and bacterial motility. J. Bacteriol. 100:512-521. 30. Veron, M., and R. Chatelain. 1973. Taxonomic study of the genus Campylobacter Sebald and Veron and designation of the neotype strain for type species, Campylobacter fetus (Smith and Taylor) Sebald and Veron. Int. J. Syst. Bacteriol. 23:122-134. 31. Williams, H. R., Jr., W. F. Verwey, G. D. Schrank, and E. K. Hurry. 1974. Proc. Ninth Joint Cholera Res. Conf., October 1-3, 1973, Grand Canyon, Arizona. Department of State Publication 8762, U.S. Government Printing Office, Washington, D.C. 32. Winter, A. J. 1966. An antigenic analysis of Vibrio fetus. III. Chemical, biologic and antigenic properties of the endotoxin. Am. J. Vet. Res. 27:653-658. 33. Wrigley, N. G. 1968. The lattice spacing of crystalline catalase as an internal standard of length in electron microscopy. J. Ultrastrct. Res. 24:454-464.
JouRNAL oF BAcrioLoGY, Apr. 1975, p. 307-315 Copyright 0 1975 American Society for Microbiology
Flagellar Ultrastructure and Flagella-Associated Antigens of Campylobacter fetus E. C. McCOY,* D. DOYLE, H. WILTBERGER, K. BURDA, AND A. J. WINTER New York State Veterinary College, Cornell University, Ithaca, New York 14853 Received for publication 3 January 1975
Ultrastructural examinations of the flagellum of Campylobacter (Vibrio) fetus were performed throughout the growth cycle. Filament diameters, exceeding 17.6 nin during the exponential phase, were substantially greater than those reported for unsheathed flagella of other genera with the exception of Pseudomonas fluorescens. Filament diameters increased during growth, reaching a mean width of 21.2 nm in middle to late stationary phase. Internal flagellar structure, principally of the parallel lined variety, was observed during the later periods of growth but not during exponential or early stationary phase. Despite the unusually large filament sizes, no evidence of a flagellar sheath was observed after selected treatments (0.01 N HCl, 6 M urea, tris(hydroxymethyl) aminomethane-hydrochloride buffer, warm water) or examination of thin sections. To
determine whether alterations in filament size and variable ability to demonwere correlated with progressive changes in serological activity, agglutination and immobilization tests were conducted with antisera directed against intact flagella, the principal flagellar antigen, the 0 antigen, and a superficial glycoprotein which has been found in association with the flagellum and the cell envelope. Significant differences in the serological activity of cells at different growth intervals were not noted with any of the sera employed. strate filament fine structure
Campylobacter (Vibrio) fetus (30), a microaerophilic gram-negative rod, is a causative agent of infertility and abortion in cattle and of abortion in sheep. Although a number of reports have been published on the epidemiology and pathogenesis of vibrionic infections, the means whereby C. fetus survives host defenses and initiates infection are not precisely defined. Similarly, the basis for virulence in other gram-negative species is incompletely understood. Recent studies have emphasized the relationship of surface structures (8, 12, 13, 28), including flagella (3, 31), to the infectious process. The bacterial flagellum is composed of a least three antigenically distinct structural units: the filament, hook, and basal disks. The flagellar filament is a tubule varying in size from 12 to 15 nm, composed of identical protein subunits in one of two configurations (1, 15). Lowy and Hanson (16, 17) demonstrated that the arrangement of subunits in intact flagella gives rise to two different types of filament configuration: an A form, which is characterized by helically connected globules, and a thick-lined B form in which helical organization is not observed.
These authors suggest that with rare exception the predominant conformational type is a constant feature for a given bacterial species. The flagellar filament is continuous with a hook structure which extends into the outer envelope layers and is anchored to the cell wall and membrane layers by basal disks (29). In several species a filament accessory structure, a flagellar sheath, has been demonstrated (7, 9, 10, 24). Sheaths vary in size and structural organization, and the presence of sheath material may obscure the demonstration of filament ultrastructure. Although continuity between sheath and cell envelope layers, as observed in Bdellovibrio bacteriovorus (24), has not been demonstrated in Vibrio species, the finding that antibody produced against purified somatic antigen is functional in flagellar immobilization of V. cholera (31) is consistent with this possibility. The present report is part of an investigation of surface antigens of C. fetus. The inability to demonstrate filament ultrastructure during the exponential phase of growth and the recognition of unusually large filament diameter measurements suggested the presence of a flagellar
307
308
McCOY ET AL.
