Isolation and characterization of Campylobacter flagellins.

Sequential acid pH dissociation, differential ultracentrifugation, and neutral pH reassociation were used to partially purify serotypically distinct flagella from three strains of Campylobacter jejuni and the two antigenic phases of flagella of Campylobacter coli VC167. Each C. jejuni flagellin and C. coli VC167 antigenic phase 1 flagellin were purified to homogeneity by reverse-phase high-performance liquid chromatography with a C8 Spheri-10 column. C. coli VC167 antigenic phase 2 was purified to homogeneity by ion-exchange chromatography with a Mono-Q column. Amino acid compositional analysis put the C. jejuni flagellin molecular weight in the range 63,200 to 63,800 and the C. coli antigenic phase 1 and 2 flagellins at 61,500 and 59,500, respectively. The amino acid compositions of the C. jejuni were similar to each other and to the C. coli VC167 antigenic phase 1 and phase 2 flagellins. One-dimensional peptide mapping of the C. jejuni flagellins by partial digestion with trypsin or chymotrypsin confirmed the structural similarities of the C. jejuni flagellins and the C. coli VC167 antigenic phase 1 flagellin and showed that C. coli VC167 antigenic phase 2 flagellin was structurally distinct from the phase 1 flagellin. The antigenic phase 2 flagellin was especially sensitive to digestion by chymotrypsin. Amino-terminal sequence analysis showed that the 20 N-terminal amino acids of the Campylobacter flagellins were highly conserved. The Campylobacter flagellins also shared limited sequence homology with the N-terminal sequences reported for Salmonella and Bacillus flagellins.     

Cloning, nucleotide sequence, and taxonomic implications of the flagellin gene of Roseburia cecicola.

The gene coding for the flagellin protein of Roseburia cecicola, an oxygen-intolerant, gram-negative, anaerobic bacterium indigenous to the murine cecum, has been cloned and sequenced. NH2-terminal amino acid sequence data from the flagellin protein were used as a basis for the synthesis of two mixed-sequence deoxyoligonucleotides. The oligonucleotides were used to identify and clone the flagellin structural gene. DNA sequence analysis of M13mp8 and mp9 subclones revealed a protein with a length of 293 amino acids and a molecular weight of 31,370. Comparisons with the sequences of flagellins of other species revealed conserved regions and suggested that although R. cecicola has structural characteristics of a gram-negative bacterium, it may be most closely related to the gram-positive bacteria.     

Evidence for posttranslational modification and gene duplication of Campylobacter flagellin.

A gene encoding a flagellin protein of Campylobacter coli VC167 has been cloned and sequenced. The gene was identified in a pBR322 library by hybridization to a synthetic oligonucleotide probe corresponding to amino acids 4 to 9 of the N-terminal sequence obtained by direct chemical analysis (S. M. Logan, L. A. Harris, and T. J. Trust, J. Bacteriol. 169:5072-5077, 1987). The DNA was sequenced and shown to contain an open reading frame encoding a protein with a molecular weight of 58,945 and a length of 572 amino acids. The deduced amino acid sequence was identical to the published N-terminal amino acid sequence of VC167 flagellin and to four internal regions whose partial sequences were obtained by direct chemical analysis of two tryptic and two cyanogen bromide peptides of VC167 flagellin. The C. coli flagellin protein contains posttranslationally modified serine residues, most of which occur within a region containing two 9-amino-acid repeating peptides separated by 34 unique amino acids. Comparisons with the sequences of flagellins from other bacterial species revealed conserved residues at the amino- and carboxy-terminal regions. Hybridization data suggest the presence of a second flagellin copy located adjacent to the first on the VC167 chromosome.     

Identification and molecular characterization of two tandemly located flagellin genes from Aeromonas salmonicida A449.

