Identification, characterization, and spatial localization of two flagellin species in Helicobacter pylori flagella.

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.     

Cloning and Characterization of Flagellin Genes and Identification of Flagellin Glycosylation from Thermophilic Bacillus Species

Flagellin glycosylation was identified in Bacillus sp. PS3 and Geobacillus stearothermophilus. In vivo complementation showed that these flagellin genes did not restore the motility of a Bacillus subtilis flagellin mutant, whereas the genes encoding non-glycosylated flagellin from Geobacillus kaustophilus and Bacillus sp. Kps3 restored motility. Moreover, four types of flagellins expressed in B. subtilis were not glycosylated. We speculate that glycosylation is required for flagellar filament assembly of these bacilli.     

Purification and characterization of a tryptic peptide of Borrelia burgdorferi flagellin, which reduces cross-reactivity in immunoblots and ELISA.

In man the early immune response in Lyme disease is primarily directed against the endoflagellin antigen. Isolated flagellar protein of Borrelia burgdorferi suggests itself as a suitable test antigen. However, cross-reactivity between flagellins of B. burgdorferi, Escherichia coli, Bacillus subtilis, Proteus mirabilis and Salmonella typhimurium was demonstrated by immunoblotting and ELISA with polyclonal rabbit-hyperimmune-sera. Tryptic cleavage of recombinant B. burgdorferi 41 kDa flagellin, expressed in E. coli, produced a peptide fragment which was recognized exclusively by antisera to Borrelia species. This peptide was designated as the 14 kDa fragment due to its migratory behaviour in SDS-PAGE. The fragment is part of the variable region of the flagellin, as proven by amino acid sequencing. The flagellin peptide was employed as an antigen in ELISA and immunoblot assays, testing the polyclonal sera mentioned above. The specificity was superior to that obtained with the intact recombinant flagellin.     

Biochemical and Immunological Analyses of the Flagellin of Clostridium tyrobutyricum ATCC 25755

The monoclonal antibody 21E7-B12 (IgG₃) can be used in a direct method of Clostridium tyrobutyricum detection based on an immunoenzymatic assay. Immunoelectron microscopy demonstrated that the 21E7-B12 antibody recognized the surface-exposed epitopes on the flagellar filaments of C. tyrobutyricum. After flagellar extraction, the purified flagellin showed an apparent molecular mass of 46 kDa with an isoelectric point of 3.6. Sugar staining, mild periodate oxidation and é-elimination experiments showed that the flagellin was glycosylated and that the 21E7-B12 epitope was located in the sugar moiety. Amino acid composition showed that the flagellar filament protein contained a high percentage of serine and threonine, while proline was absent. The first 23 residues of the N-terminal were determined and sequence homology with other flagellins was found.     

Role of the 25-, 27-, and 29-kilodalton flagellins in Caulobacter crescentus cell motility: method for construction of deletion and Tn5 insertion mutants by gene replacement.

Caulobacter crescentus incorporates two distinct, but related proteins into the polar flagellar filament: a 27-kilodalton (kDa) flagellin is assembled proximal to the hook and a 25-kDa flagellin forms the distal end of the filament. These two proteins and a third, related flagellin protein of 29 kDa are encoded by three tandem genes (alpha-flagellin cluster) in the flaEY gene cluster (S.A. Minnich and A. Newton, Proc. Natl. Acad. Sci. USA 84: 1142-1146, 1987). Since point mutations in flagellin genes had not been isolated their requirement for flagellum function and fla gene expression was not known. To address these questions, we developed a gene replacement protocol that uses cloned flagellin genes mutagenized by either Tn5 transposons in vivo or the replacement of specific DNA fragments in vitro by the antibiotic resistance omega cassette. Analysis of gene replacement mutants constructed by this procedure led to several conclusions. (i) Mutations in any of the three flagellin genes do not cause complete loss of motility. (ii) Tn5 insertions in the 27-kDa flagellin gene and a deletion mutant of this gene do not synthesize the 27-kDa flagellin, but they do synthesize wild-type levels of the 25-kDa flagellin, which implies that the 27-kDa flagellin is not required for expression and assembly of the 25-kDa flagellin; these mutants show slightly impaired motility on swarm plates. (iii) Mutant PC7810, which is deleted for the three flagellin genes in the flaEY cluster, does not synthesize the 27- or 29-kDa flagellin, and it is significantly more impaired for motility on swarm plates than mutants with defects in only the 27-kDa flagellin gene. The synthesis of essentially normal levels of 25-kDa flagellin by strain PC7810 confirms that additional copies of the 25-kDa flagellin map outside the flaEY cluster (beta-flagellin cluster) and that these flagellin genes are active. Thus, while the 29- and 27-kDa flagellins are not absolutely essential for motility in C. crescentus, their assembly into the flagellar structure is necessary for normal flagellar function.     

