RESUMEN
Purified flagella from two strains of 32P-labeled Pseudomonas aeruginosa were shown to be phosphorylated. This was confirmed by autoradiography of flagellin protein in polyacrylamide gels. Thin-layer electrophoresis and autoradiography of flagellin partial hydrolysates indicated that phosphotyrosine was the major phosphorylated amino acid. High-pressure liquid chromatographic analysis confirmed the presence of phosphotyrosine in flagellum filament protein. Preliminary data indicated that less than one tyrosine per subunit was phosphorylated. No evidence was found for phosphorylation of serine or threonine. A function related to tyrosine phosphorylation has not been determined.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Flagelos/análisis , Flagelina/aislamiento & purificación , Pseudomonas aeruginosa/análisis , Tirosina/análogos & derivados , Autorradiografía , Fraccionamiento Celular/métodos , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Flagelos/ultraestructura , Immunoblotting , Peso Molecular , Radioisótopos de Fósforo , Fosfotirosina , Tirosina/análisisRESUMEN
In high (45 mM)-phosphate medium, Methanospirillum hungatei strains GP1 and JF1 grew as very long, nonmotile chains of cells that did not possess flagella. However, growth in lower (3 or 30 mM)-phosphate medium resulted in the production of mostly single cells and short chains that were motile by means of two polar tufts of flagella, which transected the multilayered terminal plug of the cell. Electron microscopy of negatively stained whole mounts revealed a flagellar filament diameter of approximately 10 nm. Flagellar filaments were isolated from either culture fluid or concentrated cell suspensions that were subjected to shearing. Flagellar filaments were sensitive to treatment with both Triton X-100 and Triton X-114 at concentrations as low as 0.1% (vol/vol). The filaments of both strains were composed of two flagellins of Mr 24,000 and 25,000. However, variations in trace element composition of the medium resulted in the production of a third flagellin in strain JF1. This additional flagellin appeared as a ladderlike smear on sodium dodecyl sulfate-polyacylamide gels with a center of intensity of Mr 35,000 and cross-reacted with antisera produced from filaments containing only the Mr-24,000 and -25,000 flagellins. On sodium dodecyl sulfate-polyacrylamide gels, all flagellins stained by the thymol-sulfuric acid and Alcian blue methods, suggesting that they were glycosylated. This was further supported by chemical deglycosylation of the strain JF1 flagellins, which resulted in a reduction in their apparent molecular weight on sodium dodecyl sulfate-polyacylamide gels. Heterologous reactions to sera raised against the flagella from each strain were limited to the Mr-24,000 flagellins.
Asunto(s)
Euryarchaeota/ultraestructura , Flagelos/análisis , Euryarchaeota/análisis , Flagelos/ultraestructura , Flagelina/análisis , Flagelina/aislamiento & purificación , Glicosilación , Peso MolecularRESUMEN
The stoichiometries of components within the flagellar hook-(basal-body) complex of Salmonella typhimurium have been determined. The hook protein (FlgE), the most abundant protein in the complex, is present at approximately 130 subunits. Hook-associated protein 1 (FlgK) is present at approximately 12 subunits. The distal rod protein (FlgG) is present at approximately 26 subunits, while the proximal rod proteins (FlgB, FlgC and FlgF) are present at only approximately six subunits each. The stoichiometries of the proximal rod proteins and hook-associated protein 1 are, within experimental error, consistent with values of 5 or 6, and 11, respectively. Such values would correspond to either one or two turns of a helical structure with a basic helix of approximately 5.5 subunits per turn, which is the geometry of both the hook and the filament and, one supposes, the rod and hook-associated proteins. These stoichiometries may derive from rules for the heterologous interactions that occur when a helical structure consists of successive segments constructed from different proteins; the stoichiometries within the hook and the distal portion of the rod must, however, be set by different mechanisms. The stoichiometries for the ring proteins are approximately 26 subunits each for the M-ring protein (FliF), the P-ring protein (FlgI), and the L-ring protein (FlgH); the protein responsible for the S-ring feature is not known. The rings presumably have rotational rather than helical symmetry, in which case the stoichiometries would be directly constrained by the intersubunit bonding angle. The ring stoichiometries are discussed in light of other information concerning flagellar structure and function.