sheath in C. fetus. The consistent association of a glycoprotein antigen with both cell envelope and purified flagellar preparations prompted an attempt to establish the identity of this antigen as a sheath structure. MATERIALS AND METHODS Bacterial strains. C. fetus subsp. fetus (intestinalis) strain 23, maintained in lyophilized form after its isolation in 1962 from an aborted 5-month bovine fetus and characterized previously (32), was used throughout this study. The host-dependent Bdellovibrio bacteriovorus 109J was obtained from S. C. Rittenberg (Department of Bacteriology, University of California at Los Angeles), and Bacillus subtilis C U 180 was supplied by Stanley Zahler (Department of Genetics, Cornell University). Media and growth conditions. Albimi agar and broth (Difco, Detroit, Mich.) supplemented with 12.5 mg of glutathione, 37.5 mg of sodium thioglycollate, and 200 mg of cysteine hydrochloride per liter of medium were used for the cultivation of C. fetus. Cultures maintained in semisolid Albimi agar (0.16%) were used for broth overlays in Blake bottles. Cultures were incubated at 37 C in vacuum desiccator jars in an atmosphere of 87.5% N2, 10% CO2, and 2.5% 02. For growth curve studies samples from 10- to 12-h overlay cultures were introduced into flasks containing Albimi broth to give a final absorbance of 0.05 at 525 nm. Flasks were incubated in a shaker incubator (New Brunswick) at 175 rpm and continuously gassed at a rate of 1.0 cubic foot per hour with 95% N2 and 5% 02. Samples were removed at selected time intervals and processed for viable counts (18), optical density measurements and electron microscopy. Batch cultivation of C. fetus in liquid medium was conducted in the shaker incubator in two ways. In earlier studies cells were cultivated as above in Albimi broth, collected after 16 h, and used for the extraction of soluble antigens. Later, cells were grown for 3 days in a modified Stuart medium with ascending concentrations of oxygen in the gas mixture as described by Clark et al. (2). This technique was employed since it increased cell yields approximately fivefold. These organisms were used for the isolation of flagella. The cultivation method used for B. bacteriovorus was that described by Rittenberg and Shilo (23). Broth samples from 24-h cultures were used for negative staining. B. subtilis was grown in Trypticase soy broth (Difco) for 4 to 6 h prior to preparation of negative stains. Extraction of antigens from whole cells of C. fetus. Cells were washed twice with distilled water and suspended in 100 ml of 0.2 M glycine-hydrochloride buffer (pH 2.2) per 4 g of packed cells. After stirring for 15 min at ambient temperature the mixture was centrifuged for 15 min at 11,000 x g. The supernatant fluid was neutralized, dialyzed exhaustively at 5 C against deionized H20, concentrated about 20-fold by ultrafiltration, and frozen. Acrylamide gel electrophoresis was performed by the method of Davis (5). Proteins were stained by Buffalo Black and carbohydrate with the periodic acid Schiff stain.
J. BACTERIOL.
To correlate stained bands with precipitin reactions, gel rods were sliced longitudinally; one-half was stained, and the other was incubated in agar parallel to a trough containing antiserum. 0 antigen was extracted from whole cells with tri-
chloroacetic acid by the procedure described by Staub
(27).
