Vol. 138, No. 2
JOURNAL OF BACTERIOLOGY, May 1979, p. 575-583
0021-9193/79/05-0575/09$02.00/0
Purification and Characterization of a Polyhook Protein from Caulobacter crescentus MICHAEL SHEFFERY AND AUSTIN NEWTON* Department of Biology, Princeton University, Princeton, New Jersey 08540
Received for publication 26 February 1979
A polyhook-producing strain of Caulobacter crescentus was isolated, and the polyhook protein was purified. The antigenicity and morphology of the polyhook structure are similar to the wild-type hook except that the mutant strain produces a hook structure at least 10-fold the length of wild-type hooks (1.0 versus 0.1 Am). The molecular weight of the polyhook protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 72,000, and the protein has a pl of approximately 6.1. Antibodies prepared against the polyhook protein were used to show that this protein is antigenically distinct from the Caulobacter flagellins. Amino acid analysis of the polyhook protein revealed compositional similarities to other gram-negative, bacterial hook proteins.
Caulobacter crescentus is a dimorphic gramnegative bacterium in which a nonmotile stalked cell divides repeatedly to produce motile swarmer cells. During the stalked cell cycle, a flagellum, pili, and holdfast are formed at the stalk distal pole of the cell, and at division these structures become part of the new swarmer cell. The swarmer cell in turn loses motility, sheds the flagellum into the medium, and forms a stalk at the old flagellated pole (23, 26, 27). The flagellum is a convenient marker for the study of development in C. crescentus because of its periodic formation by the stalked cell and its unique location on the cell surface. An external filament, hook, and rod have been identified as components of the flagellum in these bacteria (24, 28), and recent studies have shown that the two flagellin subunits (13, 21), flagellin A and flagellin B (12, 21), and the hook protein (13) are synthesized periodically in the cell cycle. In addition, an analysis of conditional cell cycle mutants indicates that the periodicity of flagellin synthesis is controlled by DNA replication (20, 21). Nonmotile mutants can be readily isolated in C. crescentus (4, 5, 10, 12, 17), and many of these strains are useful in analyzing flagellum formation. In our examination of nonmotile mutants of strain PC1, we have identified one mutant that produces an extensive polyhook structure. This strain provides an excellent source of hook protein that can be used for chemical analysis and for the preparation of a hook-specific antiserum. We report here the characterization of the polyhook mutant and some of the properties of the polyhook protein. 575
MATERIALS AND METHODS Strains. C. crescentus strain CB15 (ATCC 19089) was obtained from the American Type Culture Collection. Strain PCI is a cysteine-requiring auxotroph derived from strain CB15 (20). Strain PCM103 was derived from strain PCI after exposure to UV light to allow 10% survival; nonmotile mutants were identified by their inability to form diffuse colonies in 0.35% peptone-yeast extract agar plates, as described previ-
ously for C. crescentus (10, 17). Nonmotile strains were purified, tested for flagellin synthesis by a radioimmunoassay (see below), and screened for morphology by electron microscopy. Media. Strain CB15 was grown in a minimal salts medium (M3 medium [23]) containing 0.2% glucose. Strain PCM103 was grown either in M3 medium supplemented with L-cysteine and Casamino Acids (Difco Laboratories) at concentrations of 10 ,ug/ml or in a peptone-yeast extract medium (23). Radioimmune precipitation assay. Flagellin or polyhook synthesis was monitored by a modified radioimmune precipitation assay (9, 21). An exponential culture that contained 108 cells was labeled with 30 ,uCi of L-[35S]methionine (550 Ci/mmol; Amersham Corp.) per ml for 5 min. Labeling was stopped by the addition of 100 jug of chloramphenicol per ml and 0.04 M sodium azide. The cell suspension was chilled on ice, and 3 x 108 unlabeled carrier cells that had been previously treated with azide and chloramphenicol were added. The cells were centrifuged at 12,000 x g for 10 min and suspended in 0.1 ml of buffer that contained 10 mM KCI, 10 mM sodium azide, and 10 mM Tris-hydrochloride (pH 8.1). Cells were lysed by adding 25 1l of lysozyme (Worthington Biochemicals Corp.; 1 mg/ml in 10 mM Tris-hydrochloride, 50 mM EDTA, pH 8.1), incubating for approximately 2 min at 37°C, and adding 0.1 ml of 10 mM KCI. Phenylmethylsulfonyl fluoride (75 ,g; Sigma Chemical Co.) was then added, and the crude lysate was incubated
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SHEFFERY AND NEWTON
with 10 pug of DNase I (Worthington Biochemicals Corp.) for 20 min at 4°C. Lysates were diluted 1:1 with 50 mM Tris-hydrochloride, pH 7.6, that contained 2 M KCI and 2% Triton X-100 and incubated with 25 pl of a preimmune rabbit serum (1.4 mg/ml) in 0.9% NaCl-20 mM Tris-hydrochloride, pH 7.6. After an incubation of at least 90 min, 25 to 50 pl of a Staphylococcus aureus cell preparation (9) was added to each tube; this and subsequent incubations were at 4°C. After 90 min the S. aureus cell complexes were removed by centrifugation at 6,000 x g for 5 min, and 25 pl of an antiflagellin or antipolyhook serum, diluted 25-fold in 0.9% NaCl-20 mM Tris-hydrochloride, pH 7.6, was added to each supernatant fraction. At equivalence, 0.1 ml of the antiflagellin serum precipitated 80 pug of purified flagellin, and 0.1 ml of the antipolyhook serum precipitated 40 pg of purified polyhook protein. After at least 90 