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A single amino acid substitution responsible for altered flagellar morphology
J. Mol. Biol. (1968) 34, 559-564
A Single Amino Acid Substitution Responsible for Altered Flagellar Morphology RAFAEL J. MARTINEZ,~BERT T. ICHZI(I, NANCY P. LUNDH AND STEVEN R. TRONICK Department of Bacteriology University of California Los Angeles, California, U.X.A. (Received 18 September 1967 and in revised form February 1968) Mutants of Bacillua subtilia 168 lacking the long-period helix in their flagella have been isolated. Purified flagella from these cells have been studied by physical, immunological and chemical techniques. Straight flagella mutants are non-motile, yet their locomotor organ&es appear to have the same fine structure and adsorption characteristics for phage PBS 1 as the wild type. Immunologically the two structures appear to be identical. A single peptide difference has been found in fingerprints of tryptic digests of flagellins from the mutants. The amino acid substitution responsible for this change was the exchange of alanine for valine in the altered dipeptide.
1. Introduction It has been well established that the nucleotide sequences of the genetic material determine the amino acid sequences of proteins. In many instances enzymic as well as structural proteins do not function as such in the cell, but exert their action in the form of aggregates of varying complexity. Relatively little is known regarding the mechanisms involved in the formation of macromolecular aggregates and especially subcellular organelles. The pioneering work on the specific aggregation of biologically active tobacco mosaic virus from its protein and ribonucleic acid components (Fraenkel-Conrat & Williams, 1955) and the more recent investigations on bacteriophage assembly (Wood & Edgar, 1967) exemplify the progress made in this area. We have selected the flagellum of Bacillus subtilis as a model system for investigations on macromolecular assemblages, because of the relative simplicity of the organelle and the ease of carrying out genetic analyses. The flagellum of B. subtilis is composed of a single type of protein subunit (flagellin) having a molecular weight’ of 40,000 (Martinez, Brown & Glazer, 1967). The protein subunits are assembled in the organelle via non-covalent forces. Normal flagella show a long-period helix with a pitch characteristic of the species (Leifson, 1960); for B. subtilis SB19 the pitch is approximately 2.5 p. Mutations leading to flagella with altered morphology have been described. The most common of these morphological alterations are the so-called “curly” mutants of Salmonella in which the pitch of the long-period helix is reduced by approximately one half of the normal (Leifson & Hugh, 1953). Leifson (1960) has observed ‘%ariants” of Listeria which produced straight flagella, i.e. lacking the long-period helix. 659
560
R. J. MARTINEZ
ET
AL.
To test the assumption that the periodicity of the long-period helix is duo to the amino acid sequence of the flagellin monomers, we have isoIated and studied mutant 5 with altered flagellar morphology. Among these were a class having straight Hag~ll,~. These mutants were non-motile; yet, investigations of the flagella produced by these cells showed that they were antigenically homologous with the flagella of the parent strain, and served as adsorption sites for the flagellotropic B. subtilis phage PBS 1 (Rairnondo, Lundh & Martinez, 1968). The data to be presented demonstrate that the altered morphology of the locomotor organelles is due to a single amino acid substitution in the primary structure of the protein subunit of the flagellum.
2. Materials and Methods B. subti& strain SB19 was grown in minimal medium (Martinez & Gordee, 1966) with 0.6% glucose and 0.1% N-Z Case (pancreatic digest of casein) on a reciprocal shaker at 37%. B. mbtilia strain 168 (trp-) and derivatives thereof were grown in the same medium supplemented with 15 pg of L-tryptophan/ml. Flagella were isolated and purified as previously described (Martinez, 1963). To detect the presence of non-motile cells in mutagen-treated cultures of B. subtilk 168, individual clones of surviving cells were selected from semi-solid medium essentially as described by Stocker, Zinder & Lederberg (1953). Nitrosoguanidine and proflavin were the mutagens giving the greatest numbers of morphologically altered flagella mutants. Ultraviolet irradiation gave rise to the greatest number ofpa- mutants, but none of the non-motile mutants obtained has been found to be flagellated. Nitrosoguanidine was used at a final concentration of 50 pg/ml. for 30 min in nutrient broth adjusted to pH 6.0. Proflavin was used at a final concentration of 5 rg/ml. for 20 min in nutrient broth adjusted to pH 7.6. Under these conditions there was approximately 0~1% survival; approximately 25% of the survivors gave rise to non-motile clones. Less than 1 to 5% of these possessed morphologically altered flagella. All mutants were found to retain the trp- marker of the parent 168 strain. The mutants possessing straight flagella were given the prefix SC and numbered sequentially. Electron microscopy was performed using an Hitachi KU 11A with a no. 3 pole piece at 75 kv. Specimens were shadowed with palladium-gold or stained with 2”/e uranyl acetate. Antiflagellar antiserum was