Purification of flagella. Freshly grown cells were separated from growth medium, washed twice in saline, and resuspended in borate buffer (0.05 M, pH 8.4) at a concentration of 1 g of packed cells per 4 ml of buffer solution. Flagella were sheared in an Omnimixer (Ivan Sorvall, Inc.) for 2 min at a predetermined speed sufficient to remove flagella without excessive fragmentation. Flagella were separated from cells by five cycles of low and high speed centrifugation, essentially as performed by Miwatani et al. (19). The final product, referred to as partially purified flagella, was devoid of cells but contained numerous globular bodies. It was stored at 5 C with toluene or NaN3 (0.02%) as preservative. Flagella were further purified by isopycnic gradient contrifugation in CsCl by the method of Shapiro and Maizel (25). Centrifugation at 25,000 rpm in a SW27 rotor (Beckman) was performed at 22 C for 2 days. Fractions were obtained by collecting drops either through a puncture in the bottom of the tube or by upward displacement, using an automatic system (ISCO Gradient Fractionator, model 182). CsCl was removed by dialysis. Acid solubilization of flagella. Aqueous suspensions of flagella were adjusted to a pH of 2.0 with HCl and held at ambient temperature with stirring for 1 h followed by centrifugation for 90 min at 70,000 x g. The pH of the supernatant fluid was adjusted to 7.0. Production of antisera. Male New Zealand white rabbits were immunized with bacterial cells, immunoprecipitates, or aqueous solutions emulsified in Freund complete adjuvant by methods previously employed (6). Two rabbits were immunized three times at 3-day intervals with 200 ,g of whole flagella by intravenous injection of aqueous suspensions. Rabbits were exsanguinated by cardiac puncture within 1 week after the last immunization. Serological reactions. Agglutination, immunoelectrophoresis, and immunodiffusion techniques were performed as previously described (6, 32). Immobilizing activity was tested by mixing bacteria with serum to obtain a final concentration of 1 x 108 cells/ml. A drop of this suspension was placed on a warmed slide and overlaid with a cover slip resting on a thin film of Vaseline. The preparation was examined by phase microscopy at a magnification of 500x. The percentage of cells retaining translational motility, either singly or clumped, was noted at intervals up to 3 min. In addition, mixtures of bacteria and antiserum were incubated at 37 C in a water bath and examined in similar fashion after 30 min. Preparation of samples and electron microscopy. Copper grids, 200 mesh, were coated with 0.18% Formvar and carbon stabilized. Samples from cultures standardized to an optical density of 0.5 at 525 nm were transferred to grids by flotation of the grid on a drop of cell suspension for 2 min. Grids were drained on filter paper and floated on distilled water for 10 to
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15 s, drained, transferred to a drop of negative stain for designated time intervals, and again drained. Staining procedures included: (i) uranyl acetate, 0.5% and 1.0% with no pH adjustment (pH 3.9) for 1 to 2 min; (ii) 1% uranyl acetate through a pH range of 3.4 to 6.8 for 2 min; (iii) 0.5 and 1.0% K and Na phosphotungstic acid (KPT or NaPT), pH 7.4, for 15S to 2 min. For short term exposure to chemicals (2 s to 2 min), grids with adfixed cells were floated on the chemical and washed prior to staining. When longer time intervals were used, the bacterial cells were mixed with the selected agent in test tubes prior to flotation of grids. Chemical agents employed included: 0.01 N HCl through a time interval of 2 s to 10 min; 6 M urea (pH 7.5) through a time interval of 2 s to 15 min; 40 C distilled water and tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (0.05 M, pH 7.5). External diameters were estimated by averaging at least three measurements along the length of negatively stained flagella, avoiding areas of curvature. Up to three individual filaments were measured per negative, and for statistical analysis a grand mean value of filament diameters obtained from one negative was taken as a single observation. Measurements were taken from negatives and converted to angstrom units using catalase crystals as an internal standard
(33). For thin-section preparation, bacteria from liquid cultures were centrifuged from the medium and enrobed in agar. The agar blocks were cut to 1-mm cubes and placed in 1% OSO4 in veronal acetate buffer, pH 6.8 (14) at room temperature for 1 h and refrigerated for an additional 45 min. After three washes in veronal acetate buffer samples were divided in half. One-half was placed on 0.5% uranyl acetate for 1 h, whereas the other was kept in buffer for the same period (26). Dehydration in graded series of alcohol and propylene oxide was followed by embedding in a modified Epon-Araldite plastic mixture (20). Sections were cut with a diamond knife on an LKB Ultratome III. Grey to silver sections were picked up on uncoated 200-mesh copper grids and stained with 2% uranyl acetate followed by lead citrate (21). Sections and negative stains were viewed in a Phillips E.M.-300 electron microscope at 80 kV.