min, 25 to 50 pl of the S. aureus cell preparation was added to each reaction, and after an additional 90-min incubation the Staphylococcus immune complexes were removed by centrifugation and washed as described by Roberts and Roberts (25). The cell pellet was suspended in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis diluent buffer (11). Samples were boiled for 3 min and allowed to cool, and the S. aureus cells were removed by centrifugation. Supernatants were generally applied to SDS-10% polyacrylamide slab gels (11). Flagellin or polyhook protein was detected by contact fluorography of dried gels, using prefogged film to insure a linear response of the film to radioactivity (15). Relative rates of protein synthesis were measured by densitometry on a Joyce-Loebel recording microdensitometer. This assay, which is performed in antibody excess (9), was linear from 150 to 1,000 p1 of lysate with respect to labeled antigen precipitated (data not shown). The intensity of non-specifically precipitated proteins (see Fig. 3E) depended on the age of the S. aureus cell preparation used; new cells were prepared approximately every 3 months. Since antigen-antibody complexes formed with S. aureus cells are quantitatively dissociated only by boiling in the presence of SDS (9), we used a carrier protein-mediated radioimmune precipitation assay (13) for two-dimensional gels. An amount of polyhook protein equivalent to 0.025 ml of antipolyhook serum was added to extracts of strains CB15 and PCM103 that had been prepared as described above. The mixtures were incubated with 0.025 ml of immune serum as described previously (13). Precipitates were washed as described previously (13), dissolved in two-dimensional gel electrophoresis sample buffer (19), and then analyzed by two-dimensional gel electrophoresis (19). Electron microscopy. Cells or protein samples were diluted, fixed with 0.5% formaldehyde, and spotted on carbon-coated, Formvar-coated grids. After 45 min, the grids were washed three times with cytochrome c (1 mg/ml in distilled water) and stained with 1% phosphotungstic acid in 0.5% ammonium acetate, pH 7.2. Protein preparations to be reacted with antisera were spotted on grids and washed with cytochrome c. The excess liquid was removed, and a drop of a 1:25 dilution of the appropriate antiserum was placed on the grid and allowed to incubate for 30 s. The grid was then washed with distilled water, the
J. BACTERIOL. excess fluid was removed, and the preparation was stained with 1% phosphotungstic acid. Grids were examined in a JEOL 100C electron microscope. Preparation of antisera. Rabbits were injected intradermally along the back with 0.5 mg of antigen emulsified in an equal volume of Freund complete adjuvant. After 4 weeks, rabbits were boosted for the next 3 weeks by intradermal injections of 0.5 mg of antigen emulsified in an equal volume of Freund incomplete adjuvant. Blood was collected 1 week after the last injection. Antipolyhook serum was prepared by using the purified polyhook protein described below as an antigen. Antiflagellin serum used in the radioimmunoassays was prepared by repeated injection of flagellin that had been purified by one reconstitution of filaments (29). Flagellin at this stage of purification contains traces of hook protein, and antiserum prepared against these antigen preparations reacted with both filaments and hooks of intact flagella, as shown by electron microscopy (data not shown). Consequently, all electron microscope experiments were performed with "preadsorbed" antiflagellin serum: hook antibodies were removed by incubating 0.1 ml of antiflagellin serum with 0.1 ml of polyhook protein (5 mg/ml) for 60 min. Polyhooks and polyhook immune complexes were removed by centrifugation at 105,000 x g for 30 min, and the supernatant was used as the preadsorbed antiflagellin serum. Preparation of polyhooks. Polyhooks were isolated from a 4-liter culture of C. crescentus strain PCM103 that had been grown to late log phase in peptone-yeast extract medium. Cells and polyhooks were removed from the culture medium by centrifugation at 13,200 x g for 25 min. The pellet was suspended in approximately 30 ml of 10 mM Tris-hydrochloride, pH 7.2, and cells were removed by two successive centrifugations at 12,000 x g for 3 min. The supematant fluid was collected, and EDTA and Triton X-100 were added to give a final concentration of 10 mM and 0.5%, respectively (13). This suspension was centrifuged at 105,000 x g for 20 min, and the pellet was suspended in 3.0 ml of distilled water. The suspension was washed two additional times by centrifugation at 105,000 x g, and the final pellet was resuspended in 1.0 ml of distilled water. Particulate matter was removed by centrifugation at 7,000 x g for 5 min. The supernatant fraction was then adjusted to pH 4.0 with HCI and centrifuged at 105,000 x g for 20 min. The pellet was suspended in 1.0 ml of 10 mM Trishydrochloride buffer, pH 7.2. Amino acid analysis. For amino acid analysis of polyhook or flagellins A and B, 200 to 400 jg of purified protein in distilled water was hydrolyzed with 6 N HCI in an evacuated, sealed tube for 24 h at 108°C. Each hydrolysate was analyzed by the method of Spackman et al. (32) on a Beckman 120B amino acid analyzer equipped with a numerical integrator for the measurement of peak areas. Peak areas were converted to the appropriate nanomole amounts by comparison with areas obtained with standard amino acid mixtures
(Beckman). Protein determination. Protein was determined by the method of Lowry et al. (16), using bovine serum albumin as a standard.