prepared according to the method of Ada, Nossal, Pye & Abbot (1964). Immobilization assays were performed as described by Nossal (1959). Purification of flagellar antibodies and iodination of the flagellar antibodies with lz51 were carried out according to the methods of Grant & Simon (1968). For the self-assembly experiments, flagellin was prepared by alkaline dissociation (Martinez, Brown & Glazer, 1967); the clear solution of protein was dialyzed for 2 hr against distilled water adjusted to pH 8.0. The dialysis bag contents were centrifuged at 40,000 rev./n& for 1 hr and the supernatant solution dialyzed against water adjusted to pH 4.0; an amorphous precipitate appeared. Upon further dialysis at pH 8.0, the prccipitate disappeared and flagella-like filaments were formed. Flagellins prepared by alkaline dissociation of flagella were sedimented in the Spinco model E analytical ultracentrifuge at 89,780 rev./min. The protein solutions were dialyzed against 2 X 10m3 rd-triethylammonium acetate buffer (pH 7.0) containing 0.1 M-NaCl prior to sedimentation. Tryptic peptide maps and acrylamide gel electrophoresis were run as already described (Martinez, Brown & Glazer, 1967). Thermal dissociation measurements were made essentially as previously described (Martinez & Rosenberg, 1964). Peptides A and B (see Plate III) from the straight flagella mutants and from strains 168 or SB19 were isolated and purified as follows: 10 to 20 mg of a tryptic digest of flagellin were applied to Whatman 3 MM paper in a line at a load of 0.7 mg of digest/cm. Guide strips containing tryptic digests were run at each side of the main application line.
AMINO
ACID
SUBSTITUTION
IN
561
FLAGELLIN
Electrophoresis was performed in pyridine-acetate bufkr (pH 4.7) (Miohl, 1951) at 50 v/cm for 100 min. The peptides were located on the guide strips after staining with cadmium-ninhydrin. Peptides A and B were cut out of the main application line and eluted with 1% acetic acid and lyophilized. The dry material wa.s dissolved in 100 ~1. of 2% acetic acid and applied to Whatman 3MM paper as above. Descending chromatography was carried out using n-butsnol-pyridine-acetic acid-water (15 : 10 : 3 : 12 by vol.) for 16 to 20 hr. Guide strips were again used to locate the desired peptides, which were eluted and lyophilized. Arniuo acid analyses were performed on a Spinco model 120 amino acid analyzer.
3. Results Non-motile colonies were picked from motility agar after incubation hours at 37°C. Exsmination of these cultures under phase microscopy
for 10 to 18 showed the
absence of translational motility. Of these clones, a small percentage (1 to 5% or less) were capable of serving as hosts for the flagellotropic B. subtilis phage PBS 1. Electron
microscopic
examination
of these
non-motile,
phage-sensitive
mutants
revealed the presence of flagellated cells having altered gross flagellar morphology, i.e. the flagella lacked the long-period helix (Plate I). Further, the numbers of flagella per cell were considerably reduced. These straight flagella, however, appear to have the same fine structure as the flagella from the parent, i.e. four or five lines parallel to the long axis of the flagellum (Lowy & Hanson, 1965). The kinetics of thermal dissociation of purified 5gella from a straight flagella mutant were compared to those of the parent strain. No significant differences were observed. Both showed a thermal dissociation temperature of 53.6”C when the experiment was conducted in 0.01 M-phosphate buffer (pH 7-O) and both showed a similar depression of the melting temperature when tested in the presence of 40% ethylene glycol. Flagellins prepared by alkaline dissociation of flagella followed by dialysis against triethylammonium acetate-NaCl do not reaggregate. Sedimentation velocity measurements were made on flagellins from the mutant strain SC6 and from SB19 under these conditions. An X20,Wvalue of 2.6~ was found for the protein from SB19 and a value of 2.7~ for that from SC6. An S,,,, value of 1.6~has recently been reported for the flagellin from SB19 when measured at pH 2 and pH 13 (Martinez, Brown & Glazer, 1967). The value of 2.6s corresponds more closely to that predicted for the folded cor@uration of the protein, and the discrepancy can be accounted for by the fact that the lower value was determined for denatured flagellin (O-01 N-HCl). Flagellins from all the straight flagella mutants were found to co-electrophorese in acrylamide gels with that of the parent strain. We were interested in determinin g whether flagellin obtained from the straight flagella mutants could undergo self-assembly to form straight flagella filaments or whether the product of self-assembly would show the long-period helix of normal flagella. The results of such an experiment are shown in Plate II. It is evident that flagellin obtained from strain SC23 reassembles spontaneously to form straight flagella-like filaments, whereas the protein from SB19 self-assembles to form filaments with a long-period helix. Immunological
analyses
were made to test the antigenic
relatedness
between
the
flagella from straight flagella mutants and the wild type. Two experimental procedures were used: immobilization of motile SB19 cells by specific flagellar antibodies before and after adsorption with flagella from mutant or wild-type cells; and specific
R. J. MARTINEZ
562
ET
AL.