RESULTS Extraction of bacteria in glycine-hydrochloride buffer. Immunodiffusion and immunoelectrophoresis with whole cell antiserum revealed the presence of 3 antigens in supernatant fluids, none of which corresponded to the 0 antigen (32). 0 antigen was present in some preparations as a faint line in immunodiffusion tests. The component present in the highest concentration has been termed [a], the anodal component adjacent to it [b], and the most cathodal one [c] (Fig la). Prior to treatment with acid buffer, C. fetus cells were inagglutinable in 0 antiserum, but after treatment agglutinability in 0 antiserum was complete,
309
indicating the removal of superficial antigens which prevent such agglutination of fresh cells. As expected, examination of negative stains disclosed that acid treatment had also caused complete removal of flagella. The presence of only very low concentrations of 0 antigen in the extracts indicated that the extraction process had not disrupted cells and that antigens [a], [b], and [c] were therefore located superficially. However, their identification as somatic or flagellar components could not be resolved. Antigen [a] has been identified in acrylamide gel electrophoresis. It migrated with a retardation factor of 0.41 and stained with protein and carbohydrate stains. CsCl separation of sheared flagella. Partially purified flagella was composed of flagellar filaments, some of which had retained hooks, and a large number of globular bodies (Fig. 2a) which resembled structures located on the bacterial cell surface (Fig. 2b). Similar structures have been reported in prior studies with C. fetus (22) and V. parahemolyticus (19). After centrifugation in CsCl gradients, two discrete but very closely spaced bands developed (Fig. lc). The upper of these, located at a specific gravity of 1.29, contained flagella almost free of globular bodies (Fig. 2c). The lower band contained flagella with somewhat greater contamination. However, the reason for the distinct separation of these bands was not established. A third broad band beneath the others contained globular bodies. Intact flagella from the topmost band produced no reactions in immunodiffusion tests. Solubilization of flagella with acid caused the release of three antigens (Fig. lb). One of these was [a]; and the other two are referred to as [d] and [e]. Antigen [d] has not been identified in immunoelectrophoresis. Antigen [e] was present in the greatest abundance, was electrophoretically heterogeneous, and migrated on each side of the origin well (Fig. la). Growth kinetics. The growth phases of C. fetus cells as determined by optical density measurements and viable counts were as follows; exponential phase, 6 to 12 h; early stationary phase 12 to 15 h; mid-stationary phase 18 to 24 h, and late stationary phase 48 h. Flagella measurements. Diameter measurements of intact flagella from overlay cultures of C. fetus in the stationary phase of growth (24 to 48 h) and of control cultures of B. bacteriovorus and B. subtilis, negatively stained for 1 min with 0.5% uranyl acetate (pH 3.9) or 0.5% NaPT (pH 6.8), are recorded in Table 1. Filament diameter did not appear to be affected by the staining method employed, although staining
310
McCOY ET AL.
J. BACTERIOL.
2 Q4
e
i
a*.fd
05
b
FIG. 1. (a) Immunoelectrophoresis patterns produced by extracts of Campylobacter fetus strain 23 cells. Anode is to the right. Top well: acid solubilized flagella; middle well: acid extract of whole cells; bottom well: trichloroacetic acid extract of whole cells (O antigen). Troughs contain a whole cell antiserum. Bands produced by antigens [a], [b], [c], [e], and 0 are labeled. (b) Immunodiffusion patterns produced with extracts of Campylobacter fetus. Well 1: acid-solubilized flagella; well 2: antigen [a] separated from an acid extract by pevikon block electrophoresis; wells 3, 4, and 5: antiserum prepared with acid solubilized flagella. Bands produced by antigens [a], [d], and [e] are labeled. (c) Cesium chloride gradient separation of sheared flagella. The two closely spaced white bands contained flagella; the diffuse band beneath these contained globular bodies.
with 0.5% uranyl acetate proved superior for demonstrating sheath-core delineation in B. bacteriovorus (Fig. 3c), as well as filament fine structure in C. fetus (Fig. 3a). Internal structure in C. fetus flagella was predominantly of the B type in which six or infrequently seven equidistant lines of stain, including the outer edges, were present. In many filaments regions of flagellar A structure were also apparent. No evidence of a sheath was observed. B. subtilis
flagella possessed B structure (Fig. 3b) as has been observed by Ichiki and Martinez (11). No internal filament fine structure was discernible in B. bacteriovorus flagella. Initial flagellar diameter measurements in C. fetus (48 h) approached in size the published values for flagella with the large sheath type (7). Further attempts were made to demonstrate a flagellar sheath in this organism despite its apparent absence in negatively stained prepara-
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CAMPYLOBACTER FETUS FLAGELLA
31
uu .2um b FIG. 2. (a) and (c) Electron micrographs of sheared flagella preparations of Campylobacter fetus, negatively stained with uranyl acetate, before (a) and after (c) fractionation in a CsCI gradient. (b) C. fetus cell from a 2-day broth overlay culture, negatively stained with uranyl acetate, showing surface structures resembling the globular bodies found. TABLE 1. Diameter measurements of negatively stained flagella Organism
Campylobacter fetus (24 h)b Camplyobacter fetus (48 h)b Bdellovibrio bacteriovorus Bacillus subtilis
No. of obser-
vationsa
11 9 9 9
Filament diameter (nm)
18.2 21.2 28.3 13.6
± ± ± ±
2.0 2.6 4.7 1.0
a The number of observations refers to the number of individual preparations examined. " Broth overlay cultures.