Two tandemly located flagellin genes, flaA and flaB, with 79% nucleotide sequence identity were identified in Aeromonas salmonicida A449. The fla genes are conserved in typical and atypical strains of A. salmonicida, and they display significant divergence at the nucleotide level from the fla genes of the motile species Aeromonas hydrophila and Aeromonas veronii biotype sobria. flaA and flaB encode unprocessed flagellins with predicted Mrs of 32,351 and 32,056, respectively. When cloned under the control of the Ptac promoter, flaB was highly expressed when induced in Escherichia coli DH5alpha, and the FlaB protein was detectable even in the uninduced state. In flaA clones containing intact upstream sequence, FlaA was barely detectable when uninduced and poorly expressed on induction. The A. salmonicida flagellins are antigenically cross-reactive with the A. hydrophila TF7 flagellin(s) and evolutionarily closely related to the flagellins of Pseudomonas aeruginosa and Vibrio anguillarum. Electron microscopy showed that A. salmonicida A449 expresses unsheathed polar flagella at an extremely low frequency under normal laboratory growth conditions, suggesting the presence of a full complement of genes whose products are required to make flagella; e.g., immediately downstream of flaA and flaB are open reading frames encoding FlaG and FlaH homologs.     

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.     

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.     

Common and variable domains of the flagellin gene, flaA, in Campylobacter jejuni

The organization of the flagellin gene locus in Campylobacter jejuni strain IN1 (Lior 7) was determined using the polymerase chain (PCR) reaction and a series of oligonucleotide primers. Two tandemly arranged flagellin genes of approximately 1.7 kb were found to be joined by an intervening segment of c.0.2kb, similar to that reported for Campylobacter coli. The 5' flagellin gene, flaA, was generated by PCR and both strands sequenced. Comparison of the deduced amino acid sequence for C. jejuni FlaA with the published sequence for C. jejuni FlaA with the published sequence for C. coli FlaA showed 77% identical amino acids between the proteins. Two common regions, C1 and C2, comprising the N-terminal 170 amino acids and C-terminal 100 amino acids, exhibit amino acids 94% and 96% identical to those of C. coli, respectively. The variable region, V1, comprising the middle of the protein, shows 61% identical residues with C. coli. Comparison of these regions with other bacterial flagellins reveals a similar pattern but with much less identity. Several areas within the V1 region correspond to predicted surface-exposed regions and may represent areas in which surface epitopes are located.     

The flagellin gene as a stable marker for detection of Pseudomonas fluorescens SBW25

Flagellin gene central regions from 111 isolates of Pseudomonas fluorescens SBW25 obtained from soil during a field release experiment were analysed using a combined PCR/RFLP technique to look for variation. In addition, a 858 bp flagellin gene sequence from the original strain and the last isolate obtained from the release site were compared. There was no variation in flagellin gene sequences indicating that the gene was stable over the period of the release, and that the flagellin gene is a suitable marker for use in the detection of bacteria in release experiments. A comparison of Pseudomonas fluorescens SBW25 flagellin with other sequenced flagellins revealed closest homology to the flagellin of Ps. putida PRS2000.     

Comparative analysis of flagellin sequences from Escherichia coli strains possessing serologically distinct flagellar filaments with a shared complex surface pattern.

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.     

Variation and predicted structure of the flagellin gene in Actinoplanes species

Members of the genus Actinoplanes are considered to be representative of motile actinomycetes. To infer the flagellar diversity of Actinoplanes species, novel degenerate primers were designed for the flagellin (fliC) gene. The fliC gene of 21 Actinoplanes strains was successfully amplified and classified into two groups based on whether they were large (type I) or small (type II). Comparison of the translated amino acid sequences revealed that this size difference could be attributed to large number of gaps located in the central variable region. However, the C- and N- terminal regions were conserved. Except for a region on the flagellum surface, structural predictions of type I and II flagellins revealed that the two flagellin types were strongly correlated with each other. Phylogenetic analysis of the 115-amino acid N-terminal sequences revealed that the Actinoplanes species formed three clusters, and type II flagellin gene containing three type strains were phylogenetically closely related each other.     

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.     