binding of lz51-labeled antibody by mutant and wild-type flagella. The results of an immobilization experiment are presented in Figure 1. The organelles from straight flagella mutants adsorb immobilizing antiserum prepared against SB19 as avidly as homologous organelles. Figure 2 gives the results of an lz5r,antibody-binding experiment. Again we see antigenic homology between the ml;cant flagella and wild-type organelles in their antibody-binding capacity. Purified flagella from the four straight flagella mutants and from SB19 were subjected to tryptic digestion after alkaline dissociation. Tryptic peptide maps were prepared and comparisons made between the mutant protein and wild-type flagellin.
0
I
I
IO
20
I
I
30 40 pg flogel IQ
I
I
50
60
Fm. 1. Adsorption of immobilizing antibody by purified flagella from SB19 (0) and SC6 (A). Anti-SB19 flagellar antibody (0.1 ml.) was treated with varying concentrations of homologous and SC6 flagella for 15 min at 37°C; 10s motile SB19 cells in 0.1 ml. were added end the percentage of residual motile cells determined by microscopy.
--I
pg flagella FIQ. 2. Binding of 1261-labeled purified antiSB19 flagellar antibody by purified flagella from SB19 and SC6 0.1 ml. of purified anti-SB19 flagellar antibody (2pg of protein-5000 &s/mm) were reacted with increasing concentrations of purified flagella from SB19 (0) and SC6 (A) for 30 min at room temperature; the mixture wae filtered through DEAE paper, washed and counted (Grant & Simon, 1968).
PLATE II. Flagella-like filaments obtained SB19 (top) and SC3 (bottom). x 40,000.
b,v rc-aggregation
of flagellinx
of H. subtilis
st,ra.ins
PLATE III. Fingerprints of tryptic digests of flagellins from H. subtilis strains SRI9 (t,op) al~(l SC3 (bottom). the negative olectrodt~ 011 tlw The lower horizontal axis is the electrophoretic phase, with right-hand side; chromatography was on the perpendicular axis.
AMINO
ACID
SUBSTITUTION
IN
FLAGELLIN
563
Representative maps for SB19 and straight flagella mutant SC3 are shown in Plate III. Careful scrutiny of the maps reveals only one pept’ide difference between the two proteins. Peptide A of strain SB19, a very basic peptide which moves ral)itll! in the electrophoretic phase and shows slow chromatographic mobility, has disappeared from the map of strain SC3. A new peptide (labeled B) appears in the map of SC3; this peptide shows slightly slower electrophoretic mobility but much greater mobility in the chromatographic phase. Such a change indicates an increase in the size and hydrophobicity of the new peptide. The rest of the map appears identical to that of the wild type. Peptide A from SB19 and peptide B from SC3 digests have been isolated and purified. Amino acid analyses of the peptides have shown that peptide A is composed of equimolar quantities of alanine and lysine (0.053 : 0.054 pmole) and peptide B is composed of equimolar quantities of valine and lysine (O-020 : 0.021 pmole). From consideration of tryptic specificity, lysine is C-terminal in these dipeptides and hence the sequence is known. Further evidence that peptide A is indeed Ala-Lys was obtained by demonstrating co-chromatography with authentic Ala-Lys in n-butanol-acetic acid-water as well as in n-butanol-pyritie-acetic acid-water. Peptide A was also shown to co-electrophorese at pH 4.7 as well as pH 1.9 with the authentic dipeptide. Similar evidence has been obtained for peptide B, which co-electrophoreses and co-chromatographs with authentic Val-Lys. The B peptides from the other three SC mutants were similarly studied and were also found to be composed of Val-Lys.