Asunto(s)
Proteínas Bacterianas/análisis , Flagelos/análisis , Salmonella typhimurium/análisis , Secuencia de Aminoácidos , Fenómenos Químicos , Química , Microscopía Electrónica , Datos de Secuencia Molecular , Salmonella typhimurium/ultraestructura , Azufre/análisisRESUMEN
The molecular composition and organization of the row of axonemal inner dynein arms were investigated by biochemical and electron microscopic analyses of Chlamydomonas wild-type and mutant axonemes. Three inner arm structures could be distinguished on the basis of their molecular composition and position in the axoneme as determined by analysis of pf30 and pf23 mutants. The three inner arm structures repeat every 96 nm and are referred to here as inner arms I1, I2, and I3. I1 is proximal to the radial spoke S1, whereas I2 and I3 are distal to spokes S1 and S2, respectively. The mutant pf30 lacks I1 whereas the mutant pf23 lacks both I1 and I2 but has a normal inner arm I3. Each of the six heavy chains that was identified as an inner dynein arm subunit has a site for ATP binding and hydrolysis. Two of the heavy chains together with a polypeptide of 140,000 molecular weight form the inner arm I1 and were extracted from the axoneme as a complex that had a sedimentation coefficient close to 21S at high ionic strength. Different subsets of two of the remaining four heavy chains form the inner arms I2 and I3. These arms at high ionic strength are dissociated as 11S particles that include one heavy chain, one intermediate chain, two light chains, and actin. These and other lines of evidence indicate that the inner arm I1 is different in structure and function from the inner arms I2 and I3.
Asunto(s)
Adenosina Trifosfatasas/análisis , Chlamydomonas/ultraestructura , Dineínas/análisis , Flagelos/análisis , Dineínas/fisiología , Electroforesis en Gel de Poliacrilamida/métodos , Flagelos/fisiología , Flagelos/ultraestructura , Microscopía Electrónica , MutaciónRESUMEN
Flagella-specific proteins of Leishmania have been identified employing the monoclonal antibody technique. Six monoclonal antibodies recognized 3 different proteins. A doublet of protein of Mr 69,000 and 74,000 Da identified by monoclonal antibodies F-3, F-4 and F-6 is continuously distributed along the flagellum by immunofluorescence. Immunocytochemical electron microscopic studies localize these molecules to the paraxial rod of the flagellum. A single protein of Mr 13,200 Da is recognized by monoclonal antibodies F-1, F-2 and F-5. The distribution of the Mr 13,200 protein appears irregular, occurring in localized patches along the length of the flagellum, especially at the flagellar tip. Immunocytochemical electron microscopic experiments show that the Mr 13,200 molecule is associated with the membrane of the flagellum. Indirect immunofluorescence experiments demonstrated these monoclonal antibodies cross-reacted with members of the Kinetoplastida family (Endotrypanum, trypanosoma, Leishmania) suggesting that these molecules may be evolutionarily conserved.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Flagelos/análisis , Leishmania/análisis , Proteínas Protozoarias/análisis , Animales , Anticuerpos Antiprotozoarios/inmunología , Especificidad de Anticuerpos , Autorradiografía , Western Blotting , Flagelos/inmunología , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Leishmania/inmunología , Leishmania/ultraestructura , Microscopía Electrónica , Pruebas de Precipitina , Proteínas Protozoarias/inmunologíaRESUMEN
A 24-kDa protein of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is recognized by antisera from both humans and experimental animals infected with this organism. Near its C terminus are two regions that have sequence similarity with several Ca2+-binding proteins and that conform to the "E-F hand" Ca2+-binding structure. We expressed a cDNA encoding this protein in Escherichia coli and showed that both the recombinant protein and the 24-kDa native trypanosome protein do indeed bind Ca2+. The protein's low Ca2+-binding capacity (less than 2 mol of Ca2+/mol of protein) and high Ca2+-binding affinity (apparent Kd less than 50 microM Ca2+) are consistent with binding of Ca2+ via the E-F hand structures. Immunofluorescence assays using a mouse antiserum directed against the fusion protein localized the native protein to the trypanosome's flagellum. The protein's abundance, Ca2+-binding property, and flagellar localization suggest that it participates in molecular processes associated with the high motility of the parasite.