POLYHOOK PROTEIN FROM C. CRESCENTUS
VOL. 138, 1979
RESULTS Electron microscopy of strain PCM103. The morphology of a series of nonmotile mutants of C. crescentus was examined by electron microscopy of negatively stained preparations. The study revealed one mutant, PCM103, with a short, thick filament attached to one cell pole instead of a flagellum (Fig. 1A). At higher magnifications of stained PCM103 swarmer cells, pili can also be observed at this pole (data not shown). The mutant structure, which was also present in the culture medium along with rare, unattached flagellar filaments, is very similar to the C. crescentus hook. The diameters measured from electron micrographs are 22.1 ± 5.7 nm for the PCM103 structure and 22.5 ± 2.9 nm for the hook. These dimensions compare with a diameter of 12.0 ± 1.7 nm for the flagellar filament of strain CB15. In addition, one end of the unattached PCM103 structure is contiguous with a smaller structure (arrow, Fig. 1B) which has approximately the same diameter (9.5 nm) as the rod that is present on the hook of C. crescentus flagella (12, 28, 29). In contrast to hooks of wild-type strains, the PCM103 structure is at least 1.0 ,um in length, or approximately 10-fold
577
that of the normal hook. Therefore, based on the structural characterization, mutant PCM103 seems to produce a hook structure of aberrant length, analogous to the polyhooks formed by Escherichia coli flaE and Salmonella flaR mutants (22, 30). This identification was confirmed by immunological studies outlined below. In contrast to the "curly" appearance of polyhooks in the E. coli mutant, however, most structures produced by PCM103 are straight, like the polyhook shown in Fig. 1B; only occasionally is a curly polyhook observed. Purification and protein composition of polyhooks. Polyhooks, like the intact flagella of C. crescentus (28), are shed into the culture medium during development (Fig. 1), presumably during formation of the stalked cell from the swarmer cell. Since filaments are not found attached to the shed polyhooks, it is possible to prepare these structures from the supernatant of PCM103 cultures by differential centrifugation (see above). Figure 2B and D show a stained one-dimensional SDS-polyacrylamide gel of this purified polyhook at different concentrations of protein. The single major band with a molecular weight of 72,000 (72K) represents 98% of the total protein in the polyhook preparation, based
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on the analysis of SDS-polyacrylamide gels with as much as 80 Mg of total protein. The polyhook protein is clearly distinct from flagellin. Figure 2A and C show, for comparison, the stained SDS-polyacrylamide gel electrophoresis pattern of flagellin that was purified by one reconstitution of disaggregated flagellar filaments (29). The two bands in the flagellin preparation correspond to the protein subunits, flagellin A (26K) and flagellin B (28K), which are in a ratio of approximately 4:1 (A/B) in intact filaments (12, 13, 29). Polyhook preparations purified by the procedure described were never contaminated by either flagellin A or flagellin B (Fig. 2), but once-reconstituted filaments were often contaminated by traces of a protein with
the same mobility as the polyhook protein (data not shown). The molecular weight of the purified polyhook protein was determined from its electrophoretic mobility relative to trypsin, ovalbumin, bovine serum albumin, flagellins A and B, and the DNA-dependent RNA polymerase from C. crescentus (1, 3). The calculated molecular weight of 72,000 is in good agreement with a previously reported value of 73,000 for the hook protein of strain CB15 (13, 14). Rates of protein synthesis by radioimmunoassay. Nonmotile mutants of C. crescentus were initially screened for flagellin synthesis by a radioimmunoassay with an antiserum prepared against once-reconstituted flagellins A and B. Compared with wild-type strain CB15, mutant PCM103 synthesizes flagellin A at a 10-foldreduced rate and flagellin B at a 4-fold-reduced rate (Fig. 3A and B). The presence of hook protein antibodies in the antiflagellin serum used in these tests (see above) also allowed the detection of the 72K polyhook protein in extracts of strain PCM103 (Fig. 3A); the hook protein from the wild-type strain is difficult to detect at comparable film exposures (Fig. 3B). When the antipolyhook protein antibodies were removed from the antiflagellin serum by preadsorption against purified polyhooks (see above), the autoradiograms were identical to those shown in Fig. 3A and B, except that the 72K protein could not be detected (data not shown). The radioimmunoassay was also carried out with a specific antipolyhook serum. The 72K polyhook protein was more intense in the extract of the PCM103 cells (Fig. 3C), and synthesis of the 72K hook protein could be identified in CB15 cells (Fig. 3D). The detection of flagellin in the labeled cell extracts of strain CB15, using antipolyhook serum, is presumably due to the structural association between the hook and the filament in these cells (13, 28, 29, 31). As shown below, there is no immunological cross-reaction between C. crescentus hook protein and flagellin. The specificity of the radioimmunoassays for flagellin, polyhook, and hook proteins is shown in Fig. 3E. When a nonimmune serum was substituted for the immune sera in the assay, most of the proteins observed (Fig. 3A to D) are present, except for those at 72K, 28K, and 26K; bands at these positions are either extremely faint or absent. The antipolyhook serum was also used to analyze immunoprecipitates from strains PCM103 and CB15 by two-dimensional gel electrophoresis (Fig. 4A and B), and the major labeled proteins precipitated were compared with purified polyhook protein and flagellin in the