tions. To this end, cells were examined in earlier phases of growth, treatments were employed which enhance the differentiation of sheath and core components, and thin sections were examined. Minimal flagellar diameter measurements were obtained from cells harvested in the exponential and early stationary phase of growth (Table 2). Examination of young cultures grown in Albimi medium with and without supplements or in brain heart infusion broth (Difco) failed to reveal any evidence of internal filament structure (Fig 4). Antigen [a] was present on the cell surface at all stages of growth, as revealed by agglutination tests with specific antiserum. Immunodiffusion tests with broth supernatants indicated that the release of some
antigen [a] had also occurred into the growth medium by mid-stationary phase. Treatment effects. Representative samples of negatively stained preparations of C. fetus and B. bacteriovorus flagella after exposure to 0.01 N HCl are included in Fig. 5. Rapid removal of sheath material was noted when B. bacteriovorus flagella were exposed to acid. However, core filaments were intact after 1 min of treatment (Fig 5d). Differential effects of a comparable nature were never observed in C. fetus. Brief exposure (2 s) of C. fetus cells to acid resulted in a loss of rigidity of the entire flagellum (Fig. 5a); upon longer treatment (10 to 15 s) complete depolymerization was observed (Figs. 5b and c). Results were the same regardless of the phase of growth examined. Treatment of the respective organisms with 6 M urea produced final results similar to those obtained with HCI. However, the initial effect noted in C. fetus cells exposed to urea for 10 to 15 s was breakage of the flagellum at the proximal end. Extraction of C. fetus cells with warm water and Tris-hydrochloride buffer (pH 7.5) resulted in an altered appearance of filaments from cells in the later phases of growth, as well as release of antigen [a] into the supernatant fluid. Internal structure could not be demonstrated in filaments after treatment. Similarly, washing stationary phase cells with distilled water prior
312
McCOY ET AL.
J. BACTERIOL.
1. ...
1
.
FIG. 3. Electron micrographs of uranyl acetate-stained flagella. (a) Campylobacter fetus filament from a 24-h broth overlay culture. Note filament substructure of the lined variety. (b) Bacillus subtilis filament from a 4-h broth culture showing filament substructure of the lined variety. (c) Bdellovibrio bacteriovorus filament from a 24-h broth culture. Note the presence of a flagellar sheath structure. TABLE 2. Characteristics of Campylobacter fetus flagella during the growth cycle No. of
Phase of grwh observagrowth tions'
Exponential (9 h) Early stationary (12 h) Stationary (15 to 24 h)
Filament diameter
(nm)
4
17.6 4 7.6
2
17.2 ± 7.6
5
18.6 ± 16.4
Itra
InternalI
stuur
None demonstrable None demonstrable Lined form
a The number of observations refers to the number of individual preparations examined.
to flotation on grids adversely affected the demonstration of filament ultrastructure. Tris treatment of Bdellovibrio flagella resulted in an apparent loosening of the sheath at intervals along the filament length. Thin sections. No evidence of a flagellar sheath continuous with the cell envelope could be demonstrated in C. fetus or B. subtilis. The results obtained with B. bacteriovorus, in agreement with the findings of Seidler and Starr (24), clearly demonstrated the relationship of the
Bdellovibrio flagellar sheath with the outer cell envelope layer. Agglutination and immobilization studies. Since flagella from cells in different phases of growth having relatively different quantities of free and cell-associated [a] antigen appeared to be structurally different by electron microscope studies, attempts were made to determine whether differences in serological behavior could be demonstrated. Agglutination and immobilization reactions were performed with cells collected from broth cultures at 9, 12, 18, 24 and 48 h. Cells were tested with antisera prepared against antigens [a], [e], whole flagella, acid-solubilized flagella, and boiled cells. No appreciable differences were noted with cells of different ages in agglutination reactions. The following effects were observed in the 3-min immobilization reaction: no effect (single, motile cells); formation of small, motile clumps; and formation of large, nonmotile clumps. The incidence of large, nonmotile clumps was low (1 to 5%) and occurred with antisera against whole flagella, solubilized flagella, and antigen [a]. On longer incubation (30 min) a marked increase in the percentage of large nonmotile
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VOL. 122, 1975
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FIG. 4. Electron micrographs of uranyl acetate-stained flagella from Campylobacter fetus broth cultures sampled at 9 h (a) 12 h (b) 24 h (c) and 48 h (d) growth time intervals. Note increases in filament width and the development of internal structure at successive stages of growth. Oblique pattern in filament substructure may be seen in Fig. 5 (arrow).