Asunto(s)
Proteínas de Unión al Calcio/análisis , Flagelos/análisis , Proteínas Protozoarias/análisis , Trypanosoma cruzi/análisis , Secuencia de Aminoácidos , Animales , Western Blotting , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Calsecuestrina/metabolismo , ADN/genética , Escherichia coli/genética , Técnica del Anticuerpo Fluorescente , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Two different affinity-purified polyclonal antibodies were prepared against A polypeptides of dynein 1 extracted from sea urchin sperm. These antibodies, named AD1 and AD2, reacted exclusively with the alpha and beta heavy chains of dynein 1. Using these antibodies, we analyzed their cross-reactivity with dynein of mammalian cells. Immunohistochemically, both AD1 and AD2 stained dynein-related structures such as cilia of rabbit tracheal epithelia and flagella of rat spermatozoa. Immunoblots of the proteins extracted from mammalian cilia and flagella revealed the presence of A polypeptide-like proteins which cross-reacted with AD1 and AD2. Immunoblot analysis showed that the cross-reactive proteins were localized to the 370-kDa band of rabbit cilia and the 390- and 350-kDa bands of rat sperms. The reaction patterns showed that there were some differences between the two antibodies. On ciliary protein immunoblots, AD1 recognized about half of the broad band region which reacted with AD2, and AD1 also recognized only the 350-kDa band of the flagella extract, suggesting that the antibody reveals only a beta-like polypeptide. Immunoprecipitation studies using the ciliary proteins and AD2 confirmed that the immunoreactive protein had ATPase activity. Given these results, we have characterized mammalian dyneins previously reported by other laboratories.
Asunto(s)
Adenosina Trifosfatasas/análisis , Anticuerpos/inmunología , Dineínas/análisis , Flagelos/análisis , Péptidos/inmunología , Erizos de Mar/citología , Espermatozoides/análisis , Adenosina Trifosfatasas/metabolismo , Animales , Anticuerpos/análisis , Cilios/análisis , Cilios/enzimología , Cilios/ultraestructura , Dineínas/inmunología , Dineínas/metabolismo , Flagelos/inmunología , Flagelos/ultraestructura , Técnica del Anticuerpo Fluorescente , Immunoblotting , Masculino , Peso Molecular , Orgánulos/análisis , Orgánulos/inmunología , Orgánulos/ultraestructura , Péptidos/análisis , Pruebas de Precipitina , Conejos , Espermatozoides/inmunología , Espermatozoides/ultraestructura , Tráquea/citología , Tráquea/ultraestructuraRESUMEN
The flgH, flgI, and fliF genes of Salmonella typhimurium encode the major proteins for the L, P, and M rings of the flagellar basal body. We have determined the sequences of these genes and the flgJ gene and examined the deduced amino acid sequences of their products. FlgH and FlgI, which are exported across the cell membrane to their destinations in the outer membrane and periplasmic space, respectively, both had typical N-terminal cleaved signal-peptide sequences. FlgH is predicted to have a considerable amount of beta-sheet structure, as has been noted for other outer membrane proteins. FlgI is predicted to have an even greater amount of beta-structure. FliF, as is usual for a cytoplasmic membrane protein of a procaryote, lacked a signal peptide; it is predicted to have considerable alpha-helical structure, including an N-terminal sequence that is likely to be membrane-spanning. However, it had overall a quite hydrophilic sequence with a high charge density, especially towards its C terminus. The flgJ gene, immediately adjacent to flgI and the last gene of the flgB operon, encodes a flagellar protein of unknown function whose deduced sequence was hydrophilic and may correspond to a cytoplasmic protein. Several aspects of the DNA sequence of these genes and their surrounds suggest complex regulation of the flagellar gene system. A notable example occurs within the flgB operon, where between the end of flgG (encoding the distal rod protein of the basal body) and the start of flgH (encoding the L-ring protein) there was an unusually long noncoding region containing a potential stem-loop sequence, which could attenuate termination of transcription or stabilize part of the transcript against degradation. Another example is the interface between the flgB and flgK operons, where transcription termination of the former may occur within the coding region of the latter.
