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1.
Flagellar hook protein from Salmonella SJ25.   总被引:17,自引:14,他引:3       下载免费PDF全文
From acid-disintegrated flagellar hooks of Salmonella SJ25 an immunochemically pure preparation of hook protein was obtained by column chromatography. The molecular weight of the protein determined by sodium dodecyl sulfate-gel electrophoresis was 43,000, whereas that of SJ25 flagellin was 56,000. The amino-terminal residue of the hook protein was determined to be seryl. The amino acid composition of the protein was determined, the results being very similar to that for an Escheria coli hook protein reported by Silverman and Simon (1972). Within a wavelength range of 200 to 250 nm, our purified preparation of hook protein gave a circular dichroism spectrum with unusually small amplitudes, suggesting that the alpha-helix content of the protein was very low.  相似文献   

2.
Formation of a flagella-like but straight polymer of Salmonella flagellin   总被引:1,自引:0,他引:1  
Salmonella flagellin (monomer) polymerizes into flagellar filaments with the addition of (NH4)2SO4 (Ada et al., 1963; Wakabayashi et al., 1969). When, however, this process was allowed to take place in the presence of a high concentration of NaCl (about 1.5 m), the product consisted of flagella-like but straight filaments. This phenomenon was common to four kinds of flagellins derived from strains SJ670, SJ25, SJ30 and SJ814. When the straight filament, suspended in 0.15 m-NaCl, was heated, it depolymerized to the monomer, which could in turn be polymerized into flagellar filaments by the addition of short fragments of flagella at room temperature. Nevertheless, attempts at direct transformation between the two types of filaments were unsuccessful. In 0.15 m-NaCl, straight filaments prepared from the four kinds of flagellins had markedly different heat stabilities, which were much lower than that of any kind of flagella. When monomeric flagellin dissolved in 3.5 m-NaCl was seeded with short fragments of straight filaments, the monomer polymerized onto the ends of the short fragments, which consequently grew into long straight filaments. In this type of experiment, monomers and seeds derived from the four strains were able to interact in any combination, suggesting that straight filaments consisting of the four kinds of flagellins have the same substructures. Whether the concentration of added NaCl was 0.15 m or 3.5 m, fragments of flagella (or straight filaments) were unable to act as seeds for the formation of straight filaments (or flagellar filaments). From this and other experimental results, it was concluded that in the two filamentous structures, flagellin molecules may be packed in different ways.  相似文献   

3.
A method for preparing bacterial flagellar hook structures is described. The method involves isolating intact flagella from a mutant which makes thermally labile flagellar filaments and heat-treating them to disaggregate the filament preferentially. The resulting hook preparation can be separated and purified by velocity and isopycnic centrifugation. The purified hooks sediment at a relative S value of 77. On acrylamide gel electrophoresis in sodium dodecyl sulfate, they show one major and a number of minor protein bands. The purified hooks can be used to immunize rabbits, and the resulting antiserum is hook-specific. These results support the notion that hooks are composed of a protein that differs from flagellin.  相似文献   

4.
Role of the flaR gene in flagellar hook formation in Salmonella spp.   总被引:14,自引:11,他引:3       下载免费PDF全文
Flagellar filaments were reconstituted by polymerization with exogenously supplied flagellin monomers at the tips of normal hooks on Salmonella cells which were missing the filaments because of mutations in either the flaL or flaU gene or the flagellin genes H1 and H2. Reconstitution did not occur at the tips of polyhooks of the flaR mutant cells. Thus, the absence of flagellar filaments in the flaR mutant cells was probably caused by the inability of the polyhooks to work as polymerization nuclei for flagellin. A Phf+ mutant which produced polyhooks with flagellar filaments was isolated from a flaR polyhook mutant. Genetic analysis of the Phf+ mutant showed that it carried an intracistronic suppressor mutation of the original flaR mutation. This result indicated that the flaR gene regulates hook length and initiates flagellin formation.  相似文献   