VOL. 138, 1979
POLYHOOK PROTEIN FROM C. CRESCENTUS
same system (Fig. 4C and D). The locations of the purified polyhook protein (Fig. 4C) and the polyhook protein immunoprecipitated from strain PCM103 (Fig. 4A) are indicated by arrows at 72K and pH 6.1. A less intensely labeled protein with the same molecular weight and isoelectric point as the polyhook protein is also precipitated from extracts of strain CB15 by the antipolyhook serum (Fig. 4B, arrow). Incomplete solubilization of proteins in the labeled cell extracts may account for the relatively heavy backgrounds in Fig. 4A and B; with the exception of flagellin and polyhook protein, which could not be detected, exactly the same patterns of contaminating spots were observed when preimmune serum was substituted for the antipolyhook serum in this assay (data not shown). The results shown in Fig. 4 also confirm the difference between the polyhook protein and flagellin. Whereas the polyhook protein is a single charge species at 72K, flagellin A is composed of three charge species and flagellin B is composed of four charge species (Fig. 4D). The three flagellin A species and two of the four flagellin B species were immunoprecipitated by antipolyhook serum from pulse-labeled extracts of strain CB15 (Fig. 4B). (The two more basic species of flagellin B were also observed in similar labeling experiments, using antiflagellin serum [unpublished data].) These pulse-label experiments suggest that the charge heterogeneity of flagellin species is present soon after the time of synthesis and that it is maintained until the filaments are shed into the medium. In vitro carbamylation of flagellin (which sequentially increases the net negative charge on a protein [2]) indicates that the charge differences between the flagellin species are greater than a single charge (data not shown). The most acidic species of flagellin has an isoelectric point of approximately 6.4, or 0.3 pH unit higher than the isoelectric point of the polyhook. Antigenicity of polyhook protein. The specific antiserum prepared against the purified PCM103 structure (Fig. 2) was used to establish that this structure is antigenically related to the hook of flagella shed by wild-type cells. The antiserum was reacted with intact flagella from C. crescentus, and the preparation was negatively stained and examined in an electron microscope, as described previously (30). Binding of antisera is visualized as an enhancement of the degree of staining and an increase in the apparent width of the bound structure (30). Figure 5A shows that the serum reacted only with the hook structure in these preparations and that neither the filament nor the rod was stained. When the same experiment was carried out with preadsorbed antiflagellin serum (see
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above), the flagellar filament of the intact wildtype flagellum reacted, but neither rods nor hooks of the flagellum showed any reaction (Fig. 5B). Figures 5C and D show the reactions of polyhooks with these antisera; polyhook preparations used for this experiment were not washed at low pH (see above) and, therefore, contained rare, unattached, flagellar filaments (arrows, Fig. 5C and D) which served as an internal control for the specificity of antibody binding. Figure 5C shows that the antipolyhook serum bound to
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70 5.0 7.0 65 60 65 5.0 6.0 FIG. 4. Two-dimensional gel electrophoresis of radioimmunoprecipitates and purified flagellar proteins. Radioimmunoprecipitates were prepared from pulse-labeled cell extracts of strains PCM103 (A) and CB15 (B), using an antipolyhook serum (see text). Approximately 400,000 cpm of each sample was analyzed by twodimensional gel electrophoresis (19) and fluorography (15). A 20-jig amount of purified polyhook protein (C) and flagellin purified by one cycle of dissaggregation and reconstitution (D) were analyzed in the same system, and the gels were stained as described in the legend to Fig. 2. The position of the 72K polyhook protein is marked by an arrow in (A) and (C); the location of the 72K, 6.1 pIprotein (see text) is marked in (B); and the positions of this protein and the polyhook protein are indicated relative to three other proteins in (A) and (B). Multiple species of flagellin B (28K) and flagellin A (26K) are marked in (B) and (D). Isoelectric focusing (IEF) and SDS-polyacrylamide gel electrophoresis (SDS) dimensions are also indicated.
polyhooks but not to the contaminating flagellar filaments. Figure 5D shows that the polyhooks did not bind antiflagellin serum but that fragments of flagellar filaments did specifically bind this antiserum. Nonimmune control serum did not react with any of the structures (data not shown). We conclude from these experiments that the short, thick filaments produced by strain PCM103 are antigenically related to the hooks of normal flagella. Thus, the morphological (Fig. 1), biochemical (Fig. 2), and immunological (Fig. 5) evidence supports the conclusion that mutant PCM103 is a polyhook-producing strain of C. crescentus.