4. Discussion The flagella from mutants lacking the long-period helix characteristic of the wild type resemble those of the parent strain in almost all the criteria examined. The fine structure of the organelles of strain SC3 appears identical in the electron microscope with that of SB19. The thermal dissociation temperature as well as the kinetics of thermal dissociation of the mutant flagella were, within experimental error, identical to those of the parent strain. These data strongly suggest that the arrangement of the subunits, as well as the forces holding the subunits together in the supermolecular structure, are very similar, if not identical. Further, phage PBS 1, a phage known to adsorb specifically to the flagella of B. subtilis, adsorbs equally well to strains SC6 and SB19. That the configuration of the subunits at the surface of the organelle in the wild-type flagella as well as the flagella from the mutant strains must be very similar, if not identical, is further substantiated by the antigenic homology between the two structures. Nevertheless, although the major body of evidence suggests identity between flagella lacking the long-period helix and wild-type organelles, three very significant differences have been observed. Self-assembly of the subunits of strain 523 gives rise to flagella-like filaments totally lacking the long-period helix, whereas the subunits of strain SB19 self-assemble to filaments possessing a long-period helix similar to that of the native organelles. This observation strongly implies that the absence of the long-period helix in the mutant flagella is not due to an altered assembly mechanism, if such an assembly mechanism truly exists. It further implies that the long-period helix is a function of the conformation of the subunits comprising the flagellum and is extremely sensitive to amino acid substitutions in certain key loci in the protein.
564
R. J. MARTINEZ
ET
AL.
Secondly, strains possessing straight flagella are non-motile. Although it is possiblr that the absence of motility in the straight-flagella mutants is due t.o a mut,atiott in the mot locus (Joys & Frankel, 1967), this appears highly unlikely since spontaneous revertants to motility invariably have regained the long-period helix. Lastly, and most significant, fingerprints of tryptic digests of flagc:llins from the straight-flagella mutants and strains SB19 or 168 differ in only one of the 33 peptides of the map. The difference resides in a substitution of valine for alanine in the altered peptide. This substitution is the only one that has been observed in the four mutants thus far analyzed, even though two of the mutants were induced by different mutagenic agents. It is striking that all of the four independently isolated straightflagella mutants show the same amino acid substitution in the same locus. This would seem to imply that the loss of the long-period helix is diagnostic of this particular amino acid interchange. It is highly probable that mutants shop ing other types of morphological aberrations will exhibit amino acid replacements characteristic of those changes. This aspect is under investigation at present. Obviously not all amino acid substitutions in flagellin will lead to gross morphological alterations; for example, the fingerprint of B. subtilis strain W23 shows a single peptide difference from that of SB19, yet the flagella from both strains are indiA ingui~hablt~ morphologically. The data presented in this communication imply that a single amino acid substitution at a critical position in the primary sequence of flagellin, a protein of molecular weight 40,000, leads to a loss of the long-period helix of the flagellum and thus to an alteration in the gross morphology of the structure. This substitution also appears to be responsible for the loss of function of the locomotor organelle. Similar observations have been made in a curly flagella mutant of Salmonella, where a single peptide has been found to be altered (Enomoto & Iino, 1966). The amino acid(s) substitution responsible for the change from normal to curly has not yet been determined. We thank Miss J. Graham for her expert technical assistance in the performance of some of these experiments. We are indebted to Dr A. Glazer for his continued stimulation and advice. This work was supported in part by National Science Foundation grant GB 4633. REFERENCES Ada, G. L., Nossal, G. J. V., Pye, J. & Abbot, A. (1964). Aust. J. E.q/. Biol. Med. Sci. 42, 267. Enomoto, M. & Iino, T. (1966). Japan. J. Genetics, 41, 131. Fraenkel-Conrat, H. & Williams, R. C. (1955). Proc. Nat. Acad. Sci., WC.&. 41, 690. Grant, G. F. & Simon, M. (1968). J. Bact. 95, 81. Joys, T. M. & Frank& R. W. (1967). J. Bact. 94, 32. Leifson, E. (1960). Atlas of Bacterial Flagellation. New York: Academic Press. Leifson, E. & Hugh, R. (1953). J. Bud. 65, 263. Lowy, J. & Hanson, J. (1965). J. MOE. Biol. 11, 293. Martinez, R. J. (1963). J. Gen. Microbial. 33, 115. Martinez, R. J., Brown, D. M. & Glazer, A. N. (1967). J. 21101. Bid. 28, 45. Martinez, R. J. & Gordee, E. Z. (1966). J. Bad. 91, 870. Martinez, R. J. & Rosenberg, E. (1964). J. Mol. Biol. 8, 702. Michl, H. (1951). Monk&. Chem. 82, 489. Nossal, G. J. V. (1959). Immunol. 2, 137. Raimondo, L. M., Lundh, N. P. & Martinez, R. J. (1968). J. Vi~irol. 2, 256. Stocker, B. A. D., Zinder, N. D. & Lederberg, J. (1953). J. Gen. Microbial. 9, 410. Wood, W. B. & Edgar, R. S. (1967). Sci. American, 217, 61.