Asunto(s)
Proteínas Bacterianas/genética , Flagelos/análisis , Genes Bacterianos , Salmonella typhimurium/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Codón , Escherichia coli/genética , Datos de Secuencia Molecular , Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de Proteínas , Conformación Proteica , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
A library of Treponema pallidum DNA was established in the direct selection vector pUN121. Six clones carrying a gene coding for a 33-kilodalton T. pallidum flagellum subunit were identified by colony hybridization with an oligodeoxynucleotide probe based on the N-terminal amino acid sequence of this subunit. An open reading frame of 286 amino acids with the expected N-terminal sequence and absence of cysteine residues was identified. The deduced protein had a calculated molecular weight of 31.3 kilodaltons. We propose to name this flagellar subunit FlaB2. FlaB2 shows a significant amino acid homology with flagellins of several remotely related bacterial species. This homology was most pronounced corresponding to the C-terminal and N-terminal parts of the protein, whereas the central region was variable.
Asunto(s)
Genes Bacterianos , Genes , Treponema pallidum/genética , Secuencia de Aminoácidos , Antígenos Bacterianos/genética , Antígenos Bacterianos/aislamiento & purificación , Secuencia de Bases , Clonación Molecular/métodos , Flagelos/análisis , Flagelos/fisiología , Datos de Secuencia Molecular , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Treponema pallidum/aislamiento & purificaciónRESUMEN
Whole-cell, outer-membrane protein, flagellum-associated antigens and partially purified urease of Campylobacter pylori were analyzed by two-dimensional gel electrophoresis. C. pylori strains were readily distinguished from strains of Campylobacter jejuni, C. coli, and C. fetus by absence of major outer membrane proteins with Mrs of 41,000 to 45,000. C. pylori strains also lacked the acidic surface-array proteins at Mr 100,000 to 149,000 identified previously in serum-resistant strains of C. fetus. Surface labeling of intact C. pylori cells with 125I revealed two common major proteins, which we have designated protein 2 (pI 5.6 to 5.8, Mr 66,000) and protein 3 (pI 5.2 to 5.5, Mr 63,000). Proteins 2 and 3 were also the major components (subunits) observed in partially purified urease. Partially purified preparations of flagella consistently contained proteins 2 and 3. Thus, urease appears to be associated with both outer membranes and flagella of C. pylori. C. pylori strains also possessed an antigen at Mr 59,000 which was cross-reactive with antiserum against flagella of C. jejuni. However, the antigen did not appear to be associated with flagella per se in C. pylori. Protein 2 was unique to C. pylori among the Campylobacter species studied. It was not recognized by antibody against whole cells of C. jejuni or C. fetus or flagella of C. jejuni. Protein 3 was cross-reactive with antiserum against whole cells of C. jejuni and C. fetus, as were several other major protein antigens. Because protein 2 is a major outer membrane protein that is apparently unique to C. pylori, development of monospecific antibodies against this antigen may be useful for the identification of C. pylori in tissues, and purified antigen may be useful for serologic tests for specific diagnosis of C. pylori infections.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Campylobacter/análisis , Electroforesis en Gel Bidimensional , Immunoblotting , Animales , Antígenos Bacterianos/aislamiento & purificación , Autorradiografía , Flagelos/análisis , Flagelos/inmunología , Humanos , Sueros Inmunes , Conejos , Ureasa/inmunología , Ureasa/aislamiento & purificaciónRESUMEN