Amino acid composition of polyhook protein. Purified preparations of the polyhook protein were hydrolyzed at 108°C in 6 N HCI for 24 h for amino acid analysis (32). Table 1 compares the amino acid composition of the PCM103 polyhook protein to purified flagellins of strain CB15. This comparison shows that the polyhook protein has a higher content of proline and tyrosine than do the flagellins of C. crescentus. This analysis also suggests a similarity between the polyhook protein of C. crescentus, the polyhook protein of E. coli (30), and the hook protein of Salmonella (8). All three of these proteins have relatively high contents of proline and ty-
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JOURNAL OF BACTERIOLOGY, May 1979, p. 575-583
0021-9193/79/05-0575/09$02.00/0
Purification and Characterization of a Polyhook Protein from Caulobacter crescentus MICHAEL SHEFFERY AND AUSTIN NEWTON* Department of Biology, Princeton University, Princeton, New Jersey 08540
Received for publication 26 February 1979
A polyhook-producing strain of Caulobacter crescentus was isolated, and the polyhook protein was purified. The antigenicity and morphology of the polyhook structure are similar to the wild-type hook except that the mutant strain produces a hook structure at least 10-fold the length of wild-type hooks (1.0 versus 0.1 Am). The molecular weight of the polyhook protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 72,000, and the protein has a pl of approximately 6.1. Antibodies prepared against the polyhook protein were used to show that this protein is antigenically distinct from the Caulobacter flagellins. Amino acid analysis of the polyhook protein revealed compositional similarities to other gram-negative, bacterial hook proteins.
Caulobacter crescentus is a dimorphic gramnegative bacterium in which a nonmotile stalked cell divides repeatedly to produce motile swarmer cells. During the stalked cell cycle, a flagellum, pili, and holdfast are formed at the stalk distal pole of the cell, and at division these structures become part of the new swarmer cell. The swarmer cell in turn loses motility, sheds the flagellum into the medium, and forms a stalk at the old flagellated pole (23, 26, 27). The flagellum is a convenient marker for the study of development in C. crescentus because of its periodic formation by the stalked cell and its unique location on the cell surface. An external filament, hook, and rod have been identified as components of the flagellum in these bacteria (24, 28), and recent studies have shown that the two flagellin subunits (13, 21), flagellin A and flagellin B (12, 21), and the hook protein (13) are synthesized periodically in the cell cycle. In addition, an analysis of conditional cell cycle mutants indicates that the periodicity of flagellin synthesis is controlled by DNA replication (20, 21). Nonmotile mutants can be readily isolated in C. crescentus (4, 5, 10, 12, 17), and many of these strains are useful in analyzing flagellum formation. In our examination of nonmotile mutants of strain PC1, we have identified one mutant that produces an extensive polyhook structure. This strain provides an excellent source of hook protein that can be used for chemical analysis and for the preparation of a hook-specific antiserum. We report here the characterization of the polyhook mutant and some of the properties of the polyhook protein. 575
MATERIALS AND METHODS Strains. C. crescentus strain CB15 (ATCC 19089) was obtained from the American Type Culture Collection. Strain PCI is a cysteine-requiring auxotroph derived from strain CB15 (20). Strain PCM103 was derived from strain PCI after exposure to UV light to allow 10% survival; nonmotile mutants were identified by their inability to form diffuse colonies in 0.35% peptone-yeast extract agar plates, as described previ-
ously for C. crescentus (10, 17). Nonmotile strains were purified, tested for flagellin synthesis by a radioimmunoassay (see below), and screened for morphology by electron microscopy. Media. Strain CB15 was grown in a minimal salts medium (M3 medium [23]) containing 0.2% glucose. Strain PCM103 was grown either in M3 medium supplemented with L-cysteine and Casamino Acids (Difco Laboratories) at concentrations of 10 ,ug/ml or in a peptone-yeast extract medium (23). Radioimmune precipitation assay. Flagellin or polyhook synthesis was monitored by a modified radioimmune precipitation assay (9, 21). An exponential culture that contained 108 cells was labeled with 30 ,uCi of L-[35S]methionine (550 Ci/mmol; Amersham Corp.) per ml for 5 min. Labeling was stopped by the addition of 100 jug of chloramphenicol per ml and 0.04 M sodium azide. The cell suspension was chilled on ice, and 3 x 108 unlabeled carrier cells that had been previously treated with azide and chloramphenicol were added. The cells were centrifuged at 12,000 x g for 10 min and suspended in 0.1 ml of buffer that contained 10 mM KCI, 10 mM sodium azide, and 10 mM Tris-hydrochloride (pH 8.1). Cells were lysed by adding 25 1l of lysozyme (Worthington Biochemicals Corp.; 1 mg/ml in 10 mM Tris-hydrochloride, 50 mM EDTA, pH 8.1), incubating for approximately 2 min at 37°C, and adding 0.1 ml of 10 mM KCI. Phenylmethylsulfonyl fluoride (75 ,g; Sigma Chemical Co.) was then added, and the crude lysate was incubated
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SHEFFERY AND NEWTON