A Single Amino Acid Substitution Responsible for Altered Flagellar Morphology RAFAEL J. MARTINEZ,~BERT T. ICHZI(I, NANCY P. LUNDH AND STEVEN R. TRONICK Department of Bacteriology University of California Los Angeles, California, U.X.A. (Received 18 September 1967 and in revised form February 1968) Mutants of Bacillua subtilia 168 lacking the long-period helix in their flagella have been isolated. Purified flagella from these cells have been studied by physical, immunological and chemical techniques. Straight flagella mutants are non-motile, yet their locomotor organ&es appear to have the same fine structure and adsorption characteristics for phage PBS 1 as the wild type. Immunologically the two structures appear to be identical. A single peptide difference has been found in fingerprints of tryptic digests of flagellins from the mutants. The amino acid substitution responsible for this change was the exchange of alanine for valine in the altered dipeptide.
1. Introduction It has been well established that the nucleotide sequences of the genetic material determine the amino acid sequences of proteins. In many instances enzymic as well as structural proteins do not function as such in the cell, but exert their action in the form of aggregates of varying complexity. Relatively little is known regarding the mechanisms involved in the formation of macromolecular aggregates and especially subcellular organelles. The pioneering work on the specific aggregation of biologically active tobacco mosaic virus from its protein and ribonucleic acid components (Fraenkel-Conrat & Williams, 1955) and the more recent investigations on bacteriophage assembly (Wood & Edgar, 1967) exemplify the progress made in this area. We have selected the flagellum of Bacillus subtilis as a model system for investigations on macromolecular assemblages, because of the relative simplicity of the organelle and the ease of carrying out genetic analyses. The flagellum of B. subtilis is composed of a single type of protein subunit (flagellin) having a molecular weight’ of 40,000 (Martinez, Brown & Glazer, 1967). The protein subunits are assembled in the organelle via non-covalent forces. Normal flagella show a long-period helix with a pitch characteristic of the species (Leifson, 1960); for B. subtilis SB19 the pitch is approximately 2.5 p. Mutations leading to flagella with altered morphology have been described. The most common of these morphological alterations are the so-called “curly” mutants of Salmonella in which the pitch of the long-period helix is reduced by approximately one half of the normal (Leifson & Hugh, 1953). Leifson (1960) has observed ‘%ariants” of Listeria which produced straight flagella, i.e. lacking the long-period helix. 659
560
R. J. MARTINEZ
ET
AL.
To test the assumption that the periodicity of the long-period helix is duo to the amino acid sequence of the flagellin monomers, we have isoIated and studied mutant 5 with altered flagellar morphology. Among these were a class having straight Hag~ll,~. These mutants were non-motile; yet, investigations of the flagella produced by these cells showed that they were antigenically homologous with the flagella of the parent strain, and served as adsorption sites for the flagellotropic B. subtilis phage PBS 1 (Rairnondo, Lundh & Martinez, 1968). The data to be presented demonstrate that the altered morphology of the locomotor organelles is due to a single amino acid substitution in the primary structure of the protein subunit of the flagellum.
2. Materials and Methods B. subti& strain SB19 was grown in minimal medium (Martinez & Gordee, 1966) with 0.6% glucose and 0.1% N-Z Case (pancreatic digest of casein) on a reciprocal shaker at 37%. B. mbtilia strain 168 (trp-) and derivatives thereof were grown in the same medium supplemented with 15 pg of L-tryptophan/ml. Flagella were isolated and purified as previously described (Martinez, 1963). To detect the presence of non-motile cells in mutagen-treated cultures of B. subtilk 168, individual clones of surviving cells were selected from semi-solid medium essentially as described by Stocker, Zinder & Lederberg (1953). Nitrosoguanidine and proflavin were the mutagens giving the greatest numbers of morphologically altered flagella mutants. Ultraviolet irradiation gave rise to the greatest number ofpa- mutants, but none of the non-motile mutants obtained has been found to be flagellated. Nitrosoguanidine was used at a final concentration of 50 pg/ml. for 30 min in nutrient broth adjusted to pH 6.0. Proflavin was used at a final concentration of 5 rg/ml. for 20 min in nutrient broth adjusted to pH 7.6. Under these conditions there was approximately 0~1% survival; approximately 25% of the survivors gave rise to non-motile clones. Less than 1 to 5% of these possessed morphologically altered flagella. All mutants were found to retain the trp- marker of the parent 168 strain. The mutants possessing straight flagella were given the prefix SC and numbered sequentially. Electron microscopy was performed using an Hitachi KU 11A with a no. 3 pole piece at 75 kv. Specimens were shadowed with palladium-gold or stained with 2”/e uranyl acetate. Antiflagellar antiserum was prepared according to the method of Ada, Nossal, Pye & Abbot (1964). Immobilization