5.
Slow motile mutant in Salmonella typhimurium   总被引:2,自引:1,他引:1       下载免费PDF全文
Enomoto, Masatoshi (National Institute of Genetics, Misima, Japan). Slow motile mutant in Salmonella typhimurium. J. Bacteriol. 90:1696-1702. 1965.-A slow motile mutant, SJ399, was isolated from a wild-type strain of Salmonella typhimurium TM2. The mutant was as motile as the wild type in broth culture at 37 C. However, on semisolid medium it produced a much narrower swarming band than TM2. The motility of this mutant was hindered by the viscosity of semisolid medium. H antigenicity and morphological characters of flagella of the mutant were the same as those of the wild type. The motility phage, chi, responded differently to SJ399 and the wild type. Plaques of SJ399 were small and cloudy, whereas on the wild type they were large and clear. The efficiency of plating on SJ399 was 0.36 as compared with 1 with the wild type. Stained preparations revealed that the mutant had about one-third the number of flagella of the wild type. The reduction of the number of flagella also was ascertained by biochemical measurement of flagellar protein which was purified after deflagellation from cells. The content of flagellin in SJ399 was about 32% of that of the wild type. Phage P22-mediated transductions from SJ399 to nonflagellated (fla(-)) and paralyzed (mot(-)) mutants showed that the mutant SJ399 complements seven fla(-) and three mot(-) strains which are representative mutants of flagellation and motility cistrons, respectively. The mutation site of SJ399 was cotransduced with both motA and B cistrons. The two point cross tests between SJ399 and mot mutants revealed that the mutation site of SJ399 is located in the motB cistron. The insertion of the genetic region containing the mutation site of SJ399 to the motB cistron is discussed in relation to intracistronic complementation.  相似文献   

6.
The flagellar filament-hook complex was removed from Campylobacter cells by shearing and was purified by differential solubilization and ultracentrifugation at pH 11 followed by cesium chloride buoyant density ultracentrifugation. Flagellar filaments were then dissociated in 0.2 M glycine-HCl (pH 2.2), and purified hooks were collected by ultracentrifugation. The hooks (105 by 24 nm) each displayed a conical protrusion at the proximal end, a concave cavity at the distal end, and helically arranged subunits. The apparent subunit molecular weight of the hook protein of seven of the eight Campylobacter strains studied was 92,500, while that of the other was 94,000. N-terminal amino acid analysis of the hook protein of two strains of Campylobacter coli and one strain of Campylobacter jejuni demonstrated that the first 15 residues were identical. Amino acid composition analysis showed that the Campylobacter hook protein contained 35.7% hydrophobic and 9.5% basic residues. Isoelectric focusing determined that the hook protein was acidic, with a pI of 4.9. Comparisons with the Salmonella and Caulobacter hook protein compositions and N-terminal amino acid sequences indicated that the Campylobacter protein was related, but more distantly than these two proteins were to each other. Immunochemical analysis with four different antisera and a panel of eight strains showed that serospecific epitopes were immunodominant. The Campylobacter hook proteins carried both cross-reactive and specific non-surface-exposed epitopes, as well as serospecific epitopes which were exposed on the surface of the assembled hook. One class of these surface-exposed hook epitopes was shared with serospecific flagellin epitopes and may involve posttranslational modification, while the second class of epitopes was hook specific and not shared with flagellin.  相似文献   

7.
Effects of galU mutation on flagellar formation in Escherichia coli.   总被引:13,自引:8,他引:5       下载免费PDF全文
Y Komeda  T Icho    T Iino 《Journal of bacteriology》1977,129(2):908-915
Two mutants of Escherichia coli strictly deficient in uridine-diphosphoglucose pyrophosphorylase activity (galU) were found to have very small numbers of flagellar filaments and hooks. In these mutants, both the rate of flagellin (flagellar protein) synthesis and the amount of messenger ribonucleic acid specific for flagellin were considerably lower than in the parental strains. Motile revertants from the galU mutants were isolated and were found to carry a suppressor mutation, which was mapped in the flaH cistron. These strains formed swarms under conditions of catabolite repression; their intracellular concentration of cyclic adenosine 5'-monophosphate was the same as that in the parental strains. These results suggest that the outer membrane affects flagellar formation through the flaH gene product.  相似文献   