The flagellin gene and protein from the brewing spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis

Flagellin genes from the anaerobic Gram-negative beer-spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis were sequenced and the flagellin proteins initially characterized. Protein microsequencing led to the design of two degenerate PCR primers that allowed the P. cerevisiiphilus flagellin gene to be partially sequenced. A combination of PCR and Bubble PCR was then used to sequence the flagellin genes of three isolates from each species. Cloning and gene expression, followed by immunoblotting, confirmed the gene identities as flagellin. Analysis of the gene sequences revealed proteins similar to other bacterial flagellins, including lengths of 446 or 448 amino acids, putative sigma 28 promoters, and a termination loop. Antibody binding studies with isolated flagella correlated with gene sequence comparisons, with both indicating that the P. cerevisiiphilus isolates studied are very similar but that the P. frisingensis isolates show greater variation. Purified flagellins were found to be glycosylated, probably through an O linkage. Phylogenetic analysis revealed greater diversity within the flagellin sequences than within the 16S rRNA genes. Despite the Gram-negative morphology of Pectinatus, this genus proved most closely related to Gram-positive Firmicutes.     

Flagellin gene polymorphism analysis of Campylobacter compared with antigen serotyping

Flagellin gene sequence polymorphisms were used to discriminate amongst 53 strains of Campylobacter jejuni and C. coli. The Campylobacter strains were made up of forty-three strains of Campylobacter jejuni and 10 strains of Campylobacter coli. The results were analysed in relation to Penner serotyping. Twenty DNA PCR-RFLP patterns (genotypes) were identified by analysis of Dde I fragment length polymorphisms in flagellin gene (fla A and fla B) polymerase chain reaction (PCR) products. Flagellin gene 13 genotype was a feature of 15% of strains, followed by flagellin gene 8 (9%). Differences in fragment patterns were observed not only between members of two species, but also between individual strains of the same species. The strains that were non-typable by the Penner serotype were distributed into 6 flagellin gene types. In conclusion, Ddc I fla typing is reproducible and offers high typability. However, when the scheme is used in combination with the Penner serotype it provides improved discrimination for the characterizing and subtyping of isolates.     

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     

Synthesis and assembly of flagellar components by Caulobacter crescentus motility mutants

Cultures of wild-type Caulobacter crescentus and strains with fla mutations representing 24 genes were pulse-labeled with 14C-amino acids and analyzed by immunoprecipitation to study the synthesis of flagellar components. Most fla mutants synthesize flagellin proteins at a reduced rate, suggesting the existence of some mechanism to prevent the accumulation of unpolymerized flagellin subunits. Two strains contain deletions that appear to remove a region necessary for this regulation. The hook protein does not seem to be subject to this type of regulation and, in addition, appears to be synthesized as a faster-sedimenting precursor. Mutations in a number of genes result in the appearance of degradation products of either the flagellin or the hook proteins. Mutations in flaA, -X, -Y, or -Z result in the production of filaments (stubs) that contain altered ratios of the flagellin proteins. In some flaA mutants, other flagellin-related proteins were assembled into the stub structures in addition to the flagellins normally present. Taken together, these analyses have begun to provide insight into the roles of individual fla genes in flagellum biogenesis in C. crescentus.     

Location of epitopes on Campylobacter jejuni flagella.

Flagella were isolated from strains of Campylobacter jejuni belonging to different heat-labile serogroups and from a strain of Campylobacter fetus, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the flagellin molecular weights (Mr) were approximately 62,000. The flagellins were cleaved by hydrolysis with cyanogen bromide, and sodium dodecyl sulfate-urea peptide gel electrophoresis showed that the C. jejuni flagellins were structurally similar, and differed from C. fetus flagellin. Immunochemical analysis by Western blotting, enzyme-linked immunosorbent assay, immune electron microscopy, and immunoprecipitation with polyclonal and monoclonal antibodies revealed the presence of both internal and surface-exposed epitopes. The internal epitopes were antigenically cross-reactive and linear, and in the case of C. jejuni flagellin were located on cyanogen bromide peptides of apparent Mr 22,400 and 11,000. Antigenically cross-reactive epitopes were also present on an Mr 43,000 cyanogen bromide peptide of C. fetus flagellin. The Mr 22,400 peptide of C. jejuni VC74 flagellin also carried closely positioned internal linear epitopes for two monoclonal antibodies. One epitope was strain specific, while the other was shared by some but not all Campylobacter flagellins. The flagella of C. jejuni VC74 also displayed both surface-exposed antigenically cross-reactive and surface-exposed serospecific epitopes. Both linear and conformational epitopes contributed to the serospecificity of C. jejuni VC74 flagella, and a linear serospecific epitope was located on a cyanogen bromide peptide of apparent Mr 4,000.     