assays were performed as described by Nossal (1959). Purification of flagellar antibodies and iodination of the flagellar antibodies with lz51 were carried out according to the methods of Grant & Simon (1968). For the self-assembly experiments, flagellin was prepared by alkaline dissociation (Martinez, Brown & Glazer, 1967); the clear solution of protein was dialyzed for 2 hr against distilled water adjusted to pH 8.0. The dialysis bag contents were centrifuged at 40,000 rev./n& for 1 hr and the supernatant solution dialyzed against water adjusted to pH 4.0; an amorphous precipitate appeared. Upon further dialysis at pH 8.0, the prccipitate disappeared and flagella-like filaments were formed. Flagellins prepared by alkaline dissociation of flagella were sedimented in the Spinco model E analytical ultracentrifuge at 89,780 rev./min. The protein solutions were dialyzed against 2 X 10m3 rd-triethylammonium acetate buffer (pH 7.0) containing 0.1 M-NaCl prior to sedimentation. Tryptic peptide maps and acrylamide gel electrophoresis were run as already described (Martinez, Brown & Glazer, 1967). Thermal dissociation measurements were made essentially as previously described (Martinez & Rosenberg, 1964). Peptides A and B (see Plate III) from the straight flagella mutants and from strains 168 or SB19 were isolated and purified as follows: 10 to 20 mg of a tryptic digest of flagellin were applied to Whatman 3 MM paper in a line at a load of 0.7 mg of digest/cm. Guide strips containing tryptic digests were run at each side of the main application line.
AMINO
ACID
SUBSTITUTION
IN
561
FLAGELLIN
Electrophoresis was performed in pyridine-acetate bufkr (pH 4.7) (Miohl, 1951) at 50 v/cm for 100 min. The peptides were located on the guide strips after staining with cadmium-ninhydrin. Peptides A and B were cut out of the main application line and eluted with 1% acetic acid and lyophilized. The dry material wa.s dissolved in 100 ~1. of 2% acetic acid and applied to Whatman 3MM paper as above. Descending chromatography was carried out using n-butsnol-pyridine-acetic acid-water (15 : 10 : 3 : 12 by vol.) for 16 to 20 hr. Guide strips were again used to locate the desired peptides, which were eluted and lyophilized. Arniuo acid analyses were performed on a Spinco model 120 amino acid analyzer.
3. Results Non-motile colonies were picked from motility agar after incubation hours at 37°C. Exsmination of these cultures under phase microscopy
for 10 to 18 showed the
absence of translational motility. Of these clones, a small percentage (1 to 5% or less) were capable of serving as hosts for the flagellotropic B. subtilis phage PBS 1. Electron
microscopic
examination
of these
non-motile,
phage-sensitive
mutants
revealed the presence of flagellated cells having altered gross flagellar morphology, i.e. the flagella lacked the long-period helix (Plate I). Further, the numbers of flagella per cell were considerably reduced. These straight flagella, however, appear to have the same fine structure as the flagella from the parent, i.e. four or five lines parallel to the long axis of the flagellum (Lowy & Hanson, 1965). The kinetics of thermal dissociation of purified 5gella from a straight flagella mutant were compared to those of the parent strain. No significant differences were observed. Both showed a thermal dissociation temperature of 53.6”C when the experiment was conducted in 0.01 M-phosphate buffer (pH 7-O) and both showed a similar depression of the melting temperature when tested in the presence of 40% ethylene glycol. Flagellins prepared by alkaline dissociation of flagella followed by dialysis against triethylammonium acetate-NaCl do not reaggregate. Sedimentation velocity measurements were made on flagellins from the mutant strain SC6 and from SB19 under these conditions. An X20,Wvalue of 2.6~ was found for the protein from SB19 and a value of 2.7~ for that from SC6. An S,,,, value of 1.6~has recently been reported for the flagellin from SB19 when measured at pH 2 and pH 13 (Martinez, Brown & Glazer, 1967). The value of 2.6s corresponds more closely to that predicted for the folded cor@uration of the protein, and the discrepancy can be accounted for by the fact that the lower value was determined for denatured flagellin (O-01 N-HCl). Flagellins from all the straight flagella mutants were found to co-electrophorese in acrylamide gels with that of the parent strain. We were interested in determinin g whether flagellin obtained from the straight flagella mutants could undergo self-assembly to form straight flagella filaments or whether the product of self-assembly would show the long-period helix of normal flagella. The results of such an experiment are shown in Plate II. It is evident that flagellin obtained from strain SC23 reassembles spontaneously to form straight flagella-like filaments, whereas the protein from SB19 self-assembles to form filaments with a long-period helix. Immunological
analyses
were made to test the antigenic
relatedness
between
the
flagella from straight flagella mutants and the wild type. Two experimental procedures were used: immobilization of motile SB19 cells by specific flagellar antibodies before and after adsorption with flagella from mutant or wild-type cells; and specific
R. J. MARTINEZ
562
ET
AL.