8.
The polymerization of bacterial flagellin protein (Salmonella strain SJ814) into flagellar filaments has been found by direct calorimetric measurement to be exothermic at 25° in .15M KCl, pH 6.8 with a ΔH of ?12.7 ± 0.6 kcal per mole of monomer polymerized. The calorimetric result at 25° contrasts sharply with the endothermic ΔH of +38 kcal/mole inferred from temperature dependence of the critical monomer concentration near 40°C. Comparison between these two values implies that unless a different mechanism of polymerization prevails at the two temperatures the heat capacity change for flagellin polymerization may be as large as 3.3 kcal/mole deg.  相似文献   

9.
The flagellar protein (flagellin) was isolated and purified from strains of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. A significant difference was observed in the molecular weight of different flagellin preparations obtained from these bacterial isolates. Antiserum prepared against S. maltophilia flagellin did not react with flagellin of P. aeruginosa or/and B. cepacia on Immunoblot or in indirect ELISA. In addition the anti-flagellin did not agglutinate P. aeruginosa and B. cepacia. No inhibition of motility of P. aeruginosa and B. cepacia was observed in presence of antiserum; though the latter inhibited the motility of S. maltophilia. The results of the present study prove that no specific relationship existed among all the studied flagellar proteins obtained from closely related bacteria.  相似文献   

10.
The genome of a halophilic archaeon Haloarcula marismortui carries two flagellin genes, flaA2 and flaB. Previously, we demonstrated that the helical flagellar filaments of H. marismortui were composed primarily of flagellin FlaB molecules, while the other flagellin (FlaA2) was present in minor amounts. Mutant H. marismortui strains with either flagellin gene inactivated were obtained. It was shown that inactivation of the flaA2 gene did not lead to changes in cell motility and helicity of the filaments, while the cells with inactivated flaB lost their motility and flagella synthesis was stopped. Two FlaB flagellin forms having different sensitivities to proteolysis were found in the flagellar filament structure. It is speculated that these flagellin forms may ensure the helical filament formation. Moreover, the flagella of a psychrotrophic haloarchaeon Halorubrum lacusprofundi were isolated and characterized for the first time. H. lacusprofundi filaments were helical and exhibited morphological polymorphism, although the genome contained a single flagellin gene. These results suggest that the mechanisms of flagellar helicity may differ in different halophilic archaea, and sometimes the presence of two flagellin genes, in contrast to Halobacterium salinarum, is not necessary for the formation of a functional helical flagellum.  相似文献   

11.
The genomic region that codes for the flagellin subunits of the complex flagellar filaments of Rhizobium meliloti was cloned and sequenced. Two structural genes, flaA and flaB, that encode 395- and 396-amino-acid polypeptides, respectively, were identified. These exhibit 87% sequence identity. The amino acid sequences of tryptic peptides suggest that both of these subunit proteins are represented in the flagellar filaments. The N-terminal methionine was absent from the mature flagellin subunits. Their derived primary structures show almost no relationship to flagellins from Escherichia coli, Salmonella typhimurium, or Bacillus subtilis but exhibit up to 60% similarity to the N- and C-terminal portions of flagellin from Caulobacter crescentus. It is suggested that the complex flagellar filaments of R. meliloti are unique in being assembled from heterodimers of two related flagellin subunits. The tandemly arranged flagellin genes were shown to be transcribed separately from unusual promoter sequences.  相似文献   