We have shown previously that the regulatory subunit (RII) of a type II cyclic AMP (cAMP)-dependent protein kinase is tightly associated with mammalian sperm flagella (J. A. Horowitz et al. (1984) J. Biol. Chem. 259, 832-838; J. A. Horowitz et al. (1988) J. Biol. Chem. 263, 2098-2104). In the present study the flagellar RII was compared to other well-characterized RIIs using biochemical and immunological methods. Flagellar polypeptides were screened by immunoblot analysis with monoclonal antibodies directed against the RII alpha and RII beta isoforms. An RII beta monoclonal antibody failed to cross-react with any flagellar polypeptide. In contrast, mAB 622, an RII alpha/RII beta monoclonal antibody, cross-reacted with a 57,000 Da polypeptide. However, another RII alpha/RII beta monoclonal antibody interacted weakly with the flagellar RII, suggesting that the epitope for this antibody is modified in flagellar RII. Partial peptide mapping of 8-azido-[32P]cAMP-labeled RIIs revealed that although heart and testis generated similar fragmentation patterns, there were differences in the maps from flagellar RII. Two-dimensional sodium dodecyl sulfate-gel electrophoresis of 8-azido-[32P]cAMP-labeled RII from rat flagella and bovine heart showed that the former possessed a considerably more acidic isoelectric point. Partial proteolysis of the flagellar RII by either endogenous or exogenous proteases resulted in the cleavage of RII to a 40,000 Mr fragment. Complete release of this fragment from the flagellum was achieved if proteolysis was performed in the presence of thiol reducing agents. In their absence, approximately 50% of the fragment remained bound to the flagellum. The soluble proteolytic fragment was shown to be monomeric by native high-resolution gel-permeation chromatography and contained a functional cAMP-binding site(s).
Asunto(s)
Proteínas Portadoras/análisis , Flagelos/enzimología , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas/análisis , Espermatozoides/enzimología , Animales , Anticuerpos Monoclonales , Bovinos , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida/métodos , Flagelos/análisis , Focalización Isoeléctrica , Masculino , Miocardio/análisis , Péptido Hidrolasas , Mapeo Peptídico , Fosforilación , Ratas , Testículo/análisisRESUMEN
Flagella of Campylobacter pylori were analyzed by electron microscopy and purified, and the molecular weight of the flagellin was determined. Isolation of flagella was performed by mechanical shearing from the cell surface, sucrose density gradient centrifugation, and Sepharose CL-4B gel chromatography. The flagella of C. pylori differ from those of other Campylobacter species and of most other bacteria by the presence of a flagellar sheath. The sheath narrows at the end and is linked to a club-shaped terminal structure. The molecular weight of C. pylori flagellin was 51,000.
Asunto(s)
Campylobacter/ultraestructura , Flagelos/ultraestructura , Campylobacter/análisis , Centrifugación por Gradiente de Densidad , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Flagelos/análisis , Flagelina/análisis , Humanos , Microscopía Electrónica , Peso MolecularRESUMEN
Two different Trypanosoma cruzi polypeptides, with masses of 70 and 68 kDa were purified and characterized in this work. These two polypeptides designated PAR 1 and PAR 2, respectively, co-purified during each step of the isolation procedure and were found to be located exclusively in T. cruzi flagella by indirect immunofluorescence. A pre-embedding immunoelectron microscopy procedure, with a gold-tagged secondary antibody, permitted direct identification of PAR 2 as a component of the T. cruzi paraflagellar rod. PAR 1 and PAR 2 were found to be immunologically distinct and showed no cross-reactivity with actin, tubulin, intermediate filament proteins, or other proteins present in mammalian cells. The results presented indicate that PAR 1 and PAR 2 are the major components of T. cruzi paraflagellar filaments, and that these filaments have no counterpart in mammalian cells.