binding of lz51-labeled antibody by mutant and wild-type flagella. The results of an immobilization experiment are presented in Figure 1. The organelles from straight flagella mutants adsorb immobilizing antiserum prepared against SB19 as avidly as homologous organelles. Figure 2 gives the results of an lz5r,antibody-binding experiment. Again we see antigenic homology between the ml;cant flagella and wild-type organelles in their antibody-binding capacity. Purified flagella from the four straight flagella mutants and from SB19 were subjected to tryptic digestion after alkaline dissociation. Tryptic peptide maps were prepared and comparisons made between the mutant protein and wild-type flagellin.
0
I
I
IO
20
I
I
30 40 pg flogel IQ
I
I
50
60
Fm. 1. Adsorption of immobilizing antibody by purified flagella from SB19 (0) and SC6 (A). Anti-SB19 flagellar antibody (0.1 ml.) was treated with varying concentrations of homologous and SC6 flagella for 15 min at 37°C; 10s motile SB19 cells in 0.1 ml. were added end the percentage of residual motile cells determined by microscopy.
--I
pg flagella FIQ. 2. Binding of 1261-labeled purified antiSB19 flagellar antibody by purified flagella from SB19 and SC6 0.1 ml. of purified anti-SB19 flagellar antibody (2pg of protein-5000 &s/mm) were reacted with increasing concentrations of purified flagella from SB19 (0) and SC6 (A) for 30 min at room temperature; the mixture wae filtered through DEAE paper, washed and counted (Grant & Simon, 1968).
PLATE II. Flagella-like filaments obtained SB19 (top) and SC3 (bottom). x 40,000.
b,v rc-aggregation
of flagellinx
of H. subtilis
st,ra.ins
PLATE III. Fingerprints of tryptic digests of flagellins from H. subtilis strains SRI9 (t,op) al~(l SC3 (bottom). the negative olectrodt~ 011 tlw The lower horizontal axis is the electrophoretic phase, with right-hand side; chromatography was on the perpendicular axis.
AMINO
ACID
SUBSTITUTION
IN
FLAGELLIN
563
Representative maps for SB19 and straight flagella mutant SC3 are shown in Plate III. Careful scrutiny of the maps reveals only one pept’ide difference between the two proteins. Peptide A of strain SB19, a very basic peptide which moves ral)itll! in the electrophoretic phase and shows slow chromatographic mobility, has disappeared from the map of strain SC3. A new peptide (labeled B) appears in the map of SC3; this peptide shows slightly slower electrophoretic mobility but much greater mobility in the chromatographic phase. Such a change indicates an increase in the size and hydrophobicity of the new peptide. The rest of the map appears identical to that of the wild type. Peptide A from SB19 and peptide B from SC3 digests have been isolated and purified. Amino acid analyses of the peptides have shown that peptide A is composed of equimolar quantities of alanine and lysine (0.053 : 0.054 pmole) and peptide B is composed of equimolar quantities of valine and lysine (O-020 : 0.021 pmole). From consideration of tryptic specificity, lysine is C-terminal in these dipeptides and hence the sequence is known. Further evidence that peptide A is indeed Ala-Lys was obtained by demonstrating co-chromatography with authentic Ala-Lys in n-butanol-acetic acid-water as well as in n-butanol-pyritie-acetic acid-water. Peptide A was also shown to co-electrophorese at pH 4.7 as well as pH 1.9 with the authentic dipeptide. Similar evidence has been obtained for peptide B, which co-electrophoreses and co-chromatographs with authentic Val-Lys. The B peptides from the other three SC mutants were similarly studied and were also found to be composed of Val-Lys.