12.
Archaeal flagella are unique motility structures, and the absence of bacterial structural motility genes in the complete genome sequences of flagellated archaeal species suggests that archaeal flagellar biogenesis is likely mediated by novel components. In this study, a conserved flagellar gene family from each of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii has been characterized. These species possess multiple flagellin genes followed immediately by eight known and supposed flagellar accessory genes, flaCDEFGHIJ. Sequence analyses identified a conserved Walker box A motif in the putative nucleotide binding proteins FlaH and FlaI that may be involved in energy production for flagellin secretion or assembly. Northern blotting studies demonstrated that all the species have abundant polycistronic mRNAs corresponding to some of the structural flagellin genes, and in some cases several flagellar accessory genes were shown to be cotranscribed with the flagellin genes. Cloned flagellar accessory genes of M. voltae were successfully overexpressed as His-tagged proteins in Escherichia coli. These recombinant flagellar accessory proteins were affinity purified and used as antigens to raise polyclonal antibodies for localization studies. Immunoblotting of fractionated M. voltae cells demonstrated that FlaC, FlaD, FlaE, FlaH, and FlaI are all present in the cell as membrane-associated proteins but are not major components of isolated flagellar filaments. Interestingly, flaD was found to encode two proteins, each translated from a separate ribosome binding site. These protein expression data indicate for the first time that the putative flagellar accessory genes of M. voltae, and likely those of other archaeal species, do encode proteins that can be detected in the cell.  相似文献   

13.
The products of the bvgAS locus coordinately regulate the expression of Bordetella virulence factors in response to environmental conditions. We have identified a phenotype in Bordetella bronchiseptica that is negatively controlled by bvg. Environmental signals which decrease (modulate) the expression of bvg-activated genes lead to flagellum production and motility in B. bronchiseptica. Wild-type (Bvg+) strains are motile and produce peritrichous flagella only in the presence of modulating signals, whereas Bvg- (delta bvgAS or delta bvgS) strains are motile in the absence of modulators. The bvgS-C3 mutation, which confers signal insensitivity and constitutive activation of positively controlled loci, eliminates the induction of motility and production of flagellar organelles. The response to environmental signals is conserved in a diverse set of clinical isolates of both B. bronchiseptica and B. avium, another motile Bordetella species; however, nicotinic acid induced motility only in B. bronchiseptica. Purification of flagellar filaments from B. bronchiseptica strains by differential centrifugation followed by CsCl equilibrium density gradient centrifugation revealed two classes of flagellins of Mr 35,000 and 40,000. A survey of clinical isolates identified only these two flagellin isotypes, and coexpression of the two forms was not detected in any strain. All B. avium strains tested expressed a 42,000-Mr flagellin. Amino acid sequence analysis of the two B. bronchiseptica flagellins revealed 100% identity in the N-terminal region and 80% identity with Salmonella typhimurium flagellin. Monoclonal antibody 15D8, which recognizes a conserved epitope in flagellins in members of the family Enterobacteriaceae, cross-reacted with flagellins from B. bronchiseptica and B. avium. Our results highlight the biphasic nature of the B. bronchiseptica bvg regulon and provide a preliminary characterization of the Bvg-regulated motility phenotype.  相似文献   

14.
The shape of the flagellar filaments of the bacterium Salmonella typhimurium under ordinary conditions is a left-handed helix. In addition to the normal wild-type filament, non-helical (i.e. straight), right-handed helical (early), or circular (semi-coiled and coiled) filaments and filament with small amplitude (fl-type) have been found in mutants or in filaments reconstituted in vitro. We analysed wild-type flagellin and flagellins from 17 flagellar-shape mutants (6 with straight filaments, 6 with curly filaments, 4 with coiled filaments and 1 with fl-type filament) by amino acid sequencing to identify the mutational sites. All mutant flagellins except that of the fl-type filament had single mutations; the fl-type flagellin had two mutations in the molecule. The sites of these mutations were localized in alpha-helical segments of the terminal regions of flagellin. A possible mechanism of the polymorphism of the flagellar filament is discussed.  相似文献   