Asunto(s)
Flagelos/análisis , Proteínas/aislamiento & purificación , Trypanosoma cruzi/análisis , Animales , Anticuerpos Monoclonales/inmunología , Western Blotting , Proteínas del Citoesqueleto/inmunología , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Peso MolecularRESUMEN
Flagellins of Halobacterium halobium are encoded in five different but homologous genes. Flagellins isolated from purified flagella were digested and the resulting peptides sequenced. The amino acid sequence data obtained prove that all five gene products are expressed and integrated into the flagellar bundle.
Asunto(s)
Proteínas Bacterianas/genética , Flagelina/genética , Halobacterium/análisis , Familia de Multigenes , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Flagelos/análisis , Halobacterium/genética , Microscopía Electrónica , Datos de Secuencia Molecular , Peso Molecular , TripsinaRESUMEN
Surface antigens of Rhizobium leguminosarum biovar viciae strain 248 were characterized by using polyclonal and monoclonal antibodies. With Western immunoblotting as the criterion, an antiserum raised against living whole cells recognized mainly flagellar antigens and the O-antigen-containing part of the lipopolysaccharide (LPS). Immunization of mice with a peptidoglycan-outer membrane complex yielded eight monoclonal antibodies, of which three reacted with LPS and five reacted with various sets of outer membrane protein antigens. The observation that individual monoclonal antibodies react with sets of related proteins is discussed. Studies of the influence of calcium deficiency and LPS alterations on surface antigenicity showed that in normally grown wild-type cells, the O-antigenic side chain of LPS blocks binding of an antibody to a deeper-lying antigen. This antigen is accessible to antibodies in cells grown under calcium limitation as well as in O-antigen-lacking mutant cells. Two of the antigen groups which can be distinguished in cell envelopes of free-living bacteria were depleted in cell envelopes of isolated bacteroids, indicating that the monoclonal antibodies could be useful tools for studying the differentiation process from free-living bacteria to bacteroids.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos , Antígenos Bacterianos/análisis , Proteínas de la Membrana Bacteriana Externa/análisis , Rhizobium/análisis , Pruebas de Aglutinación , Complejo Antígeno-Anticuerpo/análisis , Western Blotting , Membrana Celular/análisis , Pared Celular/análisis , Flagelos/análisis , Peso MolecularRESUMEN
We have shown previously that the regulatory subunit (RII) of a type II cAMP-dependent protein kinase is an integral component of the mammalian sperm flagellum (Horowitz, J.A., H. Toeg, and G.A. Orr. 1984. J. Biol. Chem. 259:832-838; Horowitz, J.A., W. Wasco, M. Leiser, and G.A. Orr. 1988. J. Biol. Chem. 263:2098-2104). The subcellular localization of this flagellum-associated RII in bovine caudal epididymal sperm was analyzed at electron microscope resolution with gold-conjugated secondary antibody labeling techniques using anti-RII monoclonal antibodies. By immunoblot analysis, the flagellum-associated RII was shown to interact with mAb 622 which cross reacts with both neural and nonneural isoforms of RII. In contrast, a neural specific monoclonal antibody (mAb 526) failed to interact with flagellar RII. In the midpiece of the demembranated sperm tail, gold label after mAb 622 incubation was primarily associated with the outer mitochondrial membrane. Although almost all specific labeling in the midpiece can be assigned to the mitochondria, in the principal piece, there is some labeling of the fibrous sheath. Labeling of the outer dense fibers and the axoneme was sparse. Specific labeling was virtually absent in the sperm head. Sections of sperm tails incubated in the absence of primary antisera or with mAb 526 showed little labeling. A beta-tubulin monoclonal antibody localized only to the 9 + 2 axoneme. These results raise the possibility that a type II cAMP-dependent protein kinase located at the outer mitochondrial membrane plays a role in the direct cAMP stimulation of mitochondrial respiration during sperm activation.