4. Discussion The flagella from mutants lacking the long-period helix characteristic of the wild type resemble those of the parent strain in almost all the criteria examined. The fine structure of the organelles of strain SC3 appears identical in the electron microscope with that of SB19. The thermal dissociation temperature as well as the kinetics of thermal dissociation of the mutant flagella were, within experimental error, identical to those of the parent strain. These data strongly suggest that the arrangement of the subunits, as well as the forces holding the subunits together in the supermolecular structure, are very similar, if not identical. Further, phage PBS 1, a phage known to adsorb specifically to the flagella of B. subtilis, adsorbs equally well to strains SC6 and SB19. That the configuration of the subunits at the surface of the organelle in the wild-type flagella as well as the flagella from the mutant strains must be very similar, if not identical, is further substantiated by the antigenic homology between the two structures. Nevertheless, although the major body of evidence suggests identity between flagella lacking the long-period helix and wild-type organelles, three very significant differences have been observed. Self-assembly of the subunits of strain 523 gives rise to flagella-like filaments totally lacking the long-period helix, whereas the subunits of strain SB19 self-assemble to filaments possessing a long-period helix similar to that of the native organelles. This observation strongly implies that the absence of the long-period helix in the mutant flagella is not due to an altered assembly mechanism, if such an assembly mechanism truly exists. It further implies that the long-period helix is a function of the conformation of the subunits comprising the flagellum and is extremely sensitive to amino acid substitutions in certain key loci in the protein.
564
R. J. MARTINEZ
ET
AL.
Secondly, strains possessing straight flagella are non-motile. Although it is possiblr that the absence of motility in the straight-flagella mutants is due t.o a mut,atiott in the mot locus (Joys & Frankel, 1967), this appears highly unlikely since spontaneous revertants to motility invariably have regained the long-period helix. Lastly, and most significant, fingerprints of tryptic digests of flagc:llins from the straight-flagella mutants and strains SB19 or 168 differ in only one of the 33 peptides of the map. The difference resides in a substitution of valine for alanine in the altered peptide. This substitution is the only one that has been observed in the four mutants thus far analyzed, even though two of the mutants were induced by different mutagenic agents. It is striking that all of the four independently isolated straightflagella mutants show the same amino acid substitution in the same locus. This would seem to imply that the loss of the long-period helix is diagnostic of this particular amino acid interchange. It is highly probable that mutants shop ing other types of morphological aberrations will exhibit amino acid replacements characteristic of those changes. This aspect is under investigation at present. Obviously not all amino acid substitutions in flagellin will lead to gross morphological alterations; for example, the fingerprint of B. subtilis strain W23 shows a single peptide difference from that of SB19, yet the flagella from both strains are indiA ingui~hablt~ morphologically. The data presented in this communication imply that a single amino acid substitution at a critical position in the primary sequence of flagellin, a protein of molecular weight 40,000, leads to a loss of the long-period helix of the flagellum and thus to an alteration in the gross morphology of the structure. This substitution also appears to be responsible for the loss of function of the locomotor organelle. Similar observations have been made in a curly flagella mutant of Salmonella, where a single peptide has been found to be altered (Enomoto & Iino, 1966). The amino acid(s) substitution responsible for the change from normal to curly has not yet been determined. We thank Miss J. Graham for her expert technical assistance in the performance of some of these experiments. We are indebted to Dr A. Glazer for his continued stimulation and advice. This work was supported in part by National Science Foundation grant GB 4633. REFERENCES Ada, G. L., Nossal, G. J. V., Pye, J. & Abbot, A. (1964). Aust. J. E.q/. Biol. Med. Sci. 42, 267. Enomoto, M. & Iino, T. (1966). Japan. J. Genetics, 41, 131. Fraenkel-Conrat, H. & Williams, R. C. (1955). Proc. Nat. Acad. Sci., WC.&. 41, 690. Grant, G. F. & Simon, M. (1968). J. Bact. 95, 81. Joys, T. M. & Frank& R. W. (1967). J. Bact. 94, 32. Leifson, E. (1960). Atlas of Bacterial Flagellation. New York: Academic Press. Leifson, E. & Hugh, R. (1953). J. Bud. 65, 263. Lowy, J. & Hanson, J. (1965). J. MOE. Biol. 11, 293. Martinez, R. J. (1963). J. Gen. Microbial. 33, 115. Martinez, R. J., Brown, D. M. & Glazer, A. N. (1967). J. 21101. Bid. 28, 45. Martinez, R. J. & Gordee, E. Z. (1966). J. Bad. 91, 870. Martinez, R. J. & Rosenberg, E. (1964). J. Mol. Biol. 8, 702. Michl, H. (1951). Monk&. Chem. 82, 489. Nossal, G. J. V. (1959). Immunol. 2, 137. Raimondo, L. M., Lundh, N. P. & Martinez, R. J. (1968). J. Vi~irol. 2, 256. Stocker, B. A. D., Zinder, N. D. & Lederberg, J. (1953). J. Gen. Microbial. 9, 410. Wood, W. B. & Edgar, R. S. (1967). Sci. American, 217, 61.