15.
We obtained two monoclonal antibodies of IgM class and IgA class of immunoglobulin prepared from mouse spleen cells immunized with crude flagellar preparation, and a polyclonal antibody raised against purified flagellin monomer of Campylobacter jejuni in a rabbit. The specificity of the reaction of these antibodies for flagellar filament was confirmed by Western blotting and by immunoelectron microscopy. These antibodies caused agglutination of the bacteria and inhibited the motility of the bacteria. When a strain of C. jejuni was treated with IgM class monoclonal antibody before being inoculated into suckling mice, it reduced colonization of the intestinal tract by this bacteria. Inhibition of the colonization by IgA class monoclonal antibody was less effective than that of IgM class, and the polyclonal antibody consisting mostly of IgG class immunoglobulin was without effect.  相似文献   

16.
Escherichia coli morphotype E flagellar filaments have a characteristic surface pattern of short-pitch loops when examined by electron microscopy. Seven of the 50 known E. coli H (flagellar antigen) serotypes (H1, H7, H12, H23, H45, H49, and H51) produce morphotype E filaments. Polymerase chain reaction was used to amplify flagellin structural (fliC) genes from E. coli strains producing morphotype E flagellar filaments and from strains with flagellar filaments representing other morphotypes. A single DNA fragment was obtained from each strain, and the size of the amplified DNA correlated with the molecular mass of the corresponding flagellin protein. This finding and hybridization data suggest that these bacteria are monophasic. fliC genes from three E. coli serotypes (H1, H7, and H12) possessing morphotype E flagellar filaments were sequenced in order to assess the contribution of conserved flagellin primary sequence to the characteristic filament architecture. The H1 and H12 fliC sequences were identical in length (1,788 bp), while the H7 fliC sequence was shorter (1,755 bp). The deduced molecular masses of the FliC proteins were 60,857 Da (H1), 59,722 Da (H7), and 60,978 Da (H12). The H1, H7, and H12 flagellins demonstrated 98 to 99% identity over the amino-terminal region (190 amino acid residues) and 89% (H7) to 99% (H1 and H12) identity in the carboxy-terminal region (100 amino acid residues). The complete primary amino acid sequences for H1 and H12 flagellins differed by only 10 amino acids, accounting for previously reported serological cross-reactions. However, the central region of H7 flagellin had only 38% identity with H1 and H12 flagellins.The characteristic morphology of morphotype E flagellar filaments is therefore not dependent on a highly conserved primary sequence within the exposed central region. Comparison of morphotype E E. coli flagellins with those from E. coli K-12, Serratia marcescens, and several Salmonella serovars supported the established concept of highly conserved terminal regions flanking a variable central region.  相似文献   

17.
Purification and antigenic analysis of flagella of Campylobacter jejuni   总被引:1,自引:0,他引:1  
The flagella of Campylobacter jejuni strain FUM158432 were purified and a flagellin preparation consisting of only a single peptide of 63,000 daltons was obtained. The peptide of 92,000 daltons usually associated with a flagellar preparation was shown to be a peptide derived from the hook region. Antiserum was prepared by immunizing a rabbit with the flagellin preparation. The reaction of the antiserum was found to be highly specific for the flagellar filament by immunoelectron microscopy and for flagellin peptide by the immunoblotting method. Seventeen of 23 clinically isolated strains of C. jejuni reacted with this antiserum but the other six strains did not, indicating the existence of antigenic variation of the flagella of C. jejuni. The flagella of a few strains of C. coli also reacted with this antiserum.  相似文献   