clumps was observed only with antisera against solubilized flagella and antigen [a]. Notable differences in immobilization attributable to phase of growth were not observed. DISCUSSION Sheathed flagella have been demonstrated in few bacterial genera which include Bdellovibrio, Vibrio, Pseudomonas, and Proteus. The flagellar sheath may contribute significantly to filament diameter (7, 9, 10, 24) or produce a minimal increment in flagellar width (17). Examinations of negatively stained and thin-sectioned preparations of C. fetus during the stationary phase of growth provided no indication of the presence of a sheath of either type. Fuerst and Hayward (9) reported that flagellar sheathing appears to be a stable characteristic and stressed the importance of the sheath as a taxonomic criterion. In this respect our results are consistent with the generic separation of C. fetus from the genus Vibrio (30). Comparisons of C. fetus flagella from different growth phases revealed several points of interest. The diameters of filaments from exponential phase cultures of C. fetus were smaller than those from stationary phase but exceeded diameters reported for unsheathed species with
the exception of P. fluorescens, which has a mean width of 17.3 nm (17). Demonstration of filament fine structure was not achieved in C. fetus during expontial phase and could not be enhanced by manipulation of the growth environment or suspending milieu. However, in flagella from stationary phase cells filament fine structure, resembling that in P. fluorescens with respect to the number of internal lines in the B filament, was obscured by distilled water washing or suspension in Tris buffer. Czajkowski et al. (4) reported the failure to demonstrate internal structure in intact flagella of P. mirabilis and B. subtilis which were grown overnight and water washed by centrifugation. They suggested that linear patterns, associated with an increase in filament diameter, could be ascribed to filament degradation. Our findings with B. subtilis, in which young cells were not washed prior to grid preparation, disagree with those of Czajkowski et al., and experiments with C. fetus cells in stationary phase revealed that washing prior to grid preparation caused the loss of filament substructure. The cellular localization and function of antigens [a], [b], and [c] are not fully clarified, and the identification of flagellar components [d] and [e] with specific flagellar structures re-
314
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J. BACTrERIOL.
McCOY ET AL.
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FIG. 5. Electron micrographs of uranyl acetate-stained preparations of flagella following exposure to 0.01 N HCI. (a) Campylobacter fetus flagellum exposed to acid for 2 s. (b) C. fetus flagellum after a 5-s exposure to acid. (c) C. fetus flagellum exposed to acid for 15 s. (d) Bdellovibrio bacteriovorus flagellum after a 5-s exposure to acid. Note the delineation of sheath and core components.
mains to be established. The location of component [a] on the cell body has recently been confirmed (E. C. McCoy et al., submitted for publication). Its detection in flagellar prepara-
tions may have been the result of minute contamination of those products with globular bodies, although the possibility that this antigen represents a superficial constituent of both
VOL. 122, 1975
CAMPYLOBACTER FETUS FLAGELLA
the cell body and flagellum cannot be excluded. The latter possibility is supported by the observation that antiserum prepared against intact flagella is unable to effect significant immobilization of cells possessing [a] antigen. However, the same antiserum is extremely effective in immobilizing a mutant of strain 23 which lacks [a] antigen (E. C. McCoy, unpublished data). The possibility that [a] antigen covers a site on the flagellum critical for immobilization is under study. ACKNOWLEDGMENTS We would like to acknowledge the advice and assistance of John Telford in electron microscopy and Rafael Martinez for critical review of the manuscript. This study was supported by Public Health Service grant AI 11160 from the National Institute of Allergy and Infectious Diseases.
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