Flagellin genes of Methanococcus vannielii : amplification by the Polymerase Chain Reaction, demonstration of signal peptides and identification of major components of the flagellar filament

The highly conserved nature of the 5′-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (M_r= 30 800 and 28 600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins. Received: 27 November 1997 / Accepted: 19 March 1998     

Enzymatic characterization of a nonmotile,nonsolventogenicClostridium acetobutylicum ATCC 824 mutant

Decreased motility has been correlated with lower solvent yields in fermentations withClostridium acetobutylicum. A spontaneous mutant ofC. acetobutylicum was found to be nonmotile as evidenced by bright-field microscopy and motility-agar plates. The loss of motility was accompanied by the production of an altered flagellin. The mutant flagellin was much smaller than the wild-type (32 vs 43 kDa), although the NH₂-terminal amino acid sequences of both flagellins were identical. This mutant was simultaneously incapable of producing the solvents acetone and butanol. In vitro enzyme activity analyses demonstrated the absence of three enzymes directly involved in solvent production: acetoacetate decarboxylase (EC 4.1.1.4), acetoacetyl-coenzyme A:acetate/butyrate coenzyme A-transferase (EC 2.8.3.9), and NADP-dependent butyraldehyde dehydrogenase (EC 1.2.1.10).     

Isolation and characterization of flagella and flagellin proteins from the Thermoacidophilic archaea Thermoplasma volcanium and Sulfolobus shibatae.

Isolated flagellar filaments of Sulfolobus shibatae were 15 nm in diameter, and they were composed of two major flagellins which have M(r)s of 31,000 and 33,000 and which stained positively for glycoprotein. The flagellar filaments of Thermoplasma volcanium were 12 nm in diameter and were composed of one major flagellin which has an M(r) of 41,000 and which also stained positively for glycoprotein. N-terminal amino acid sequencing indicated that 18 of the N-terminal 20 amino acid positions of the 41-kDa flagellin of T. volcanium were identical to those of the Methanococcus voltae 31-kDa flagellin. Both flagellins of S. shibatae had identical amino acid sequences for at least 23 of the N-terminal positions. This sequence was least similar to any of the available archaeal flagellin sequences, consistent with the phylogenetic distance of S. shibatae from the other archaea studied.     

N-terminal amino acid sequence of the Borrelia burgdorferi flagellin

Abstract The 41 kDa flagellar protein of Borrelia burgdorferi appears to be an immunodominant antigen producing an early and strong response in most, if not all, individuals during infection in humans. It would represent a very good antigen for serodiagnosis of Lyme disease, if its crossreactivity with flagella of other bacteria was low. To gain information on this point we isolated the B. burgdorferi flagellin by preparative two-dimensional electrophoresis for N-terminal amino acid analysis. By comparing the N-terminal amino acid sequences of flagellar proteins from other eubacteria we found that the first six out of twenty nine amino acids were identical to the Treponema pallidum and Treponema phagedenis 'class B' flagellins. All 29 N-terminal residues exhibited a moderate inter-genus homology (44–55%), in contrast to the high degree (67–95%) of inter-species conservation of the treponemal 'class B' flagellar N-terminal sequences. There was little similarity to other flagellins except the B. subtilis flagellar protein.     

Differences in the envelope proteins ofChlamydia pneumoniae,Chlamydia trachomatis,andChlamydia psittaci shown by two-dimensional gel electrophoresis

Analysis by two-dimensional gel electrophoresis of theN-laurylsarkosinate(Sarkosyl)-insoluble envelope complexes ofl-^[35]S-cysteine-iabeled elementary bodies ofChlamydia pneumoniae strain IOL-207,Chlamydia trachomatis serovar LGV2, D, and F, andChlamydia psittaci strain 6BC showed differences in the molecular charges of chlamydial outer membrane proteins. The apparent isoelectric point (pI) of the major outer membrane protein ofC. pneumoniae strain IOL-207 was 6.4, whereas the pI of the major outer membrane protein of theC. trachomatis andC. psittaci strains differed little from one another, ranging from 5.3 to 5.5. The 60-kDa cysteinerich protein ofC. pneumoniae was the only 60-kDa chlamydial protein with a pI value (5.9) more acidic than that of the corresponding major outer membrane protein. As a general rule, the charges of both the 60-kDa and the lowmolecular-mass (12–15 kDa) cysteine-rich proteins were widely variable, depending on the strain. However, in cach individual strain, the variation of the charge of the 60-kDa protein had a compensatory change in the lowmolecular-mass cysteine-rich protein.     