18.
Flagellar motility in Rhodobacter sphaeroides is notably different from that in other bacteria. R. sphaeroides moves in a series of runs and stops produced by the intermittent rotation of the flagellar motor. R. sphaeroides has a single, plain filament whose conformation changes according to flagellar motor activity. Conformations adopted during swimming include coiled, helical, and apparently straight forms. This range of morphological transitions is larger than that in other bacteria, where filaments alternate between left- and right-handed helical forms. The polymorphic ability of isolated R. sphaeroides filaments was tested in vitro by varying pH and ionic strength. The isolated filaments could form open-coiled, straight, normal, or curly conformations. The range of transitions made by the R. sphaeroides filament differs from that reported for Salmonella enterica serovar Typhimurium. The sequence of the R. sphaeroides fliC gene, which encodes the flagellin protein, was determined. The gene appears to be controlled by a sigma(28)-dependent promoter. It encodes a predicted peptide of 493 amino acids. Serovar Typhimurium mutants with altered polymorphic ability usually have amino acid changes at the terminal portions of flagellin or a deletion in the central region. There are no obvious major differences in the central regions to explain the difference in polymorphic ability. In serovar Typhimurium filaments, the termini of flagellin monomers have a coiled-coil conformation. The termini of R. sphaeroides flagellin are predicted to have a lower probability of coiled coils than are those of serovar Typhimurium flagellin. This may be one reason for the differences in polymorphic ability between the two filaments.  相似文献   

19.
In Caulobacter crescentus biogenesis of the flagellar organelle occurs during one stage of its complex life cycle. Thus in synchronous cultures it is possible to assay the sequential synthesis and assembly of the flagellum and hook in vivo with a combination of biochemical and radioimmunological techniques. The periodicity of synthesis and the subcellular compartmentation of the basal hook and filament subunits were determined by radioimmune assay procedures. Unassembled 27,000-dalton (27K) flagellin was preferentially located in isolated membrane fractions, whereas the 25K flagellin was distributed between the membrane and cytoplasm. The synthesis of hook began before that of flagellin, although appreciable overlap of the two processes occurred. Initiation of filament assembly coincided with the association of newly synthesized hook and flagellin subunits. Caulobacter flagella are unusual in that they contain two different flagellin subunits. Data are presented which suggest that the ratio of the two flagellin subunits changes along the length of the filament. Only the newly synthesized 25K flagellin subunit is detected in filaments assembled during the swarmer cell stage. By monitoring the appearance of flagellar hooks in the culture medium, the time at which flagella are released was determined.  相似文献   

20.
Flagellar filaments were isolated from Helicobacter pylori by shearing, and flagellar proteins were further purified by a variety of techniques, including CsCl density gradient ultracentrifugation, pH 2.0 acid disassociation-neutral pH reassociation, and differential ultracentrifugation followed by molecular sieving with a Sephacryl S-500 column or Mono Q anion-exchange column, and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to an Immobilon membrane. Two flagellin species of pI 5.2 and with apparent subunit molecular weights (Mrs) of 57,000 and 56,000 were obtained. N-terminal amino acid analysis showed that the two H. pylori flagellin species were related to each other and shared sequence similarity with the N-terminal amino acid sequence of Campylobacter coli, Bacillus, Salmonella, and Caulobacter flagellins. Analysis of the amino acid composition of the predominant 56,000-Mr flagellin species isolated from two strains showed that it was comparable to the flagellins of other species. The minor 57,000-Mr flagellin species contained a higher content of proline. Immunoelectron microscopic studies with polyclonal monospecific H. pylori antiflagellin antiserum and monoclonal antibody (MAb) 72c showed that the two different-Mr flagellin species were located in different regions of the assembled flagellar filament. The minor 57,000-Mr species was located proximal to the hook, and the major 56,000-Mr flagellin composed the remainder of the filament. Western immunoblot analysis with polyclonal rabbit antisera raised against H. pylori or Campylobacter jejuni flagellins and MAb 72c showed that the 56,000-Mr flagellin carried sequences antigenetically cross-reactive with the 57,000-Mr H. pylori flagellin and the flagellins of Campylobacter species. This antigenic cross-reactivity did not extend to the flagellins of other gram-negative bacteria. The 56,000-Mr flagellin also carried H. pylori-specific sequences recognized by two additional MAbs. The epitopes for these MAbs were not surface exposed on the assembled inner flagellar filament of H. pylori but were readily detected by immunodot blot assay of sodium dodecyl sulfate-lysed cells of H. pylori, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of this important pathogen.  相似文献   

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