Asunto(s)
Flagelos/análisis , Proteínas Quinasas/análisis , Adenosina Trifosfatasas/fisiología , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Inmunohistoquímica , Membranas Intracelulares/análisis , Masculino , Mitocondrias/análisis , Espermatozoides/citologíaRESUMEN
The periplasmic flagella of many spirochetes contain multiple proteins. In this study, two-dimensional electrophoresis, Western blotting (immunoblotting), immunoperoxidase staining, and N-terminal amino acid sequence analysis were used to characterize the individual periplasmic flagellar proteins of Treponema pallidum subsp. pallidum (Nichols strain) and T. phagedenis Kazan 5. Purified T. pallidum periplasmic flagella contained six proteins (Mrs = 37,000, 34,500, 33,000, 30,000, 29,000, and 27,000), whereas T. phagedenis periplasmic flagella contained a major 39,000-Mr protein and a group of two major and two minor 33,000- to 34,000-Mr polypeptide species; 37,000- and 30,000-Mr proteins were also present in some T. phagedenis preparations. Immunoblotting with monospecific antisera and monoclonal antibodies and N-terminal sequence analysis indicated that the major periplasmic flagellar proteins were divided into two distinct classes, designated class A and class B. Class A proteins consisted of the 37-kilodalton (kDa) protein of T. pallidum and the 39-kDa polypeptide of T. phagedenis; class B included the T. pallidum 34.5-, 33-, and 30-kDa proteins and the four 33- and 34-kDa polypeptide species of T. phagedenis. The proteins within each class were immunologically cross-reactive and possessed similar N-terminal sequences (67 to 95% homology); no cross-reactivity or sequence homology was evident between the two classes. Anti-class A or anti-class B antibodies did not react with the 29- or 27-kDa polypeptides of T. pallidum or the 37- and 30-kDa T. phagedenis proteins, indicating that these proteins are antigenically unrelated to the class A and class B proteins. The lack of complete N-terminal sequence homology among the major periplasmic flagellar proteins of each organism indicates that they are most likely encoded by separate structural genes. Furthermore, the N-terminal sequences of T. phagedenis and T. pallidum periplasmic flagellar proteins are highly conserved, despite the genetic dissimilarity of these two species.
Asunto(s)
Proteínas Bacterianas/análisis , Flagelos/análisis , Treponema pallidum/análisis , Treponema/análisis , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/análisis , Proteínas Bacterianas/inmunología , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Flagelos/inmunología , Inmunoensayo , Técnicas para Inmunoenzimas , Datos de Secuencia Molecular , Peso Molecular , Homología de Secuencia de Ácido Nucleico , Treponema/inmunología , Treponema pallidum/inmunologíaRESUMEN
The periplasmic flagella of Spirochaeta aurantia were isolated and were found to be ultrastructurally and biochemically complex. Generally, flagellar filaments were 18 to 20 nm in diameter and appeared to consist of an 11 to 13-nm-wide inner region and an outer layer. The hook-basal body region consisted of two closely apposed disks connected to a hook by a rod. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified flagella together with a Western blot analysis of a motility mutant that produces hooks and basal bodies but not flagellar filaments revealed that the filaments were composed of three major polypeptides of 37,500, 34,000, and 31,500 apparent molecular weight (37.5K, 34K, and 31.5K polypeptides) and three minor polypeptides of 36,000, 33,000, and 32,000 apparent molecular weight (36K, 33K, and 32K polypeptides). Purified hook-basal body preparations were greatly enriched in three polypeptides in the range of 62,000 to 66,000 apparent molecular weight. Immunogold labeling experiments with a monoclonal antibody specific for the 37.5K flagellin and one that reacts with an epitope common to the 36K, 34K, 33K, 32K, and 31.5K flagellins revealed that the 37.5K major polypeptide was a component of the outer layer, whereas one or more of the other polypeptides constituted the core.