Effects of components of theBacillus sphaericus toxin on mosquito larvae and mosquito-derived tissue culture-grown cells

Bacillus sphaericus 2362 produces a parasporal crystal containing 42 and 51 kilodalton (kDa) proteins. Both of these proteins are required for toxicity to mosquito larvae; neither is toxic alone. When overexpressed inB. subtilis, these two proteins accumulate as amorphous inclusions (AIs). Bioassays involving larvae ofCulex pipiens and different ratios of these AIs indicated that maximal toxicity was observed at a ratio of approximately one 42-kDa protein to one 51-kDa protein. Purified preparations of these proteins, as well as derivatives similar to those which accumulate in the gut of mosquito larvae, were also toxic when combined, but not toxic singly. Different results were obtained when the toxicity of these preparations was tested for tissue culture-grown cells ofC. quinquefasciatus. Under these conditions, the 39-kDa derivative of the 42-kDa protein was alone sufficient for toxicity, which was not increased by the addition of the 51-kDa protein or its derivatives. These results indicate that theB. sphaericus larvicide acts as a binary toxin in mosquitos, whereas only the 39-kDa protein is required for full toxicity to tissue culture-grown cells.     

Flagellin genes of Methanococcus vannielii : amplification by the Polymerase Chain Reaction, demonstration of signal peptides and identification of major components of the flagellar filament

The highly conserved nature of the 5′-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (M_r= 30 800 and 28 600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins.     

Characterization and functional analysis of seven flagellin genes in Rhizobium leguminosarum bv. viciae. Characterization of R. leguminosarum flagellins

Background

Rhizobium leguminosarum bv. viciae establishes symbiotic nitrogen fixing partnerships with plant species belonging to the Tribe Vicieae, which includes the genera Vicia, Lathyrus, Pisum and Lens. Motility and chemotaxis are important in the ecology of R. leguminosarum to provide a competitive advantage during the early steps of nodulation, but the mechanisms of motility and flagellar assembly remain poorly studied. This paper addresses the role of the seven flagellin genes in producing a functional flagellum.

Results

R. leguminosarum strains 3841 and VF39SM have seven flagellin genes (flaA, flaB, flaC, flaD, flaE, flaH, and flaG), which are transcribed separately. The predicted flagellins of 3841 are highly similar or identical to the corresponding flagellins in VF39SM. flaA, flaB, flaC, and flaD are in tandem array and are located in the main flagellar gene cluster. flaH and flaG are located outside of the flagellar/motility region while flaE is plasmid-borne. Five flagellin subunits (FlaA, FlaB, FlaC, FlaE, and FlaG) are highly similar to each other, whereas FlaD and FlaH are more distantly related. All flagellins exhibit conserved amino acid residues at the N- and C-terminal ends and are variable in the central regions. Strain 3841 has 1-3 plain subpolar flagella while strain VF39SM exhibits 4-7 plain peritrichous flagella. Three flagellins (FlaA/B/C) and five flagellins (FlaA/B/C/E/G) were detected by mass spectrometry in the flagellar filaments of strains 3841 and VF39SM, respectively. Mutation of flaA resulted in non-motile VF39SM and extremely reduced motility in 3841. Individual mutations of flaB and flaC resulted in shorter flagellar filaments and consequently reduced swimming and swarming motility for both strains. Mutant VF39SM strains carrying individual mutations in flaD, flaE, flaH, and flaG were not significantly affected in motility and filament morphology. The flagellar filament and the motility of 3841 strains with mutations in flaD and flaG were not significantly affected while flaE and flaH mutants exhibited shortened filaments and reduced swimming motility.

Conclusion

The results obtained from this study demonstrate that FlaA, FlaB, and FlaC are major components of the flagellar filament while FlaD and FlaG are minor components for R. leguminosarum strains 3841 and VF39SM. We also observed differences between the two strains, wherein FlaE and FlaH appear to be minor components of the flagellar filaments in VF39SM but these flagellin subunits may play more important roles in 3841. This paper also demonstrates that the flagellins of 3841 and VF39SM are possibly glycosylated.     