with 10 pug of DNase I (Worthington Biochemicals Corp.) for 20 min at 4°C. Lysates were diluted 1:1 with 50 mM Tris-hydrochloride, pH 7.6, that contained 2 M KCI and 2% Triton X-100 and incubated with 25 pl of a preimmune rabbit serum (1.4 mg/ml) in 0.9% NaCl-20 mM Tris-hydrochloride, pH 7.6. After an incubation of at least 90 min, 25 to 50 pl of a Staphylococcus aureus cell preparation (9) was added to each tube; this and subsequent incubations were at 4°C. After 90 min the S. aureus cell complexes were removed by centrifugation at 6,000 x g for 5 min, and 25 pl of an antiflagellin or antipolyhook serum, diluted 25-fold in 0.9% NaCl-20 mM Tris-hydrochloride, pH 7.6, was added to each supernatant fraction. At equivalence, 0.1 ml of the antiflagellin serum precipitated 80 pug of purified flagellin, and 0.1 ml of the antipolyhook serum precipitated 40 pg of purified polyhook protein. After at least 90 min, 25 to 50 pl of the S. aureus cell preparation was added to each reaction, and after an additional 90-min incubation the Staphylococcus immune complexes were removed by centrifugation and washed as described by Roberts and Roberts (25). The cell pellet was suspended in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis diluent buffer (11). Samples were boiled for 3 min and allowed to cool, and the S. aureus cells were removed by centrifugation. Supernatants were generally applied to SDS-10% polyacrylamide slab gels (11). Flagellin or polyhook protein was detected by contact fluorography of dried gels, using prefogged film to insure a linear response of the film to radioactivity (15). Relative rates of protein synthesis were measured by densitometry on a Joyce-Loebel recording microdensitometer. This assay, which is performed in antibody excess (9), was linear from 150 to 1,000 p1 of lysate with respect to labeled antigen precipitated (data not shown). The intensity of non-specifically precipitated proteins (see Fig. 3E) depended on the age of the S. aureus cell preparation used; new cells were prepared approximately every 3 months. Since antigen-antibody complexes formed with S. aureus cells are quantitatively dissociated only by boiling in the presence of SDS (9), we used a carrier protein-mediated radioimmune precipitation assay (13) for two-dimensional gels. An amount of polyhook protein equivalent to 0.025 ml of antipolyhook serum was added to extracts of strains CB15 and PCM103 that had been prepared as described above. The mixtures were incubated with 0.025 ml of immune serum as described previously (13). Precipitates were washed as described previously (13), dissolved in two-dimensional gel electrophoresis sample buffer (19), and then analyzed by two-dimensional gel electrophoresis (19). Electron microscopy. Cells or protein samples were diluted, fixed with 0.5% formaldehyde, and spotted on carbon-coated, Formvar-coated grids. After 45 min, the grids were washed three times with cytochrome c (1 mg/ml in distilled water) and stained with 1% phosphotungstic acid in 0.5% ammonium acetate, pH 7.2. Protein preparations to be reacted with antisera were spotted on grids and washed with cytochrome c. The excess liquid was removed, and a drop of a 1:25 dilution of the appropriate antiserum was placed on the grid and allowed to incubate for 30 s. The grid was then washed with distilled water, the
J. BACTERIOL. excess fluid was removed, and the preparation was stained with 1% phosphotungstic acid. Grids were examined in a JEOL 100C electron microscope. Preparation of antisera. Rabbits were injected intradermally along the back with 0.5 mg of antigen emulsified in an equal volume of Freund complete adjuvant. After 4 weeks, rabbits were boosted for the next 3 weeks by intradermal injections of 0.5 mg of antigen emulsified in an equal volume of Freund incomplete adjuvant. Blood was collected 1 week after the last injection. Antipolyhook serum was prepared by using the purified polyhook protein described below as an antigen. Antiflagellin serum used in the radioimmunoassays was prepared by repeated injection of flagellin that had been purified by one reconstitution of filaments (29). Flagellin at this stage of purification contains traces of hook protein, and antiserum prepared against these antigen preparations reacted with both filaments and hooks of intact flagella, as shown by electron microscopy (data not shown). Consequently, all electron microscope experiments were performed with "preadsorbed" antiflagellin serum: hook antibodies were removed by incubating 0.1 ml of antiflagellin serum with 0.1 ml of polyhook protein (5 mg/ml) for 60 min. Polyhooks and polyhook immune complexes were removed by centrifugation at 105,000 x g for 30 min, and the supernatant was used as the preadsorbed antiflagellin serum. Preparation of polyhooks. Polyhooks were isolated from a 4-liter culture of C. crescentus strain PCM103 that had been grown to late log phase in peptone-yeast extract medium. Cells and polyhooks were removed from the culture medium by centrifugation at 13,200 x g for 25 min. The pellet was suspended in approximately 30 ml of 10 mM Tris-hydrochloride, pH 7.2, and cells were removed by two successive centrifugations at 12,000 x g for 3 min. The supematant fluid was collected, and EDTA and Triton X-100 were added to give a final concentration of 10 mM and 0.5%, respectively (13). This suspension was centrifuged at 105,000 x g for 20 min, and the pellet was suspended in 3.0 ml of distilled water. The suspension was washed two additional times by centrifugation at 105,000 x g, and the final pellet was resuspended in 1.0 ml of distilled water. Particulate matter was removed by centrifugation at 7,000 x g for 5 min. The supernatant fraction was then adjusted to pH 4.0 with HCI and centrifuged at 105,000 x g for 20 min. The pellet was suspended in 1.0 ml of 10 mM Trishydrochloride buffer, pH 7.2. Amino acid analysis. For amino acid analysis of polyhook or flagellins A and B, 200 to 400 jg of purified protein in distilled water was hydrolyzed with 6 N HCI in an evacuated, sealed tube for 24 h at 108°C. Each hydrolysate was analyzed by the method of Spackman et al. (32) on a Beckman 120B amino acid analyzer equipped with a numerical integrator for the measurement of peak areas. Peak areas were converted to the appropriate nanomole amounts by comparison with areas obtained with standard amino acid mixtures
(Beckman). Protein determination. Protein was determined by the method of Lowry et al. (16), using bovine serum albumin as a standard.