A new type of flagellin gene in Pseudomonas putida

Previously established PCR amplification and Southern hybridization procedures were developed for the isolation of the 0.8-kb flagellin gene in Pseudomonas putida. The deduced protein sequence has significant homology to the N- and C-terminal sequences of other bacterial flagellins. We propose that P. putida flagellin genes can be divided at least into three size groups: type I (2.0 kb), type II (1.4 kb), and type III (0.8 kb). Type I and type II flagellin genes have been reported. The new 0.8-kb type III gene was expressed in E. coli, and the resulting protein was purified and used to raise polyclonal antibody to study whether this small gene encodes flagellin. The antiserum reacted with purified flagellin monomers from representatives of each flagellin type, as well as proteins of the same sizes in lysates of these organisms, on Western immunoblots. This antiserum was determined to be functional in a motility inhibition assay. Similar results were obtained from antiserum directed against purified type III flagellin, indicating that a new type of flagellin gene in P. putida has been found. Preliminary electron microscopic study revealed that P. putida isolate with the smaller flagellin gene type appeared to have a thinner flagellar filament.     

Analysis of the flagellin (hag) gene of alkalophilic Bacillus sp. C-125.

Motility of the alkalophilic Bacillus sp. C-125, a flagellate bacterium, was demonstrated to be Na(+)- and pH-dependent. Flagellin protein from this strain was purified to homogeneity and the N-terminal sequence determined. Using the hag gene of Bacillus subtilis as a probe, the hag gene of Bacillus sp. C-125 was identified and cloned into Escherichia coli. Sequencing of this hag gene revealed that it encodes a protein of 272 amino acids (M(r) 29,995). The predicted N terminal sequence of this protein was identical to that determined by N-terminal sequencing of the flagellin protein from strain C-125. The alkalophilic Bacillus sp. C-125 flagellin shares homology with other known flagellins in both the N- and C-terminal regions. The middle portion, however, shows considerable differences, even from that of flagellin from the related species, B. subtilis.     

Cloning and Comparison of fliC Genes and Identification of Glycosylation in the Flagellin of Pseudomonas aeruginosa a-Type Strains

Pseudomonas aeruginosa a-type strains produce flagellin proteins which vary in molecular weight between strains. To compare the properties of a-type flagellins, the flagellin genes of several Pseudomonas aeruginosa a-type strains, as determined by interaction with specific anti-a monoclonal antibody, were cloned and sequenced. PCR amplification of the a-type flagellin gene fragments from five strains each yielded a 1.02-kb product, indicating that the gene size is not likely to be responsible for the observed molecular weight differences among the a-type strains. The flagellin amino acid sequences of several a-type strains (170018, 5933, 5939, and PAK) were compared, and that of 170018 was compared with that of PAO1, a b-type strain. The former comparisons revealed that a-type strains are similar in amino acid sequence, while the latter comparison revealed differences between 170018 and PAO1. Posttranslational modification was explored for its contribution to the observed differences in molecular weight among the a-type strains. A biotin-hydrazide glycosylation assay was performed on the flagellins of three a-type strains (170018, 5933, and 5939) and one b-type strain (M2), revealing a positive glycosylation reaction for strains 5933 and 5939 and a negative reaction for 170018 and M2. Deglycosylation of the flagellin proteins with trifluoromethanesulfonic acid (TFMS) confirmed the glycosylation results. A molecular weight shift was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis for the TFMS-treated flagellins of 5933 and 5939. These results indicate that the molecular weight discrepancies observed for the a-type flagellins can be attributed, at least in part, to glycosylation of the protein. Anti-a flagellin monoclonal antibody reacted with the TFMS-treated flagellins, suggesting that the glycosyl groups are not a necessary component of the epitope for the human anti-a monoclonal antibody. Comparisons between a-type sequences and a b-type sequence (PAO1) will aid in delineation of the epitope for this monoclonal antibody.