A Model of the Quaternary Structure of Enolases, Based on Structural and Evolutionary Analysis of the Octameric Enolase from Bacillus subtilis

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pH_o) for activity is 8.1–8.2, and the K _m for 2-PGA is approximately 0.67 mM. Using the dimeric C structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86–96) and the loop between helix L and strand 1 (HL–S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL–S1 loop is truncated by 4–6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL–S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.     

A common allergenic epitope of Bermuda grass pollen shared by other grass pollens

The present study disclosed the cross-reactivity between Bermuda grass pollen (BGP) and other grass pollens using monoclonal antibodies (MAbs) and polyclonal antiserum. MAb 9–13, directed against a group of minor allergens of BGP (Cyn d Bd68K, 48K, 38K) was found to cross-react with extracts of ten other grass pollens. Immunoblotting assays illustrated that MAb 9–13 cross-reacted with multiple components of most of these pollens, and the major cross-reactive components had molecular weights of 29–36 kD. The cross-reactivity between BGP andLol pI, the group I allergen of rye grass pollen, was further evaluated;Lol pI was recognized by MAb 9–13, but not by our MAbs/polyclonal antiserum againstCyn dI, the major allergen of BGP. These results suggest that the epitope recognized by MAb 9–13 is a common (C) epitope shared byLol pI andCyn d Bd68K, 48K, 38K, andCyn dI does not share significant antigenicity withLol pI. In a modified radio-allergosorbent test, IgE antibodies in the serum of BGP-allergic patients reacted mildly with C-epitope-bearing components of both BGP and rye grass pollens, and this binding could be blocked specifically by MAb 9–13. This suggests that in addition to an antigenic cross-reaction, the C epitope can also lead to an allergenic cross-reaction.     

Actinoplanes capillaceus sp. nov., a new species of the genus Actinoplanes

Two motile actinomycete strains, K95–5561^T and K95–5562, were isolated from a soil sample collected at Sayama City, Saitama Prefecture, Japan. They produced bell shaped spore vesicles (sporangia) with hairy surfaces on substrate hyphae. When released into water, the sporangiospores became motile by a tuft of polar flagella. The chemotaxonomic and morphological characteristics together with 16S rRNA gene sequence data indicated that the two isolates belonged to the genus Actinoplanes. The two strains were assigned to a single species on the basis of phenotypic, notably cultural, morphological and physiological characteristics, and DNA-DNA pairing data. The two strains were distinguished from representatives of all validly described species of Actinoplanes using a combination of genotypic and phenotypic properties. It is, therefore, proposed that strains K95–5561 and K95–5562 be recognized as a new species of the genus Actinoplanes with the name Actinoplanes capillaceus sp. nov. The type strain of the species is strain K95–5561^T (=JCM 10268^T =IFO 16408^T). The invalidly proposed species `Ampullariella cylindrica', `Ampullariella pekinensis' and `Ampullariella pilifera' were assigned to Actinoplanes capillaceus on the basis of genotypic and phenotypic data.     

Cellulosome-like entities inBacteroides cellulosolvens

Cellulosome-like complexes were identified in the broth and sonic extracts of cellobiose-and cellulose-grown cells ofBacteroides cellulosolvens. The extracellular fractions contained three to four major polypeptides and several minor polypeptide bands that were localized in two major gel filtration peaks indicating average molecular weights of about 700 kDa and >10 MDa. A relatively large molecular weight component (M_r 230 kDa) was found to contain carbohydrate, but no apparent enzymatic activity of its own could be detected. The cell sonicate displayed a more complicated polypeptide profile, and glycosylated polypeptides were larger (ca. 310 and 290 kDa) than that of the extracellular fraction. The 230-kDa extracellular component interacted strongly with the GSI isolectin fromGriffonia simplicifolia, exhibited immunochemical cross-reactivity with the S1 subunit of the cellulosome fromClostridium thermocellum, and displayed anomalous pH- and salt-dependent migratory behavior in SDS-PAGE. Taken together, this evidence strongly suggests a structural similarity between the glycoconjugates of these two distinct cellulolytic bacteria. A major 84-kDa polypeptide was identified as a xylanase, and a 50-kDa polypeptide displayed endoglucanase activity. Additional biochemical and cytochemical evidence indicated that cellulosome-like cellulolytic complexes are associated with the cell surface in this bacterium.