POLYHOOK PROTEIN FROM C. CRESCENTUS
VOL. 138, 1979
RESULTS Electron microscopy of strain PCM103. The morphology of a series of nonmotile mutants of C. crescentus was examined by electron microscopy of negatively stained preparations. The study revealed one mutant, PCM103, with a short, thick filament attached to one cell pole instead of a flagellum (Fig. 1A). At higher magnifications of stained PCM103 swarmer cells, pili can also be observed at this pole (data not shown). The mutant structure, which was also present in the culture medium along with rare, unattached flagellar filaments, is very similar to the C. crescentus hook. The diameters measured from electron micrographs are 22.1 ± 5.7 nm for the PCM103 structure and 22.5 ± 2.9 nm for the hook. These dimensions compare with a diameter of 12.0 ± 1.7 nm for the flagellar filament of strain CB15. In addition, one end of the unattached PCM103 structure is contiguous with a smaller structure (arrow, Fig. 1B) which has approximately the same diameter (9.5 nm) as the rod that is present on the hook of C. crescentus flagella (12, 28, 29). In contrast to hooks of wild-type strains, the PCM103 structure is at least 1.0 ,um in length, or approximately 10-fold
577
that of the normal hook. Therefore, based on the structural characterization, mutant PCM103 seems to produce a hook structure of aberrant length, analogous to the polyhooks formed by Escherichia coli flaE and Salmonella flaR mutants (22, 30). This identification was confirmed by immunological studies outlined below. In contrast to the "curly" appearance of polyhooks in the E. coli mutant, however, most structures produced by PCM103 are straight, like the polyhook shown in Fig. 1B; only occasionally is a curly polyhook observed. Purification and protein composition of polyhooks. Polyhooks, like the intact flagella of C. crescentus (28), are shed into the culture medium during development (Fig. 1), presumably during formation of the stalked cell from the swarmer cell. Since filaments are not found attached to the shed polyhooks, it is possible to prepare these structures from the supernatant of PCM103 cultures by differential centrifugation (see above). Figure 2B and D show a stained one-dimensional SDS-polyacrylamide gel of this purified polyhook at different concentrations of protein. The single major band with a molecular weight of 72,000 (72K) represents 98% of the total protein in the polyhook preparation, based
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FIG. 1. Electron micrograph of strain PCM103 and released polyhook. Cultures of PCM103 cells were stained with phosphotungstic acid. (A) Swarmer cell with an attached polyhook and a detached polyhook in the same field. (B) Higher magnification of a shed polyhook. The location of the rod is indicated by an arrow. Bars = I ,m (A) and 0.1 ,tm (B).
578
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on the analysis of SDS-polyacrylamide gels with as much as 80 Mg of total protein. The polyhook protein is clearly distinct from flagellin. Figure 2A and C show, for comparison, the stained SDS-polyacrylamide gel electrophoresis pattern of flagellin that was purified by one reconstitution of disaggregated flagellar filaments (29). The two bands in the flagellin preparation correspond to the protein subunits, flagellin A (26K) and flagellin B (28K), which are in a ratio of approximately 4:1 (A/B) in intact filaments (12, 13, 29). Polyhook preparations purified by the procedure described were never contaminated by either flagellin A or flagellin B (Fig. 2), but once-reconstituted filaments were often contaminated by traces of a protein with
the same mobility as the polyhook protein (data not shown). The molecular weight of the purified polyhook protein was determined from its electrophoretic mobility relative to trypsin, ovalbumin, bovine serum albumin, flagellins A and B, and the DNA-dependent RNA polymerase from C. crescentus (1, 3). The calculated molecular weight of 72,000 is in good agreement with a previously reported value of 73,000 for the hook protein of strain CB15 (13, 14). Rates of protein synthesis by radioimmunoassay. Nonmotile mutants of C. crescentus were initially screened for flagellin synthesis by a radioimmunoassay with an antiserum prepared against once-reconstituted flagellins A and B. Compared with wild-type strain CB15, mutant PCM103 synthesizes flagellin A at a 10-foldreduced rate and flagellin B at a 4-fold-reduced rate (Fig. 3A and B). The presence of hook protein antibodies in the antiflagellin serum used in these tests (see above) also allowed the detection of the 72K polyhook protein in extracts of strain PCM103 (Fig. 3A); the hook protein from the wild-type strain is difficult to detect at comparable film exposures (Fig. 3B). When the antipolyhook protein antibodies were removed from the antiflagellin serum by preadsorption against purified polyhooks (see above), the autoradiograms were identical to those shown in Fig. 3A and B, except that the 72K protein could not be detected (data not shown). The radioimmunoassay was also carried out with a specific antipolyhook serum. The 72K polyhook protein was more intense in the extract of the PCM103 cells (Fig. 3C), and synthesis of the 72K hook protein could be identified in CB15 cells (Fig. 3D). The detection of flagellin in the labeled cell extracts of strain CB15, using antipolyhook serum, is presumably due to the structural association between the hook and the filament in these cells (13, 28, 29, 31). As shown below, there is no immunological cross-reaction between C. crescentus hook protein and flagellin. The specificity of the radioimmunoassays for flagellin, polyhook, and hook proteins is shown in Fig. 3E. When a nonimmune serum was substituted for the immune sera in the assay, most of the proteins observed (Fig. 3A to D) are present, except for those at 72K, 28K, and 26K; bands at these positions are either extremely faint or absent. The antipolyhook serum was also used to analyze immunoprecipitates from strains PCM103 and CB15 by two-dimensional gel electrophoresis (Fig. 4A and B), and the major labeled proteins precipitated were compared with purified polyhook protein and flagellin in the
VOL. 138, 1979
POLYHOOK PROTEIN FROM C. CRESCENTUS
same system (Fig. 4C and D). The locations of the purified polyhook protein (Fig. 4C) and the polyhook protein immunoprecipitated from strain PCM103 (Fig. 4A) are indicated by arrows at 72K and pH 6.1. A less intensely labeled protein with the same molecular weight and isoelectric point as the polyhook protein is also precipitated from extracts of strain CB15 by the antipolyhook serum (Fig. 4B, arrow). Incomplete solubilization of proteins in the labeled cell extracts may account for the relatively heavy backgrounds in Fig. 4A and B; with the exception of flagellin and polyhook protein, which could not be detected, exactly the same patterns of contaminating spots were observed when preimmune serum was substituted for the antipolyhook serum in this assay (data not shown). The results shown in Fig. 4 also confirm the difference between the polyhook protein and flagellin. Whereas the polyhook protein is a single charge species at 72K, flagellin A is composed of three charge species and flagellin B is composed of four charge species (Fig. 4D). The three flagellin A species and two of the four flagellin B species were immunoprecipitated by antipolyhook serum from pulse-labeled extracts of strain CB15 (Fig. 4B). (The two more basic species of flagellin B were also observed in similar labeling experiments, using antiflagellin serum [unpublished data].) These pulse-label experiments suggest that the charge heterogeneity of flagellin species is present soon after the time of synthesis and that it is maintained until the filaments are shed into the medium. In vitro carbamylation of flagellin (which sequentially increases the net negative charge on a protein [2]) indicates that the charge differences between the flagellin species are greater than a single charge (data not shown). The most acidic species of flagellin has an isoelectric point of approximately 6.4, or 0.3 pH unit higher than the isoelectric point of the polyhook. Antigenicity of polyhook protein. The specific antiserum prepared against the purified PCM103 structure (Fig. 2) was used to establish that this structure is antigenically related to the hook of flagella shed by wild-type cells. The antiserum was reacted with intact flagella from C. crescentus, and the preparation was negatively stained and examined in an electron microscope, as described previously (30). Binding of antisera is visualized as an enhancement of the degree of staining and an increase in the apparent width of the bound structure (30). Figure 5A shows that the serum reacted only with the hook structure in these preparations and that neither the filament nor the rod was stained. When the same experiment was carried out with preadsorbed antiflagellin serum (see
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above), the flagellar filament of the intact wildtype flagellum reacted, but neither rods nor hooks of the flagellum showed any reaction (Fig. 5B). Figures 5C and D show the reactions of polyhooks with these antisera; polyhook preparations used for this experiment were not washed at low pH (see above) and, therefore, contained rare, unattached, flagellar filaments (arrows, Fig. 5C and D) which served as an internal control for the specificity of antibody binding. Figure 5C shows that the antipolyhook serum bound to
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70 5.0 7.0 65 60 65 5.0 6.0 FIG. 4. Two-dimensional gel electrophoresis of radioimmunoprecipitates and purified flagellar proteins. Radioimmunoprecipitates were prepared from pulse-labeled cell extracts of strains PCM103 (A) and CB15 (B), using an antipolyhook serum (see text). Approximately 400,000 cpm of each sample was analyzed by twodimensional gel electrophoresis (19) and fluorography (15). A 20-jig amount of purified polyhook protein (C) and flagellin purified by one cycle of dissaggregation and reconstitution (D) were analyzed in the same system, and the gels were stained as described in the legend to Fig. 2. The position of the 72K polyhook protein is marked by an arrow in (A) and (C); the location of the 72K, 6.1 pIprotein (see text) is marked in (B); and the positions of this protein and the polyhook protein are indicated relative to three other proteins in (A) and (B). Multiple species of flagellin B (28K) and flagellin A (26K) are marked in (B) and (D). Isoelectric focusing (IEF) and SDS-polyacrylamide gel electrophoresis (SDS) dimensions are also indicated.
polyhooks but not to the contaminating flagellar filaments. Figure 5D shows that the polyhooks did not bind antiflagellin serum but that fragments of flagellar filaments did specifically bind this antiserum. Nonimmune control serum did not react with any of the structures (data not shown). We conclude from these experiments that the short, thick filaments produced by strain PCM103 are antigenically related to the hooks of normal flagella. Thus, the morphological (Fig. 1), biochemical (Fig. 2), and immunological (Fig. 5) evidence supports the conclusion that mutant PCM103 is a polyhook-producing strain of C. crescentus.
Amino acid composition of polyhook protein. Purified preparations of the polyhook protein were hydrolyzed at 108°C in 6 N HCI for 24 h for amino acid analysis (32). Table 1 compares the amino acid composition of the PCM103 polyhook protein to purified flagellins of strain CB15. This comparison shows that the polyhook protein has a higher content of proline and tyrosine than do the flagellins of C. crescentus. This analysis also suggests a similarity between the polyhook protein of C. crescentus, the polyhook protein of E. coli (30), and the hook protein of Salmonella (8). All three of these proteins have relatively high contents of proline and ty-
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